Pyrimidinone antibiotics - Heterocyclic analogues with improved antibacterial spectrum

Article abstract of DOI:10.1016/S0960-894X(03)00578-X

We report the synthesis and pharmacological evaluation of new derivatives of the natural dipeptide antibiotic TAN 1057 A,B containing heterocycles either in the β-amino acid side chain or as mimics of the urea function. In the course of this program, we identified novel analogues that display activity towards a broader panel of Gram-positive bacteriae.

Full text of DOI:10.1016/S0960-894X(03)00578-X

Bioorganic & Medicinal Chemistry Letters 13 (2003) 2641–2645
Pyrimidinone Antibiotics—Heterocyclic Analogues with Improved
Antibacterial Spectrum
Michael Brands,* Yolanda Cancho Grande, Rainer Endermann, Reinhold Gahlmann,
Jochen Kruger and Siegfried Raddatz
BAYER AG, Business Group Pharma, Research, D-42096 Wuppertal, Germany
Received 17 February 2003; revised 2 June 2003; accepted 3 June 2003
Abstract—We report the synthesis and pharmacological evaluation of new derivatives of the natural dipeptide antibiotic TAN 1057
A,B containing heterocycles either in the b-amino acid side chain or as mimics of the urea function. In the course of this program,
we identified novel analogues that display activity towards a broader panel of Gram-positive bacteriae.
# 2003 Elsevier Ltd. All rights reserved.
Introduction
Further optimisation was aimed towards expanding the
limited antibacterial spectrum of TAN 1057 A,B to
cover in particular Staphylococcus pneumoniae subtypes.
The appearance of resistant pathogens such as Multi-
Resistant Staphylococcus Aureus (MRSA) as well as
Vancomycin Resistant Enterococcus Faecium (VRE)
constitutes a severe problem particularly in hospital
intensive care units. It is generally agreed that the
introduction of novel antibiotic classes would con-
tribute to the combat of the ever lasting problem of
bacterial resistance.1 However, despite world wide
efforts, only one new antibacterial class (oxazolidinones,
namely linezolide) has reached the market within the
last 15 years.
In this communication we summarise the synthesis and
antibacterial properties of new TAN 1057 A,B ana-
logues focusing in particular on the introduction of
heterocycles into the b-amino acid side chain as well as
in the urea part of the TAN 1057 A,B heterocycle.
Chemistry
Some examples of a-amino acids containing constrained
basic side chains have been reported,5 but no precedents
are known for b-amino acids. We now report the
synthesis of novel enantiomerically pure analogues of
b-homolysine (n=1) and b-bishomolysine (n=2) which
contain a thiazole ring (9, 10 in Scheme 1), as well as the
synthesis of a racemic b-homolysine derivative 17
containing an isoxazol ring (Scheme 2).
In search of promising lead structures for the synthesis
of new antibacterials, we have focused our attention on
unexplored natural products with reported antibacterial
activity. For the natural dipeptide antibiotic TAN 1057
A,B (1) (Fig. 1) excellent Minimal Inhibitory Con-
centrations (MIC) against staphylococci including
MRSA were reported.2 Two independent total synth-
eses of the natural compound3 and some analogues were
published recently.4 In particular the b-lysine and b-
homolysine derivatives 2 and 3 were found to exhibit
interesting properties such as improved tolerability.4c
Key synthetic intermediate for the formation of thia-
zoles 9/10 (Scheme 1) is the a-bromo ketone 6 derived
*Corresponding author at present address: Bayer Corp., 400 Morgan
Lane, West Haven, CT 06516-4175; fax: +1-203-812-6182; e-mail:
michael.brands.b@bayer.com
Figure 1. Natural dipeptide antibiotic TAN 1057 A,B (1) and syn-
thetic analogues.
0960-894X/03/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/S0960-894X(03)00578-X

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M. Brands et al. / Bioorg. Med. Chem. Lett. 13 (2003) 2641–2645
Scheme 1. Synthesis of 9 and 10, reagents and conditions (yields are
comparable for n=1and 2): (a) iBuOC(O)Cl, DIPEA, CH2Cl2,
À20 ꢀC; (b) CH2N2; (c) HBr, AcOH; (d) N-Z-glycine thioamide or N-
Z-b-alanine thioamide, DMF, 100 ꢀC; (e) HATU, DIPEA, DMF,
TAN-heterocycle; (f) CF3SO3H, anisole, CH2Cl2.
