Functionalised bicyclic tetramates derived from cysteine as antibacterial agents

Article abstract of DOI:10.1039/c9ob01076a

Routes to bicyclic tetramates derived from cysteine permitting ready incorporation of functionality at two different points around the periphery of a heterocyclic skeleton are reported. This has enabled the identification of systems active against Gram-positive bacteria, some of which show gyrase and RNA polymerase inhibitory activity. In particular, tetramates substituted with glycosyl side chains, chosen to impart polarity and aqueous solubility, show high antibacterial activity coupled with modest gyrase/polymerase activity in two cases. An analysis of physicochemical properties indicates that the antibacterially active tetramates generally occupy physicochemical space with MW of 300-600, clog D_7.4 of -2.5 to 4 and rel. PSA of 11-22%. This work demonstrates that biologically active 3D libraries are readily available by manipulation of a tetramate skeleton.

Full text of DOI:10.1039/c9ob01076a

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Panduwawala, S. Iqbal, A. L. Thompson, M. Genov, A. Pretsch, D. Pretsch, S. Liu, R. H. Ebright, A. Howells
and T. Maxwell, Org. Biomol. Chem., 2019, DOI: 10.1039/C9OB01076A.
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Page 1 of 27
Organic & Biomolecular Chemistry
Functionalised Bicyclic Tetramates Derived from Cysteine as Antibacterial Agents
View Article Online
DOI: 10.1039/C9OB01076A
†
Tharindi D. Panduwawala,† Sarosh Iqbal,
Amber L. Thompson, Miro Genov,‡ Alexander
†
Pretsch,‡ Dagmar Pretsch,‡ Shuang Liu,§ Richard H. Ebright,§ Alison Howells,¥ Anthony Maxwell&
and Mark G. Moloney*,†
†Department of Chemistry, Chemistry Research Laboratory, University of Oxford, 12 Mansfield
Road, Oxford, UK.
‡Oxford Antibiotic Group, The Oxford Science Park, Magdalen Centre, Oxford OX4 4GA, UK.
§Waksman Institute and Department of Chemistry, Rutgers University, Piscataway, NJ 08854, USA.
&Dept. Biological Chemistry, John Innes Centre, Norwich Research Park, Norwich NR4 7UH, UK.
¥Inspiralis Limited, Innovation Centre, Norwich Research Park, Colney Lane, Norwich NR4 7GJ, UK.
e-mail: mark.moloney@chem.ox.ac.uk
_______________________________________________________________________________
Routes to bicyclic tetramates derived from cysteine permitting ready incorporation of functionality at
two different points around the periphery of heterocyclic skeleton are reported. This has enabled the
identification of systems active against Gram-positive bacteria, some of which show gyrase and RNA
polymerase inhibitory activity. In particular, tetramates substituted with glycosyl side chains, chosen
to impart polarity and aqueous solubility, show high antibacterial activity coupled with modest
gyrase/polymerase activity in two cases. An analysis of physicochemical properties indicates that the
antibacterially active tetramates generally occupy physicochemical space with MW of 300-600,
clogD7.4 of -2.5 to 4 and rel. PSA of 11-22%. This work demonstrates that biologically active 3D
libraries are readily available by manipulation of a tetramate skeleton.
Introduction
Tetramates are of interest1-4 principally for their antibacterial activity,5, 6 and we have reported
recently that systems derived from the amino acids, serine 1a,7 threonine 1b,8 and cysteine 1c9 provide
useful templates for application in synthetic and medicinal chemistry, which in some cases exhibit
potent antibacterial activity. Amongst various methodologies to these derivatives,10 our route makes
use of the preferential formation of the malonamides of t-butyl oxazolidine/thiazolidine templates,
formed as the cis-2,5-isomer 2a-c rather than the alternative trans-2,5- 3a-c, which mimises the steric

Current address: Department of Applied Chemistry, Government College University, Faisalabad,
Faisalabad-38000, Pakistan.
1

Organic & Biomolecular Chemistry
Page 2 of 27
impact of the bulky t-butyl group, to direct a very efficient chemoselective Dieckmann ring closure
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(Route a, Scheme 1) in preference to the alternative (Route b, Scheme 1).11 Of interest, though, is that
DOI: 10.1039/C9OB01076A
the analogous thiazolidine system derived from cysteine and an aromatic aldehyde behaves differently
(Scheme 2), not least because there is a shift in preference away from the cis-thiazolidine 6 to the
trans- isomer 7, the latter of which now preferentially cyclizes to give tetramate 8, equivalent to Route
b in Scheme 1; this shift is, however, not complete and some of the cis-isomer 6, after epimerization at
C5, closes under these conditions leading to the enantiomer of 8, and therefore giving some erosion of
enantioselectivity.12 The overall outcome is that acyl tetramates 8 (Scheme 2),13 known for their
metal chelating14 and antibacterial activity,14 are accessed directly, rather than by acylation of an
unfunctionalised tetramate nucleus.15 Since we had earlier explored in detail the structure activity
relationship of tetramates substituted at C-7 but limited to t-butyl substitution at C-2, these
thiazolidine compounds provided access for the first time to compounds with an aryl substituent at C-
2 and this gave the opportunity to explore the scope of antibacterial bioactivity at this position. The
results of that work are reported here.
Results and Discussion
Synthesis of the required tetramate proceeded from cysteine 1c to a mixture of the cis- and
trans- diastereomeric malonamides 6a-g and 7a-g, with a preference for the latter trans- isomers in
three cases, followed by Dieckmann cyclisation to 7-ethoxcarbonyltetramates 8a-g (Scheme 2); there
is up to 20% attrition of e.e. during this process, arising from cyclisation of the minor but unseparated
cis-isomer 6a-g.12 These esters were expected to be suitable for conversion to the amide by direct
aminolysis, a process which had been used successfully previously,16 and this was immediately
successful for synthesis of systems 9a-v (see Table 1) despite the high level of functional group
density around the ring system which might not have given a chemoselective process, although
elevated temperature was needed for successful reaction.
2

