Novel antibiotics for the treatment of Gram-positive bacterial infections

Article abstract of DOI:10.1021/jm0111191

The natural dipeptide antibiotic TAN 1057 A,B displays excellent antibacterial activity against staphylococci including methicillin resistant Staphylococcus aureus. However, the in vitro activity against additional Gram-positive strains, in particular pne

Full text of DOI:10.1021/jm0111191

4246
J . Med. Chem. 2002, 45, 4246-4253
Novel An tibiotics for th e Tr ea tm en t of Gr a m -P ositive Ba cter ia l In fection s
Michael Brands,† Rainer Endermann,† Reinhold Gahlmann,† J ochen Kru¨ger,*,† Siegfried Raddatz,†
J u¨rgen Stoltefuâ,† Vladimir N. Belov,‡ Shamil Nizamov,‡ Viktor V. Sokolov,‡ and Armin de Meijere*,‡
Business Group Pharma, Research, BAYER AG, Wuppertal, D-42096, Germany, and Institut fu¨r Organische Chemie,
Georg-August-Universita¨t Go¨ttingen, Tammannstrasse 2, D-37077, Go¨ttingen, Germany
Received December 10, 2001; Revised Manuscript Received May 16, 2002
The natural dipeptide antibiotic TAN 1057 A,B displays excellent antibacterial activity against
staphylococci including methicillin resistant Staphylococcus aureus. However, the in vitro
activity against additional Gram-positive strains, in particular pneumococci and Enterococcus
faecalis, proved to be considerably lower. We report the synthesis and pharmacological
evaluation of new derivatives of this natural product that displayed increased antibacterial
potency against staphylococci and were also active against pneumococci. In particular, the
analogues bearing modified â-homoarginine side chains with methylated guanidine moieties
were shown to be significantly more potent than the natural product TAN 1057 A,B.
In tr od u ction
The emergence and spread of bacterial resistance
represent a severe global problem.1 The escalating
resistance has led to the appearance of multiresistant
staphylococci, enterococci, and pneumococci in nosoco-
F igu r e 1. Natural dipeptide antibiotic TAN 1057 A,B.
mial- and community-acquired infections. Therefore,
there is an urgent need for novel chemical entities that
are particularly effective against Gram-positive patho-
gens including the multiresistant strains.
Ta ble 1. MIC Values against S. aureus 133 and IC50 Values
for Prokaryotic Translation for Compounds 4a -c, 8a -b, 14,
and 18
Natural products have always been a valuable source
for the identification of new antibacterial entities.2 A
natural antibiotic that has received only moderate
attention in the past years is the dipeptide TAN 1057
A,B (1) (Figure 1), which was first isolated and char-
acterized by researchers at the Takeda company.3 The
natural product is an inhibitor of the bacterial transla-
tion,3a and it displays excellent minimal inhibitory
concentrations (MIC) against staphylococci including
methicillin resistant Staphylococcus aureus strains
(MRSA).3
Moreover, a promising in vivo activity against S.
aureus was observed in a murine sepsis model after ip
or sc administration of the natural product.3 However,
it proves to be less active in vitro against Enterococcus
feacalis and pneumococci.3a Our own results as depicted
in Table 2 confirm these results for TAN 1057 A,B.4
Additionally, TAN 1057 A,B suffers from toxic side
effects (LD50 in the mouse: 50 mg/kg, ip and 100 mg/
kg, iv),3 which are in our view prohibitive for the
therapeutic use in humans.
MIC, S. aureus
133 (µg/mL)
IC50, prokaryotic
translation (µM)
compd
R1
R2
4a
4b
4c
8a
8b
14
18
MeNH
EtNH
i-PrNH
Me
i-Pr
H2N
H
H
H
H
H
Me
Me
0.8
6.3
12.5
0.2
3.2
0.025
0.4
0.5
12.5
12.5
0.1
not determined
0.06
0.4
MeNH
Ch em istr y
Two independent total syntheses of TAN 1057 A,B
have been disclosed to date6 setting the stage for a facile
construction of analogues. Primarily, we focused our
attention on alterations of the â-amino acid side chain
with a special emphasis on modified guanidine deriva-
tives. In this regard, a convergent synthesis strategy
that allowed the introduction of a modified guanidine
or amidine at a late stage in the process was elaborated.
As illustrated in Scheme 1, the synthesis commenced
with orthogonally protected â-lysine 2,7 which was
coupled with the heterocyclic core 38 using a cocktail of
water soluble N-(3-dimethylaminopropyl)-N′-ethylcar-
bodiimide (EDC) and N-hydroxybenzotriazole (HOBt) as
the coupling reagent. The coupling product from this
reaction could conveniently be purified by crystalliza-
tion. Subsequently, the BOC group was removed under
acidic conditions to yield the amine hydrochloride salt
5 in quantitative yield.
The promising antibiotic activity and the unique
molecular architecture of TAN 1057 A,B prompted us
to launch a chemical optimization program.5 Our goal
was to identify analogues that are better tolerated and
display improved antibiotic activity toward a broader
panel of Gram-positive pathogens including enterococci
and pneumococci.
* To whom correspondence should be addressed. J .K.: Tel: +49 202
368644. Fax: +49 202 364061. E-mail: joachim.krueger.jk2@
bayer-ag.de. A.M.: Tel: +49 551 393231. Fax: +49 551 399475.
E-mail: Armin.deMeijere@chemie.uni-goettingen.de.
† BAYER AG.
‡ Georg-August-Universita¨t Go¨ttingen.
10.1021/jm0111191 CCC: $22.00 © 2002 American Chemical Society
Published on Web 08/10/2002

Novel Antibiotics for Gram-Positive Bacterial Infections
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 19 4247
Ta ble 2. MIC (µg/mL) for TAN 1057 A,B and Its Analogues 4a , 8a , 14, and 18 against a Panel of Gram-Positive Pathogens
compd
TAN-1057
4a
8a
14
18
R1
R2
R1
R2
H
R1
R2
H
R1
R2
R1
R2
bacterial strains
H
H
MeNH
Me
H2N
Me
MeNH
Me
S. aureus 133
0.25
0.25
8
2
>16
16
0.8
0.13
4
16
64
16
0.2
0.2
1.0
16
>64
16
0.025
0.12
0.5
2
64
4
0.5
0.5
8
8
>64
32
S. epidermis 18570
Str. Pyogenes Wacker
E. faecium L4001
E. faecalis 18531
S. pneumoniae G9a
Sch em e 1a
Sch em e 2a
a
Reagents and conditions: (a) EDC, HOBt, 60%. (b) 4 M HCl
in dioxane, quant. (c) Compound 6a , diisopropyl ethylamine
(DIPEA), 50%. (d) Compound 6b or 6c, HgCl2, Et3N, DMF, 36-
51%. (e) PdCl2, H2, MeOH, 82-100%. (f) Compound 7, DIPEA, 44-
57%.
a
Reagents and conditions: (a) 4 M HCl in dioxane, 100%. (b)
CF3CO2Me, MeOH, Et3N, 81%. (c) t-BuOAc, 70% aqueous HClO4.
(d) MeI, Cs2CO3, DMF, 80 °C, 50 h or K2CO3, TBAB, acetone,
reflux, 100 h, 97%. (e) 1 M LiOH, MeOH, 0 °C, 95%. (f) Ether, 4
M HCl in dioxane, 73%. (g) N,N′-Bis-Z-1-amidinopyrazole, DIPEA,
CH2Cl2, reflux. (h) TFA, Et3SiH, CH2Cl2, 0 °C. (i) Compound 3,
HATU, DIPEA, DMF, 82%. (j) H2/PdCl2, MeOH, 95%.
The methylguanidine moiety was installed by treat-
ment of 5 with N-methylated bis-Z-S-methylisothiourea
6a in the presence of an excess of diisopropylethylamine
at 60 °C.9 The yield for this transformation was moder-
ate (50%) due to the inherent instability of the hetero-
cycle toward basic reaction conditions at elevated tem-
peratures. For the use of sterically more encumbered
S-methylthioureas 6b,c, the reaction even failed to
furnish the corresponding guanidines. In these cases,
the use of HgCl2 as promoter for the guanylation proved
to be beneficial, and the desired intermediates were
obtained in acceptable yields (36% for 6b and 51% for
6c).10 At the final step of the synthesis, all Z groups
were removed by hydrogenation in MeOH using PdCl2,
and the target molecules 4a -c were obtained as a 1:1
mixture of diastereomers in good yields (82-100%).11
to provide the target molecules 8a ,b in 47 and 30% yield
(for two steps), respectively.
In TAN 1057 A,B, the â-homoarginine side chain
represents a key structural element. A related â-amino
acid motif is found in the naturally occurring antibiotic
Blasticidin S, which bears a Nδ-methyl-â-arginine side
chain and acts as an inhibitor of protein synthesis.12 In
analogy, we considered decorating TAN 1057 A,B with
an Nꢀ-methyl-â-homoarginine amino acid fragment
exploiting the structural and functional resemblance
with Blasticidin. For the synthesis of such a arginine
analogue with a methyl group on the proximal guani-
dine nitrogen, the initial strategy had to be extended
(Schemes 2 and 3).13
In close analogy to the derivatives with substituted
guanidine moieties, the corresponding amidine deriva-
tives of TAN 1057 were prepared starting from 5 using
S-methylthioamides as electrophiles. The amidine for-
mation as depicted in Scheme 1 worked with reasonable
yields, and the resulting intermediates were deprotected
Starting from the same orthogonally protected â-ly-
sine 2,7 the terminal tert-butoxycarbonyl protecting
group was changed to a trifluoroacetyl residue,14 and

4248 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 19
Brands et al.