Scheme 4. Synthesis of 31 and 32, reagents and conditions: (a) ace-
tone, 0 ꢀC; (b) aq NaOH, 90 ꢀC; (c) MeI, CH3CN, 40 ꢀC; (d) 3-Amino-
2-(N-Z-N-methylamino) methyl propionate hydrochloride, NaOAc,
CH3CN, 70 ꢀC; (e) HBr, AcOH; (f) HATU, DIPEA, DMF, Nb,No-
bis-Z-L-b-homo lysine; (g) HBr, AcOH; (h) aq NH3; (i) MeOH/HCl.
hydrogenolysis was not applicable for removal of the Z-
group presumably due to catalyst poisoning caused by
sulfur.9 However, trifluoromethansulfonic acid in the
presence of anisole as a scavenger was successfully used
for the cleavage.10
For the isoxazol containing b-amino-acid 16, a [3+2]
cycloaddition11 of Z-protected propargylamin and the
corresponding nitrile oxide was used as a key step fol-
lowed by introduction of the b-amino group through a
Michael addition of ammonia to a,b-unsaturated ester
14 (Scheme 2). This strategy required the transforma-
tion of ester 12 via reduction and subsequent Swern
oxidation of the corresponding primary alcohol (yields
not optimised) followed by a Wittig–Horner olefination.
The amino group was protected, the ester was saponi-
fied providing the b-amino acid 16 which was coupled
with the TAN 1057 heterorocycle. Finally, removal of
the Z-group by catalytic hydrogenolysis yielded the
desired product 17.
Scheme 2. Synthesis of 17, reagents and conditions: (a) NO2CH2-
CO2Et, PhNCO, Et3N, benzene, reflux; (b) DIBAL, CH2Cl2, À78 ꢀC;
(c) DMSO, (COCl)2, Et3N, À78 ꢀC; (d) EtO2(O)PCH2CO2Et,
NaHMDS, THF, 0 ꢀC; (e) NH3/EtOH (satd.), 100 ꢀC; (f) ZCl, Et3N,
CH2Cl2; (g) CH2Cl2, KOSiMe3; (h) HATU, DIPEA, DMF, TAN-
heterocycle; (i) PdCl2, H2, MeOH.
In order to tune physicochemical parameters (e.g., lipo-
philicity), a sulfide and a sulfone bridge, respectively,
were introduced into to the b-amino acid side chain
(Scheme 3). For this purpose, S-alkylation of N-Boc
cysteine using the mesylate of BOC-ethanolamine 19
was performed. The b-amino acid 21 obtained by stan-
dard Arndt–Eistert homologation of the corresponding
acid 20 was coupled with the TAN 1057 heterocycle.
Finally, removal of the BOC-groups yielded 23. Com-
pound 24 was obtained following the same synthetical
route starting from the oxidised compound 22, albeit in
poor yield.
Scheme 3. Synthesis of 23 and 24, reagents and conditions: (a) Boc2O,
NaHCO3, MeOH; (b) MsCl, pyridine; (c) N-Boc-cysteine methyl car-
boxylate, Cs2CO3, DMF; (d) CH2Cl2, KOSiMe3; (e) iBuOC(O)Cl,
DIPEA, CH2Cl2, À20 ꢀC; (f) CH2N2; (g) hv, dioxane/water; (h)
HATU, DIPEA, DMF, TAN-heterocycle; (i) HCl, dioxane; (j)
CH2N2; (k) mCPBA, CH2Cl2.
from the non-natural enantiomer of aspartic acid. Poly-
functional intermediate 6 already bears the b-amino
acid moiety as well as an a-bromo ketone which can
function as a thiazole precursor. Diazoketone 5 was
obtained from commercially available N-(benzyloxy-
carbonyl) tert-butyl aspartate [d-Z-Asp(OtBu)-OH, 4]
by a known procedure.6 The conversion of diazoketone
5 to a-bromo ketone is reported to proceed without
cleavage of the tert-butyl ester.7 However in all the cases
we obtained the corresponding acid. Condensation of 6
with the required thioamides, prepared from the corre-
sponding carboxamides by treatment with Lawesson’s
reagent,8 yielded the desired thiazole b-amino-acids 7/8
which were coupled in the next step with the TAN 1057
heterocycle3b under standard conditions. Catalytic
So far, changes in the periphery of the dihydro pyri-
midinone part of TAN 1057 A,B have only rarely been
described.4a We envisaged to exchange the urea part by
amino heterocycles which were believed to appro-
priately mimic the donor/acceptor properties of the urea
group.
