Total synthesis of the anti methicillin-resistant Staphylococcus aureus peptide antibiotics TAN-1057A-D

Article abstract of DOI:10.1021/ja972670j

TAN-1057A-D, dipeptides isolated from bacteria Flexibacter sp. PK-74 and PK-176, are new antibiotics with potent antibacterial activity against methicillin-resistant Staphylococcus aureus. We describe, in detail, the total synthesis of TAN-1057A-D by a co

Full text of DOI:10.1021/ja972670j

J. Am. Chem. Soc. 1997, 119, 11777-11784
11777
Total Synthesis of the Anti Methicillin-Resistant Staphylococcus
aureus Peptide Antibiotics TAN-1057A-D
Chenguang Yuan and Robert M. Williams*
Contribution from the Department of Chemistry, Colorado State UniVersity,
Fort Collins, Colorado 80523
ReceiVed August 4, 1997X
Abstract: TAN-1057A-D, dipeptides isolated from bacteria Flexibacter sp. PK-74 and PK-176, are new antibiotics
with potent antibacterial activity against methicillin-resistant Staphylococcus aureus. We describe, in detail, the
total synthesis of TAN-1057A-D by a convergent route featuring a new method to construct the cyclic amidinourea
functional group.
Introduction
Scheme 1
Nosocomial infections caused by methicillin-resistant Sta-
phylococcus aureus (MRSA) have become a very serious
clinical problem.1-5 MRSA has developed resistance to most
â-lactam antibiotics as well as numerous other antibiotics due
to presence of the mec A gene.1 MRSA produces an altered
penicillin-binding protein, PBP2a, for which most clinically
significant â-lactam antibiotics have low affinity. A desperate
search has very recently been initiated to find so-called fourth
generation cephalosporins that possess affinity for PBP2a.
Screening programs aimed at discovering new structural drug
motifs that are efficacious against MRSA have therefore become
increasingly significant.
Recently, Takeda Chemical Co., Japan, described the isolation
of four new compounds identified as TAN-1057A-D from
Flexibacter sp. PK-74 and PK-176 (Chart 1).6,7 These com-
pounds were found to be dipeptide antibiotics with potent
activity against MRSA. TAN-1057A-D displayed better
activity against Gram-positive bacteria than against Gram-
negative bacteria. TAN-1057A and -D, which have the S-
configuration in the heterocyclic portion of the molecule, were
more active than TAN-1057B and -C which possess the
R-configuration. There was no cross-resistance between TAN-
1057 and methicillin, erythromycin, and gentamycin. It is
significant to note that TAN-1057A displays potent activity
against all of the MRSA strains evaluated and was found to
compare very favorably to vancomycin.6 The Takeda group
concluded that the therapeutic effects of TAN-1057A, as
determined in mice, were superior to vancomycin and imipenem,
especially against MRSA. The preliminary acute toxicity (LD50)
data obtained for TAN-1057A was ca. 100 mg/kg upon
intraperitoneal injection and 50 mg/kg upon intravenous injec-
tion in mice.
tions below the minimal inhibitory concentration (MIC). In
addition, poly-A and poly-U-directed protein synthesis was
inhibited in an E. coli cell-free system at 40 and 10 µg/mL,
respectively. TAN-1057A did not inhibit aminoacyl-tRNA
synthetase; thus, this drug appears to interfere with protein
biosynthesis after the formation of aminoacyl-tRNA. There is
no published data concerning the morphological characteristics
of susceptible strains treated with TAN-1057; thus, it is not
presently known if TAN-1057 inhibits bacterial cell wall protein
biosynthesis.
TAN-1057A and -B are dipeptides consisting of â-homoargi-
nine and a unique heterocyclic amidinourea derivative of 2,3-
diaminopropionic acid. TAN-1057A and -B gradually lost their
antibacterial activities in basic aqueous solutions due to
hydrolytic opening of the six-membered ring system (Scheme
1).7 Hydrolysis of TAN-1057A occurs in both acidic and basic
media, to afford the acyclic form (5) with attendant racemization
of the R-amino acid stereogenic center. It was also reported7
that the acyclic form of the molecule can be converted back
into the cyclic form via the methyl ester intermediate resulting
in a diastereomeric mixture (1:1) of TAN-1057A and -B.
Due to the unique functionality present in these structures
and the possibility for the discovery of new therapeutically
useful biochemical targets through mechanism of action studies
on these substances, we have developed the first synthesis of
TAN-1057A-D featuring new methodology for constructing
the cyclic amidinourea and report these findings here.
TAN-1057A did not inhibit the incorporation of tritiated
thymidine and uridine into S. aureus FDA 209P or Escherichia
coli LD-2. However, TAN-1057A inhibited the incorporation
of leucine into macromolecules in these organisms at concentra-
X Abstract published in AdVance ACS Abstracts, December 1, 1997.
(1) Cohen, M. L. Science 1992, 257, 1050.
(2) Bloom, B. R.; Murray, C. J. L. Science 1992, 257, 1055.
(3) Neu, H. C. Science 1992, 257, 1064,
(4) Krause, R. M. Science 1992, 257, 1073.
(5) Kuntz, I. D. Science 1992, 257, 1078.
(6) Katayama, N.; Fukusumi, S.; Funabashi, Y.; Iwahi, T.; Ono, H. J.
Antibiot. 1993, 46, 606.
(7) Funabashi, Y.; Tsubotani, S.; Koyama, K.; Katayama, N.; Harada,
S. Tetrahedron 1993, 49, 13.
Results and Discussion
A very simple and straightforward retrosynthetic analysis
consisting of dissecting the molecule into the â-homoarginine
S0002-7863(97)02670-X CCC: $14.00 © 1997 American Chemical Society

11778 J. Am. Chem. Soc., Vol. 119, No. 49, 1997
Yuan and Williams
Chart 1
subunit and the 2,3-diaminopropionic acid portion was initially
considered. Peptide coupling, followed by heterocycle forma-
tion and urea homologation, was expected to give the desired
product. Several variants of this approach have been extensively
investigated; a successful approach, predicated on new meth-
odology to construct the cyclic amidinourea is described herein.
In planning alternate synthetic routes, the choice of the
protecting groups was viewed as being critical to an ultimately
successful approach. In addition, the unusual and labile cyclic
amidino urea was targeted to be constructed in the late stages
of the synthesis. It was deemed crucial to orchestrate the
selective assembly of blocking groups such that (1) the
guanidino group of the homoarginine moiety could be kept inert,
(2) the amino group at C5 could be methylated selectively, and
(3) the 3′- and 1-amino groups could be differentially protected
and sequentially unmasked. Commercially available tri-N-CBZ-
L-arginine (6) (CBZ ) benzyloxycarbonyl) was chosen as the
core starting material, since it was expected that catalytic
hydrogenation would be compatible with the final unmasking
of the fully derivatized, protected structure. The phthalimido
group was chosen to block N1, since this group should permit
the selective N-methylation and be removable under relatively
mild conditions.
Synthesis of the â-Homoarginine Subunit. Tri-N-CBz-L-
arginine (6) was first converted to the corresponding ethoxy-
carbonic anhydride in the presence of N-methylmorpholine and
ethyl chloroformate.8-10 The mixed anhydride was immediately
allowed to react with diazomethane to furnish the corresponding
diazoketone. Wolff rearrangement in methanol gave tri-N-CBz-
â-homoarginine methyl ester as a white solid, which was
saponified in 2 N LiOH to give the carboxylic acid derivative.