Sch em e 3a
Compound 12 was further used as a precursor for
ester 15 with two methyl groups on the guanidine
moiety (Scheme 3). A Mitsunobu protocol may be used
for the methylation of the urethane-protected NH
groups in guanidines.19 It turned out, however, that the
separation of the resulting ester 15 from the byproduct
triphenylphosphine oxide by chromatography proved to
be tedious, and it was difficult to obtain the ester 15 in
a pure form. Therefore, the previously described ap-
proach for the methylation of trifluoroacetylamine 10,
namely, a 2.3-fold excess of MeI and 2 equiv of K2CO3
in the presence of catalytic amounts of TBAB in
anhydrous acetone, was used. In this case, the methy-
lation reaction proceeded faster than for compound 10,
and the reaction was complete in 2 days. The ester 15
was purified by chromatography and deprotected, and
the acid 16 was coupled with heterocycle 3. Finally,
hydrogenolytic cleavage of the Z groups from the
coupling product 17 delivered the double-methylated
guanidine derivative 18 in 65% yield.
a
Reagents and conditions: (a) MeI, K2CO3, Bu4NBr, acetone,
Resu lts a n d Discu ssion
60 °C, 2 d. (b) TFA, Et3SiH, CH2Cl2, 0 °C. (c) Compound 3, HATU,
DIPEA, DMF. (d) H2/PdCl2, MeOH.
All synthesized TAN 1057 A,B analogues were tested
for their antibacterial properties employing a prokary-
otic translation assay (cf. Table 1). In this regard, we
have used a Gram-negative transcription-translation
assay based on Escherichia coli since a well-established
Gram-positive in vitro translation assay is not available.
Nevertheless, we consider our IC50 values in Table 1 as
meaningful because the translation machineries of
Gram-positive and Gram-negative bacteria have been
highly conserved during evolution as indicated by the
large number of available broad spectrum translation
inhibitors.
Furthermore, we determined the MIC values of all
TAN 1057 A,B analogues against S. aureus 133.
In general, the data presented in Table 1 show that
a good activity in the translation assay translates into
a good MIC for the given compound. In turn, less active
inhibitors of the bacterial translation display a reduced
efficacy in the whole cell MIC assay. From these
observations, we conclude that antibacterial activity of
our analogues refers to an inhibition of the bacterial
translation confirming the results reported by the
Takeda group for TAN 1057 A,B.3a
More important, a comprehensive structure-activity
relationship (SAR) can be extracted from the data shown
in Table 1. When a substituent is introduced at the
terminal guanidine nitrogen, the antibacterial activity
decreases rapidly with the size of the substituent.
Merely the terminal methylation led to a highly active
analogue 4a , whereas for compounds bearing slightly
larger groups such as ethyl (4b) or isopropyl (4c) a
drastic loss in activity was observed.
Interestingly, the guanidine moiety can be replaced
by an amidine without loss of the biological activity
(compounds 8a ,b). Thus, a guanidine is not essential
for activity and may be replaced by other basic sur-
rogates. In parallel to the SAR of the guanidine deriva-
tives, lower activities were found for substrates with
sterically more demanding groups such as isopropyl in
the amidine series (compound 8b).
subsequently, the carboxyl function in compound 9 was
blocked as a tert-butyl ester. For the latter step, several
methods were tried out, but reasonably good yields
(81%) were achieved only upon treatment of the acid
with tert-butyl acetate in the presence of 70% aqueous
HClO4.15 Total conversion of the starting material 9 was
achieved when this operation was performed twice, and
the reisolated unreacted acid was recycled. The trifluo-
roacetylamino group in the protected â-amino acid ester
10 was selectively methylated by treatment with methyl
iodide in the presence of either Cs2CO3 in dimethyl
formamide (DMF) at 80 °C or K2CO3 in refluxing
acetone.16 Both procedures gave good yields of the crude
product 10-Me; however, an impurity containing a Nâ-
MeZ-group resulting from 2-fold methylation was some-
times detected (up to 7%). Because 10-Me was obtained
as an oil, an additional purification step was necessary.
Subsequently, the trifluoroacetyl group was removed
and the amino ester 11 was converted into its hydro-
chloride salt, which was purified by recrystallization.
An impurity containing two methyl groups was easily
removed by a single crystallization. Several guanidiny-
lation agents were scrutinized to obtain a high yield of
the protected guanidino ester 12. In our hands, the best
yield (87%) was achieved with N,N′-bis-Z-1-amidinopy-
razole17 in dichloromethane in the presence of (i-Pr)2NEt
under reflux. Subsequently, liberation of the carboxylic
acid was performed under mild conditions employing
trifluoroacetyl (TFA) and Et3SiH. The latter reagent has
been reported to facilitate the cleavage of tert-butyl
esters.18 Indeed, without addition of Et3SiH, one of the
two terminal Z groups was partially cleaved off by TFA.
Finally, tris-Z-Nꢀ-methyl-â-(S)-arginine 13 was coupled
with the heterocycle 3 of TAN-1057 A,B, and after it
was deprotected, the dipeptide 14 was obtained. Its
structure was confirmed by NMR spectroscopy. Un-
equivocal assignment of all signals, including those of
the quaternary carbon atoms, was based on the regu-
larities found in the two-dimensional (2D) 13C-1H cor-
relation spectroscopy (COSY) and COLOC spectra.
The most active analogues were those with a methyl
group at the proximal guanidine nitrogen. Accordingly,

Novel Antibiotics for Gram-Positive Bacterial Infections
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 19 4249
calibrated against tetramethylsilane as an internal standard
(δ ) 0) or the signals of the residual protons of deuterated
solvents: 7.26 for CHCl3, 2.50 for [D6]DMSO, and 3.30 for [D4]-
MeOH. Coupling constants (J ) are given in Hz. Electron impact
mass spectrometry (EI-MS) were recorded on Finnigan MAT
95 and Varian CH 5 spectrometers (70 eV). High-resolution
compounds 14 and 18 effectively inhibit the bacterial
translation process and, moreover, they exhibit excellent
MIC values against S. aureus 133. For further assess-
ment, the most active TAN 1057 A,B derivatives were
tested for their activity toward a broader panel of Gram-
positive pathogens. In this regard, MIC values against
a range of Gram-positive bacteria including Enterococ-
cus faecium, E. faecalis, and Streptococcus pneumoniae
were recorded (Table 2).
Derivatives 4a , 8a , and 18 showed similar antibiotic
activity against Gram-positive bacteria as the natural
product 1. In contrast, analogue 14 bearing a methyl
group at the proximal guanidine nitrogen displayed
significantly improved in vitro potency. In comparison
to the natural product, analogue 14 was superior in the
overall antibiotic activity, and more important, a drastic
increase in the in vitro potency against S. pneumoniae
was observed. Finally, compound 14 was tested in vivo
using a murine S. aureus sepsis model and an excellent
ED100 of e0.5 mg/kg (route iv) was found underscoring
the high antibacterial potency of derivative 14.
MS (HRMS) were aquired on
a Micromass LCT. High-
performance liquid chromatography (HPLC)-MS spectra were
recorded on a Hewlett-Packard 1100 (Micromass Platform
LCZ); UV detection was at 210 nm. HPLC-MS parameters:
column symmetry C18, 50 mm × 2.1 mm, 3.5 µm; eluent A,
MeCN + 0.1% HCOOH; B, 0.3 g of 30% aqueous HCl in 1 L of
H2O; 0-4 min, from 10 to 90% A; 4-6 min, 90% A, 0.5 mL/
min, 40 °C. HPLC, method A: Kromasil RP-18, 125 mm × 4
mm, eluent A: heptanesulfonic acid (WAT 084282 Fa. Waters)
low UV, 4 bottles/L H2O; B: MeCN; gradient, 0-1 min 90%
A; 1-9 min from 90% A to 90% B; 9-13 min 90% B; 2 mL/
min; room temperature. HPLC, method B: Kromasil RP-18,
50 mm × 2.1 mm, eluent A: MeCN + 0.1% HCOOH; B: H2O
+ 0.1% HCOOH; gradient, 0-4 min from 90% B to 90% A;
4-6 min 90% A; 0.5 mL/min; 40 °C. HPLC, method C:
Kromasil RP-18, 60 mm × 2.1 mm, eluent A: H2O + 5 mL
HClO4/L; B: MeCN; gradient, 0-4.5 min from 98% A to 90%
B; 4.5-6.5 min 90% B; 0.75 mL/min; 30 °C. Capillary electro-
phoresis (CE) was performed on an ALIGENT 3D CE device
with 64.5 cm capillary (50 µ). HP-CE chromatograms were
recorded with sodium citrate buffer (pH 4, 20 mM) + car-
boxymethyl-R-CD, 40 mM, CY-E-1006; 25 °C; voltage 20 kV;
detection 214 nm.
Con clu sion
A new class of antibiotics based on the natural
dipeptide antibiotic TAN 1057 A,B have been made
available by novel synthetic routes leading to analogues
with alterations at the terminal guanidine moiety. In
the course of this program, derivatives of the natural
product that exhibit good to excellent in vitro and in
vivo efficacy against a broad panel of Gram-positive
pathogens were identified. In particular, compound 14
was found to be a promising new candidate for the
treatment of bacterial infections caused by Gram-
positive pathogens. Further in-depth pharmacological
characterization of 14 is being conducted.
