SAR studies on dihydropyrimidinone antibiotics

Article abstract of DOI:10.1016/j.bmcl.2011.01.099

There is an urgent need for the development of novel antimicrobial agents that offer effective treatment against MRSA. Using a new class of dipeptide antibiotic TAN-1057A/B as lead, we designed, synthesized and evaluated analogs of TAN-1057A/B. Several novel dihydropyrimidinone antibiotics demonstrating comparable antibiotic efficacy while possessing favorable selectivity were identified.

Full text of DOI:10.1016/j.bmcl.2011.01.099

Bioorganic & Medicinal Chemistry Letters 21 (2011) 1670–1674
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
SAR studies on dihydropyrimidinone antibiotics
⇑
Lianhong Xu , Lijun Zhang, Robert Jones , Clifford Bryant , Nina Boddeker , Eric Mabery, Gina Bahador,
Julia Watson , Jeffery Clough , Murty Arimilli , Wendy Gillette , Dorothy Colagiovanni , Keyu Wang,
Craig Gibbs, Choung U. Kim
Gilead Sciences Inc., 333 Lakeside Drive, Foster City, CA 94404, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
There is an urgent need for the development of novel antimicrobial agents that offer effective treatment
against MRSA. Using a new class of dipeptide antibiotic TAN-1057A/B as lead, we designed, synthesized
and evaluated analogs of TAN-1057A/B. Several novel dihydropyrimidinone antibiotics demonstrating
comparable antibiotic efficacy while possessing favorable selectivity were identified.
Ó 2011 Elsevier Ltd. All rights reserved.
Received 5 January 2011
Revised 20 January 2011
Accepted 21 January 2011
Available online 31 January 2011
Keywords:
TAN-1057A/B
Antimicrobial agent
Dipeptide antibiotic
Many antibacterial products are available on the world market,
but successful treatment of bacterial infections is becoming
increasingly problematic as resistance to current agents becomes
more widespread. Methicillin-resistant Staphylococcus aureus
NH
CH3
N
*
H N
N
NH
1
O
2
5
H
3
NH2
O
O
N
N
NH2
H
(
MRSA) is one of the leading causes of severe infections in both
1
a TAN-1057A *S
1,2
the hospital and community environments.
Although, several
1b TAN-1057B *R
new classes of antimicrobial agents have reached the market in re-
cent years with world wide efforts, there is still an urgent need for
the development of novel agents that offer effective treatment
against MRSA.
TAN-1057A/B (1) (Fig. 1), isolated from bacteria Flexibacter by
scientist at Takeda Chemical Co. in 1993, is a dipeptide antibiotic
consisting a mixture of two equilibrate epimeric isomer 1a and
Figure 1. Dipeptide antibiotics TAN-1057A/B.
activity against both prokaryotic and eukaryotic translation using
5
6
biochemical translation assays and MIC were obtained respec-
tively to evaluate selectivity and potency of the designed analogs.
Chemistry was developed to enable the detailed studies of each
portion of the lead TAN-1057. In general, analogs were assembled
according to route depicted in Scheme 1, utilizing the strategy used
1
2
b. It represents a novel antibacterial class, containing a unique
,5-diamino-5,6-1H-dihydropyrimidin-4-one scaffold and a b-
homoarginine side chain. TAN-1057A/B inhibits the bacterial
translation, and displays potent minimal inhibitory concentration
7
in the total synthesis of TAN-1057A/B. Dihydropyrimidinone 4
was constructed by treatment of appropriately substituted amine
with isothiuronium salt 7. Coupling of diazoketone 3, prepared
from the corresponding amino acid 5, with dihydropyrimidinone
4
amine in the presence of AgClO followed by necessary deprotec-
(
MIC) against staphylococci including MRSA.3 However, TAN-
6
1
057A/B has low selectivity, inhibiting both prokaryotic and
eukaryotic translation, it also has significant toxicity in vivo (LD50
in the mouse: 50 mg/kg ip and 100 mg/kg iv). In addition, TAN-
tion afforded compound 2.
1
057A/B has reduced antibiotic activity against other pathogens,
We initiated our efforts by examining the SAR around the dihy-
dropyrimidinone core, the results are displayed in Table 1. Replace-
such as enterococci and pneumococci.
