Toward a library synthesis of the natural dipeptide antibiotic TAN 1057 A,B

Article abstract of DOI:10.3390/70600469

The natural dipeptide antibiotic TAN 1057 A,B represents a promising new antibiotic entity. In this communication we report a novel approach for the synthesis of TAN 1057 A,B analogs bearing variations in the β-arginine side chain. This approach involves a combination of liquid and solid phase methods and allows for a library synthesis of analogs of the natural product.

Full text of DOI:10.3390/70600469

Molecules 2002, 7, 469-474
molecules
ISSN 1420-3049
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Toward a Library Synthesis of the Natural Dipeptide Antibiotic
TAN 1057 A,B
Nuria Aguilar and Jochen Krüger*
Bayer-AG, Business Group Pharma, Research, 42096 Wuppertal, Germany, Tel. +49-202-368644, Fax +49
202/364061.
* Author to whom correspondence should be addressed; e-mail: Joachim.Krueger.JK2@bayer-ag.de
Received: 2 May 2002; in revised form: 10 June 2002 / Accepted: 14 June 2002/ Published: 30 June 2002
Abstract: The natural dipeptide antibiotic TAN 1057 A,B represents a promising new
antibiotic entity. In this communication we report a novel approach for the synthesis of
TAN 1057 A,B analogs bearing variations in the β-arginine side chain. This approach
involves a combination of liquid and solid phase methods and allows for a library
synthesis of analogs of the natural product.
Keywords: TAN 1057 A, B, dipeptide antibiotics, combinatorial chemistry.
Introduction
The natural dipeptide antibiotic TAN 1057 A,B (1, Figure 1) was first isolated and characterized by
Takeda [1]. To date, two independent total syntheses of this natural product have been disclosed [2]. TAN
1057 A,B displays excellent in vitro and in vivo activity against staphylococci including Methicillin
Resistant Staphylococcus Aureus (MRSA) and represents a promising new antibiotic entity [3]. However,
TAN 1057 A,B suffers from strong toxic side effects (LD50 = 50 mg/Kg) which are in our view prohibitive
for therapeutic use in humans. Therefore, we launched a synthesis program with the goal of identifying

Molecules 2002, 7
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analogs with significantly improved tolerance and concomitant retention of the promising antibiotic activity.
In this communication we report a new strategy that allows for the parallel synthesis of novel derivatives of
the natural product focusing on alteration of the β-arginine moiety.
Figure 1: TAN 1057 A, B (1).
H
NH
HN
NH
O
N
N
NH2
NH2
O
NH
O
N
Me
Results and Discussion
The salient molecular architecture of TAN 1057 A,B defines its unique physical and chemical properties:
the natural product is highly water soluble, displays almost no retention on RP-HPLC material under
standard conditions [4] and proves unstable towards nucleophiles and strong bases at elevated temperatures.
This instability presumably results from the dihydropyrimidinone heterocycle which decomposes readily
under the aforementioned conditions. However, early structure activity relationship (SAR) studies revealed
that the heterocyclic core was essential for biological activity. Therefore, our program was primarily focused
on alterations of the β-arginine side chain.
For the parallel synthesis of TAN 1057 analogs we envisioned a convergent strategy combining solid and
liquid phase chemistry. Our synthesis commenced with the construction of amine 4 as outlined in Scheme 1.
Accordingly, orthogonally protected β-lysine 2 was coupled with the dihydropyrimidinone core 3 [2b]
employing standard EDC-peptide coupling conditions followed by HCl mediated deprotection of the
terminal BOC-group.
Scheme 1: Synthesis of amine 4.
H
O
N
N
NH2
CbzHN
O
+
BOCHN
NH
O
OH
HN
1. EDC (60%)
2. HCl, dioxane
(quant.)
Me
3
2
H
O
N
N
NH2
CbzHN
O
HCl x
H2N
NH
O
N
Me
4