Analytical thin-layer chromatography (TLC) was performed
on Macherey-Nagel ready-to-use plates AluGram Sil G/UV254
.
Detection was under a UV lamp at 254 nm, and development
was with molybdatophosphoric acid solution (5% in EtOH) or
ninhydrin solution in butanol/glacial acetic acid mixture.
Elemental analyses were obtained at the Mikroanalytisches
Laboratorium des Instituts fu¨r Organische Chemie in Go¨ttin-
gen (Georg-August Universita¨t). Solvents were purified ac-
cording to standard procedures. Organic solutions were dried
over MgSO4, unless otherwise stated.
(3′S,5RS)-5-[N-(3-Ben zyloxyca r bon yla m in o-6-ter t-b u -
toxyca r bon yla m in o)h exa n oyl-N-m eth yla m in o]-5,6-d ih y-
d r o-2-u r eid op yr im id -4(1H)-on e (5-Boc). To a solution of
32.0 g (84.2 mmol) of Nâ-Z-Nꢀ-BOC-â-lysine (2) in 800 mL of
DMF was added 19.4 g (101 mmol) of EDC and 15.5 g (101
mmol) of HOBt (monohydrate), and the mixture was stirred
at room temperature for 1 h. Subsequently, 17.1 g (92.4 mmol)
of rac-3,4,5,6-tetrahydro-5-methylamino-2-ureidopyrimidin-4-
one (3) and 13.1 g (101 mmol) of N,N-diisopropylethylamine
were added, and the mixture was maintained at room tem-
perature for 16 h. To the reaction mixture was added 5.6 L of
water, and the aqueous layer was extracted three times with
2.1 L each of ethyl acetate. The combined organic layers were
washed twice with 1.6 L each of water. During the washing
process, the product precipitated. It was filtered off and washed
with 100 mL of cold methanol. The organic layer was dried,
and the solvent was removed in vacuo. The resulting oil was
treated with 640 mL of methanol, and the precipitated product
was filtered off and subsequently washed with 160 mL of
methanol followed by washing with 160 mL of diethyl ether.
From the filtrate, another crop of solids precipitated upon
standing. It was filtered and washed as described before. In
total, 28.0 g (60%) of the desired coupling product was isolated.
HPLC (method B): Rt 3.05 min, peak area 100%. 1H NMR (300
MHz, [D6]DMSO): δ 1.36 (s, 9 H), 1.40 (m, 4 H), 2.79 (m, 2
H), 2.89 (m, 2H), 3.12 (m, 3 H), 3.36-3.90 (m, 5 H), 5.00 (m,
3 H), 6.76 (m, 3 H), 7.12 (m, 1 H), 7.34 (m, 5 H). 13C NMR
(125 MHz, CDCl3/[D4]MeOH, 1:1): δ 176.5, 172.2, 172.1, 159.7,
157.3, 156.3 (2 C), 156.2, 136.5, 136.4, 128.4, 128.3, 128.0,
127.9, 127.8, 127.7 (2 C), 127.6, 79.2, 77.2, 66.4, 66.3, 52.3,
51.6, 48.1, 47.7, 39.8, 38.5, 38.3, 37.7, 33.7, 33.1, 31.5, 31.2,
28.2 (3 C), 26.5. HRMS (ESI) calcd for C25H36N7O7 [M+ - H],
546.2676; found, 546.2665.
Exp er im en ta l Section
P h a r m a cology. P r ok a r yotic Tr a n sla tion Assa y. The
inhibition of the bacterial translation was assayed by using
an E. coli transcription-translation assay with a luciferase
reporter as read out based on published and commercially
available methods.20
MIC Deter m in a tion . The MICs were determined by the
microdilution method using AOAC broth (Difco) supplemented
with 4% dextrose (staphylococci, enterococci, and Streptococcus
pyogenes) or 1:10 diluted BHI broth (Oxoid) supplemented with
10% bovine serum (S. pneumoniae). Microtiter plates contain-
ing the test compounds (concentration range: 0.125-64
µg/mL) were inoculated with 1:50 (streptococci, enterococci)
or 1:500 (staphylococci) dilutions of overnight cultures of the
test bacteria. Plates were incubated in the presence of 8-10%
CO2 at 37 °C and read after approximately 20 h. The MIC was
considered to be the first concentration with no visible growth.
System ic In fection w ith S. a u r eu s 133. Staphylococci
from an overnight culture were grown to the logarithmic
growth phase in BHI broth (Oxoid). The log-culture was
precipitated and washed twice with phosphate-buffered saline.
Bacteria were suspended in phosphate-buffered saline contain-
ing 5% mucin to a cell density of 4 × 106 cells/mL. Female
CFW1-mice (20 g) were infected with 0.25 mL of the bacterial
suspension. The test compounds were dosed iv at 30 min
postinfection. The survival in each treatment group was
monitored for 6 days.
Ch em ist r y. Gen er a l. Melting points (uncorrected) were
determined in capillaries using an apparatus manufactured
by Bu¨chi. Routine NMR spectra were recorded using Bruker
AM 250 (250 MHz), Varian UNITY 300 (300 MHz), Bruker
DRX400, or Bruker AC500 instruments. All spectra were
(3′S,5RS)-5-[N-(6-Am in o-3-b en zyloxyca r b on yla m in o-
h exan oyl)-N-m eth ylam in o]-5,6-dih ydr o-2-u r eidopyr im id-

4250 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 19
Brands et al.
4(1H)-on e Hyd r och lor id e (5). A solution of 31.0 g (56.7
mmol) of the coupling product in 566 mL of 4 M HCl in dioxane
was stirred at room temperature for 2.5 h. All volatile
components were evaporated in vacuo, and the resulting
product 5 (28.4 g, quantitative) was used without further
purification and characterization.
39.0, 37.9, 37.5, 31.9, 26.9, 16.0. HRMS (ESI) calcd for
C15H28N9O3 [M+ - H], 382.2315; found, 382.2321.
(3′S,5RS)-5-[N-[3-Ben zyloxyca r b on yla m in o-6-[2,3-d i-
(b en zyloxyca r b on yl)-2(3)-isop r op ylgu a n id in -1-yl]h ex-
a n oyl]-N-m et h yla m in o]-5,6-d ih yd r o-2-u r eid op yr im id -
4(1H)-on e (tr is-Z-4c). The synthesis was conducted in analogy
to the synthesis of tris-Z-4b on a 207 mg (0.428 mmol) scale
of 5 using 6c (171 mg, 0.428 mmol) as an electrophilic
component. The guanylation yielded 174 mg (51%) of tris-Z-
4c. HPLC (method C): Rt 4.40 min, peak area 100%. 1H NMR
(500 MHz, [D4]MeOH): δ 1.18-1.69 (m, 10 H), 2.50 (m, 2 H),
3.08 (m, 3 H), 3.25-3.40 (m, 2 H), 3.66 (m, 1 H), 3.80 (m, 1
H), 3.92 (m, 1 H), 3.90 (m, 1 H), 4.11 (m, 1 H), 4.96-5.24 (m,
(3′S,5RS)-5-[N-[3-Ben zyloxyca r b on yla m in o-6-[2,3-d i-
(ben zyloxycar bon yl)-2(3)-m eth ylgu an idin -1-yl]h exan oyl]-
N -m e t h yla m in o]-5,6-d ih yd r o-2-u r e id op yr im id -4(1H )-
on e (tr is-Z-4a ). A solution of 37.6 g (77.8 mnmol) of 5, 34.7 g
(93.2 mmol) of 6a , and 20.1 g (26.6 mL, 155 mmol) of N,N-
diisopropylethylamine in 710 mL of ethanol was heated to 60
°C for 16 h. The solvent was evaporated in vacuo, and the
residue was purified by flash chromatography using a gradient
of dichloromethane with increasing polarity to dichloromethane/
methanol (95:5) to yield 30.0 g (50%) of the desired tris-Z-4a .
HPLC (method A): Rt 6.08 min, peak area 92%. 1H NMR (200
MHz, CDCl3): 1.5 (m, 4 H), 2.60 (m, 2 H), 3.14 (s, 3 H), 3.39-
4.05 (m, 5 H), 5.01 (m, 1 H), 5.06-5.18 (m, 6 H), 7.31 (m, 15
H), 9.25 (m, 1 H), 10.34 (m, 1 H). HRMS (ESI) calcd for
7 H), 7.32 (m, 15 H). HRMS (ESI) calcd for C40H48N9O9 [M+
H], 798.3575; found, 798.3525.
-
(3′S,5RS)-5-[N-[3-Am in o-6-[2(3)-isop r op ylgu a n id in -1-
yl]h exa n oyl]-N-m et h yla m in o]-5,6-d ih yd r o-2-u r eid op y-
r im n id -4(1H)-on e Dih yd r och lor id e (4c). The removal of the
Z groups was conducted as described for 4a on a 50 mg (0.07
mmol) scale of tris-Z-4c. The hydrogenation yielded 32 mg
(97%) of 4c. HPLC (method A): Rt 4.64 min, peak area 100%.
HP-CE: injection time 20 s; Rt 24.91 and 26.19 min (two
diastereomers), peak areas 51.66 and 48.34%, respectively
C
38H44N9O9 [M+ - H], 770.3262; found, 770.3254.
