Synthesis of de novo designed small-molecule inhibitors of bacterial RNA polymerase

Article abstract of DOI:10.1016/j.tet.2008.08.037

The de novo molecular design program SPROUT has been used in conjunction with the X-ray crystal structure of RNA polymerase (RNAP) from Thermus aquaticus to produce a novel enzyme inhibitor scaffold. A?short and efficient synthesis of molecules corresponding to this scaffold has been developed and in keeping with the design predictions, the resulting inhibitors displayed useful levels of inhibition of Escherichia coli RNAP.

Full text of DOI:10.1016/j.tet.2008.08.037

Tetrahedron 64 (2008) 10049–10054
Contents lists available at ScienceDirect
Tetrahedron
journal homepage: www.elsevier.com/locate/tet
Synthesis of de novo designed small-molecule inhibitors of bacterial
RNA polymerase
*
*
Anil K. Agarwal, A. Peter Johnson , Colin W.G. Fishwick
School of Chemistry, University of Leeds, Leeds LS2 9JT, UK
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 28 May 2008
Received in revised form 27 July 2008
Accepted 14 August 2008
Available online 16 August 2008
The de novo molecular design program SPROUT has been used in conjunction with the X-ray crystal
structure of RNA polymerase (RNAP) from Thermus aquaticus to produce a novel enzyme inhibitor
scaffold. A short and efficient synthesis of molecules corresponding to this scaffold has been developed
and in keeping with the design predictions, the resulting inhibitors displayed useful levels of inhibition of
Escherichia coli RNAP.
Ó 2008 Elsevier Ltd. All rights reserved.
1. Introduction
functionally distinct and essential sites, which may be amenable to
inhibition by antibacterial drugs. Bacterial RNAP is structurally
The global emergence of bacterial resistance to existing antibi-
otics is a serious public health issue. Indeed, the World Health Or-
ganization estimates that infection by antibiotic resistant bacteria
more than doubles the risk of death.1,2 Consequently, there remains
an urgent need to discover and develop new antibiotics to coun-
teract resistance. Traditionally, the pharmaceutical industry has
adopted analogue improvement programmes to develop new an-
distinct from mammalian RNAPs7 permitting selective design of
bacterial inhibitors. Antisense knock-down technology demon-
strates that RNAP is essential for bacterial growth and survival and
the potent bactericidal antibiotic rifampicin, which specifically
targets bacterial RNAP, has been in medical use for over 40 years to
treat infectious diseases.8,9 Rifampicin binds close to the active site
of the catalytic
b subunit and inhibits initiation of RNA synthesis.
tibiotics whereby derivatives of established classes (e.g.,
b
-lactams,
Indeed, rifampicin, which is a bactericidal antibiotic, is an impor-
tant component of anti-TB therapies and is also used to treat
staphylococcal infections.10 These observations provide proof of
principle that inhibition of RNAP can lead to successful chemo-
therapeutic intervention of infections, including those caused by
Mycobacterium tuberculosis and Staphylococcus aureus. Although
there is still interest in developing members of the rifamycin class
for the chemotherapy of bacterial infections11–15 the progressive
emergence of RNAP mutants resistant to rifamycins makes it more
appealing to search for novel RNAP inhibitors not subject to cross-
resistance with existing rifamycin antibiotics. However, a recent
report, from a major pharmaceutical company, summarising their
work in antibacterial drug discovery has indicated that RNAP is
a challenging target in terms of hit identification using traditional
high throughput screening of large compound libraries.16
quinolones, macrolides, tetracyclines, sulfonamides) with improved
activity against existing resistant isolates have been introduced.3
However, opportunities for further chemical modification of existing
classes are now diminishing and a consensus is also emerging that
new antibiotics should not simply be analogues of existing classes
since pre-existing bacterial mechanisms of resistance rapidly evolve
to confer resistance to new derivatives.4
Bacterial RNA polymerase (RNAP) is an attractive drug target.5,6
It plays an essential role in transcription in all bacteria and conse-
quently drugs that inhibit this enzyme are likely to have a broad
spectrum of activity affecting both Gram-positive and Gram-neg-
ative pathogens.6 Bacterial core RNAP is a stable, noncovalent as-
sembly of four essential polypeptide chains with the subunit
composition b, b, 2a.
The roles of the individual subunits have been defined and
functional sites, or regions of importance for subunit interactions,
have been identified.7 Promoter-specific initiation of transcription
requires both core RNAP and a sigma factor, which together com-
As part of a structure-based design and synthesis program for
the discovery of new enzyme inhibitors, we have previously
described the computer-aided molecular design, synthesis and
biological evaluation of novel inhibitors targeted at the bacterial
prise the holoenzyme. Thus, RNAP contains
a
number of
D-ala-D D
-ala ligase,17 the bacterial -glu adding ligase MurD18 and the
Plasmodium dihydroorotate dehydrogenase19,20 enzymes, re-
spectively. Here, we describe the first non-macrocyclic bacterial
RNAP inhibitors, designed to target the rifampicin binding site,
which were created using a fragment-based de novo design
approach.
* Corresponding authors. Tel.: þ44 (0) 113 343 6510; fax: þ44 (0) 113 343 6565.