However, one N-CBz group on the guanidine was lost during
saponification and NR,Nγ-di-N-CBz-â-homoargine was thus
obtained. Alternatively, tri-N-CBz-â-homoarginine (7) was
prepared through a modified Arndt-Eistert synthesis with a 1:1
mixture of tert-butyl alcohol and water as solvent. This
permitted the preparation of the free acid without a saponifica-
tion step, obviating loss of one N-CBz group (Scheme 2).
Synthesis of the 2,3-Diaminopropionic Acid Subunit. (2S)-
N2-CBz-N2-methyl-N3-phthalimido-2,3-diaminopropionic acid
(8) was prepared according to the literature procedure11 using
L-N-CBz-asparagine as starting material. The acid (8) was
converted into the corresponding tert-butyl ester (9) using EDCI/
t-BuOH in excellent yield. However, under these conditions,
it was found that complete racemization of the substrate
occurred. Optically pure 9 could be obtained by an alternate
procedure employing concentrated sulfuric acid and isobutene.
Reductive removal of the N-CBz group provided the amine
component (10, Scheme 3).
Scheme 2
Scheme 3
Compound 10 proved to be labile to racemization on
attempted silica gel column chromatographic purification; this
method of purification was thus avoided. As will be discussed
below, racemic 10 was ultimately used in the synthesis due to
the ubiquitous lability of the R-stereogenic center of this amino
acid to epimerization.
Construction of the Amidinourea Subunit. The success
of developing a general synthetic approach to this new class of
peptide antibiotics relies heavily on an efficient preparation of
the unusual, cyclic amidinourea moiety. Only one report in
the literature has described this unusual ring system,12 and
recently, a related five-membered cyclic amidinourea was found
in an antitumor antibiotic NA22598.13 All of the published
methods, which includes the reaction of guanidines with
isocyanates,14 hydrogenation of 5,3-diamino-1,2,4-oxadiazoles,15
or the hydrolysis of cyanoguanidines under strongly acidic
conditions,16 are either inefficient, involve harsh reaction
conditions, or require numerous steps.12 We have examined
and subsequently determined that none of these methods were
suitable for accessing the labile TAN-1057 amidinourea sub-
structure. Recently, we reported17 a new and efficient method
for the preparation of acyclic amidinoureas using N-(benzy-
loxycarbonyl)ureido-N′-(benzyloxycarbonyl)-(S)-methylisothio-
urea (21) under very mild conditions.18-20 As a model system,
(12) Garratt, P. J.; Hobbs, C. J.; Wrigglesworth, R. J. Org. Chem. 1989,
54, 1062-1069.
(13) Kuwahara, A.; Nishikiori, T.; Shimada, N.; Nakagawa, T.; Fuka-
zawa, H.; Mizuno, S.; Uehara, Y. J. Antibiot. 1997, 50, 712-713.
(14) Tilley, J. W.; Blount, J. F. HelV. Chim. Acta 1980, 63, 841-859.
(15) Tilley, J. W.; Blount, J. F. HelV. Chim. Acta 1980, 63, 832-840.
(16) Wagenaar, F. L.; Kerwin, J. F., Jr. J. Org. Chem. 1993, 58, 4331-
4338.
(8) Wakamiya, T.; Uratani, H.; Teshima, T.; Shiba, T. Bull. Chem. Soc.
Jpn. 1975, 48 (8), 2401-2.
(9) Nomoto, S.; Shiba, T. Chem. Lett. 1978, 589-590.
(10) Podlech, J.; Seebach, D. Angew. Chem., Int. Ed. Engl. 1995, 34
(4), 471-2.
(11) Waki, M.; Kitajima, Y. and Izumiya, N. Synthesis 1981, 266.
(17) Yuan, C.; Williams, R. M. Tetrahedron Lett. 1996, 37, 1945.

Total Synthesis of TAN-1057A-D
J. Am. Chem. Soc., Vol. 119, No. 49, 1997 11779
Scheme 4
Scheme 5
the direct preparation of cyclic amidinoureas from peptide
substrates was examined, as shown in Scheme 4. N-(Benzy-
loxycarbonyl)ureido-N′-(tert-butyloxycarbonyl)-(S)-methylisothio-
urea (35) was prepared from mono-(tert-butyloxycarbonyl)-(S)-
methylisothiourea and (benzyloxycarbonyl)isocyanate21 (THF,
100%). Treatment of compound 35 with trifluoroacetic acid
(TFA) gave 13.
Next, carboxylic acid 11 was prepared from protected 2,3-
diaminopropionic acid (9) following standard protecting group
exchange. Coupling of the (S)-methylisothiourea derivative 13
with 12 using EDCI in the presence of 4-(dimethylamino)-
pyridine (DMAP) gave 14 in 41% yield. Treatment of 14 with
TFA removed the tert-butyoxycarbonyl (BOC) group to furnish
the crude amine-TFA salt 15. When 15 was treated with 2
mol equiv of triethylamine in DMF, an intramolecular cycliza-
tion reaction ensued to furnish the desired amidinourea (17) in
92% yield. However, in the presence of 4 mol equiv of
triethylamine under the same conditions, the formation of an
insoluble substance with spectral characteristics consistent with
the bicyclic structure 16 was observed. Careful control of the
reaction time and the amount of base added for this cyclization
permitted the reproducible construction of the desired mono-
cyclic amidinourea system 17.
This methodology was first applied to the construction of
the fully functionalized acyclic hydrolysis product (5) of TAN-
1057A, as shown in Scheme 5. The peptide coupling reaction
between NR,Nγ-di-N-CBz-â-homoarginine 18 and 10 with BOP-
Cl gave the desired peptide 19 in good yield. The phthaloyl
group was removed by treatment with hydrazine in methanol
to provide the free amine 20, which was condensed with
(18) Tian, Z.; Edwards, P.; Roeske, R. W. Int. J. Pept. Protein Res. 1992,
40, 119-126.
(19) Bergeron, R. J.; Mcmanis, J. S. J. Org. Chem. 1987, 52, 1700-
1703.
(20) Su, W. Synth. Commun. 1996, 26, 407-413.
(21) Grehn, L.; Almeida, L. S.; Ragnarsson, U. Synthesis 1988, 992-
994.

11780 J. Am. Chem. Soc., Vol. 119, No. 49, 1997
Yuan and Williams
Scheme 6
N-(benzyloxycarbonyl)ureido-N′-(benzyloxycarbonyl)-(S)-me-
thylisothiourea (21) to give the desired product 22 and triaz-
inedione byproduct 23. The best results were obtained by using
1 equiv of triethylamine and stopping the reaction before
completion, where the attendant appearance of the byproduct
(23) could be minimized (4-5 h, as monitored by TLC).
The tetra-N-CBz-protected TAN-1057 precursor 22 was
deprotected by catalytic hydrogenation following TFA removal
of the tert-butyl ester to give the linear compound 5 in excellent
yield. Despite extensive effort, we were unable to effect the
clean conversion of 5 into TAN-1057A/B.7 Although this route
can technically be considered a formal total synthesis on the
basis of the Takeda work,7 we turned our attention to a reliable
and versatile method to construct the biologically active, cyclic
amidinourea-containing natural product.