Previously, limited structure–activity relationship (SAR) results
on TAN-1057A/B have been published.4 In this Letter, we report
systematic SAR studies of TAN-1057A/B series analogs. Inhibitory
2
ment of methyl (R ) with ethyl (compound 8) significantly
improved the selectivity but led to a reduction of both MIC and
the inhibitory activity against translation. While acetyl amide
replacing the urea moiety at the 2-position attaching to the hetero-
cyclic ring (compound 9) provided mild improvement in both
activity against bacterial translation and selectivity, other modifi-
⇑
Corresponding author. Tel.: +1 650 522 5828.
E-mail address: lianhong.xu@gilead.com (L. Xu).
These authors are no longer with Gilead Sciences.
8
3
cations at R only yielded analogs with reduced potency
0
960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.01.099

L. Xu et al. / Bioorg. Med. Chem. Lett. 21 (2011) 1670–1674
1671
R2
N
R2
HN
O
R1
*
NH
iii
N2
*
R3
R1
+
NH
NH2
O
R3
O
N
N
NHCbz
O
N
N
H
2
3
4
H
i
ii
R2
HN
O
R1
OH
SMe
NH
2
R3
+
NHCbz
5
H N
N
O
OMe
2
6
7
Scheme 1. Synthesis of 2. Reagents : (i) ClCOOEt, TEA, CH
2
Cl
2
2
; CH N
2 4
; (ii) NaOAc; (iii) AgClO TEA, DMF.
Table 1
SAR of the heterocyclic core region
NH
CH3
R2
N
N
*
*
H N
N
NH
1
O
2
5
H
Het
O
3
NH2
TAN-1057A/B
R3
O
N
N
NH2
H
1
*
Compounda
R2
R3
MIC9
(l
g/mL) S. aureus
Prokaryotic
translation IC50
Eukaryotic translation
IC50 M)
Selective indexb
Het
(lM)
(l
*
NH
1
8
9
1
1
1
(TAN1057 A/B)
CH
3
–NHCONH
2
2
8
256
8
0.3
1.8
0.1
<5
0.2
0.67
O
O
O
O
O
O
O
N
N
N
N
N
N
N
*
NH
NH
NH
NH
NH
NH
Et
–NHCONH
45
25
*
*
*
*
*
CH
3
CH
3
CH
3
CH
3
–NHCOCH
3
0.2
2
0
–NHCOEt
16
<5
1
2
3
–N(CH )COCH
3
64
6.8
8.8
21
3.1
3.4
–NHCON(CH
3
)
2
>500
19.7
O
1
1
3
4
CH
3
3
>500
>500
>500
>500
>500
—
N
NH
CH3
*
N
CH
–NHCONH
–NHCONH
2
>500
>500
—
—
O
O
N
*
N
1
5
CH
3
2
>500
>500
N
CH3
*
NH
1
610
CH
3
3
–NHCONH
–NHCONH
2
2
>256
>500
121
—
—
—
N
1
710
CH
>500
56
O
O
N
H
*
NH
1
1
811
CH
3
3
–NHCOCH
3
>500
>500
>500
51
>500
56
*
NH
9
CH
–NHCONH
2
1.1
O
N
a
⁄
⁄
Each compound is an equilibrate epimeric mixture of both R and S at 5-position.
b
In vitro selectivity index (IC50 eukaryotic transl/prokaryotic transl).