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471
For the requisite installation of the guanidine moiety we employed the method published by Dodd and
Wallace [5] who described the use of immobilized isothioureas 6 as convenient guanylation agents (cf.
Scheme 2). Isothioureas 6 are readily available by a Mitsunobu reaction starting from commercially
available resin 5.
For method evaluation we selected six structurally diverse primary alcohols R1OH (see Table 1) and
submitted them to the Mitsunobu protocol as outlined in Scheme 2. Subsequently we used these polymeric
intermediates 6 without further characterization to install the guanidine moiety at the terminus of 4. To our
dismay, the conditions originally described to effect guanylation (excess amine, 50°C, DMF) failed in our
hands to deliver protected intermediates 7. This was presumably due to the inherent instability of the
heterocyclic moiety towards the given reaction conditions.
Scheme 2: Parallel synthesis of TAN 1057 A,B analogs.
1. 4, DBU
R1
BOCN
R1OH, THF
Ph3P, DIAD
DMF, r.t.
3 days
H
NBOC
NBOC
NBOC
NHBOC
NBOC
O
N
N
NH2
HNCbzO
S
S
HN
NH
O
6 examples
2. Filtration,
RP-HPLC
R1
N
6
5
Me
7
R1
N
H
NH
O
N
N
NH2
NH2
O
HN
NH
O
1. HBr, HOAc
2. recrystallisation
N
Me
8 x 2HBr
Thus, we surveyed a range of additional bases such as ethyl diisopropylamine, N-methylmorpholine,
DBU, Schwesinger type bases or proton sponges to promote the desired transformation under milder
conditions. Finally, the optimal conditions for the guanylation included the use of a slight excess of resin 6
with respect to the hydrochloride 4, DMF as solvent of choice, and DBU as base at room temperature for 3
days. The addition of transition metal salts like silver triflate or mercury chloride did not enhance the
reaction rate significantly.
Subsequently, with an optimized coupling protocol in hand, all six polymeric isothioureas 6 were each
reacted with 4 in a parallel setup. LC/MS analyses of the crude reaction mixtures revealed that protected
intermediates 7 were formed as main products in all cases, however, the crude mixtures also contained
byproducts arising from degradation of the dihydropyrimidinone. Purification of the crude products was
achieved by automated preparative HPLC to deliver a range of modified natural product precursors 7 in
acceptable yields around 40% (over 2 steps starting from 5) and sufficient HPLC-purities (>90% in all

Molecules 2002, 7
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cases). Finally, the target molecules 8 were obtained from 7 upon deprotection of the amino groups using
HBr in acetic acid followed by precipitation of the products from acetone-methanol mixtures.
The results for all TAN 1057 A,B analogs are depicted in Table 1. In general, the overall yields (21 -
32%, 3 steps from 5) were acceptable taking into account that the dihydropyrimidinone core proved
susceptible towards decomposition in the guanylation event. Moreover, HPLC analyses revealed that the
majority of products were isolated in essentially pure form.
Table 1: Analogs of TAN 1057 A,B by parallel synthesis.
alcohol
R1OH
overall yield HPLC
entry
1
producta
b
from 5 (%)b
purity (%)
H
H
N
NH
O
N
N
NH2
94
97
88
80
84
97
OH
OH
32
NH2
O
HN
NH
O
N
8a
Me
H
H
N
NH
NH
NH
O
N
N
NH2
N
NH2
O
O
O
N
30
26
22
21
26
2
3
4
5
6
HN
NH
O
N
Me
8b
H
N
H
N
O
N
N
N
NH2
NH2
NH2
OH
NH2
HN
HN
HN
NH
NH
NH
O
O
O
N
8c
Me
OAc
OMe
NH2
OAc
H
N
H
N
O
OH
OH
NH2
N
Me
8d
NH2
8e
OMe
H
N
H
N
NH
NH
O
O
O
N
Me
NHBOC
OH
H
N
H
N
O
N
NH2
NH2
HN
NH
O
N
8f
Me
(a) All products were obtained as HBr salts, (b) see Experimental section for details.