Total Synthesis of TAN-1057A/B. Drawing on the observa-
tions from the model study, the approach shown in Scheme 6
was devised. Coupling of tri-N-CBz-homoarginine (7) and d,l-
10 with BOP-Cl gave the desired peptide 25 in good yield.
Attempts to deprotect the phthalimido group with hydrazine in
methanol resulted in loss of one of the N-CBz groups from the
guanidine. An alternative procedure using methylamine in
ethanol followed by treatment with TFA gave the corresponding
N-methylamide (26).
When peptide 25 was treated with 30% methylamine in EtOH
for more than 5 min, one of the N-CBz groups was removed.
However, if the reaction was stopped after 5 min., the partially
deprotected derivative was obtained without concomitant loss
of the N-CBz groups. The monoacylated amine was next
converted to the corresponding BOC-protected amine 27 by
treatment with excess triethylamine in the presence of BOC-
ON in dioxane/water.
Initially, we utilized optically pure 10 for coupling with 7.
Under the same conditions, no detectable racemization ac-
companied this condensation. However, during the removal of
the phthalimido group under various conditions, complete
epimerization of the C5 stereogenic center was observed. The
Takeda group had reported that HPLC-purified TAN-1057A
rapidly epimerizes at C5 to a mixture of TAN-1057A + TAN-
1057B; as such, it was decided to carry the synthesis forward
with racemic 10. All intermediates from compound 25 forward
are therefore 1:1 mixtures of two optically pure diastereomers.
Attempted chromatographic separation of each diastereomeric
pair proved unsuccessful. Since the lability of this stereogenic
center renders the final compounds, in practice, to be handled
(and tested biologically) as diastereomeric mixtures, the route
chosen turned out to be the most practical and economical.
Compound 27 was coupled to N-(benzyloxycarbonyl)ureido-
(S)-methylisothiourea 13 to give 2822 in 52% yield. Deprotec-
tion of the N-t-BOC group with TFA produced the correspond-
ing TFA salt. When this substance was treated with triethylamine
(2.0 equiv), cyclization ensued to furnish the desired, fully
protected TAN-1057A derivative 2922 in 67% yield (two steps).
This substance proved to be labile undergoing a second
cyclization to a substance corresponding to 16 (Scheme 4).
Fortunately, this problem can be circumvented by converting
29 into TAN-1057A/B immediately after PTLC purification.
The deprotection was very efficient with hydrogen (1 atm) and
PdCl2 in methanol for 30 min. Pure TAN-1057A/B was
obtained as a ∼1:1 mixture after simple workup. The synthetic
1
material proved to be identical to the natural sample23 by H
NMR, mass spectrometric fragmentation (ES+), IR, mobility
by HPLC, and bioassay against S. aureus.
Total Synthesis of TAN-1057C/D. TAN-1057C and TAN-
1057D are also labile substances that were reported7 to rapidly
convert to a mixture of TAN-1057A/B upon standing in water.
Utilizing the methodology developed for the synthesis of TAN-
1057A/B reported above, the synthesis of these seven-membered
ring isomers was undertaken as shown in Scheme 7. L-NR-t-
BOC-Nδ,Nω-di-CBz-â-homoarginine (31) was prepared from
commercially available 30 in the same manner as that used for
compound 7. Coupling of 31 with d,l-10 in the presence of
BOP-Cl gave the desired peptide 32 in 56% yield.22 Treatment
of 32 with TFA effected removal of the BOC group and tert-
butyl ester; cyclization was effected with TBTU furnishing 3322
in 43% overall yield. In addition to the desired cyclic peptide,
a macrocyclic dimer was also isolated in approximately the same
molar ratio to that of 33; this substance was easily removed by
preparative thin layer chromatography. Dilution of the reaction
(22) This material was obtained as an inseparable mixture of diastere-
omers epimeric at C5.
(23) An authentic sample of natural TAN-1057A/B was kindly furnished
by Takeda Pahrmaceutical Co., Japan.

Total Synthesis of TAN-1057A-D
J. Am. Chem. Soc., Vol. 119, No. 49, 1997 11781
Scheme 7
tert-butyl dicarbonate; BOC-ON ) 2-(tert-butoxycarbonyloxyimino)-
2-phenylacetonitile; BOP-Cl ) bis(2-oxo-3-oxazolidinyl)phosphinic
chloride (Aldrich Chemical Co.); EDCI ) 1-(3-(dimethylamino)propyl)-
3-ethylcarbodiimide hydrochloride (Aldrich Chemical Co.); NMM )
4-methylmorpholine (Aldrich Chemical Co.); TBTU ) O-benzotriazol-
1-yl-N,N,N′,N′-tetramethyluronium tetrafluoroborate (Aldrich Chemical
Co.); MNNG ) 1-methyl-3-nitro-1-nitrosoguanidine (Aldrich Chemical
Co.).
mixture for the peptide coupling did not, unfortunately, signifi-
cantly diminish the amount of dimer formed.
Cleavage of the phthaloyl group by the sequential methy-
lamine ring-opening and BOC-ON protocol gave 34 in 30%
overall yield. Treatment of this substance with TFA followed
by guanidylation with reagent 35 gave the desired, fully
protected product 3622 in 30% yield accompanied by the
unexpected substance 37 (39%). These compounds were
separated by chromatography, and sequential deprotection of
36 with TFA followed by catalytic hydrogenation gave TAN-
1057C (3) and TAN-1057D (4) as an inseparable mixture.
Further attempted HPLC purification of the synthetic material
corroborated the findings of the Takeda group,7 namely, that
rapid conversion of the initial mixture of 3 and 4 to 1 and 2
occurs. Authentication of the synthetic material was provided
by comparing 1H NMR, mass spectrometric fragmentation
(ES+), IR, mobility by HPLC, and bioassay against S. aureus
with that of natural TAN-1057A/B.23
Coupling Product 14. To a solution of 12 (246 mg, 0.7 mmol, 1.0
equiv), DMAP (171 mg, 1.4 mmol, 2.0 equiv), and EDCI (148 mg,
0.77 mmol, 1.1 equiv) in CH2Cl2 (2.0 mL) was added 13 (300 mg,
0.84 mmol, 1.2 equiv). The resulting mixture was stirred overnight at
room temperature, diluted with CH2Cl2, washed with brine, dried over
anhydrous Na2SO4, filtered, and concentrated. Purification by column
chromatography (silica gel, 4:1:0.1 CH2Cl2:EtOAc:MeOH) provided
172 mg (41%) of 14 as a semi-solid. 1H NMR (300 MHz, CD3COOD
vs TMS, 333K): δ 1.45 (9H, s), 2.34 (3H, s), 3.06 (3H, s), 3.65 (1H,
m), 3.81 (1H, dd, J ) 5.4, 5.6 Hz), 4.72 (1H, m), 5.19 (2H, s), 5.26
(2H, s), 7.38 (10H, m). IR (NaCl, film): 3357, 3184, 2976, 1769,
1713, 1534, 1486, 1456, 1403, 1315, 1164, 1066, 1002, 733 cm-1
.
The synthetic route devised herein should open the way for
a systematic study of the structure/function relationships in this
new class of peptide antibiotics. In particular, the synthesis of
radiolabeled versions of the natural product for receptor-binding
studies is currently being investigated. The cyclization approach
described herein to construct the unusual amidinourea moiety
provides a general and flexible method for accessing a poten-
tially important generation of anti-MRSA antibiotics. Applica-
tions of this methodology toward these goals are presently under
active investigation in these laboratories and will be reported
in due course.