1
672
L. Xu et al. / Bioorg. Med. Chem. Lett. 21 (2011) 1670–1674
Table 2
SAR of the side chain
NH
CH3
CH3
N
N
*
R1
*
H N
N
NH
O
1NH
O
2
5
1
5
H
O
3
O
3
NH2
TAN-1057A/B
O
N
N
NH2
O
N
N
NH2
1
H
H
Compounda
R1
MIC9
S. aureus
(
l
g/mL)
Prokaryotic translation
IC50 M)
Eukaryotic translation
IC50 M)
Selective indexb
(l
(l
NH
1
(TAN1057 A/B)
H N
N
8
0.3
0.2
0.67
2
H
NH2
H N
2
2
2
2
2
2
0
64
64
9
<5
<0.6
6
NH2
H N
2
1
2
3
4
6.5
5
39.8
10
5
NH2
H N
2
16
2
NH2
H N
2
128
>500
25
0.2
—
NHCH3
H N
2
>500
>500
>500
NHCHO
O
25
26
27
28
29
H N
N
>500
>500
>256
8
>500
7
—
2
H
NH2
NH
O
H N
N
226
106
0.03
—
2
H
NH2
NH
AcHN
N
—
H
NH2
NH
H C
3
N
H
N
0.2
1.3
6.5
—
H
NH2
NH2
NH
Et
N
N
>256
2.4
1.3
4.2
8.2
4.2
27.4
26.3
20.6
23.2
35
H
H
H C
3
N
H C
30
31
32
33
3
256
64
20
N
H
N
H
NH2
NH
H C
3
N
CH3
N
H
4.9
2.8
8.3
NH2
HO
N
64
H N
N
H
2
NH2
NH
HN
N
128
NH2

L. Xu et al. / Bioorg. Med. Chem. Lett. 21 (2011) 1670–1674
1673
Table 2 (continued)
Compounda
R1
MIC9
S. aureus
(
l
g/mL)
Prokaryotic translation
IC50 M)
Eukaryotic translation
IC50 M)
Selective indexb
(l
(l
NH
3
4
256
0.15
1.9
10
H N
N
2
H
NH2
NH2
HN
3
3
3
5
6
7
512
64
0.51
6.6
12.3
6.6
8.2
H N
2
1
HN
HN
NH2
N
NH2
16
0.11
0.75
6.8
NH2
N
N
NH2
3
3
8
9
64
16
3.6
53
14.7
1.3
H C
3
NH2
HN
HN
N
NH2
0.18
0.23
NH2
H
4
4
0
1
64
2.0
3.9
0.6
3.4
0.3
0.9
NH2
NH2
H
128
HN
HN
H
4
2
8
0.17
0.45
2.6
N
NH2
H N
2
a
⁄
⁄
Each compound is an equilibrate epimeric mixture of both R and S at 5-position.
b
In vitro selectivity index (IC50 eukaryotic transl/prokaryotic transl).
CH3
CH3
O
HN
N
N
2
*
ii
*
NH
O
NH
NH
O
O
+
HN
NH
O
CbzN
CbzN
NHBoc
O
N
N
NH2
Boc
S
O
N
N
N
H
NH2
4
4
3
45
H
46
iii
i
BocHN
N
NHBoc
4
7
CH3
O
N
*
OH
NHBoc
HN
NH2
O
O
N
H
NH2
NH2
3
7
4
Scheme 2. Synthesis of 37. Reagents : (i) ClCOOEt, TEA, CH
2
Cl
2
; CH
2 2
N ; (ii) AgClO
4
2
, TEA, DMF; H , Pd/C; (iii) HgCl
2
, DMF; TFA.
(
(
compounds 10–13). Methyl substitution at the heterocyclic ring
14 and 15) abolished the antibacterial activity. It has been re-
activity. The intolerability of the core towards any modifications
that remove its acetylating ability may indicate that acetylation
of the protein with TAN-1057A/B contributes to its mechanism of
action. Lastly, inserting a methylene between methyl amine and
the core (19) also removed the activity.
We next turned our attention to the b-homoarginine side chain,
investigating the effect of b-amine, terminal guanidine and linker
between them on the SAR. Representative SAR results are summa-
ported that TAN-1057A/B gradually loses its antibacterial activity
in basic aqueous solution due to the hydrolysis of the acetyl ami-
dine and opening of the heterocyclic ring. The heterocyclic core
of compounds 16–18 was designed10,11 to be stable toward hydro-
lysis while causing minimal structure disturbance. Unfortunately,
these modifications resulted in a complete loss of antibacterial

1
674
L. Xu et al. / Bioorg. Med. Chem. Lett. 21 (2011) 1670–1674
Table 3
Pharmacological profile
In summary, novel analogs of dipeptide antibiotics TAN-1057A/
B were designed and synthesized. SARs of the dihydropyrimidi-
none core and side chains were established. Our studies showed
that the dihydropyrimidinone core is the phamacophore of this no-
vel class of antibiotics and is highly sensitive to modifications.
Optimizations of the side chain provided several analogs with
favorable safety while maintain the potency against MRSA. Further
work need to be carried out to identify drug candidates that can be
used to treat MRSA infections.