(3′S,5RS)-5-[N-[3-Am in o-6-[2(3)-m eth ylgu a n id in -1-yl]-
h exan oyl]-N-m eth yylam in o]-5,6-dih ydr o-2-u r eidopyr im id-
4(1H )-on e Dih yd r och lor id e (4a ). A solution of tris-Z-4a
(13.8 g, 17.9 mmnol) and 6.34 g (35.8 mmol) of palladium(II)
chloride in 700 mL of methanol (4 L flask) was stirred in a
hydrogen atmosphere for 1 h. Because TLC (dichloromethane/
methanol, 9:1) indicated complete conversion, the reaction
mixture was filtered through a plug of Celite, and it was
washed thoroughly with methanol. The filtrate was concen-
trated, and the crude product was treated with acetone. The
solid was filtered off (D2-frit) and dried in vacuo to yield 8.3 g
(quantitative) of 4a . HPLC (method A): Rt 3.52 min, peak area
98%. HPLC (method C): Rt 0.70 min, peak area 97%. HP-CE:
injection time 8 s; Rt 17.90 and 18.77 min (two diastereomers),
1
(100% in total). H NMR (400 MHz, [D4]MeOH): δ 1.23 (d, 6
H), 1.75 (m, 4 H), 2.76 (dd, 1 H), 2.97 (dt, 1 H), 3.13 (s, 3 H),
3.26 (m, 2 H), 3.60 (mn, 1 H), 3.72 (m, 1 H), 3.88 (m, 1 H),
4.01 (dt, 1 H), 5.19 (m, 1 H). 13C NMR (125 MHz, D2O): δ
172.8, 167.6, 155.1, 152.8, 54.6, 49.2, 48.5, 44.2, 40.8, 38.9, 35.4,
35.1, 29.5, 24.5, 21.8 (2C). HRMS (ESI) calcd for C16H30N9O3
[M+ - H], 396.2472; found, 396.2508.
(3′S,5RS)-5-[N-[6-Acetim id oyla m in o-3-(ben zyloxyca r -
b on yla m in o)h exa n oyl]-N-m et h yla m in o]-5,6-d ih yd r o-2-
u r eid op yr im id -4(1H)-on e (Z-8a ). A solution of 500 mg (1.03
mmol) of 5, 175 mg (1.39 mmol) of 7a , and 400 mg (3.10 mmol)
of N,N-diisopropylethylamine in 10 mL of ethanol was stirred
at 40 °C for 16 h. The solvent was evaporated in vacuo, and
the crude product was purified by flash chromatography on
silica gel using dichloromethane/methanol/concentrated aque-
ous NH3 (85:15:3) to yield 287 mg (57%) of the desired product
Z-8a . HPLC (method C): Rt 3.4 min, peak area 94%. 1H NMR
(500 MHz, [D4]MeOH): δ 1.51-1.73 (m, 4 H), 2.20 (m, 3 H),
2.64 (m, 2 H), 3.02 (m, 3 H), 3.27 (m, 2 H), 3.49 (m, 1 H), 3.68
(m, 2 H), 4.02 (m, 1 H), 5.06 (m, 3 H), 7.31 (m, 5 H), 8.45 (s, 1
H). HRMS (ESI) calcd for C22H31N8O5 [M+ - H], 487.2417;
found, 487.2431.
1
peak areas 48.85 and 48.93%, respectively (98% in total). H
NMR (400 MHz, [D4]MeOH): δ 1.70 (m, 4 H), 2.72-2.94 (m,
2 H), 2.84 (s, 3 H), 3.14 (s, 3 H), 3.23 (m, 2 H), 3.61 (m, 1 H),
3.90 (m, 1 H), 4.02 (dt, 1 H), 5.15 (m, 1 H). 13C NMR (125 MHz,
CDCl3/[D4]MeOH, 1:1): δ 173.2, 173.1, 167.2, 157.9, 157.0,
155.5, 153.9, 55.6, 55.4, 41.9, 40.2, 36.1, 35.9, 30.6, 28.5, 25.7.
HRMS (ESI) calcd for C14H26N9O3 [M+ - H], 368.2159; found,
368.2169.
(3′S,5RS)-5-[N-[3-Ben zyloxyca r b on yla m in o-6-[2,3-d i-
(ben zyloxyca r bon yl)-2(3)-eth ylgu a n id in -1-yl]h exa n oyl]-
N -m e t h yla m in o]-5,6-d ih yd r o-2-u r e id op yr im id -4(1H )-
on e (tr is-Z-4b). A solution of 300 mg (0.780 mmol) of
S-methylisothiourea 6b, 376 mg (0.778 mmol) of 5, 231 mg
(0.85 mmol) of HgCl2, and 471 mg (4.66 mmol) of triethylamine
in 4 mL of DMF was stirred at room temperature for 72 h.
The solvent was evaporated in vacuo, and the residue was
redissolved in dichloromethane and filtered through a plug of
Celite. The solvent was evaporated, and the crude product was
purified by flash chromatography on silica gel (dichloromethane/
methanol/concentrated aqueous NH3, 90:10:1) to yield 225 mg
(36%) of the desired tris-Z-4b. HPLC (method C): Rt 4.30 min,
(3′S,5RS)-5-[N-(6-Acetim idoylam in o-3-am in oh exan oyl)-
N -m e t h yla m in o]-5,6-d ih yd r o-2-u r e id op yr im id -4(1H )-
on e Dih yd r och lor id e (8a ). The removal of the Z group was
conducted as described for 4a on a 66 mg (0.14 mmol) scale of
Z-8a . The hydrogenation yielded 47 mg (82%) of 8a . HPLC
(method A): Rt 4.36 min, peak area 94%. HP-CE: injection
time 8 s; Rt 16.45 and 17.21 min (two diastereomers), peak
areas 55.39 and 44.61%, respectively (100% in total). 1H NMR
(400 MHz, [D4]MeOH): δ 1.81 (m, 4 H), 2.26 (s, 3 H), 2.80
(dd, 1 H), 2.99 (m, 1 H), 3.17 (s, 3 H), 3.32 (m, 2 H), 3.62 (m,
2 H), 3.90 (m, 1 H), 4.04 (m, 1 H), 5.18 (m, 1 H). 13C NMR
(125 MHz, D2O): δ 175.2, 169.8, 167.6, 157.4, 155.0, 57.1, 50.8,
44.2, 41.3, 37.9, 37.5, 32.0, 25.6, 21.1. HRMS (ESI) calcd for
1
peak area 93%. H NMR (CDCl3, 500 MHz): δ 1.18 (m, 3 H),
1.55 (m, 4 H), 2.57 (m, 2 H), 2.92 (m, 3 H), 3.12 (m, 2 H), 3.22
(m, 2 H), 3.39-3.65 (m, 3 H), 3.71 (m, 2 H), 3.90 (m, 1 H),
5.04-5.26 (m, 7 H), 5.80 (m, 1H), 7.25-7.40 (m, 15 H), 9.19
(m, 1 H), 10.28 (m, 1 H). HRMS (ESI) calcd for C39H46N9O9
[M+ - H], 784.3419; found, 784.3412.
C
14H25N8O3 [M+ - H], 353.2050; found, 353.2020.
(3′S,5R,S)-5-[N-[3-Ben zyloxyca r b on yla m in o-6-(isob u -
tyr im idoylam in o)h exan oyl]-N-m eth ylam in o]-5,6-dih ydr o-
2-u r eid op yr im id -4(1H)-on e (Z-8b). The synthesis was con-
ducted in analogy to the synthesis of Z-8a with 400 mg (0.83
mmol) of 5 using 7b as an electrophilic component. The
reaction yielded 190 mg (44%) of Z-8b. HPLC (method C): Rt
(3′S,5RS)-5-[N-[3-Am in o-6-[2(3)-eth ylgu an idin -1-yl]h ex-
a n oyl]-N-m et h yla m in o]-5,6-d ih yd r o-2-u r eid op yr im id -
4(1H)-on e Dih yd r och lor id e (4b). According to the procedure
described for 4a , 109 mg (0.14 mmol) of tris-Z-4b was depro-
tected to yield 63 mg (99%) of the desired product 4b. HPLC
(method A): Rt 4.54 min, peak area 98%. HP-CE: injection
time 8 s; Rt 21.24 and 22.21 min (two diastereomers), peak
areas 48.65 and 51.35%, respectively (100% in total). 1H NMR
(500 MHz, D2O): δ 1.19 (t, 3 H), 1.74 (m, 4 H), 2.85 (m, 1 H),
3.02 (m, 1 H), 3.14 (s, 3 H), 3.23 (m, 4 H), 3.68 (m, 1 H), 4.00
(m, 2 H), 5.11 (m, 1 H). 13C NMR (125 MHz, CDCl3/[D4]MeOH,
1:1): δ 175.2, 169.7, 158.2, 157.4, 154.9, 57.0, 50.9, 43.1, 41.3,
1
3.57 min, peak area 79%. H NMR (500 MHz, [D4]MeOH): δ
1.25 (m, 6 H), 1.68 (m, 4 H), 2.56-2.82 (m, 2 H), 3.02 (m, 3
H), 3.26 (m, 2 H), 3.49 (m, 1 H), 3.69 (m, 2 H), 4.02 (m, 1 H),
4.91-5.11 (m, 3 H), 7.31 (m, 5H). HRMS (ESI) calcd for
C
24H35N8O5 [M+ - H], 515.2730; found, 515.2643.