HRMS: calcd for (C28H35N5O8S + H) ) 602.2285; found (M + H) )
602.2285.
Cyclization Product 17. Peptide 14 (30 mg, 0.05 mmol) was treated
with anisole (0.1 mL) and TFA (2.0 mL) at 0 °C. The mixture was
stirred for 5 h at room temperature and concentrated, and the crude 15
was immediately dissolved in THF (1.0 mL). To this solution was
added Et3N (14 µL, 0.1 mmol, 2.0 equiv). The mixture was stirred for
4.5 h at room temperature and poured into CH2Cl2/brine. The organic
phase was separated, washed with brine, dried over anhydrous Na2-
SO4, filtered, concentrated, and triturated with ether to give 21 mg (75%)
of 17 as a semi-solid. 1H NMR (300 MHz, DMSO-d6 vs TMS): δ
2.83/2.88 (3H, s), 3.56 (1H, m), 3.70 (1H, q, J )12.2 Hz), 4.80 (1H,
m), 5.11 (4H, s), 7.37 (10H, m). IR (NaCl, film): 3226, 2952, 1766,
1662, 1635, 1542, 1498, 1455, 1390, 1324, 1249, 1198, 1071 cm-1
.
Experimental Section
HRMS: calcd for (C22H23N5O6 + H) ) 454.1727; found (M + H) )
454.1730.
General. General procedures and instrumentation have been previ-
ously described.24 Mass spectra were obtained on a 1992 Fisons VG
AutoSpec. HPLC analysis of TAN-1057 was carried out using a Waters
6000 pump equipped with a UV detector, utilizing an ODS, YMC Pack
A-312 column and 0.1 M phosphate buffer (pH 5.0) as the mobil phase.
All of the amino acids used as starting material were purchased from
BACHEM Inc. Abbreviations not defined in the text: (BOC)2O ) di-
L-Nr,Nδ,Nω-Tri-CBz-â-homoarginine Methyl Ester. To a solution
of tri-N-CBz-L-arginine (BACHEM, Inc.) (1.15 g, 2.0 mmol, 1.0 equiv)
in EtOAc (30 mL) was added NMM (250 µL) and ethyl chloroformate
(200 µL) at 0 °C. The resulting mixture was stirred for 3 h at 0 °C.
The precipitated amine hydrochloride was rapidly filtered off in the
cold. To this clear solution was added CH2N2/ether solution (generated
from MNNG). The solution was stirred overnight at room temperature
(24) Williams, R. M.; Yuan, C. J. Org. Chem. 1994, 59, 6190-6193.

11782 J. Am. Chem. Soc., Vol. 119, No. 49, 1997
Yuan and Williams
and concentrated to give an oily diazoketone. The diazoketone was
dissolved in MeOH (30 mL), and to this solution were added silver
benzoate (250 mg, 1 mmol, 0.5 equiv) and triethyl amine (1 mL). The
resulting mixture was stirred overnight in the dark at room temperature
and then concentrated in Vacuo. The residue was dissolved in ethyl
acetate, and insoluble material was filtered off. The filtrate was washed
with saturated sodium bicarbonate solution, saturated sodium chloride
solution, 1 M hydrochloric acid, and then finally saturated sodium
chloride solution. The organic layer was dried over anhydrous sodium
sulfate, filtered, and concentrated. Purification via column chroma-
tography (silica gel, 5:4:0.2 methylene chloride:EtOAc:MeOH) yielded
873 mg (74%) of L-NR,Nδ,Nω-tri-CBz-â-homoarginine methyl ester as
(silica gel, CH2Cl2:ethyl acetate:MeOH, 75:20:5) to afford 167 mg
(33.8%) of 22 as a semi-solid. 1HNMR (300 MHz, CDCl3/D2O vs
TMS): δ 1.44 (9H, m), 1.56 (4H, m), 2.56 (1H, m), 2.75 (1H, m),
2.91/2.94 (3H, s), 3.12 (2H, m), 4.11(3H, m), 5.11 (9H, m), 7.31 (20H,
m). IR (NaCl, film): 3307, 2946, 1715, 1629, 1449, 1373, 1293, 1218,
1152, 1097, 731, 695 cm-1. HRMS: calcd for (C49H59N9O12 + H) )
966.4361; found (M + H) ) 966.4361.
Carboxylic Acid 24. To a mixture of 22 (167 mg, 0.18 mmol, 1.0
equiv) and anisole (0.1 mL) was added TFA (3.0 mL) at 0 °C. The
mixture was stirred for 4 h at room temperature, concentrated, and dried
in Vacuo. Trituration with dry ethyl ether provided 153 mg (97%) of
24 as an amorphous powder. 1H NMR (300 MHz, CD3COOD vs
TMS): δ 1.64 (4H, m), 2.70 (2H, m), 3.15 (3H, m), 3.30 (2H, m),
3.96 (3H, m), 5.13 (9H, m), 7.32 (20H, m). IR (NaCl, film): 3310,
25
a white solid: mp 139.5-140.5 °C; [R]D ) -1.43 (c 0.7, CH2Cl2).
1H NMR (300 MHz, CDCl3 vs TMS): δ 1.49 (2H, m), 1.53 (2H, m),
2.48 (2H, d, J ) 5.40 Hz), 3.58 (3H, s), 3.93 (1H, m), 5.04 (2H, s),
5.10 (2H, s), 5.19 (2H, s), 5.43 (1H, d, J ) 7.0 Hz, D2O exchanged),
7.31 (15H, m), 9.30 (1H, br, D2O exchanged), 9.46 (1H, br, D2O
exchanged). IR (NaCl, film): 3342, 2936, 1697, 1525, 1455, 1248,
1056, 1024, 667 cm-1. Anal. Calcd for C32H36N4O8: C, 63.57; H,
6.00; N, 9.27. Found: C, 63.71; H, 5.81; N, 9.20.
2934, 1734, 1700, 1636, 1494, 1454, 1383, 1260, 1187, 1097, 695 cm-1
.
Anal. Calcd for C45H51N9O12H2O: C, 58.25; H, 5.76; N, 13.58.
Found: C, 58.25; H, 5.76; N, 13.71.
TAN-1057A/B Acyclic Hydrolysis Product 5. To a solution of
24 (110 mg, 0.123 mmol, 1.0 equiv) in MeOH (2 mL)/THF (2 mL)
was added PdCl2 (20 mg). The reaction vessel was charged with H2,
and the mixture was hydrogenated at 60 psi for 24 h. The mixture
was then purged with nitrogen and filtered to remove the catalyst. The
filtrate was concentrated and dried in Vacuo to give a 2HCl salt of 5
(46 mg, 100% yield) as an amorphous solid. 1HNMR (300 MHz,
D2O): δ 1.57 (4H, m), 2.66 (1H, m), 2.79 (1H, m), 2.88/2.89 (3H, s),
3.05 (2H, t, J ) 5.7 Hz), 3.53 (2H, m), 3.73 (1H, m), 4.79 (1H, m). IR
L-Nr,Nω-Di-CBz-â-homoarginine (18). To a solution of L-NR,Nδ,Nω-
tri-CBz-â-homoarginine methyl ester (873 mg, 1.44 mmol) in dioxane
(16 mL) was added 2 M NaOH (16 mL). The resulting mixture was
stirred for 1 h at room temperature, diluted with water, and extracted
with ethyl acetate (50 mL). The aqueous layer was adjusted to pH )
5 with 1 M HCl solution and extracted with dichloromethane (2 ×
100 mL). The combined organic extract was washed with saturated
ammonium chloride, dried over MgSO4, filtered, and concentrated to
give a colorless oil. After trituration with anhydrous ethyl ether, 656
(KBr, pellet): 3350, 3179, 2955, 1696, 1622, 1395, 1205, 1135 cm-1
.