For this purpose, different derivatives were synthesised
through the route outlined in Scheme 4. Applying
known procedures,12 2-amino-pyrimidine (26) was con-
verted into thiourea 27 which was subsequently trea-
ted13 with methyl iodide to yield the corresponding
isothiouronium salt 28. The coupling reaction between
the salt 28 and 3-amino-2-(N-Z-N-methylamino) methyl

M. Brands et al. / Bioorg. Med. Chem. Lett. 13 (2003) 2641–2645
2643
propionate hydrochloride3b provided the protected
dihydropyrimidinone 29. After removal of the Z-pro-
tecting group, the heterocycle 30 was coupled with
appropriately protected b-homolysine. Subsequent
removal of the Z-protecting group was accomplished
using HBr in acetic acid generating dihydrobromide 31,
from which the corresponding dihydrochloride 32 is
available by consecutive treatment with ammonia fol-
lowed by hydrochloric acid. Alternatively, tris-Z-pro-
tected b-homoarginine can be used for the peptide
coupling finally yielding derivative 43 (cf. Table 2).
As can be deduced from Table 1, introduction of thia-
zole (9/10) and isoxazole (17) heterocycles into the b-
amino acid side chain leads to considerably less active
compounds. Also the incorporation of sulfide/sulfone
groups leads to loss of activity (compounds 23/24). This
is surprising since b-lysine and b-homolysine derivatives
are highly active. The loss of activity observed for deri-
vatives 23 and 24 might be explained by changes in
lipophilicity influencing for example, up-take by the
bacteriae rather than by stereo-electronical reasons.
Variations in the urea part of the TAN 1057 A,B het-
erocycle led to a completely different picture: derivatives
with strong basic nitrogen atoms in ortho-position of
the amino group show high anti-staphylococcal activity.
Examples are the 2-pyridyl derivatives 33 and 44, the 2-
pyrimidyl derivatives 32 and 43, the 2-pyrazine deriva-
tive 37 as well as the 2-quinolyl derivatives 34 and 45.
For some of those examples (e.g., derivatives 34, 37, 43,
45), an improved target activity compared to TAN 1057
A,B (1) is observed. Heterocycles with a less basic
nitrogen atom in ortho position to the exocyclic amino
group such as thiazole (35), benzothiazole (36) or ben-
zimidazole (39) show considerably weaker effects. Also
the shift of the nitrogen atom position from the ortho to
the meta position (41) or the transfer of the pyrimidyl
residue to tertrahydropyrimidyl (42) significantly
reduces the activity. Finally, replacement of the het-
erocycle by a phenyl ring abolishes the activity (40).
Those observations underline that the nitrogen atom
By analogous procedures, numerous amino or methyl
amino heterocycles were introduced yielding novel
dihydropyrimidinone antibiotics 32–45 (cf. Table 2).
Pharmacology
On a first level of in vitro assays, the novel derivatives
were tested for their abilities to inhibit the growth of
Staphylococcus aureus bacteriae. Since TAN 1057 A,B
(1) is a known inhibitor of the bacterial translation
machinery,2 respective translation assays14 were used in
parallel to gain in-sight into the level of selectivity of the
procaryotic versus the eucaryotic translation. This
selectivity delivers a first clue about the toxicity of the
samples. As a reference compound, b-homolysine deri-
vative 3 showing markedly improved selectivity in
favour of the eucaryotic translation was used.4c
Table 1. Pharmacological profile of novel TAN 1057 A,B analogues with modified b-amino acid side chain
Compd
Structuresa
MIC (mg/mL)
S. aureus 133
IC50 (mM)
procaryotic translation
IC50 (mM)
eucaryotic translation
Selectivity
indexb
1
0.10
0.05
0.30
0.50
32
0.17
2.8
0.57
5.3
3
9
50
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
10
17c
23
24
100
16
>100
100
>64
n.d.
n.d.