Page 3 of 27
Organic & Biomolecular Chemistry
MeO2C
MeO2C
CO2Me
SH
(i) ArCHO, Et3N
5
5
View Article Online
DOI: 10.1039/C9OB01076A
EtO
N
Cl-H3N+
2
S
EtO
N
2
+
S
(ii) EtOC(O)CH2C(O)OH,
DCC
1c
O
O
O
O
Ar
Ar
6a-g
7a-g
a Ar = Ph
b Ar = p-BrC6H4
c Ar = m-BrC6H4
d Ar = p-FC6H4
e Ar = p-NO2C6H4
f Ar = 2-Cl,4-FC6H3
g Ar = 2-C4H3O
KOt-Bu
O
O
EtO
OH
H
RHN
OH
H
RNH2 (1.2 eq)
8
5
8
N
5
O
O
N
THF/Toluene
reflux, 12-16 h
2
2
S
S
Ar
Ar
9a-v
see Table 1
8a-g
Scheme 2
Table 1: Synthesis of tetramates 9a-v according to Scheme 2.
Compound
9a
Ar
R
Yield (%)
45
Tautomeric ratioa
2.7:1
Ph
1-Adamantyl
9b
9c
p-BrC6H4
m-BrC6H4
p-FC6H4
p-NO2C6H4
2Cl-4-FC6H3
2-C4H3O
Ph
70
2.6:1
38
2:1
9d
9e
56
2.7:1
40
2.4:1
9f
60
2.5:1
9g
92
3:1
9h
9i
4-Cyclohexylphenyl
54
15:1
p-FC6H4
p-NO2C6H4
2Cl-4-FC6H3
Ph
45
13:1
9j
59
12.5:1
9k
9l
73
15:1
4-Chloro-2-methylphenyl 53
only 1 form
only 1 form
only 1 form
only 1 form
only 1 form
only 1 form
only 1 form
9m
9n
9o
p-FC6H4
p-NO2C6H4
p-BrC6H4
m-BrC6H4
2Cl-4-FC6H3
p-BrC6H4
40
37
40
38
35
9p
9q
9r
4-Morpholinophenyl
69
75
9s
2-C4H3O
only 1 form
4-Aminotetrahydropyranyl
9t
p-BrC6H4
70
5.2:1
3

Organic & Biomolecular Chemistry
Page 4 of 27
9u
9v
Ph
Cyclohexyl
49
40
3.1:1
View Article Online
DOI: 10.1039/C9OB01076A
3.1:1
2Cl-4-FC6H3
a Major form is tautomer pair AB -see Figure 4 and Table 2.
Chemoselective formation of the amide product, and not the alternative enamine arising by attack at
the ketone, was evident by loss of the ester function, and also confirmed by HMBC correlation
(Figure 1); thus, C-6 correlates with H-4 and H-5, while C-8 correlates with H-2 and H-5 as expected.
For compounds 9s and 9t, C-9 correlates with the β-CH in the amine with respect to C-9, confirming
the assignment of the carbonyl functional groups.
HO
HO
HO
H
H
H
O
N
O
N
O
N
4 S
4 S
4 S
6
6
6
5
5
5
N
2
9
9
9
8
8
2
8
2
N
N
Cl
H
H
H
Cl
H
H
O
O
H
O
O
F
9g
9h
9q
HO
O
HO
HO
H
H
H
O
N
O
O
4 S
4 S
4 S
2
6
6
6
5
5
5
2
2
9
8
9
9
8
8
N
N
N
N
H
N
O
H
H
H
Cl
H
H
O
O
F
Br
9k
9t
9u
Figure 1. HMBC correlation data of selected carboxamide tetramates.
HO
O
H
HO
O
H
HO
O
H
H
H
N
H
N
H
H
H
O
O
O
4
2
5
S
S
S
N
NH
NH
NH
Cl
H
H
H
H
H
9d
9h
9m
F
F
HO
H
HO
H
H
N
H
N
H
H
O
O
S
S
NH
NH
O
N
H
H
Cl
O
O
H
9k
9r
F
Br
Figure 2. NOE analysis of selected carboxamide tetramates.
4