MS (ES+): calcd (C13H28N9O4 + H) ) 374.2; found (M + H) ) 374.2,
(M - CONH2) ) 331.2.
25
mg (100%) of 18 was obtained as a white solid: mp 95-98 °C; [R]D
Peptide 25. To a mixture of the acid 7 (1.50 g, 2.54 mmol, 1.0
equiv) and NMM (464 µL, 1.2 mmol, 1.3 equiv) in CH2Cl2 (3 mL)
was added BOP-Cl (306 mg, 1.2 mmol, 1.2 equiv) at 0 °C. After 10
min of stirring, the amine d,l-10 (970 mg, 3.19 mmol, 1.26 equiv) in
CH2Cl2 (3 mL) was added. The resulting mixture was stirred overnight,
diluted with CH2Cl2 (200 mL), washed with brine, dried over anhydrous
Na2SO4, filtered, and concentrated. Purification via column chroma-
tography (silica gel, methylene chloride:EtOAc, 8:2) provided 1.22 g
(55%) of 25 as an oil: [R]D25 ) -36.1 (c 0.7, CH2Cl2) (this data is for
the optically pure diastereomer obtained with (S)-10). 1H NMR (300
MHz, CD3OD): δ 1.20 (2H, m), 1.39 (2H, m), 1.43 (9H, s), 2.21 (1H,
m), 2.53 (1H, m), 2.92 (3H, s), 3.72 (3H, m), 4.67 (2H, d, J ) 7.8
Hz), 4.95 (2H, s), 5.08 (2H, s), 5.20 (2H, s), 5.22 (1H, m), 7.33 (15H,
m), 7.64 (2H, m), 7.74 (2H, m). IR (NaCl, film): 3389, 2936, 1774,
1716, 1646, 1609, 1505, 1395, 1370, 1251, 1100, 1008, 722 cm-1. Anal.
Calcd for C47H52N6O11: C, 64.37; H, 5.98; N, 9.58. Found: C, 63.38;
H, 6.08; N, 9.60.
Peptide 26. To a solution of 25 (500 mg, 0.57 mmol, 1.0 equiv) in
CH2Cl2 (10 mL) was added 2 N methylamine in MeOH (5.0 mL). The
mixture was stirred for 5 min and concentrated. Purification via column
chromatography (silica gel, methylene chloride:EtOAc:MeOH, 4:1:0.3)
provided 500 mg (97%) of the methylamine adduct as an oil. 1H NMR
(300 MHz, CD3OD): δ 1.43 (9H, s), 1.61 (4H, m), 2.55 (1H, m), 2.64
(1H, m), 2.85 (3H, m), 3.03/3.04 (3H, s), 3.70 (1H, m), 3.86 (4H, m),
4.94 (3H, m), 5.09 (2H, s), 5.21 (2H, s), 7.37 (19H, m). IR (NaCl,
film): 3383, 3280, 3067, 2935, 1717, 1646, 1507, 1456, 1373, 1252,
1098, 697 cm-1. Anal. Calcd for C48H57N7O11: C, 63.49; H, 6.33; N,
10.80. Found: C, 63.51; H, 6.38; N, 10.81. The crude material
obtained above (500 mg, 0.55 mmol, 1.0 equiv) was treated with anisole
(0.2 mL) and TFA (5.0 mL) at 0 °C. The resulting mixture was stirred
for 1 h at room temperature, concentrated, and triturated in dry ether
to give 26 (437 mg, 93%) as an oil. 1H NMR (300 MHz, CD3OD): δ
1.59 (4H, m), 2.53 (1H, m), 2.80 (1H, m), 2.84 (3H, s), 3.03/3.05 (3H,
s), 3.87 (5H, m), 4.94 (3H, m), 5.13 (2H, s), 5.22 (2H, s), 7.34 (19H,
m). IR (NaCl, film): 3396, 3290, 2945, 1717, 1684, 1615, 1540, 1378,
1254, 1099 cm-1. Anal. Calcd for (C44H49N7O11‚3H2O): C, 58.33;
H, 6.12; N, 10.82. Found: C, 58.22; H, 5.93; N, 10.85.
) -14.0 (c 1.0, CH3OH). 1H NMR (300 MHz, CD3OD, vs TMS) δ
1.60 (4H, m), 2.37 (2H, m), 3.20 (2H, m), 3.94 (1H, m), 5.07 (2H, s),
5.16 (2H, s), 7.32 (10H, m). IR (NaCl, film): 3314, 2947, 1693, 1613,
1503, 1402, 1259, 1065 cm-1. Anal. Calcd for C23H28N4O6: C, 60.52;
H, 6.18; N, 12.27. Found: C, 60.80; H, 5.80; N, 12.06.
Peptide 19. To a mixture of the acid 18 (540 mg, 1.19 mmol, 1.0
equiv) and NMM (200 µL, 1.43 mmol, 1.2 equiv) in CH2Cl2 (2 mL)
was added BOP-Cl (456 mg, 1.82 mmol, 1.2 equiv) at 0 °C. The
reaction mixture was stirred for 10 min at 0 °C. To the resulting
mixture was added amine 10 (360 mg, 1.19 mmol, 1.0 equiv) in CH2-
Cl2 (2 mL). The mixture was stirred overnight at room temperature,
diluted with CH2Cl2 (200 mL), washed with brine, dried over anhydrous
Na2SO4, and concentrated. Purification via column chromatography
(silica gel, methylene chloride:EtOAc:MeOH, 5:4:0.2) provided 650
mg (74%) of 19 as an oil. 1H NMR (300 MHz, DMSO-d6, 393K vs
TMS): δ 1.43 (13H, m), 2.38 (2H, m), 2.93 (3H, s), 3.05 (1H, m),
3.15 (1H, m), 3.77 (1H, m), 4.04 (2H, m), 5.00 (2H, s), 5.03 (2H, s),
5.15 (1H, m), 7.32 (10H, m), 7.82 (4H, m). IR (NaCl, film): 3396,
2939, 1774, 1716, 1636, 1395, 1287, 1155, 1065 cm-1
. HRMS
(FAB): calcd for (C39H46N6O9 + H) ) 743.3405; found (M + H) )
743.3439.
Amine 20. To a solution of 19 (700 mg, 0.94 mmol, 1.0 equiv) in
MeOH (20 mL) was added hydrazine (0.4 mL, 12.5 mmol, 13 equiv)
at 0 °C. The mixture was stirred overnight at room temperature,
concentrated, and treated with ethyl acetate (200 mL) and saturated
NaHCO3 (50 mL). The organic phase was separated, washed with
brine, dried over anhydrous MgSO4, filtered, and concentrated.