E-mail addresses: p.johnson@leeds.ac.uk (A.P. Johnson), colinf@chem.leeds.
ac.uk (C.W.G. Fishwick).
0040-4020/$ – see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2008.08.037

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A.K. Agarwal et al. / Tetrahedron 64 (2008) 10049–10054
The structure-based design of small-molecule enzyme in-
hibitors is powerful tool in drug discovery, particularly
2. Results and discussion
a
when there is an X-ray crystal structure of the target enzyme
available. In particular, fragment-based de novo methods offer
a rapid and powerful approach to inhibitor design, which can
complement other in silico approaches such as virtual high
throughput screening.21–24
The X-ray crystal structure of the complex formed between
core RNAP from Thermus aquaticus and rifampicin has been
reported to 3.3 Å resolution.7 This structure reveals that rifampi-
cin binds to the enzyme within a cleft located approximately
12.1 Å away from the active site in a region in which the newly
synthesised RNA polymer leaves the complex. Rifampicin inhibits
RNAP by preventing formation of RNA beyond the addition of two
to three nucleotides. Additionally, since rifampicin binds remotely
from the active site and in a region of bacterial RNAP that is
structurally different from that in the human RNAP II, the antibi-
otic has low toxicity.
The binding site of rifampicin is revealed to be a fairly open
cavity, which is substantially exposed to solvent. Rifampicin makes
several specific hydrogen bond contacts to residues within the
RNAP including S411, R409, H406, F394 and Q390 (Fig. 1, Escherichia
coli numbering used throughout). Additionally, several residues
have important roles in contributing to hydrophobic interactions
with rifampicin including R405, H52, L391, L413, Q390, S392, D396
and F394.
In terms of the design of new inhibitors, the rifampicin binding
site poses a number of challenges, mainly due to the rather solvent-
exposed nature of the binding site, its relatively large surface area,
and the general lack of compact binding pockets within which to
locate small-molecule inhibitors. We reasoned, therefore, that
a fragment-based modular design approach, such as that used
within the SPROUT25–27 molecular design software, would offer
good prospects for the production of novel RNAP inhibitors tar-
geting this binding cavity.
Analysis, using SPROUT, of the inhibitor binding potential within
this cavity revealed the presence of additional binding sites not
utilised by rifampicin (Fig. 2). It was reasoned that a cluster of five
specific hydrogen-bonding sites (involving residues Q390, F394,
R405, Q567 and Q633), in addition to a region of relatively high
hydrophobicity (located near to Q390 and R405), offered good
prospects for the design of small-molecule inhibitors.
The fragment-based molecular design approach utilised within
SPROUT typically involves the docking of small molecular frag-
ments into each of the chosen binding sites followed by joining of
these docked fragments to yield sets of designed inhibitor tem-
plates. These are then ranked using a number of criteria including
predicted binding affinity, molecular complexity and synthetic ac-
cessibility, to reveal the best inhibitor candidates for laboratory
synthesis and biological evaluation. Application of this fragment-
based inhibitor design approach to the six chosen binding sites
within the rifampicin binding cavity of RNAP yielded a simple,
synthetically attractive molecular template 1 based on a 1,2,4,6-
tetrasubstituted aromatic core (Fig. 2).
In addition to the highly substituted aromatic core, the design
template required the hydrogen bond donor (shown as an N–H in
structure 1 in Scheme 1 below), which makes contact with the
backbone carbonyl group of F394 and, which is connected to
a stereocentre with ‘R’ absolute stereochemistry. In addition to two
substituents designed to accept hydrogen bonds from the side-
chains of residues R405 and Q567, respectively, the designed
template 1 includes a second aromatic ring designed to reside
HO
O
O
O
OH
OH OH
O
NH
N
O
N
O
OH
O
N
O
F394
O
H
N
H
H2N
HN
OH
Hydrophobic
region
O
O
O
O
N
O
H
H
NH
NH
Q567
O
Q390
H
O
9
HN
HN
O
NH
R405
Figure 2. (Top) SPROUT-analysis of designed inhibitor template 1 bound within RNAP.
The hydrophobic region and the H-bonding sites utilised in the design are shown as
shaded regions; (bottom) schematic showing bonding interactions of SPROUT-de-
signed inhibitor 9.
Figure 1. (Top) Structure of rifampicin; (bottom) important H-bond contacts of
rifampicin to RNAP.

A.K. Agarwal et al. / Tetrahedron 64 (2008) 10049–10054
10051
O
O
H
F394
O
H
R
N
H
H
N
R
O
OR
OR
O
+
H
R
O
N
H
N
H
NH2
O
hydrophobic
region
HN
N
O
+
-
3
R
OMe
CHO
O
O
Me
Q390
O
O
O
O
O
N
O
H
+
-
NH
HN
O
F
H
R
HO
H
H
+
Me
2
O
+
Q567
O2N
1
H2N
NH
R405
OMe
4
Scheme 1. (Left) SPROUT-generated RNAP inhibitor template 1 and synthetic equivalent 2; (right) synthetic approach to inhibitors 2.
within the hydrophobic region flanked by residues Q390 and R405.
This ring also incorporates a para-substituent designed to make
a hydrogen bond to the side chain of Q390 (Scheme 1).
by the nitro group position. In order to explore this alternative H-
bonding possibility, the nitro groups in inhibitors 9 and 10 were
selectively reduced in the presence of the benzyl amine moiety to
the corresponding amines 11 and 12 using PtO2 as the catalyst
(Scheme 3).