Molecules 2002, 7
473
Conclusions
We have demonstrated an efficient and convergent route to TAN 1057 A,B analogs via a parallel
synthesis approach. Key features of this strategy include the use of a diverse set of polymer bound
isothioureas originating from a solid phase Mitsunobu protocol, optimized guanylation conditions,
automated HPLC purification of the protected intermediates, and elaboration of a crystallization protocol for
the final products. An application of this methodology for multi-component library synthesis and in depth
pharmacological investigations of the synthesized compounds are currently underway.
Experimental
General procedure for the synthesis of 8 (90 µmol scale) as outlined in Scheme 2: To a suspension of 1.5 eq
of functionalized resin 5 (purchased from Novabiochem) in dry THF (3 mL), triphenylphosphine (9.4 eq)
and the corresponding alcohol (9.4 eq) were added. Subsequently DIAD (9.0 eq.) was added dropwise and
the resulting orange suspension was shaken at room temperature overnight. The solvent was filtered off and
the resin was thoroughly washed with DMF (4 x 5mL), THF (3 x 5mL), MeOH (3 x 5mL) and
dichloromethane (3x 5 mL).
The functionalized resin 6 thus obtained was suspended in DMF (2.5 mL) and the hydrochloride 4 (1.0
eq) was added in one portion. Finally, DBU (5 eq) was also added dropwise and the suspension was shaken
at room temperature for 3 days. After filtration the resin was rinsed with DMF and the solvent was removed
in vacuo. The crude products were purified using a Gilson automated preparative HPLC (conditions:
Kromasil 100 C18, 5 µm, 50 x 20 mm; gradient: acetonitrile / water 15:85 to 90:10, room temperature, flow
25 mL / min, detection @ 220 nm) to yield 7 (35-45% yield, >90% HPLC purity in all cases).
Finally, at 0°C HBr (30% solution in acetic acid, 1 mL) was added to a solution of 7 in dichloromethane
(2 mL) and the mixture was stirred at 0°C for 1 hour. The solvent was removed in vacuo. In the final step the
products were redissolved in methanol (0.5 mL) and acetone was added until the hydrobromide salts
precipitated from the solution. After filtration compounds 8 were obtained as white powders. The products
were analyzed by HPLC (conditions: Kromasil 100 C18, 25 x 4 mm; gradient: A = water containing PIC B7
low UV, B = acetonitrile, 0-1 min 90% A, 1-9 min 90% A to 90% B, 9-13 min 90% B; room temperature,
flow 2 mL / min, detection @ 210 nm), 1H-NMR (D4-methanol) and MS.
References and Notes
1. Funabashi, Y.; Tsubotani, S.; Koyama K.; Katayama, N.; Harada, S. Tetrahedron 1993, 49, 13-28.
2. (a) Williams, R. M.; Yuan, C. J. Am Chem. Soc. 1997, 119, 11777-11784 (b) Sokolov, V. V.;
Kozhushkov, S. I.; Nikolskaya, S.; Belov, V. N.; Es-Sayed, M.; de Meijere, A. Eur. J. Org. Chem.

Molecules 2002, 7
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1998, 777-783 (c) For a recent synthesis of the heterocyclic core see: Ganesan, A.; Lin, P. Synthesis
2000, 2127-2130.
3. (a) Katayama, N.; Fukusumi, S.; Funabashi, Y.; Iwahi, T.; Ono, H. J. Antibiotics 1993, 46, 606-613. (b)
Williams, R. M.; Yuan, C.; Lee, V. J.; Chamberland, S. J. Antibiotics 1998, 51, 189-201.
4. For analytical purposes we analyzed the natural product and derivatives thereof by ion-pair
chromatography, however, effective conditions for preparative RP-HPLC were not found.
5. Dodd, D. S.; Wallace, O. B. Tetrahedron Lett. 1998, 39, 5701-5704.
Sample Availability: Samples of entries 1, 5, 7 and 11 are available from MDPI
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