>100
aObtained as one pair of diastereomers.
bIn vitro selectivity index (IC50 eucaryotic translation/procaryotic translation).
cObtained as a mixture of diastereomers.

2644
M. Brands et al. / Bioorg. Med. Chem. Lett. 13 (2003) 2641–2645
incorporated in the heterocycle functions as a hydrogen
bond acceptor and might mimic the carbonyl group of
the urea moiety. On the other hand, the bridging amino
group most likely displays hydrogen donor properties,
since N-methyl derivative 38 is inactive.
parent compound TAN 1057 A,B (1) they were tested
on a panel of Gram-positive pathogens (Table 3).
Although activity on S. aureus and Enterococcus faecium
remains in the range of the TAN 1057 A,B level, the
first considerable activity against S. pneumoniae strains
was observed for compounds 44 and 45. E. faecalis
constitutes the only significant gap in the Gram-positive
spectrum.
In-depths investigations were carried out with the most
active compounds 44 and 45. Back to back with the
Table 2. Pharmacological profile of novel TAN 1057 A, B analogues 32–45
Compd
Structuresa
MIC (mg/mL)
S. aureus 133
IC50 (mM)
procaryotic translation
IC50 (mM)
eucaryotic translation
Selectivity
indexb
32
33
34
35
36
37
38
39
40
41
42
1.6
0.2
1
0.1–0.5
0.05
0.38
0.2
0.38
0.2
6.3
0.05
n.d.
1
50
1.0
n.d.
n.d.
25
3.2–3.6
<0.1
n.d.
n.d.
<0.1
>100
50
<0.1n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
1–2
n.d.
100
50
n.d.
n.d.
>64
n.d.
n.d.
>100
n.d.
43
44
45
0.4
<0.05
0.10
0.07
>2
0.05
0.10.1
n.d.
n.d.
01.1
aObtained as one pair of diastereomers.
bIn vitro selectivity index (IC50 eucaryotic translation/procaryotic translation).

M. Brands et al. / Bioorg. Med. Chem. Lett. 13 (2003) 2641–2645
Table 3. Antibacterial activity of derivatives 1, 44 and 45
2645
References and Notes
Compd
1
44
0.5
0.25
2
45
0.5
0.25
8
1. (a) Weber, D. J.; Raasch, R.; Rutala, W. A. CHEST
1999, 115, 34. (b) Bodi, M.; Ardanuy, C.; Rello, J. Crit. Care
Med. 2001, 29, 82. (c) Fluit, A. C.; Schmitz, F. J.; Verhoef, J.
Int. J. Antimicrob. Agents 2001, 18, 147. (d) Jeljaszewicz, J.;
Mlynarczyk, G.; Mlynarczyk, A. Int. J. Antimicrob. Agents
2001, 16, 473. (e) McGowan, J. E., Jr. Crit. Care Med. 2001,
29, 69.
2. (a) Katayama, N.; Fukusumi, S.; Funabashi, Y.; Iwahi, T.;
Ono, H. J. Antibiot. 1993, 46, 606. (b) Funabashi, Y.; Tsubo-
tani, S.; Koyama, K.; Katayama, N.; Harada, S. Tetrahedron
1993, 49, 1 3.
MIC S. aureus 133 (mg/mL)
0.25
0.25
2
MIC S. epidermidis 18570 (mg/mL)
MIC E. faecium L 4001(mg/mL)
MIC E. faecalis 18531 (mg/mL)
MIC S. pneumoniae G9a (mg/mL)
MIC S. pneumoniae 1707/4 (mg/mL)
ED100 S. aureus 133 (mg/kg)
>16
16
32
>64
2
>16
n.d.
2
1n.d.
8
0.25
3. (a) Williams, R. M.; Yuan, C. J. Am. Chem. Soc. 1997, 119,
11777. (b) Sokolov, V. V.; Kozhushkov, S. I.; Nikolskaya, S.;
Belov, V. N.; Es-Sayed, M.; de Meijere, A. Eur. J. Org. Chem
1998, 777.
4. (a) Williams, R. M.; Yuan, C.; Lee, V. J.; Chamberland, S.