Structural analysis of the designed template 1 with respect to
synthetic accessibility led to the identification of amino acid-de-
rived systems 2 (Scheme 1). It was envisaged that these systems
would be easily prepared, in optically active form, via reductive
amination of a suitably functionalised 2,4,6-trisubstituted benzal-
dehyde 3 (available via O-arylation of phenols 4 with 4-fluoroni-
trobenzene) with readily available amino acid amides (Scheme 1).
Two representative examples of inhibitors 1 were readily pre-
pared using this synthetic approach (Scheme 2).
As shown below (Scheme 2), the first step involves an ipso
substitution reaction between 4-fluoronitrobenzene and 3, 5-
dimethoxyphenol to yield ether 5, which, on Vilsmeier formylation,
yielded aldehyde 6.
O
O
H2N
H2N
HN
HN
R
R
H2, PtO2
EtOAc
O
OAc
O
OAc
9, R = OH
10, R = H
O2N
11, R = OH
12, R = H
H2N
OMe
OMe
Scheme 3. Preparation of amino-derivatives.
The selective demethylation of 6 worked well using the litera-
ture method28 giving phenol 7 in good yield, which was then
acetylated to yield the key aldehyde 8 (Scheme 2). Reductive ami-
nation of aldehyde 8, using commercially available amino acid
amides, yielded the target molecules (9 and 10).
Inhibitors 9 and 10 were screened for their ability to inhibit E.
coli RNAP using the SYBR Green assay.29 Preliminary experiments
revealed that compounds 9 and 10 inhibited RNAP activity with IC50
Inhibitor 11 was threefold less active than the corresponding
nitro version 9 and inhibitor 12 was found to be inactive in the SYBR
Green assay. These results suggest that the H-bonding between
Q390 and the designed inhibitors are as shown in Figure 2. It is
interesting to note, however, that Diederich et al. have suggested
that nitro groups are relatively poor H-bond acceptors.30,31
values of 70
m
M and 62
mM, respectively. In addition to enable the
3. Conclusions
facile synthesis of inhibitors 9 and 10, the nitro group present in
these systems was included so as to contact Q390. Although, it was
reasoned that this would involve donation of an H-bond from the
side chain NH2 of Q390, at the resolution of the crystal structure, we
reasoned that it was possible for the carbonyl oxygen and the NH2
moieties of this side chain to be reversed. This would require an H-
bond donor within the inhibitors at the position currently occupied
In summary, a fragment-based molecular design approach to-
gether with a very short and efficient synthesis has been used, for
the first time, to produce a novel type of inhibitor, designed to
target the rifampicin binding cavity of bacterial RNAP. Although
preliminary experiments indicate that these molecules inhibit the
operation of bacterial RNAP, clearly more work is needed in order to
CHO
F
OMe
HO
OMe
O
OMe
O
i
ii
+
O2N
O2N
O2N
OMe
OMe
OMe
6
5
O
H2N
CHO
OMe
HN
R
OR
O
v
iii, iv
O
OAc
O2N
O2N
9, R = OH
10, R = H
7, R = H
8, R = Ac
OMe
Scheme 2. Reagents and conditions: (i) K2CO3, DMF, 40 ꢀC, 93%; (ii) DMF, POCl3, 80 ꢀC, 58%; (iii) BCl3, DCM, 70 ꢀC, 95%; (iv) AcCl, Et3N, DCM, 0 ꢀC, 80%; (v) L-tyrosinamide or L-
phenylalaninamide, AcOH, DCM/THF (1:1), NaBH(OAc)3, 53%.

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A.K. Agarwal et al. / Tetrahedron 64 (2008) 10049–10054
probe the details of this inhibitory activity. Additionally, although
we appreciate that this type of inhibitor is not really ‘drug-like’, the
power of this approach in the rapid identification of bacterial RNAP
inhibitors is underlined by the recently reported failure of classical
HTS methods to identify RNAP inhibitors.16 We believe that our
approach to ligand design, where specific small-molecule frag-
ments are matched to particular features within the protein, is
particularly advantageous when applied to rather open and sol-
vent-exposed binding cavities such as the rifampicin binding re-
gion of bacterial RNAP. Additionally, via careful selection of the
contacting sites within the protein to be used by the designed in-
hibitor, it should be possible to design such inhibitors that do not
rely on making contacts to residues prone to resistance mutations
and may yield inhibitors active against bacteria resistant to existing
antibiotics such as rifampicin. We are currently extending these
findings to develop more drug-like inhibitors possessing such novel
antibacterial activity and will report these results elsewhere.