Purification via column chromatography (silica gel, methylene chloride:
MeOH, 10:1-10:4) provided 300 mg (52%) of 20 as an oil. 1H NMR
(300 MHz, CDCl3/D2O vs TMS): δ 1.43 (9H, s), 1.51 (4H, m), 2.56
(2H, m), 2.84 (1H, m), 2.92 (3H, s), 3.06 (1H, m), 3.18 (2H, m), 3.67
(1H, m), 4.85 (1H, m), 5.06 (4H, s), 7.32 (10H, m). IR (NaCl, film):
3374, 3310, 2933, 1718, 1636, 1595, 1285, 1149, 1061 cm-1. HRMS
(FAB): calcd for (C31H44N6O7 + H) ) 613.3350; found (M + H) )
613.3366.
Condensation Product 22. To a mixture of 20 (315 mg, 0.79 mmol,
1.5 equiv) in DMF (5 mL) was added 21 (320 mg, 0.52 mmol, 1.0
equiv) and triethylamine (73 µL, 0.52 mmol, 1.0 equiv). The resulting
mixture was stirred for 4 h at room temperature. The reaction mixture
was concentrated and dried in Vacuo to give a white solid. The crude
product was triturated with EtOAc, and the solid was filtered off. The
filtrate was concentrated and separated via column chromatography
Carboxylic Acid 27. To a solution of 26 (437 mg, 0.51 mmol, 1.0
equiv) in H2O/dioxane (8 mL, 1:1) and Et3N (715 µL, 5.1 mmol, 10
equiv) was added BOC-ON (376 mg, 1.53 mmol, 3.0 equiv). The
mixture was stirred overnight and extracted with EtOAc(2 × 100 mL).
The organic phase was dried over anhydrous Na2SO4, filtered, and
concentrated. Purification via column chromatography (silica gel, CH2-

Total Synthesis of TAN-1057A-D
J. Am. Chem. Soc., Vol. 119, No. 49, 1997 11783
Cl2:MeOH, 9:1) provided 320 mg (79%) of 27 as a semi-solid. 1H
NMR (300 MHz, CD3OD): δ 1.35 (9H, s), 1.65 (4H, m), 2.50 (2H,
m), 2.86 (3H, m), 3.60 (3H, m), 3.95 (3H, m), 5.00 (2H, m), 5.10 (2H,
s), 5.24 (2H, s), 7.32 (15H, m). IR (NaCl, film): 3388, 2927, 1714,
1609, 1513, 1454, 1382, 1253, 1173, 1098, 1006, 697 cm-1. HRMS
(FAB): calcd for (C40H50N6O11 + H) ) 791.3616; found (M + H) )
791.3616.
(S)-Methylisothiourea 28. To a solution of 27 (70 mg, 0.066 mmol,
1.0 equiv), DMAP (25 mg, 0.13 mmol, 2.0 equiv), and EDCI‚HCl (21
mg, 0.07 mmol, 1.1 equiv) in CH2Cl2 (0.5 mL) was added 13 (45 mg,
0.1 mmol, 1.5 equiv). The resulting mixture was stirred overnight at
room temperature, diluted with CH2Cl2, washed with brine, dried over
anhydrous Na2SO4, filtered, and concentrated. Purification via column
chromatography (silica gel, CH2Cl2:EtOAc:MeOH, 4:1:0.1) provided
36 mg (52%) of 28 as a semi-solid. 1H NMR (300 MHz, CD3OD): δ
1.39 (9H, s), 1.64 (4H, m), 2.28 (3H, s), 2.60 (1H, m), 2.75 (1H, m),
3.05 (3H, m), 3.47 (1H, m), 3.69 (1H, m), 3.91 (3H, m), 4.94 (3H, m),
5.08 (2H, s), 5.20 (2H, s), 5.23 (2H, s), 7.31 (20H, m). IR (NaCl,
film): 3388, 2969, 1770, 1713, 1647, 1609, 1499, 1251, 1175, 1099
cm-1. Anal. Calcd for C51H61N9O13S: C, 58.89; H, 5.91; N, 12.12.
Found: C, 59.03; H, 6.12; N, 12.19.
MHz, CD3OD): δ 1.39 (9H, s), 1.42 (2H, m), 1.63 (2H, m), 2.36 (2H,
m), 3.83 (1H, m), 3.93 (2H, t, J ) 7.2 Hz), 5.12 (2H, s), 5.27 (2H, s),
7.36 (10H, m). IR (NaCl, film): 3386, 3286, 2975, 1718, 1610, 1508,
1456, 1380, 1253, 1174, 1098, 1006, 738, 698 cm-1. Anal. Calcd for
C28H36N4O8: C, 60.42; H, 6.52; N, 10.07. Found: C, 60.30; H, 6.62;
N, 10.06.
Peptide 32. To the mixture of the acid 31 (1.0 g, 1.79 mmol, 1.0
equiv) and NMM (255 µL, 2.33 mmol, 1.3 equiv) in CH2Cl2 (2 mL)
was added BOP-Cl (593 mg, 2.33 mmol, 1.3 equiv) at 0 °C. After for
10 min of stirring, d,l-10 (708 mg, 2.33 mmol, 1.3 equiv) in CH2Cl2 (3
mL) was added. The resulting mixture was stirred overnight, diluted
with CH2Cl2 (200 mL), washed with brine, dried over anhydrous Na2-
SO4, and concentrated. Purification via column chromatography (silica
gel, methylene chloride:EtOAc, 8:2) provided 838 mg (56%) of 32 as
an oil. 1H NMR (300 MHz, CD3OD): δ 1.16 (2H, m), 1.35 (9H, s),
1.43 (2H, m), 1.45 (9H, s), 2.10-2.55 (2H, m), 2.87/2.91 (3H, s), 3.72
(3H, m), 4.08 (1H, m), 5.10 (2H, s), 5.17 (2H, m), 5.24 (2H, m), 7.38
(10H, m), 7.67 (2H, m), 7.74 (2H, m). IR (NaCl, film): 3390, 2974,
1716, 1647, 1609, 1507, 1368, 1252, 1169, 1099, 1006, 719, 696 cm-1
.
Anal. Calcd for C44H54N6O11: C, 62.70; H, 6.46; N, 9.97. Found: C,
62.60; H, 6.31; N, 9.79.
Cyclic Peptide 33. Compound 32 (179 mg, 0.212 mmol, 1.0 equiv)
was treated with anisole (0.1 mL) and TFA (2.0 mL) at 0 °C. The
mixture was stirred for 2 h at room temperature, concentrated, and
triturated in dry ether to give 140 mg of product as a white solid. This
solid was taken up into CH2Cl2 (350 mL). To this solution was added
TBTU (84 mg, 0.26 mmol, 1.5 equiv) and N,N-diisopropylethylamine
(DIEA) (92 µL, 0.51 mmol, 3.0 equiv). The resulting mixture was
stirred 48 h, washed with brine, dried over anhydrous Na2SO4, filtered,
and concentrated. Purification by PTLC (silica gel, CH2Cl2:EtOAc:
MeOH, 4:1:0.1) provided 50 mg (43%) of 33 as a semi-solid. 1H NMR
(300 MHz, CD3OD): δ 1.40 (1H, m), 1.55 H, m), 1.68 (2H, m), 2.64
(1H, m), 2.81/2.86 (3H, s), 3.13 (1H, m), 3.59 (1H, m), 3.93 (2H, m),
4.18 (2H, m), 4.45 (1H, m), 5.09/5.11 (2H, s), 5.26/5.30 (2H, s), 7.34
(10H, m), 7.78 (4H, m). IR (NaCl, film): 3389, 3280, 2948, 1773,
Cyclization Product 29. To a mixture of 28 (70 mg, 0.067 mmol,
1.0 equiv) and anisole (0.1 mL) was added TFA (1.0 mL). The resulting
mixture was stirred for 15 min at room temperature. The TFA was
evaporated and coevaporated with CH2Cl2 to dryness. The resulting
residue was dried in Vacuo for 2 h and triturated with ethyl ether to
give a white solid. This white solid was dissolved in THF (1.5 mL).