J. Antibiot. 1998, 51, 189. (b) Brands, M.; Endermann, R.;
Gahlmann, R.; Kruger, J.; Raddatz, S.; Stoltefuß, J.; Belov,
V. N.; Nizamov, S.; Sokolov, V. V.; de Meijere, A. J. Med.
Chem. 2002, 45, 4246. (c) Brands, M.; Endermann, R.; Gahl-
mann, R.; Kruger, J.; Raddatz, S. Bioorg. Med. Chem. Lett.
2003, 13, 241.
5. (a) Patt, W. C.; Hamilton, H. W.; Taylor, M. D.; Ryan,
M. J.; Taylor, D. G.; Connolly, C. J. C.; Doherty, A. M.;
Klutchko, S. R.; Sircar, L.; Steinbaugh, B. A.; Batley, B. L.;
Painchaud, C. A.; Rapundalo, S. T.; Michniewicz, B. M.;
Olson, S. J. Med. Chem. 1992, 35, 2562. (b) Hardy, K. D.;
Harrington, F. P.; Stachulski, A. V. J. Chem. Soc., Perkin
Trans. 1 1984, 1227. (c) Farloni, M.; Giacomelli, G.; Spanu, E.
Tetrahedron Lett. 1998, 39, 9241.
In an S. aureus mouse sepsis model (iv application),
derivative 44 shows a slightly lower activity than TAN
1057 A,B. The protective effect remains comparable to
penicillin G (ED100 1mg/kg) which was used as standard.
The cytoxicity potential of 44 and 45 was estimated
using macrophage cell lines. Cytotoxic concentrations
(CC50 values) on those cells are in the same range as
observed for TAN 1057 A,B which is known to display
a relatively low LD50 value in mice of 50 mg/kg iv. The
toxicity potential can also be predicted from the selec-
tivity index for the heterocyclic derivatives (Table 2) in
comparison to well tolerated derivative 3. Thus, further
optimization of broad spectrum antibacterial activity
and tolerability are needed.
6. Kranzt, A.; Copp, L. J.; Coles, P. J.; Smith, S. J.; Heard,
S. B. Biochemistry 1991, 30, 4678. In according with this pub-
lication, no epimerisation for the diazoketone 5 is observed.
7. Dolle, R. E.; Hoyer, D.; Prasad, C. V. C.; Schmidt, S. J.;
Helaszek, C. T.; Miller, R. E.; Ator, M. A. J. Med. Chem.
1994, 37, 563.
8. Irako, N.; Hamada, Y.; Shiori, T. Tetrahedron 1995, 46,
12731.
9. Meinhofer, J.; Kuromizu, K. Tetrahedron Lett. 1974, 37, 3259.
10. Yahima, H.; Nobutaka, E.; Ogawa, H.; Kawatani, H. J.
Chem. Soc., Chem. Commun. 1974, 107.
Summary
Whereas the incorporation of heterocycles in the b-
amino acid side chain cuts down activity significantly,
aromatic ortho-amino nitrogen heterocycles are suitable
replacements of the urea group. In addition, consider-
able anti-streptococcal activity was observed for amino
pyridyl derivative 44 and amino quinolyl derivative 45.
However, the antibacterial spectrum and concomitant
tolerability need to be improved further.
11. Pei, Y.; Moss, W. H. Tetrahedron Lett. 1984, 25, 5831.
12. (a) Hurst, D.-T. Aust. J. Chem. 1988, 1221. (b) Kaplan,
G. M.; Frolov, A. N.; El’tsov, A. V. J. Org. Chem. USSR
1991, 27, 201(Engl. Transl.).
13. Rasmussen, C. R. Synthesis 1998, 456.
Acknowledgements
14. (a) Procaryotic translation assay: E. coli S30 Extract Sys-
tem for Circular DNA, Technical Bulletin 092, Promega Cor-
poration.
(b) Eucaryotic translation assay: FlexiR Rabbit Reticulocyte
Lysate System L 4540, Technical Bulletin #TB127, Promega
Corporation.
We thank F.-U. Geschke, D. Habich and H. Labi-
schinski for helpful discussions. We are grateful to A. de
Meijere and V. N. Belov (Univ. Gottingen, Germany)
for a very fruitful co-operation.