water (50 ml) and stirred vigorously for 2 h to coagulate any pre-
cipitate, which was filtered off. The precipitate was washed with
water (50 ml), dried and chromatographed [DCM/EtOAc, 99:1] to
yield the title compound 6 (1.9 g, 6.3 mmol, 58% yield) as colourless
plates. Mp 115–116 ꢀC. Rf 0.83 (DCM/EtOAc, 95:5). 1H NMR
(300 MHz, CDCl3):
Ar–H), 7.00 (dd, 2H, JHH 7.0, 2.1, Ar–H), 6.39 (d, 1H, JHH 2.1, Ar–H),
6.18 (d, 1H, JHH 2.1, Ar–H), 3.95 (s, 3H, OMe), 3.85 (s, 3H, OMe). 13
d 10.25 (s, 1H, Ar–CHO), 8.20 (dd, 2H, JHH 7.0, 2.1,
C
NMR (75 MHz, CDCl3): 186.77 (Ar–CHO), 166.51, 164.79, 163.33,
157.93, 143.26, 126.38, 117.21, 112.12, 99.85, 96.14, 56.69 (OMe),
56.32 (OMe). nmax/cmꢁ1: 2985, 2883, 2794, 1690 (CO), 1608, 1486,
1414,1335,1242,1147; m/z (ES): 304 (Mþþ1); Found: C, 59.3; H, 4.4;
N, 4.45%. C15H13NO6 requires: C, 59.41; H, 4.32; N, 4.62%.
4.1.3. 2-Hydroxy-4-methoxy-6-(4-nitrophenoxy) benzaldehyde (7)
Boron trichloride (8.5 ml, 1 M solution in DCM, 8.5 mmol) was
added during 20 min to a stirred solution of 2,4-dimethoxy-6-(4-
nitrophenoxy) benzaldehyde 6 (1.6 g, 5.3 mmol) in DCM (20 ml)
cooled to ꢁ70 ꢀC. The resulting pale yellow solution was allowed to
warm to rt, stirred at this temperature for 3 h, and then poured
with stirring into ice-cold 1 M HCl (50 ml). The DCM layer was
separated, washed with water (3ꢂ30 ml) and brine (30 ml), dried
(MgSO4) and evaporated to dryness. The crude solid was chroma-
tographed to yield the title compound 7 (1.45 g, 5.0 mmol, 95%
yield) as colourless needles. Mp 150–151 ꢀC. Rf 0.4 (petrol/EtOAc,
4. Experimental
4.1. General
Infrared spectra were recorded using a Nicolet Avatar 300 FT-IR
spectrophotometer. The vibrational frequencies are reported in
wave numbers (cmꢁ1). 1H and 13C NMR spectra were measured on
Bruker DPX300 Fourier transform spectrometer using an internal
deuterium lock using tetramethylsilane as the internal standard.
Coupling constants (J) are quoted in hertz (Hz). Low and high res-
olution mass spectra were recorded on a VG Autospec mass spec-
trometer, operating at 70 eV, using either chemical ionisation (CI),
electron impact (EI) or fast atom bombardment (FAB) ionisation
techniques. All accurate mass measurements were performed using
a ZMD mass spectrometer using ES. Optical rotations were de-
termined using an Optical Activity AA1000 polarimeter with cells of
path lengths 0.25 or 1 dm. Melting points were determined using
a Reichert hot stage and are uncorrected. Microanalyses were
performed by the University of Leeds Microanalytical service using
an Elemental Analyser 1106. Flash column chromatography was
carried out using Merck Kieselgel 60 (230–400 mesh). Solvents
were either obtained dry from commercial sources or dried prior to
use using standard methods.
8:2). 1H NMR (300 MHz, CDCl3):
d 10.07 (s, 1H, Ar–CHO), 8.28 (dd,
2H, JHH 7.0, 2.1, Ar–H), 7.18 (dd, 2H, JHH 7.0, 2.1, Ar–H), 6.27 (d,1H, JHH
2.2, Ar–H), 5.94 (d, 1H, JHH 2.2, Ar–H), 3.82 (s, 3H, OMe). 13C NMR
(75 MHz, CDCl3): 191.66 (Ar–CHO), 168.10, 166.67, 161.76, 159.89,
144.36,126.60, 119.33, 107.98, 98.81, 97.45, 56.45 (OMe). nmax/cmꢁ1
:
3080, 2928, 2447, 1921, 1782 (CO), 1748, 1509, 1336, 1156, 1107; m/z
(ES): 290 (Mþþ1); Found: C, 58.4; H, 4.1; N, 4.75%. C14H11NO6 re-
quires: C, 58.13; H, 3.83; N, 4.84%.