(3′S,5R,S)-5-[N-(3-Am in o-6-isobu tyr im id oyla m in oh ex-
a n oyl)-N-m et h yla m in o]-5,6-d ih yd r o-2-u r eid op yr im iid -
4(1H)-on e Dih yd r och lor id e (8b). The removal of the Z
group was conducted as described for 4a on a 130 mg (0.25

Novel Antibiotics for Gram-Positive Bacterial Infections
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 19 4251
mmol) scale of Z-8b. The hydrogenation yielded 95 mg (83%)
of 8b. HPLC (method A): Rt 4.52 min, peak area 100%. HP-
CE: injection time 20 s; Rt 20.01 and 21.01 min (two diaster-
eomers), peak areas 47.32 and 45.87%, respectively (93% in
total). 1H NMR (400 MHz, D2O): δ 1.24 (d, 6 H), 1.76 (m, 4
H), 2.76 (m, 3 H), 2.92 (m, 1 H), 3.05 (m, 3 H), 3.31 (m, 3 H),
3.62 (m, 1 H), 3.74 (m, 2 H), 4.87 (m, 1 H). HRMS (ESI) calcd
for C16H29N8O3 [M+ - H], 381.2363; found, 381.2396.
(62.9 MHz, CDCl3): δ 170.8, 155.3 (q, J ) 37), 156.2, 136.3,
128.5, 128.2, 128.1, 116.3 (q, J ) 286), 81.5, 66.8, 47.4, 40.2,
39.4, 32.0, 27.3, 24.9. EI-MS m/z (rel intensity, %): 376 (31),
359 (10), 269 (11), 241 (9), 178 (14), 108 (39), 91 (100). DCI
(NH3)-MS m/z (rel intensity, %): 450 (M + NH4+, 31), 299
(100). Anal. (C20H27F3N2O5) C, H, N.
Nâ-Z-NE-m eth yl-NE-tr iflu or oa cetyl-(S)-â-lysin e ter t-Bu -
tyl Ester (10-Me). A mixture of the compound 10 (2.64 g, 6.11
mmol), Cs2CO3 (3.98 g, 12.2 mmol), and MeI (1.91 mL, 4.33 g,
30.5 mmol) in 30 mL of DMF was heated at 80 °C with rapid
stirring in a closed reactor for 50 h. (A round-bottom cylindrical
glass reactor with thick walls was used, volume 200 mL.)
Nâ-Ben zyloxyca r bon yl-NE-t r iflu or oa cet yl-(S)-â-lysin e
(9). To a suspension of Nâ-Z-Nꢀ-Boc-(S)-â-lysine (2) (30.4 g, 80
mmol) in 10 mL of anhydrous dioxane was added dropwise
with stirring 90 mL of 4 M HCl in dioxane at 20-25 °C. The
solution was kept at +5 °C overnight, the dioxane was
evaporated in vacuo, and the crystalline mass was washed
twice with anhydrous ether. The hydrochloride of Nâ-Z-(S)-â-
lysine was dried in vacuo and dissolved in 40 mL of anhydrous
MeOH, and triethylamine (33.2 mL, 24.2 g, 0.24 mol) was
added dropwise with stirring while the reaction flask was
cooled with ice water. The thick precipitate, which appeared
soon was gradually dissolved by adding methyl trifluoroacetate
(9.6 mL, 12.3 g, 96 mmol) at +5 °C. The reaction mixture was
warmed to room temperature and was stirred overnight. All
volatile materials were removed in vacuo, the semisolid residue
was dissolved in water (250 mL), and 20% aqueous HCl was
added to it with stirring and cooling until the pH value was
1-2. The precipitated product was extracted with ethyl acetate
(4 × 150 mL). The combined organic solutions were washed
with water (2 × 40 mL) and brine (100 mL), and then dried
and concentrated to yield a solid, which was recrystallized from
ethyl acetate/hexane mixture in two crops. The first crop (21.9
After it was cooled to ambient temperature, the reaction
mixture was filtered, and the filter cake was washed with ether
(200 mL). The combined liquids were filtered once more to
remove the precipitated salts, and the solution was concen-
trated in vacuo, first at about 10 Torr and then at 1 Torr with
a cold trap (-78 °C) and a warm water bath (<40 °C). The
residual oil was taken up in ether (250 mL), and the solution
was washed with water (3 × 30 mL) and brine (30 mL) and
dried. The TLC of the crude title compound displayed a single
spot with Rf 0.70 (EtOAc/hexane, 1:1), yield 2.7 g (100%). IR
(neat): 3338, 2978, 2941, 1694, 1530, 1454, 1368, 1247, 1146
1
cm-1. H NMR (250 MHz, CDCl3): δ 1.42 (s, 9 H), 1.50-1.63
(m, 4 H), 2.33-2.46 (m, 2 H), 2.94 and 3.05 (2s, total 3 H),
3.39-3.47 (m, 2 H), 3.96 (m, 1 H), 5.08 (s, 2 H), 5.38 (m, 1 H),
7.33-7.42 (m, 5 H). 13C NMR (62.9 MHz, CDCl3): δ (signals
of the major isomer are marked*) 170.6, 156.6 (q, J ) 35),
155.9, 136.5, 128.4, 128.1, 128.0, 116.4 (q, J ) 286), 81.4/81.3*,
66.64/66.56*, 49.0/48.9*, 47.8*/47.6, 40.3/40.2*, 34.6* (q, J )
3.8)/34.2, 31.5*/31.3, 27.9, 24.75, 23.1. The progress of the
methylation reaction was monitored by mass spectroscopy. The
product of the selective Nꢀ-methylation has two characteristic
peaks in the EI-MS: a more intensive one with m/z ) 390 (M+
- C4H8) and 373 (M+ - C4H8 - OH). For the starting material
10, they are found at m/z ) 376 and 359 and at for the
“overmethylated” product at 404 and 387. As a rule, when the
conversion is complete, the reaction mixture contains about
7% of the impurity with an NMeZ group.
20
g) with mp 122-124 °C was used in the next step; [R]D ) -
6.4° (c ) 1.05, EtOAc). Total yield, 24.5 g (81%). IR (KBr):
3307, 3108, 2955, 1700, 1542, 1456, 1213, 1183, 1069 cm-1
.
1H NMR (250 MHz, [D4]MeOH): δ 1.55-1.61 (m, 4 H), 2.46
(d, J ) 7.5, 2 H), 3.28 (m, 2 H), 3.95 (br. m, 1 H), 5.06 (s, 2 H),
7.28-7.33 (m, 5 H). 13C NMR (62.9 MHz, [D4]MeOH): δ 173.0,
158.7, 156.5 (q, J ) 35), 138.7, 129.7, 129.2, 129.0, 116.3 (q, J
) 286), 67.6, 49.5, 41.0, 40.7, 33.2, 26.8. HRMS (EI) calcd for
C
16H19F3N2O5, 376.1247; found, 376.1246.
Nâ-Ben zyloxyca r bon yl-NE-m eth yl-(S)-â-lysin e ter t-Bu -
tyl Ester Hyd r och lor id e (11). The crude methylation prod-
uct 10-Me (19.7 g, 44 mmol) was dissolved in MeOH (100
mnL), and a solution of LiOH‚H2O (2.1 g, 50 mmol) in MeOH
(150 mL) was added carefully at about +5 °C. The mixture
was allowed to warm to ambient temperature, and after 3 h,
it was evaporated in vacuo. The residue was dissolved in ether
(500 mL), washed with water (3 × 30 mL) and brine (50 mL),
and dried. Evaporation of the solvent provided 14.6 g of the
Nâ-Ben zyloxyca r bon yl-NE-t r iflu or oa cet yl-(S)-â-lysin e
ter t-Bu tyl Ester (10). Compound 9 (21.9 g, 58.2 mmol) was
suspended in t-BuOAc (830 mL), and 4.5 mL of 70% aqueous
HClO4 was added. Stirring in the closed reaction flask was
continued for 2 days at ambient temperature. The flask was
opened carefully, the solution was diluted with ether (1 L),
and the mixture was carefully washed with many portions of
saturated aqueous KHCO3, until no gas evolution was ob-
served, and TLC of the organic layer (CH2Cl2/MeOH/AcOH,
100:5:1) displayed no spot of 9 (Rf 0.15) any more. The
combined aqueous solutions were extracted once with ethyl
acetate (200 mL) and carefully acidified with 20% aqueous HCl
with cooling. Unreacted 9 was extracted with ethyl acetate (3
× 150 mL), and the combined organic layers were washed with
brine (100 mL) and dried. Evaporation of the solvent in vacuo
left 8.4 g of the crude acid 9. The organic solution that
contained ether was washed with brine and dried. Evaporation
of the solvents in vacuo yielded a two phase liquid residue.