To this solution was added triethylamine (20 µL, 0.14 mmol, 2.0 equiv).
After 10 min of stirring, the solvent was evaporated, and the resulting
residue was immediately purified by PTLC (silica gel, CH2Cl2:EtOAc:
MeOH, 4:1:0.5) to give 40 mg (67%) of 29 as a white solid. This
product was unstable with a tendency to form a bicyclic byproduct
(t1/2 is about 1 day) and was used for next step immediately. 1H NMR
(300 MHz, CD3Cl/D2O vs TMS): δ 1.62 (4H, m), 2.45 (2H, m), 2.76/
2.78 (3H, s), 3.32 (1H, m), 3.70 (1H, m), 3.93 (4H, m), 5.17 (6H, m),
7.32 (20H, m). IR (NaCl, film): 3381, 3258, 2934, 1765, 1713, 1646,
1608, 1504, 1452, 1252, 1186, 1096, 1063 cm-1. MS (ES+): calcd
for (C45H49N9O11 + H) ) 892.4; found (M + H) ) 892.4, (M + H -
108) ) 784.3.
1716, 1659, 1608, 1513, 1394, 1253, 1097, 1006, 910, 724 cm-1
.
HRMS (FAB): calcd for (C35H36N6O8 + H) ) 669.2672, found (M +
H) ) 669.2690.
t-BOC-Amine 34. To a solution of 33 (100 mg, 0.15 mmol, 1.0
equiv) in CH2Cl2 (2.0 mL) was added 2 N methylamine/MeOH (2.0
mL) at 0 °C. The mixture was stirred for 10 min at 0 °C, concentrated,
and separated by column chromatography (silica gel, methylene
chloride:EtOAc:MeOH, 4:1:0.3) to give 84 mg of an oil. To a mixture
of the oil obtained above in H2O/dioxane (1 mL, 1:1) and Et3N (217
µL, 1.5 mmol, 10 equiv) was added BOC-ON (106 mg, 0.45 mmol,
3.0 equiv). The mixture was stirred overnight at room temperature
and extracted with EtOAc (2 × 100 mL). The organic phase was dried
over anhydrous Na2SO4, filtered, concentrated, and purified by column
chromatography (silica gel, eluted with CH2Cl2:MeOH, 9:1) to give
35 mg (38%) of 34 as a semi-solid. 1H NMR (300 MHz, CD3OD): δ
1.42 (9H, s), 1.47 (2H, m), 1.66 (2H, m), 2.63 (1H, m), 2.85 (1/2H, m),
2.90/2.93 (3H, s), 3.05 (1/2H, m), 3.52 (2H, m), 3.72 (1H, m), 3.92
(2H, m), 4.25 (1H, m), 5.11 (2H, s), 5.26/5.27 (2H, s), 7.35 (10H, m).
IR (NaCl, film): 3386, 3289, 2934, 1716, 1652, 1609, 1507, 1456,
1366, 1252, 1175, 1095, 1006, 910, 734, 698 cm-1. HRMS (FAB):
calcd for (C32H42N6O8 + H) ) 639.3142, found (M + H) ) 639.3157.
Condensation Products 36 and 37. Compound 34 (33 mg, 0.052
mmol) was treated with anisole (0.1 mL) and TFA (1.0 mL) at 0 °C.
The mixture was stirred for 1 h at room temperature, concentrated,
and triturated with dry ether to give 32 mg (99%) of the corresponding
amine as an oil. A solution of the crude amine (30 mg, 0.046 mmol,
1.0 equiv), triethylamine (20 µL, 0.14 mmol, 3.0 equiv), and 35 (37
mg, 0.092 mmol, 2.0 equiv) in CH2Cl2 (1.0 mL) was stirred for 4 h at
room temperature. The resulting mixture was poured into EtOAc,
washed with brine, dried over anhydrous Na2SO4, filtered, concentrated,
and separated on PTLC (silica gel, CH2Cl2:EtOAc, 7:3) to give 12 mg
(30%) of 36 as a semi-solid and 13 mg (39%) of 37 as an oil. Data
for 36. 1H NMR (300 MHz, CD3OD): δ 1.42 (2H, m), 1.48 (9H, s),
1.67 (2H, m), 2.70 (1H, m), 2.83/2.89 (3H, s), 2.98 (1H, m), 3.62 (2H,
m), 3.91 (3H, m), 4.67 (1H, m), 5.10 (2H, s), 5.14 (2H, s), 5.26 (2H,
s), 7.35 (15H, m). IR (NaCl, film): 3384, 3293, 2932, 1716, 1722,
3(S),5′(S/R)-3-Amino-6-[(aminoiminomethyl)amino]-N-[2-[(ami-
nocarbonyl) amino]-1,4,5,6-tetrahydro-4-oxo-5-pyrimidinyl)-N-me-
thylhexanamide, TAN-1057A/B. To a solution of 29 (40 mg, 0.045
mmol, 1.0 equiv) in MeOH (1.5 mL)/CH2Cl2 (0.5 mL) was added PdCl2
(40 mg). The reaction flask was degassed and charged with H2 (1
atm). The mixture was stirred for 30 min. The mixture was then
purged with nitrogen and filtered to remove the catalyst. The filtrate
was concentrated and dried in Vacuo to give a 2HCl salt of TAN-
1057A/B (1/2) (1:1, 20 mg, 100% yield) as an amorphous solid. This
product was identical in mobility by HPLC, NMR, and antibiotic
activity by bioassay to authentic TAN-1057A/B (Takeda). 1H NMR
(300 MHz, D2O): δ 1.77 (4H, m), 2.85 (1H, dd, J ) 18, 9.3 Hz), 3.00
(1H, dd, J ) 18, 4.0 Hz), 3.17 (3H, s), 3.27 (2H, t, J ) 6.0 Hz), 3.70
(1H, m), 3.99 (2H, m), 5.14 (1H, dd, J ) 12, 8.5 Hz). IR (KBr
pellet): 3350, 3179, 2955, 1696, 1622, 1395, 1205, 1135 cm-1. MS
(ES+): calcd (C13H25N9O3 + H) ) 356.2; found (M + H) ) 356.4.