4.1.4. 2-Formyl-5-methoxy-3-(4-nitrophenoxy) phenylacetate (8)
A solution of 2-hydroxy-4-methoxy-6-(4-nitrophenoxy) benz-
aldehyde 7 (0.4 g, 1.4 mmol) and triethylamine (0.4 ml, 2.8 mmol)
in dry DCM (10 ml) was stirred under nitrogen at 0 ꢀC and treated
drop wise with acetyl chloride (0.1 ml, 1.5 mmol). After 1 h, the
solution was allowed to warm to rt and stirred at rt for 6 h, diluted
with DCM (20 ml) and washed successively with water (20 ml) and
brine (20 ml). The organic layer was separated, dried (MgSO4) and
evaporated to dryness. The crude product was chromatographed
(petrol/EtOAc, 85:15) to yield the title compound 8 (365 mg,
1.1 mmol, 80% yield) as yellow plates. Mp 136–137 ꢀC. Rf 0.23
4.1.1. 1,3-Dimethoxy-5-(4-nitrophenoxy) benzene (5)
K2CO3 (4.0 g, 28.4 mmol) was added to a solution of 3,5-dime-
thoxyphenol (2.2 g, 14.2 mmol) in DMF (20 ml) and stirred for
15 min. A solution of 1-fluoro-4-nitrobenzene (2.0 g, 14.2 mmol) in
DMF (10 ml) was added and the reaction mixture was then stirred
at 40 ꢀC for 6 h. The reaction mixture was cooled, poured into
crushed ice (50 g) and stirred for 30 min. The precipitate was fil-
tered, washed with water (20 ml) and dried in vacuo to yield the
title compound 5 (3.5 g, 12.7 mmol, 90% yield) as colourless plates,
which needed no further purification. Mp 117–118 ꢀC. Rf 0.66
(petrol/EtOAc, 7:3). 1H NMR (300 MHz, CDCl3):
d 10.15 (s, 1H, Ar–
CHO), 8.27 (dd, 2H, JHH 7.0, 2.1, Ar–H), 7.14 (dd, 2H, JHH 7.0, 2.1, Ar–
H), 6.53 (d, 1H, JHH 2.3, Ar–H), 6.39 (d, 1H, JHH 2.3, Ar–H), 3.85 (s, 3H,
OMe), 2.40 (s, 3H, OCOMe). 13C NMR (75 MHz, CDCl3): 185.30 (Ar–
CHO), 169.15 (CO), 165.53, 161.80, 159.99, 153.10, 143.77, 126.24,
118.25, 114.08, 106.45, 103.92, 56.14 (OMe), 20.97 (OCOMe). nmax
/
cmꢁ1: 3346, 3118, 3073, 2893, 1755 (CO), 1679 (CO), 1615, 1485,
(petrol/EtOAc, 8:2). 1H NMR (300 MHz, CDCl3):
d
8.20 (dd, 2H, JHH
1437, 1367; m/z (ES): 332 (Mþþ1); Found: C, 57.8; H, 4.0; N, 4.3%.
7.0, 2.0, Ar–H), 7.04 (dd, 2H, JHH 7.1, 2.1, Ar–H), 6.35 (t, 1H, JHH 2.2,
Ar–H), 6.23 (d, 2H, JHH 2.2, Ar–H), 3.78 (s, 6H, OMe). 13C NMR
(75 MHz, CDCl3): 163.46, 162.34, 156.81, 143.11, 126.69, 117.72,
99.33, 97.83, 55.97 (OMe). nmax/cmꢁ1: 2843, 1601, 1459, 1342, 1208,
1154, 1116, 1048; m/z (ES): 276 (Mþþ1); Found: C, 60.8; H, 4.95; N,
5.3%. C14H13NO5 requires: C, 61.09; H, 4.76; N, 5.09%.
C16H13NO7 requires: C, 58.01; H, 3.96; N, 4.23%.
4.1.5. (S)-2-((1-Amino-3-(4-hydroxyphenyl)-1-oxopropan-2-
ylamino)methyl)-5-methoxy-3-(4-nitrophenoxy) phenylacetate (9)
NaBH(OAc)3 (200 mg, 0.9 mmol) was added to a stirred solution of
2-formyl-5-methoxy-3-(4-nitrophenoxy)phenylacetate
8 (100 mg,
0.3 mmol) with -tyrosinamide (54 mg, 0.3 mmol) and acetic acid
L
4.1.2. 2,4-Dimethoxy-6-(4-nitrophenoxy) benzaldehyde (6)
(5 ml) in DCM/THF (20 ml) and the resulting mixture was stirred at
rt for 15 h. The reaction mixture was diluted with EtOAc (50 ml) and
washed with water (20 ml) and brine (20 ml). The organic phase
was separated, dried (MgSO4) and concentrated by rotary evapo-
ration. Purification using chromatography (DCM/MeOH, 99:1 to
95:5) yielded the title compound 9 (80 mg, 0.16 mmol, 53% yield) as
Phosphoryl chloride (5.0 ml, 55.0 mmol) was carefully added to
ice-cold DMF (4.2 ml, 55.0 mmol) at 0 ꢀC. A solution of 1, 3-dime-
thoxy-5-(4-nitrophenoxy) benzene 5 (3.0 g, 11.0 mmol) in DMF
(10 ml) was added and the resulting mixture was heated at 80 ꢀC
for 3 h. The dark viscous mixture was then poured into ice-cold

A.K. Agarwal et al. / Tetrahedron 64 (2008) 10049–10054
10053
pale yellow prisms. Mp 129–130 ꢀC. Rf 0.31 (DCM/MeOH, 95:5). [
a
]
3.69, 55.90 (OMe), 45.37, 34.16, 22.96 (COMe). nmax/cmꢁ1: 3351,
1673 (CO), 1586, 1501, 1206, 1142, 1057, 825; m/z (ES): 466 (Mþþ1).