The upper oily layer (diisobutylene) was decanted, and the
lower layer was triturated with pentane (150 mL), seeded with
10, and stirred. More pentane was added, when the crystal-
lization proceeded. The mixture was kept for 2 h at +5 °C,
and the colorless powder was collected on a filter, washed with
cold pentane, and dried to yield 14.1 g of 10 with mp 44-47
°C. The yield was 91% based on the converted acid 9. A second
run was conducted with the recovered starting material (8.4
g), and a second crop of 9 was obtained in the previous
experiment (2.6 g). t-BuOAc (700 mL) and 2.8 mL of 70%
aqueous HClO4 were used, and 2.8 g of unreacted acid 9 was
isolated; the yield of 10 was found to be 6.9 g (73% based on
1
free amine (95% crude yield). H NMR (250 MHz, CDCl3): δ
1.41 (s, 9 H), 1.53 (m, 4 H), 2.03 (br. s, 1 H), 2.39 (s, 3 H), 2.42
(d, J ) 5.8, 2 H), 2.56 (br. t, 2 H), 3.97 (br. s, 1 H), 5.06 (s, 2
H), 5.58 (br. s, 1 H), 7.32 (m, 5 H). 13C NMR (62.9 MHz,
CDCl3): δ 170.8, 155.8, 136.6, 128.4, 128.1, 127.9, 81.0, 66.4,
51.4, 48.1, 40.2, 36.1, 32.1, 28.0, 25.9. ESI-MS m/z, positive
ions, 351 (M+ + H, 100): 295 (38). The oil was dissolved in
anhydrous ether (200 mL), and 11 mL of 4 M HCl in dioxane
was slowly added at +5 °C with vigorous stirring. The
suspension was kept at +5 °C for 2 h, and the solid was filtered
off, washed with anhydrous ether, and dissolved in a minimal
amount of boiling 2-propanol. Anhydrous ether was added until
a precipitate began to appear, and then, the solution was left
overnight at room temperature. The salt 11 was collected on
a filter, washed with anhydrous ether (200 mL), and dried to
20
yield 11.8 g (73%) of hydrochloride 11 with mp 143 °C; [R]D
) -10.6° (c ) 1.15, MeOH). IR (KBr): 3343, 2948, 2867, 2793,
1719, 1694, 1539, 1267 cm-1. 1H NMR (250 MHz, [D4]MeOH):
δ 1.40 (s, 9 H), 1.53-1.79 (m, 4 H), 2.29-2.42 (m, 2 H), 2.64
(s, 3 H), 3.00 (m, 2 H), 3.96 (m, 1 H), 5.05/5.07 (AB-q, J )
12.5, 2 H), 7.27-7.34 (m, 5 H). 13C NMR (62.9 MIHz, [D4]-
MeOH): δ 172.4, 158.6, 138.6, 129.8, 129.3, 129.1, 82.3, 67.6,
50.2, 49.5, 42.5, 33.8, 33.2, 28.6, 24.0. Anal. (C19H31N2O4Cl)
C, H, N.
20
the reacted acid 9); [R]D ) -8.0° (c ) 1.03, EtOAc). IR
(KBr): 3336, 2944, 1701, 1681, 1534, 1165 cm-1. 1H NMR (250
MHz, CDCl3): δ 1.42 (s, 9 H), 1.52-1.64 (m, 4 H), 2.46 (t, J )
5.8, 2 H), 3.37 (m, 2 H), 3.96 (br. m, 1 H), 5.08 (s, 2 H), 5.37 (d,
J ) 9, 1 H), 7.10 (br. m, 1 H), 7.26-7.35 (m, 5 H). 13C NMR
Nâ,Nω,Nω′-Tr is(b en zyloxyca r b on yl)-NE-m et h yl-(S)-[â-
h om oa r gin in e ter t-Bu tyl Ester (12). A mixture of compound

4252 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 19
Brands et al.
11 (1.48 g, 3.83 mmol) and N,N′-bis-Z-1-guanidinylpyrazole17
(1.51 g, 3.98 mmol) in 10 mL of CH2Cl2 and 3 mL of Et3N was
heated overnight under reflux. The product was isolated by
chromatography on basic aluminum oxide (200 g, activity
grade I) eluting with CH2Cl2/MeOH/40% NH3 in MeOH (100:
1:0.5); Rf 0.75; yield 2.21 g (87%) of a glasslike foam. IR
(neat): 3330, 3033, 2941, 1723, 1615, 1498, 1455, 1404, 1368,
nitrogen and then with H2. Hydrogenation under normal
pressure was continued for 1.5 h, while the reaction mixture
was stirred vigorously, and a slow stream of hydrogen was
passed above the solution. The catalyst (coagulated Pd-black)
was removed by filtering the solution through Celite. The filter
cake was washed with anhydrous methanol (15 mL), and the
combined solutions were evaporated to dryness. The residue
was dissolved in MeOH (1.5 mL), anhydrous acetone (20 mL)
was added carefully with stirring, and the precipitated salt
was filtered off and washed with anhydrous ether to yield 60
mg of a hygroscopic colorless solid. HPLC-MS: Rt 0.29 min,
peak area 98%. HPLC (method A): Rt 3.52 min, peak area
97%. HP-CE: injection time 8 s; Rt 24.52 and 25.79 min (two
diastereomers), peak areas 51.46 and 46.88%, respectively
(98% in total). ESI-MS m/z: positive ions, 370 (M + H, 100);
1
1214, 1065 cm-1. H NMR (250 MHz, CDCl3): δ 1.41 (s, 9 H),
1.48 (m, 2 H), 1.67 (m, 2 H), 2.41 (t, J ) 6.5, 2 H), 2.98 (s, 3
H), 3.52 (m, 2 H), 3.98 (m, 1 H), 4.72 (s, 1 H), 5.06 (s, 2 H),
5.15 (s, 4 H), 5.41 (m, 1 H), 7.23-7.38 (m, 15 H). 13C NMR
(62.9 MHz, CDCl3): δ 170.7, 156.3, 156.0, 136.6, 128.5, 128.2,
128.0, 127.9, 127.4, 126.8, 81.0, 67.6 (br), 67.2, 66.5, 51.0 (br),
48.0, 40.5, 37.3 (br), 31.3, 28.0, 23.5. ESI-MS m/z: positive ions,
683 (M + Na, 100), 661 (M + H, 92); negative ions, 659 (M -
H, 100).
1
yield was calculated to be 95% (by 98% purity). H NMR (400
Nâ,Nω,Nω′-Tr is(ben zyloxyca r bon yl)-NE-m eth yl-(S)-â-h o-
m oa r gin in e (13). To a solution of the ester 12 (2.35 g, 3.56
mmol) and Et3SiH (1.03 g, 8.86 mmol) in 6 mL of CH2Cl2 was
added TFA (3.5 mL) carefully with stirring at + 5 °C. The
reaction mixture was left overnight at the same temperature
and concentrated in vacuo. The oily residue was washed with
anhydrous ether (3 × 30 mL with sonification followed by
decantation) and coevaporated with CH2Cl2 (3 × 30 mL). After
it was dried at 0.1 Torr overnight, 2.1 g of the crude title
product was obtained; Rf 0.20 (hexane/EtOAc/AcOH, 4:2:1).
The purity was found to be 80% (HPLC-MS) corresponding to
a yield of 78%. All attempts to obtain the crystalline dicyclo-
hexyl-ammonium salt failed. Purification by chromatography
on silica gel (100 g) eluting with a hexane/EtOAc/AcOH
mixture (1:1:1) was accompanied by losses: 1.27 g (59%) were
MHz, [D4]MeOH): δ 1.62 (br. s, 4 H, 4′/5′-H), 2.76 (dd, J )
8.8 and 17.5, 1 H, 2′-H), 2.97 (dt, J ) 2.7 and 17, 1 H, 2′-H),
3.04 (s, 3 H, 6′-NMe), 3.12 (s, 3 H, CONMe), 3.38 (br. s, 2 H,
6′-H), 3.60 (br.s, 1 H, 3′-H), 3.87 (m, 1 H, 6-H), 4.02 (m, 1 H,
6-H), 5.14 (m, 1 H, 5-H). 13C NMR (400 MHz, [D4]MeOH, two
rotamers, ratio ca. 1:1): δ 173.3 (C-1′), 173.4 (C-1′), 168.0
(C-2), 158.5 (C-guanidino), 155.2 (C-ureido), 154.2 (C-4), 55.5
(C-5), 51.0 (C-6′), 49.0 (C-3′), 40.2 (C-6), 36.8 (C-6′-N-Me), 36.1
(C-2′), 35.6 (C-2′), 35.4 (CONMe), 30.6 (C-5′), 30.5 (C-5′), 24.0
(C- 4′).
Nâ,Nω,Nω′-Tr is(ben zyloxyca r bon yl)-NE,Nω-d im eth yl-(S)-
â-h om oa r gin in e ter t-Bu tyl Ester (15). Dry, finely powdered
K2CO3 (362 mg, 2.62 mmol) was added to a solution of the ester
12 (866 mg, 1.31 mmol), MeI (428 mg, 3.02 mmol), and TBAB
(21 mg, 66 µmol) in anhydrous acetone (10 mL). The suspen-
sion was vigorously stirred in a closed screw-cap Pyrex bottle
at 60 °C for 2 days. After the bottle was cooled, the inorganic
salts were filtered off and washed with anhydrous acetone,
and the filtrate was evaporated. The residue was dissolved in
AcOEt (50 mL) and washed with 5% aqueous KHSO4, water,
saturated aqueous NaHCO3, water, and brine and dried. The
product was isolated by chromatography on silica gel (hexane/
EtOAc, 1:1, Rf 0.31), yielding 781 mg (88%) of a colorless oil.