L-Nr-t-BOC-Nδ,Nω-di-CBz-â-homoarginine (31). To a solution of
NR-t-BOC-di-CBz-L-arginine 30 (BACHEM) (2.72 g, 5.0 mmol, 1.0
equiv) in THF (50 mL) was added NMM (604 µL, 5.5 mmol, 1.1 equiv)
and ethyl chloroformate (524 µL, 5.5 mmol, 1.1 equiv) at 0 °C. The
resulting mixture was stirred for 3 h at 0 °C. The precipitated amine
hydrochloride was rapidly filtered off cold. To this clear solution was
added CH2N2/ether solution (generated from MNNG). The solution
was stirred overnight at room temperature and concentrated to give an
oily diazoketone. The diazoketone was dissolved in t-BuOH/H2O (60
mL, 1:1), and to this solution were added silver benzoate (1.0 g) and
triethylamine (5 mL). The resulting mixture was stirred overnight in
the dark at room temperature and then concentrated in Vacuo. The
residue was treated with a ethyl acetate/saturated NaH2PO4 aqueous
solution. The organic layer was separated, dried over anhydrous sodium
sulfate, and concentrated. Purification via column chromatography
(silica gel, CH2Cl2:EtOAc:MeOH, 4:4:0.5) provided 1.80 g (65%) of
1651, 1491, 1254, 1144, 1008 cm-1
(C42H51N9O11 + H) ) 858.3786; found (M + H) ) 858.3763. Data
. HRMS (FAB): calcd for
25
31 as a semi-solid: [R]D ) -1.8 (c 2.0, CH2Cl2). 1H NMR (300

11784 J. Am. Chem. Soc., Vol. 119, No. 49, 1997
Yuan and Williams
for 37. 1H NMR (300 MHz, CD3OD): δ 1.45 (2H, m), 1.67 (2H, m),
2.73 (1H, m), 2.82 (1H, m), 2.85/2.91 (3H, s), 3.54 (1H, m), 3.70 (1H,
m), 3.91 (3H, m), 4.44 (1H, t, J ) 9 Hz), 5.10 (2H, m), 5.14 (2H, m),
5.27 (2H, m), 7.35 (15H, m). IR (NaCl, film): 3385, 3272, 2931, 1715,
1651, 1494, 1455, 1379, 1241, 1092 cm-1. HRMS (FAB): calcd for
(C36H41N7O9 + H) ) 716.3044; found (M + H) ) 716.3021.
1072, 1056 cm-1. Anal. Calcd for C16H21N3O5S: C, 52.30; H, 5.76;
N, 11.44. Found: C, 52.15; H, 5.73; N, 11.38.
N-(Benzyloxycarbonyl)ureido-(S)-methylisothiourea (13). Com-
pound 35 (401 mg, 1.0 mmol) was treated with TFA (1.0 mL) and
was stirred for 30 min at room temperature, evaporated to dryness,
dried under reduced pressure for 2 h, and triturated with anhydrous
Et2O to give 400 mg of 13 as a solid. This crude product was carried
on without further purification. 1H NMR (300 MHz, CD3OD): δ 2.64
(3H, m), 5.21 (2H, m), 7.31 (5H, m). IR (NaCl, CH2Cl2): 3252, 2959,
1790, 1682, 1203, 1137, 1025, 778, 722 cm-1.
N-(Benzyloxycarbonyl)ureido-N′-(benzyloxycarbonyl)-(S)-meth-
ylisothiourea (21). To a solution of (benzyloxycarbonyl)-(S)-meth-
ylisothiourea18 (1.12 g, 5.0 mmol, 1.0 equiv) in THF (20 mL) was added
(benzyloxycarbonyl)isocyanate (1.06 mL, 6.0 mmol, 1.2 equiv) at room
temperature. After 20 min, the solvent was evaporated and the residue
was triturated in anhydrous ethyl ether two times to afford 2.0 g (100%)
of 21 as a white solid: mp 165-166 °C (recrystallized CH2Cl2/EtOAc).
1HNMR (300 MHz, DMSO-d6 vs TMS): δ 2.29 (3H, s), 5.15 (2H, s),
5.22 (2H, s), 7.40 (10H, m). IR (NaCl, film): 3226, 3159, 2925, 1749,
1715, 1558, 1469, 1261, 1205 cm-1. Anal. Calcd for C10H12N2O2S:
C, 56.85; H, 4.77; N, 10.47; S, 7.99. Found: C, 57.00; H, 4.97; N,
10.36; S, 7.76.
[[[[5-[3-[(Aminoiminomethyl)amino]propyl]hexahydro-2(S/R),5-
(S)-1-methyl-3,7-dioxo-1H-1,4-diazepin-2-yl]methyl]amino]iminom-
ethyl]urea, TAN-1057C/D (3 and 4). Compound 36 (10 mg, 0.012
mmol) was treated with anisole (0.1 mL) and TFA (1.0 mL) at 0 °C.
The mixture was stirred for 1 h at room temperature, concentrated,
and purified by PTLC (silica gel, CH2Cl2:MeOH, 6:1) to give 6.0 mg
of a semi-solid. To a solution of this substance (6 mg, 0.0066 mmol,
1.0 equiv) in MeOH (1.0 mL)/CH2Cl2 (0.5 mL) was added PdCl2 (8.0
mg). After degassing with N2, the reaction mixture was charged with
H2 (1 atm) and the mixture was hydrogenated for 10 min. The mixture
was then purged with nitrogen and filtered to remove the catalyst. The
filtrate was concentrated and dried in Vacuo to give the 2HCl salt of
TAN-1057 C/D (1:1, 3 mg, ∼100% yield) as an amorphous solid. 1H
NMR (300 MHz, DMSO-d6 vs TMS): δ 1.51 (4H, m), 2.82 (3H, s),
2.70-2.90 (2H, m), 3.09 (2H, m), 3.43 (1H, m), 3.79 (2H, m), 4.66
(1/2H, m), 4.75 (1/2H, m), 7.26 (6H, m), 7.86 (1H, br), 8.06 (1H, br),
8.70 (2H, br), 9.15 (1H, br), 10.35 (1H, br). MS (FAB): calcd for
(C13H25N9O3 + H) ) 356.2; found (M + H) ) 356.3, (M + 2H)++
)
Acknowledgment. This paper is dedicated to Professor
Yoshito Kishi on the occaision of his 60th birthday. This work
was supported by the NSF and Microcide Pharmaceutical Co.
We thank Takeda Chemical Co., Japan, for the generous gift
of an authentic specimen of TAN-1057A/B.
178.7. Upon standing in 0.1 M phosphate buffer at pH ) 5, the mixture
of 3 and 4 had reverted to a mixture of 1 and 2.
N-(Benzyloxycarbonyl)ureido-N′-(tert-butyloxycarbonyl)-(S)-me-
thylisothiourea (35). To a solution of (tert-butyloxycarbonyl)-(S)-
methylisothiourea17,18 (1.69 g, 8.91 mmol, 1.0 equiv) in THF (40 mL)
was added N-(benzyloxycarbonyl)isocyanate (1.90g, 10.73 mmol, 1.2
equiv). The resulting mixture was stirred for 10 min at room
temperature and concentrated. Purification via column chromatography
(silica gel, CH2Cl2:EtOAc:MeOH, 4:4:0.5) provided 3.25 g (100%) of
35 as a white solid: mp 120-3 °C. 1H NMR (300 MHz, CDCl3 vs
TMS): δ 1.48 (9H, s), 2.32 (3H, s), 5.22 (2H, s), 7.37 (5H, m), 7.50
(1H, br, D2O exchanged), 11.96 (1H, br, D2O exchanged). IR (NaCl,
film): 3270, 2979, 1747, 1664, 1570, 1476, 1370, 1275, 1207, 1138,
Supporting Information Available: Experimental proce-
dures for the synthesis of compounds 7, 9, 10, and 12 plus HPLC
data on the synthetic and natural TAN-1057A/B samples (5
pages). See any current masthead page for ordering and Internet
access instructions.
JA972670J