D
ꢁ136 (c 0.10, MeOH). 1H NMR (300 MHz, MeOD):
d 8.11 (dd, 2H, JHH
7.1, 2.0, Ar–H), 6.87 (dd, 2H, JHH 7.1, 2.0, Ar–H), 6.75 (d, 2H, JHH 8.4,
Ar–H), 6.46 (d, 2H, JHH 8.4, Ar–H), 6.22 (d, 1H, JHH 2.3, Ar–H), 5.95 (d,
1H, JHH 2.3, Ar–H), 4.22 (d, 1H, JHH 14.5, Ar–CH2–NH), 3.71 (dd, 1H,
JHH 9.9, 5.3, NH–CH–CONH2), 3.57 (s, 3H, OMe), 3.51 (d, 1H, JHH
14.5, Ar–CH2–NH), 3.15 (dd, 1H, JHH 13.8, 10.0, Ar–CH2–CH–CONH2),
3.06 (dd, 1H, JHH 13.8, 5.3, Ar–CH2–CH–CONH2), 2.14 (s, 3H,
CO2Me). 13C NMR (75 MHz, MeOD): 176.00 (CONH2), 174.26
(COMe), 164.06, 163.41, 160.34, 157.25, 156.76, 144.69, 131.60,
130.71, 127.34, 118.89, 116.65, 108.29, 100.20, 99.09, 64.24, 56.29
Calculated accurate mass: 488.1798;
488.1783.
C25H27N3O6Na requires:
4.1.8. (S)-2-((1-Amino-1-oxo-3-phenylpropan-2-ylamino)methyl)-
3-(4-amino phenoxy)-5-methoxy phenylacetate (12)
A slurry of PtO2 (0.95 mg, 4.0 mol % of catalyst) was stirred in
EtOAc (5 ml) while bubbling nitrogen through the mixture. A so-
lution
of
(S)-2-((1-amino-1-oxo-3-phenylpropan-2-ylamino)
methyl)-5-methoxy-3-(4-nitrophenoxy) phenylacetate 10 (50 mg,
0.1 mmol) in EtOAc (10 ml) was added and the purging continued.
Hydrogen gas was then purged into the flask and the resulting
mixture was stirred at rt for 15 h. After completion of reaction
(TLC), reaction mixture was filtered through a pad of Celite and
taken to dryness by rotary evaporation. The crude product was
chromatographed (DCM/MeOH, 9:1) to yield the title compound 12
(25 mg, 0.06 mmol, 54% yield) as colourless needles. Mp 106–
(OMe), 45.82, 34.08, 23.14 (COMe). nmax/cmꢁ1
: 3298, 1673
(CO), 1600, 1514, 1398, 1236, 1142, 1110, 1056; m/z (ES): 496
(Mþþ1). Calculated accurate mass: 518.1539; C25H25N3O8Na re-
quires: 518.1542.
4.1.6. (S)-2-((1-Amino-1-oxo-3-phenylpropan-2-ylamino)methyl)-
5-methoxy-3-(4-nitrophenoxy) phenylacetate (10)
NaBH(OAc)3 (384 mg, 1.8 mmol) was added to a stirred solution
of 2-formyl-5-methoxy-3-(4-nitrophenoxy) phenylacetate
108 ꢀC. Rf 0.47 (DCM/MeOH, 9:1). [
(300 MHz, MeOD): d 7.05 (dd, 2H, JHH 5.0, 1.6, Ar–H), 7.00 (m, 3H,
a]
D ꢁ116 (c 0.10, MeOH). 1H NMR
8
(200 mg, 0.6 mmol) with -phenylalanine amide (100 mg,
L
Ar–H), 6.67 (m, 4H, Ar–H), 6.01 (d, 1H, JHH 2.3 Hz, Ar–H), 5.63 (d, 1H,
JHH 2.3 Hz, Ar–H), 4.27 (d, 1H, JHH 14.3 Hz, NH–CH–CONH2), 3.95 (t,
1H, JHH 7.5 Hz, Ar–CH2–CH-CONH2), 3.69 (d, JHH 14.3 Hz, Ar–CH2–
CH-CONH2), 3.50 (s, 3H, OMe), 3.26 (d, 2H, JHH 7.3 Hz, Ar–CH2–NH),
2.17 (s, 3H, Me). 13C NMR (75 MHz, MeOD): 176.12 (CONH2), 174.09
(COMe), 162.99, 160.99, 159.75, 148.85, 145.96, 140.19, 130.86,
129.71, 127.69, 122.27, 118.03, 105.30, 96.66, 95.45, 63.47, 55.91
(OMe), 45.30, 35.11, 22.96 (COMe). nmax/cmꢁ1: 3353, 1673 (CO),
1594, 1503, 1429, 1364, 1207, 1143, 1058; m/z (ES): 472 (MþNaþ).
0.6 mmol) and acetic acid (2 ml) in DCM/THF (20 ml) and the
resulting mixture was stirred at rt for 15 h. The reaction mixture
was diluted with EtOAc (50 ml) and washed with water (20 ml) and
brine (20 ml). The organic phase was separated, dried (MgSO4) and
concentrated by rotary evaporation. Purification using chroma-
tography (DCM/MeOH, 99:1 to 95:5) yielded the title compound 10
(90 mg, 0.19 mmol, 31% yield) as colourless plates. Mp 193–195 ꢀC.
Rf 0.37 (DCM/MeOH, 9:1). [
(300 MHz, MeOD):
a
]
ꢁ72 (c 0.10, MeOH). 1H NMR
D
d
8.15 (dd, 2H, JHH 7.0 and 2.1, Ar–H), 7.03 (m, 5H,
Calculated accurate mass: 472.1848;
472.1840.