HPLC (method C): Rt 5.14 min, peak area 99%. IR (neat):
3054, 2977, 1724, 1591, 1508, 1422, 1265, 1156 cm-1. 1H NMR-
(300 MHz, 60 °C, C2D2Cl4): δ 1.43 (s, 9 H), 1.40-1.68 (m, 4
H), 2.36 (m, 2 H), 2.91 (br. s, 3 H), 2.97 (s, 3 H), 3.20-3.52 (m,
2 H), 3.83-3.96 (m, 1 H), 5.06 (s, 6 H), 5.15-5.40 (m, 1 H),
7.24-7.42 (m, 15 H). 13C NMR (75.5 MHz, 60 °C, C2D2Cl4): δ
170.2, 159.9, 156.3, 155.5, 153.4, 136.55, 136.50, 135.7, 128.30,
128.25, 128.2, 128.1, 127.9, 127.8, 127.7, 127.6, 80.9, 67.6 (br),
67.7, 67.1, 66.3, 49.9 (br), 47.8, 40.3, 35.6 (br), 34.4, 31.4, 27.9,
22.9 (br). DCI (NH3)-MS m/z: 675 (M + H, 62), 183 (100). HR
LC-MS (EI) calcd for C37H47N4O8 [M+ + H], 675.3394; found,
675.3370.
1
recovered. H NMR (300 MHz, 50 °C, C2D2Cl4): δ 1.43 (m, 2
H), 1.62 (m, 2 H), 2.43 (m, 2 H), 2.95 (s, 3 H), 3.44 (m, 2 H),
3.96 (m, 1 H), 5.05/5.12 (s, 6 H), 5.47 (m, 1 H), 7.25-7.38 (m,
15 H), 8.8 (br. s, 2 H). 13C NMR (75.5 MHz, 35 °C, C2D2Cl4):
δ 175.2, 157.7, 156.0, 155.7, 136.3, 135.6, 128.4, 128.3, 128.2,
128.1, 127.9, 127.6, 67.4, 66.4, 50.2, 47.8, 39.6, 36.9, 30.8, 23.3.
ESI-MS m/z: positive ions, 1231 (2M + Na, 14), 649 (M - H
+ 2Na, 26), 627 (M + Na, 100), 605 (M + H, 12), 519 (M -
PhCH2OH + Na, 12); negative ions, 1207 (2M - H, 80), 625
(M - 2H + Na, 50), 603 (M - H, 50), 495 (M - PhCH2OH -
H, 100).
(3′S,5RS)-5-[N-[3-Ben zyloxyca r b on yla m in o-6-[2,3-d i-
(ben zyloxyca r bon yl)-1-m eth ylgu a n id in -1-yl]h exa n oyl]-
N -m e t h yla m in o]-5,6-d ih yd r o-2-u r e id op yr im id -4(1H )-
on e (tr is-Z-14). Under an argon atmosphere, the acid 13 (1.20
g, 1.98 mmol) was dissolved in DMF (20 mL), and the
heterocycle 3 (368 mg, 1.98 mmol) was added followed by
HATU (1.13 g, 2.98 mmol) and DIPEA (385 mg, 0.510 mL,
2.98 mmol), which was added dropwise with stirring at room
temperature. The mixture was stirred overnight at room
temperature, DMF was evaporated in vacuo (1 Torr), and the
residue was dissolved in dichloromethane (200 mL). This
solution was washed with water (20 mL), 0.5 aqueous H2SO4
(10 mL), water (20 mL), saturated aqueous NaHCO3 (10 mL),
and brine (10 mL). After the solvent was dried and evaporated,
the coupling product was isolated by repeated chromatography
on silica gel eluting with CH2Cl2/MeOH (9:1, Rf ) 0.27), EtOAc/
EtOH/AcOH (90:10:5), and CH2Cl2/MeOH (100:7); yield 1.26
g (82%) tris-Z-14 as a colorless foam. HPLC (method C): Rt
4.3 min, peak area 98%. IR (KBr): 3410, 2945, 1714, 1616,
Nâ,Nω,Nω′-Tr is(ben zyloxyca r bon yl)-NE,Nω-d im eth yl-(S)-
â-h om oa r gin in e (16). TFA (0.857 mL) was added dropwise
with stirring to a solution of the compound 15 (577 mg, 0.856
mmol) and Et3SiH (0.344 mL, 2.14 mmol) in CH2Cl2 (2 mL) at
+5 °C. The reaction mixture was allowed to warm to room
temperature, and after 6 h, volatiles were removed in vacuo,
and the oily residue was dissolved in EtOAc (40 mL), washed
with water (3 × 10 mL) and brine, and dried. Evaporation in
vacuo gave 500 mg (95%) of the title compound as an off-white
foam. IR (KBr): 3330, 3034, 2953, 1792, 1723, 1588, 1521,
1576, 1456, 1378, 1273, 1225 cm-1
.
1H NMR (200 MHz,
1327, 1174 cm-1 1H NMR (300 MHz, 50 °C, [D6]DMSO): δ
.
CDCl3): δ 1.60 (m, 4 H), 2.45-2.70 (m, 2 H), 2.87 and 2.92 (2
s, total 3 H), 2.99 (s, 3 H), 3.35-3.52 (m, 4 H), 3.97 (m, 1 H),
4.88 (m, 1 H), 5.04 (s, 2 H), 5.11 (s, 4 H), 5.68-5.90 (m, 1 H),
7.38 (m, 15 H), 9.28 (br. s, 1 H), 10.26 (br. s, 1H), 10.41 (br. s,
1H), 11.43 (br. s, 1H). HPLC-MS: Rt 2.31 min. ESI-MS m/z:
positive ions, 772 (M + H, 100), 407 (69), 387 [(M + 2H)/2,
78]; negative ions, 770 (M - H, 100).
(3′S,5R,S)-5-[N-[3-Am in o-6-(1-m eth ylgu a n id in -1-yl)h ex-
a n oyl]-N-m et h yla m in o]-5,6-d ih yd r o-2-u r eid op yr im id -
4(1H)-on e Dih yd r och lor id e (14). Tris-Z-14 (110 mg, 0.143
mmol) was dissolved in MeOH/CH2Cl2 (1:1, 5 mL), PdCl2 (38
mg, 0.21 mmol) was added, the solution was flushed with
1.39 (m, 2 H), 1.48 (m, 2 H), 2.23-2.42 (m, 2 H), 2.87 (s, 6 H),
3.33 (m, 2 H), 3.81 (m, 1 H), 4.98 (s, 2 H), 5.05 (s, 4 H), 7.08
(d, 1 H), 7.24-7.38 (m, 15 H). 13C NMR (75.5 MHz, 50 °C, [D6]-
DMSO): δ 172.0, 158.9, 155.7, 155.4, 152.8, 137.1, 136.7, 135.9,
128.11, 128.08, 128.05, 127.75, 127.54, 127.45, 127.3, 67.0,
66.2, 65.0, 49.6, 47.5 (br), 40.3, 35.1 (br), 31.2, 22.9 (br). DCI
(NH3)-MS m/z (rel intensity, %): 619 (M + H+, 38), 183 (64),
143 (60), 126 (100).
(3′S,5RS)-5-[N-[3-Ben zyloxyca r b on yla m in o-6-[2,3-d i-
(ben zyloxycar bon yl)-2,3-dim eth ylgu an idin -1-yl)h exan oyl]-
N -m e t h yla m in o]-5,6-d ih yd r o-2-u r e id o-4(1H )-p yr im i-
d on e (17). HATU (494 mg, 1.30 mmol) was added to the

Novel Antibiotics for Gram-Positive Bacterial Infections
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 19 4253
biot. 1998, 51, 189-201. (b) Brands, M.; Es-Sayed. M.; Ha¨bich,
D.; Raddatz, S.; Kru¨ger, J .; Endermann, R.; Gahlmann, R.; Kroll,
H.-P.; Geschke, F.-U.; deMeijere, A.; Belov, V.; Sokolov, V.;
Kozhushkov, S.; Kordes, M. TAN-1057 Derivatives. WO 00/
12484.
solution of the heterocycle 3 (118 mg, 0.637 mmol) and the
acid 16 (360 mg, 0.582 mmol) in DMF (3 mL). Then, DIPEA
(0.225 mL, 1.30 mmol) was added dropwise, and the reaction
mixture was stirred at room temperature overnight. After the
workup (similar to tris-Z-14), the title compound was iso-
lated by chromatography on silica gel (50 mL), eluting with
CH2Cl2/MeOH (10:1), Rf 0.50; yield 0.350 g (76%) of a glasslike
foam. HPLC (method C): Rt 4.42 min, peak area 94%. IR
(KBr): 3399, 3336, 3151, 2942, 1717, 1577, 1497, 1456, 1375,
(6) (a) Williams, R. M.; Yuan, C. Total synthesis of the anti-MRSA
peptide antibiotics TAN- 1057 A-D. J . Am. Chem. Soc. 1997,
119, 11777-11784. (b) Sokolov, V. V.; Kozhushkov, S. I.; Nikol-
skaya, S.; Belov, V. N.; Es-Sayed, M.; de Meijere, A. Total
Synthesis of TAN-1057 A/B, a New Dipeptide Antibiotic from
Flexibacter sp. PK-74. Eur. J . Org. Chem. 1998, 777-783.
(7) Purchased from EMKA Chemical Enterprise, Ltd.
(8) Heterocycle 3 was synthesized as a racemic mixture according
to ref 6b.
(9) For guanylations including the use of S-methylisothioureas, see
Ku¨hle, E.; Guanidine aus S-Alkylisothioharnstoffen mit Aminen.