Compound
iv PD50 (mg/kg)
MIC (
lg/mL)
CC50 (lg/mL)
1
(TAN1057 A/B)
0.7
9.6
3.3
2.4
1.5
12.8
0.8–2
32
16
16–32
6
—
131
62
—
Vancomycin
2
3
4
8
7
2
rized in Table 2. Replacement of terminal guanidine with primary
amines (compounds 20–22, 36, 40 and 41) cause a significant
reduction in inhibitory activity against bacterial translation, yet
the reduction of MIC was minor. We speculate that this result
may attribute to the better permeability of an amino analog com-
paring with its corresponding guanidino one. Substitution on the
b-amine is detrimental to the potency and selectivity (23 and
Acknowledgments
The authors wish to thank John Wolf, Greg Biesecker and Frank
Richardson for their contributions.
References and notes
2
4). Reduction of the basicity of the terminal guanidine group
1. Bertrand, X. Therapy 2010, 7, 169 and references cited there in.
2.
Zetola, N.; Francis, J. S.; Nuermberger, E. L., et al Lancet Infect. Dis. 2005, 5, 275
and references cited there in.
(
compounds 25–27) diminished antibacterial activity, demonstrat-
ing the importance of basicity of the terminal moiety for this series
of antibacterial in order to interact with the target protein. Moder-
ate selectivity can be achieved through alkylation of the terminal
guanidine, yet the potency is sensitively associated with the posi-
tions and sizes of the alkyls (28–33). Compound 28 is the best of
this series in terms of selectivity and potency. Modifying the linker
also proved to be fruitful. Many analogs incorporated an aromatic
ring as a spacer with different orientations and distances were de-
signed and synthesized, two most potent ones (34 and 35) were
shown in Table 2. Although possessing potent inhibitory activity
against the bacterial translation and favorable selectivity, both 34
and 35 failed to provide desirable MIC. However, when using satu-
rated a heterocycle as the spacer, compound 37 and 42 displayed
better inhibitory activities against bacterial translation and favor-
able selectivity, and also showed comparable MIC with TAN-
3
.
(a) Katayama, N.; Fukusumi, S.; Funabashi, Y.; Iwahi, T.; Ono, H. J. Antibiot.
1993, 46, 606; (b) Funabashi, Y.; Tsubotani, S.; Koyama, K.; Katayama, N.;
Harada, S. Tetrahedron 1993, 49, 13.
(a) William, R. M.; Yuan, C. J. Am. Chem. Soc. 1997, 119, 11777; (b) Williams, R.
M.; Yuan, C.; Lee, V.; Chamberland, S. J. Antibiot. 1998, 51, 189; (c) Brands, M.;
Endermann, R.; Gahlmann, R.; Krüger, J.; Raddatz, S. Bioorg. Med. Chem. Lett.
4
.
2002, 13, 241; (d) Brands, M.; Endermann, R.; Gahlmann, R.; Krüger, J.; Raddatz,
S.; Stoltefu, J.; Belov, V. N.; Nizamov, S.; Sokolov, V. V.; de Meijere, A. J. Med.
Chem. 2002, 45, 4246; (e) Brands, M.; Grande, Y. C.; Endermann, R.; Gahlmann,
R.; Krüger, J.; Raddatz, S. Bioorg. Med. Chem. Lett. 2003, 13, 2641; (f) Kordes, M.;
Brands, M.; Es-Sayed, M.; de Meijere, A. Eur. J. Org. Chem. 2005, 14, 3008.
IC50s for prokaryotic translation were determined using E. coli S30 extracts, see
5
.
.
(
a) Zubay, G. Annu. Rev. Genet. 1973, 7, 267; IC50s for eukaryotic translation
were determined using rabbit reticulocytes, see: (b) Pelham, H. R.; Jackson, R. J.
Eur. J. Biochem. 1976, 67, 247; (c) Walter, P.; Blobel, G. Methods Enzymol. 1983,
9
6, 84.
6
National Committee for Clinical Laboratory Standards 1997 Methods for
dilution antimicrobial susceptibility tests for bacteria that grow aerobically.
NCCLS Document M7-A4+ Villanova, PA: National Committee for Clinical
Laboratory Standards.