C25H27N3O5Na requires:
Ar–H), 6.92 (dd, 2H, JHH 7.0 and 2.1, Ar–H), 6.22 (d, 1H, JHH 2.3, Ar–
H), 5.96 (d, 1H, JHH 2.3, Ar–H), 4.22 (d, 1H, JHH 14.5, Ar–CH2–NH), 3.8
(dd, 1H, JHH 9.9 and 5.3, NH–CH–CONH2), 3.59 (s, 3H, OMe), 3.47 (d,
1H, JHH 14.5, Ar–CH2–NH), 3.24 (d, 2H, JHH 4.1 Hz, Ar–CH2–CH–
CONH2), 2.15 (s, 3H, CO2Me). 13C NMR (75 MHz, MeOD): 175.75
(CONH2), 174.26 (COMe), 164.03, 163.46, 160.35, 156.80, 140.19,
130.64, 129.71, 127.76, 127.32, 118.97, 108.20, 100.14, 98.95, 63.95,
56.23 (OMe), 45.67, 35.01, 30.97, 23.14 (COMe). nmax/cmꢁ1: 3431,
3086, 2940, 1673 (CO), 1622, 1514, 1434, 1343, 1236, 1143; m/z (ES):
502 (MþNaþ). Calculated accurate mass: 502.1590; C25H25N3O7Na
requires: 502.1577.
Acknowledgements
The authors gratefully acknowledge the financial support pro-
vided by the Dorothy Hodgkin Postgraduate Award from Research
Council UK (to A.K.A.), and I. Chopra, A. O’Neill, K. Miller, J. Hurdle
and A. Al-Omar for help and advice.
References and notes
1. Binder, S.; Levitt, A. M.; Sacks, J. J.; Hughes, J. M. Science 1999, 284, 1311–1313.
2. Schmidt, F. R. Appl. Microbiol. Biotechnol. 2004, 63, 335–343.
3. Moellering, R. C. Clin. Infect. Dis. 1998, 27, S135–S140.
4. Chopra, I. Curr. Opin. Microbiol. 1998, 1, 495–501.
5. Record, M. T. J. Cellular and Molecular Biology; ASM: Washington, DC, 1996, pp
792–820.
6. Villain-Guillot, P.; Bastide, L.; Gualtieri, M.; Leonetti, J.-P. Drug Discovery Today
2007, 12, 200–208.
7. Campbell, E. A.; Korzheva, N.; Mustaev, A.; Murakami, K.; Nair, S.; Goldfarb, A.;
Darst, S. A. Cell 2001, 104, 901–912.
8. Mitchison, D. A. Int. J. Tuber. Lung Dis. 2000, 4, 796–806.
9. Sensi, P. Rev. Infect. Dis. 1983, 5, S402–S406.
10. Schrenzel, J.; Harbarth, S.; Schockmel, R.; Genne, D.; Bregenzer, T.; Flueckiger,
U.; Petignat, C.; Jacobs, F.; Francioli, P.; Zimmerli, W.; Lew, D. P. Clin. Infect. Dis.
2004, 39, 1285–1292.
11. Barluenga, J.; Aznar, F.; Garcia, A. B.; Cabal, M. P.; Palacios, J. J.; Menendez, M. A.
Bioorg. Med. Chem. Lett. 2006, 16, 5717–5722.
12. Murphy, C. K.; Mullin, S.; Osburne, M. S.; van Duzer, J.; Siedlecki, J.; Yu, X.;
Kerstein, K.; Cynamon, M.; Rothstein, D. M. Antimicrob. Agents Chemother. 2006,
50, 827–834.
13. Rothstein, D. M.; Farquhar, R. S.; Sirokman, K.; Sondergaard, K. L.; Hazlett, C.;
Doye, A. A.; Gwathmey, J. K.; Mullin, S.; van Duzer, J.; Murphy, C. K. Antimicrob.
Agents Chemother. 2006, 50, 3658–3664.
14. Rothstein, D. M.; Shalish, C.; Murphy, C. K.; Sternlicht, A.; Campbell, L. A. Expert
Opin. Investig. Drugs 2006, 15, 603–623.
15. Zorov, S. D.; Yuzenkova, J. V.; Severinov, K. V. Mol. Biol. 2006, 40, 875–884.
16. Payne, D. J.; Gwynn, M. N.; Holmes, D. J.; Pompliano, D. L. Nat. Rev. Drug Dis-
covery 2007, 6, 29–40.
17. Besong, G. E.; Bostock, J. M.; Stubbings, W.; Chopra, I.; Roper, D. I.; Lloyd, A. J.;
Fishwick, C. W. G.; Johnson, A. P. Angew. Chem., Int. Ed. 2005, 44, 6403–6406.
18. Horton, J. R.; Bostock, J. M.; Chopra, I.; Hesse, L.; Phillips, S. E. V.; Adams, D. J.;
Johnson, A. P.; Fishwick, C. W. G. Bioorg. Med. Chem. Lett. 2003, 13, 1557–1560.