In Methoden der Organischen Chemie (Houben-Weyl); Hageman,
H., Ed.; Georg-Thieme Verlag: Stuttgart, New York, 1983; E4,
pp 614-615.
(10) For the use of HgCl2 in guanidinylation reactions, see (a) Guo,
Z.-X.; Cammidge, A. N.; Horwell, D. C. A Convenient and
Versatile Method for the Synthesis of Protected Guanidines.
Synth. Commun. 2000, 30, 2933-2943. (b) Kim, K. S.; Qian, L.
Improved Method for the Preparation of Guanidines. Tetrahe-
dron Lett. 1993, 34, 7677-7680. (c) Levallet, C.; Lerpiniere, J .;
Ko, S. Y. The HgCl2-Promoted Guanylation Reaction: The Scope
and Limitations. Tetrahedron 1997, 53, 5291-5304.
(11) Further studies revealed that the stereogenic center residing in
the heterocyle was highly prone to epimerization. Accordingly,
the final products were synthesized as a 1:1 mixture of diaste-
reoisomers. TAN 1057 A,B was isolated from the natural source
as a 1:1 mixture of diastereomers (ref 3).
(12) Yamaguchi, H.; Yamamoto, C.; Tanaka, N. Inhibition of Protein
Synthesis by Blasticidin S. J . Biochem. 1965, 57, 667-677.
(13) For the synthesis of Nδ-methyl-â-ornithine, see Nomoto, S.;
Shimoyama, A. First Synthesis of Blasticidic Acid, a Component
Amino Acid in an Antibiotic Blasticidin S. Tetrahedron Lett.
2001, 42, 1753-1755.
(14) Curphey, T. J . Trifluoroacetylation of Amino Acids and Peptides
by Ethyl Trifluoroacetate. J . Org. Chem. 1979, 44, 2805-2807.
(15) Antonjuk, D. J .; Boadle, D. K.; Cheung, H. T. A.; Tran, T. Q.
Synthesis of Monoamides of Methotrexate from L-Glutamic Acid
Monoamide tert-Butyl Esters. J . Chem. Soc., Perkin Trans. 1
1984, 1989-2003.
(16) For N-methylation of N-trifluoroacetamides with MeI and
Cs2CO3 or K2CO3, see, for example, (a) Wyatt, P. G.; Coomber,
B. A.; Evans D. N.; J ack, T. I.; Fulton, H. E.; Wonacott, A. J .;
Colman, P.; Varghese, J . Sialidase Inhibitors Related to Zan-
amivir. Further SAR Studies of 4-Amino-4H-pyran-2-carboxylic
acid 6-propylamides. Bioorg. Med. Chem. Lett. 2001, 11, 669-
673. (b) Takeda, Y.; Kawagoe, K.; Yokomizo, A.; Yokomizo, Y.;
Hosokami, T.; et al. Synthesis of Phenoxyacetic Acid Derivatives
as Highly Potent Antagonists of Gastrin/Cholecystokinin-B
Receptors. II. Chem. Pharm. Bull. 1998, 46, 951-961.
(17) Bernatowicz, M. S.; Wu, Y.; Matsueda, G. R. Urethane Protection
Derivatives of 1-Guanylpyrazoles for the Mild and Efficient
Preparation of Guanidines. Tetrahedron Lett. 1993, 34, 3389-
3392.
(18) Mehta, A.; J aouhari, R.; Benson, T. J .; Douglas, K. T. Improved
Efficiency and Selectivity in Peptide Synthesis: Use of Trieth-
ylsilane as a Carbocationic Scavenger in Deprotection of t-Butyl
Esters and t-Butoxycarbonyl-protected Sites. Tetrahedron Lett.
1992, 33, 5441-5444.
(19) (a) Dodd, D. S.; Kozikowski, A. P. Conversion of Alcohols to
Protected Guanidines Using the Mitsunobu Protocol. Tetrahe-
dron Lett. 1994, 35, 977-980. (b) Feichtinger, K.; Sings, H. L.;
Baker, T. J .; Matthews, K.; Goodman, M. Triurethane-protected
Guanidines and Triflyldiurethane-protected Guanidines: New
reagents for Guanidinylation Reaction. J . Org. Chem. 1998, 63,
8432-8439.
1338, 1273, 1143, 1055 cm-1 1H NMR (500 MHz, CD3OD/
.
CDCl3, 1:1): δ 1.58 (m, 4 H), 2.52 (m, 2 H) 2.79-3.00 (m, 9
H), 3.26-4.01 (m, 5 H), 4.98 (m, 7 H), 7.29 (m, 15 H). ESI-MS
m/z: positive ions, 808 (M + Na, 100); negative ions, 784 (M
- H, 100). HR LC-MS (EI) calcd for C39H48N9O9 [M + H],
786.3575; found, 786.3575.
(3′S,5RS)-5-[N-[3-Am in o-6-(1,2-d im eth ylgu a n id in -1-yl)-
h exan oyl]-N-m eth ylam in o]-5,6-dih ydr o-2-u r eidopyr im id-
4(1H)-on e Dih yd r och lor id e (18). A suspension of 17 (306
mg, 0.389 mmol) in MeOH (15 mL) with PdCl2 (69.1 mg, 0.390
mmol) was hydrogenated and worked-up as described for
compound 14. After the methanol evaporated, the residue was
triturated with several portions of anhydrous ether (ca. 10 mL
each) to yield 160 mg (90%) of white powder. HPLC (method
C): Rt 1.15 min, peak area 93%. HPLC (method A): Rt 4.44
min, peak height 94%. IR (KBr): 3375, 3194, 2941, 1716, 1622,
1
1569, 1275 cm-1. H NMR (500 MHz, [D4]MeOH): δ 1.73 (br
s, 4 H, 4′/5′-H), 2.79 (dd, J ) 9 and 17, 1 H, 2′-H), 2.82 (s,
NMe), 2.89 (s, NMe), 2.96 (dt, J ) 4 and 17, 1 H, 2′-H), 3.05
(s, NMe), 3.08 (s, total intensity of all N-Me singlets is 12 H),
3.40 (br s, 2 H, 6′-H), 3.62 (m, 1 H, 3′-H), 3.73 (dd, J ) 8 and
13, 1 H, 6-H), 3.86 (t, J ) 13, 1 H, 6-H), 5.21 (dd, J ) 8 and
13, 1 H, 5-H). 13C NMR (250 MHz, [D4]MeOH): δ 173.4 (C-
1′), 158.9, 158.3, 158.0, 54.6 (C-5), 51.4 (C-6′), 50.1 (C-3′), 40.3
(C- 6), 39.2 (NMe), 37.2 (NMe), 36.5 (C-2′), 34.5 (NMe), 30.8
(C-5′), 29.6 (NMe), 24.5 (C-4′). ESI-MS m/z: positive ions, 384
(M + H, 100); negative ions, 382 (M - H, 100). HR LC-MS
(EI) calcd for C15H28N9O3 [M - H], 382.2315; found, 382.2325.
Ack n ow led gm en t. This work was supported by the
BAYER AG and the Fonds der Chemischen Industrie.
We thank Dr. B. Knieriem, Go¨ttingen, for his careful
reading of the manuscript and M. Graeser, Marburg,
for his contributions in the synthesis of TAN 1057 A,B
analogues.
Refer en ces
(1) For recent publications dealing with antibacterial resistance, see
(a) Ehlert, K. Methicillin-Resistance in Staphylococcus aureus
- Molecular Basis, Novel Targets and Antibiotic Therapy. Curr.
Pharm. Des. 1999, 5, 45-55. (b) Kotra, L. P.; Golemi, D.;
Vakulenko, S.; Mobashery, S. Bacteria fight back. Chem. Ind.
(Berlin) 2000, 341-344. (c) Marchese, A.; Schito, G. C.; Debbia,
E. A. Evolution of Antibiotic Resistance in Gram-Positive
Pathogens. J . Chemother. 2000, 12, 459-462. (d) Levy, S. B. The
future of antibiotics: facing antibiotic resistance. Clin. Microbiol.
Infect. 2000, 6, 101-106.
(2) For a recent review, see Singh, M. P.; Greenstein, M. Antibacte-
rial leads from microbial natural products discovery. Curr. Opin.
Drug Discovery Dev. 2000, 3, 167-176 and references therein.
(3) (a) Katayama, N.; Fukusumi, S.; Funabashi, Y.; Iwahi, T.; Ono,
H. TAN-1057 A-D. New Antibiotics With Potent Antibacterial
Activity Against Methicillin-resistant Staphylococcus aureus. J .
Antibiot. 1993, 46, 606-613. (b) Funabashi, Y.; Tsubotani, S.;
Koyama K.; Katayama, N.; Harada, S. A New Anti-MRSA
Dipeptide, TAN-1057 A. Tetrahedron 1993, 49, 13-28.
(20) (a) Mu¨ller, M.; Blobel, G. Protein Export in Escherichia coli
Requires a Soluble Activity. Proc. Natl. Acad. Sci. U.S.A. 1984,
81, 7737-7741. (b) E. coli S30 Extract System for Circular DNA,
Technical Bulletin 092, Promega Corporation.
(4) All MIC data reported in Tables 1 and 2 were measured by in
house resources (see Experimental Section for details).
(5) For the synthesis of analogues of TAN 1057 A,B, see (a) Williams,
R. M.; Yuan, C.; Lee, V. J .; Chamberland, S. Synthesis and
Antimicrobial Evaluation of TAN-1057 A/B Analogues. J . Anti-
J M0111191