National Committee for Clinical Laboratory Standards 1998 Performance
Standard for Antimicrobial Susceptibility Testing. NCCLS Document M100-S8
Villanova, PA: National Committee for Clinical Laboratory Standards
(a) Sokolov, V. V.; Kozhushkov, S. I.; Nikolskaya, S.; Belov, V. N.; Es-Sayed, M.;
de Meijere, A. Eur. J. Org. Chem. 1998, 63, 777; (b) Zhang, L.; Xu, L.; Kim, C. U.
Tetrahedron Lett. 2003, 44, 5871.
1
057A/B.
Synthesis of this group of analogs was outlined in Scheme 2,
represented by compound 37.12 Protected amino acid 43, obtained
from commercial sources, was converted to diazoketone 44 by
treatment with ClCOOEt followed by diazomethane. Dihydropyri-
midinone amine 45, prepared following the procedure disclosed
7
.
8. For synthesis of some 2-position analogs, see: Xu, L.; Zhang, L.; Bryant, C. M.;
7
Kim, C. U. Tetrahedron. Lett. 2003, 44, 2601.
by de Meijere and co-workers, was then reacted with diazoketone
9. The value of MIC is shown to be media dependent.
4
4
4 in the presence of AgClO to give the coupling product. Removal
1
0. The cores of compounds 16 and 17 are obtained from 2,4-diamine pyrimidine
and 3,6-diamine pyridine. Compounds 16 and 17 were prepared following the
same procedure as described in Scheme 1.
of the Cbz protecting group with hydrogenolysis provided the
piperidine 46. Treatment of the piperidine with Boc-protected
thiourea 47 followed by deprotection provided compound 37.
1
1
1. Zhang, L.; Kim, C. U.; Xu, L. Tetrahedron Lett. 2007, 48, 3273.
2. For the synthesis of compound 46, please see Ref. 7. For the preparation of
Compounds 28, 37 and 42 were further evaluated. Their CC50
s
compound 37:
compound 47 (30.9 mg, 0.112 mmol) in DMF (2 mL) was added HgCl
36.4 mg, 0.134 mmol) followed by TEA (31.2 L, 0.224 mmol). The mixture
a solution of compound 46 (50.6 mg, 0.112 mmol) and
1
3
was obtained. The data supported that these compounds are
more selective than TAN-1057A/B. They were also studied in
mouse septicemia model against MRSA, and their PD50 was listed
in Table 3. In this model, 28, 37 and 42 are more efficacious than
vancomycin.
2
(
l
was stirred at room temp for 12 h before it was filtered through celite. The
residue was washed with CH Cl2 (Â3). The filtrate and washings were
2
combined and evaporated to dryness. The residue was then taken into
partition between CH
aqueous layer was extracted with CH
combined, dried (Na SO ), filtered and evaporated to dryness. Flash
chromatography (2–8% CH OH/ CH Cl ) gave 37.8 mg compound (49%).
2
Cl
2
and brine. The CH
2
Cl
2
layer was separated and
2
Cl
2
(Â2). The CH
2
Cl layers were
2
Previous studies have shown that TAN-1057A/B inhibits the
peptidyl transferase reaction which leads to inhibition of transla-
tion but does not directly bind to the A or P tRNA binding sites.
Analysis of drug resistant mutants shows two classes of mutants
depending on selection method. The first class of mutants may
2
4
3
2
2
The mixture of above compound (15 mg) and TFA (1 mL) was stayed at room
temperature for 1 h and evaporated to dryness. The mixture was then co-
evaporated with toluene (Â2). The resulting solid was then dissolved in water,
4 2 3
pH of the resulting solution was adjusted to 5.5–6.0 with (NH ) CO aqueous
1
4
solution. The mixture was purified with C-18 column to give 7.4 mg of
compound after lypholization.
be efflux mutants. This finding is supported by MIC studies done
with minimal media in the presence or absence of dipeptides,
which suggests that TAN-1057A/B is actively transported into bac-
terial cells via a dipeptide transport mechanism. The second class
of mutants is ribosomal mutations of unknown function which
confer resistance to TAN-1057A/B.15
13. Mosmann, T. J. Immunolog. Methods 1983, 65, 55.
14. Boeddeker, N.; Bahador, G.; Gibbs, C.; Mabery, E.; Wolf, J.; Xu, L.; Watson, J. RNA
002, 8, 1120.
5. Limburg, E.; Gahlmann, R.; Kroll, H. P.; Beyer, D. Antimicrob. Agents Chemother.
004, 48, 619.
2
1
2