4.1.7. (S)-2-((1-Amino-3-(4-hydroxyphenyl)-1-oxopropan-2-
ylamino)methyl)-3-(4-aminophenoxy)-5-methoxy
phenylacetate (11)
A slurry of PtO2 (1.1 mg, 4.0 mol % of 81% catalyst) was stirred in
EtOAc (5 ml) while bubbling nitrogen through the mixture. A so-
lution of (S)-2-((1-amino-3-(4-hydroxyphenyl)-1-oxopropan-2-
ylamino)methyl)-5-methoxy-3-(4-nitrophenoxy) phenylacetate 9
(60 mg, 0.12 mmol) in EtOAc (10 ml) was added and the purging
continued. Hydrogen gas was then purged into the flask and the
resulting mixture was stirred at rt for 15 h. After completion of
reaction (TLC), reaction mixture was filtered through a pad of
Celite and taken to dryness by rotary evaporation. The crude
product was chromatographed (DCM/MeOH, 95:5) to yield the title
compound 11 (28 mg, 0.06 mmol, 50% yield) as off white prisms.
Mp 132–133 ꢀC. Rf 0.25 (DCM/MeOH, 95:5). [
a
]
ꢁ120 (c 0.10,
D
MeOH). 1H NMR (300 MHz, MeOD):
d
6.92 (d, 2H, JHH 8.3, Ar–H),
6.79 (s, 4H, Ar–H), 6.61 (d, 2H, JHH 8.3, Ar–H), 6.13 (d, 1H, JHH 2.0,
Ar–H), 5.76 (d, 1H, JHH 2.0, Ar–H), 4.41 (d, 1H, JHH 14.3, Ar–CH2–NH),
4.01 (dd, 1H, JHH 9.9 and 5.3, NH–CH–CONH2), 3.89 (d, 1H, JHH 14.3,
Ar–CH2–NH), 3.62 (s, 3H, OMe), 3.15 (dd, 1H, JHH 13.8 and 10.0, Ar–
CH2–CH–CONH2), 3.06 (dd, 1H, JHH 13.8 and 5.3, Ar–CH2–CH–
CONH2), 2.28 (s, 3H, CO2Me). 13C NMR (75 MHz, MeOD): 176.33
(CONH2), 174.06 (COMe), 162.97, 161.01, 159.75, 157.23, 148.94,
145.89, 131.78, 130.71, 122.26, 118.06, 116.33, 105.43, 96.66, 95.47,

10054
A.K. Agarwal et al. / Tetrahedron 64 (2008) 10049–10054
19. Heikkila, T.; Ramsey, C.; Davies, M.; Galtier, C.; Stead, A. M. W.; Johnson, A. P.;
Fishwick, C. W. G.; Boa, A. N.; McConkey, G. A. J. Med. Chem. 2007, 50, 186–
191.
20. Heikkila, T.; Thirumalairajan, S.; Davies, M.; Parsons, M. R.; McConkey, A. G.;
Fishwick, C. W. G.; Johnson, A. P. Bioorg. Med. Chem. Lett. 2006, 16, 88–92.
21. Grzybowski, B. A.; Ishchenko, A. V.; Kim, C. Y.; Topalov, G.; Chapman, R.;
Christianson, D. W.; Whitesides, G. M.; Shakhnovich, E. I. Proc. Natl. Acad. Sci.
U.S.A. 2002, 99, 1270–1273.
24. Schneider, G.; Clement-Chomienne, O.; Hilfiger, L.; Schneider, P.; Kirsch, S.;
Bohm, H. J.; Neidhart, W. Angew. Chem., Int. Ed. 2000, 39, 4130–4133.
25. Gillet, V.; Johnson, A. P.; Mata, P.; Sike, S.; Williams, P. J. Comput. Aided Mol. Des.
1993, 7, 127–153.
26. Gillet, V. J.; Myatt, G.; Zsoldos, Z.; Johnson, A. P. Perspect. Drug Discovery Des.
1995, 3, 34–50.
27. Gillet, V. J.; Newell, W.; Mata, P.; Myatt, G.; Sike, S.; Zsoldos, Z.; Johnson, A. P.
J. Chem. Inf. Comput. Sci. 1994, 34, 207–217.
22. Han, Q.; Dominguez, C.; Stouten, P. F. W.; Park, J. M.; Duffy, D. E.; Galemmo, R.
A.; Rossi, K. A.; Alexander, R. S.; Smallwood, A. M.; Wong, P. C.; Wright, M. M.;
Luettgen, J. M.; Knabb, R. M.; Wexler, R. R. J. Med. Chem. 2000, 43, 4398–
4415.
23. Honma, T.; Hayashi, K.; Aoyama, T.; Hashimoto, N.; Machida, T.; Fukasawa, K.;
Iwama, T.; Ikeura, C.; Ikuta, M.; Suzuki-Takahashi, I.; Iwasawa, Y.; Hayama, T.;
Nishimura, S.; Morishima, H. J. Med. Chem. 2001, 44, 4615–4627.
28. Broadhurst, M. J.; Hassall, C. H.; Thomas, G. J. J. Chem. Soc., Perkin Trans. 1 1977,
2502–2512.
29. Ohmichi, T.; Maki, A.; Kool, E. T. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 54–59.
30. Paulini, R.; Lerner, C.; Diederich, F.; Jakob-Roetne, R.; Zurcher, G.; Borroni, E.
Helv. Chim. Acta 2006, 89, 1856–1887.
31. Paulini, R.; Lerner, C.; Jakob-Roetne, R.; Zurcher, G.; Borroni, E.; Diederich, F.
Chembiochem 2004, 5, 1270–1274.