Page 5 of 27
Organic & Biomolecular Chemistry
As anticipated, the stereochemical relationship of the starting bicyclic tetramate esters 8 was
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conserved in the amide systems, and a trans- relationship across the bicyclic ring between H-2 and H-
DOI: 10.1039/C9OB01076A
5 was determined from NOE analysis (Figure 2); this outcome was further confirmed by single crystal
X-ray diffraction studies of adamantyl derivative 9f (Figure 3).17
Figure 3: The structure of 9f from single crystal X-ray diffraction studies. Displacement ellipsoids
drawn at 50% probability.
The products were generally found to exist as tautomeric forms, a phenomenon which has been
observed previously, and which could be assigned from careful NMR examination.15, 18 In their NMR
spectra, the internal tautomeric pairs (A and B, C and D) could not distinguished and were observed as
13
an averaged hybrid, while the external tautomeric pairs (AB and CD) had distinct C chemical shift
differences of C-6, C-8 and C-9 (Figure 4), so that the tautomeric forms could be readily distinguished
(Table 2).
Table 2. 13C chemical shifts of tautomers of selected examples of carboxamide tetramates.
Compound Solvent
13C chemical shifts (ppm)
Tautomeric form
5

Organic & Biomolecular Chemistry
Page 6 of 27
Number
9a
C-6
C-8
C-9
View Article Online
DOI: 10.1039/C9OB01076A
187.8
191.1
188.4
191.1
188.7
191.0
189.5
185.2
191.5
184.7
186.6
191.6
172.2
178.0
172.4
178.2
172.0
177.8
175.2
171.7
178.2
171.7
172.5
178.8
165.8
166.4
165.9
166.4
166.0
166.5
166.9
164.3
165.4
164.3
165.9
166.5
major (AB)
minor (CD)
major (AB)
minor (CD)
major (AB)
minor (CD)
CDCl3
CDCl3
9b
9f
CDCl3
MeOD
CD2Cl2
CD2Cl2
CD2Cl2
only 1 form (AB)
major (AB)
9k
9q
9t
minor (CD)
only 1 form (AB)
major (AB)
minor (CD)
13
The C chemical shifts of C-6, C-8 and C-9 of the minor tautomers were invariably more downfield
than their major tautomers, with the smallest ∆δ13C observed for the exocyclic amide C-9. The
tautomeric behaviour is also solvent dependent, as seen from the different chemical shift values of 9f
in CDCl3 and CD3OD. In polar solvents (e.g. methanol-d4, acetone-d6, DMSO-d6 and CD3CN), only
one set of resonances was observed for each carbon, and appears to be that of an averaged hybrid
structure arising from facile equilibrium of the tautomeric forms. NOESY data obtained for 9k in dry
CD2Cl2 revealed that the major tautomer for 9k was A (Figure 4), based on the correlation from H-5
to proximal enol O-H, which would not have been possible in other tautomeric forms B, C and D
(Figure 4). This conclusion is supported by single crystal X-ray diffraction studies of tetramate 9f
(Figure 3, vide supra), which was found to exist in the endoenolic form A. Since the chemical shift
pattern observed for the major/minor tautomers is similar for all compounds, they are all assumed to
adopt the major tautomeric form A, and this finding is similar to that found in related systems.15
Minimum energy conformations were generated for the four tautomeric forms A-D for compounds 9k
using MM2 methodology and confirmed tautomer A to be the thermodynamically more stable (Table
3 and Table 1, SI).
6

Page 7 of 27
Organic & Biomolecular Chemistry
View Article Online
DOI: 10.1039/C9OB01076A
3.660 Å
H
4.304 Å
O
O
H
O
O
H
H
O
H
N
H
N
H
H
O
4
S
2
5
S
NH
NH
H
H
Cl
Cl
H
H
9k-A
9k-B
F
F
O
H
H
O
H
H
N
H
H
NH
NH
S
S
N
O
O
H
H
H
Cl
Cl
O
O
H
5.122 Å
4.810 Å
9k-C
9k-D
F
F
Figure 4. NOESY correlation data for 9k suggesting tautomer A as the major tautomer. The distance between H-5
and enol O-H for each tautomer was calculated for the MM2-energy minimized molecular model using Chem3D v.15.
Table 3. MM2-minimized energy of tautomeric forms A-D for selected examples 9f, 9k and 9q.
Compound Tautomeric Minimum Energy of tautomeric forms (kcal/mol)
Number
ratio
2.5:1
15:1
A
B
C
D
9f
18.93
6.93
39.86
31.28
25.54
26.83
13.04
7.05
39.32
30.75
26.23
9k
9q
only 1 form 0.80
Having demonstrated the generality and selectivity of this approach, of interest was its
application to more elaborate side chain systems; to this end, the products 9w-g’ were accessed by
direct aminolysis of esters 8a, b and f using the required amines (Scheme 2 and Table 4), mostly
available from commercial sources. In order to incorporate a higher level of polarity, mono-Fmoc
protected 10b, prepared from dapsone 10a, an inhibitor of dihydropteroate synthetase,19 was used for
the aminolysis of tetramate ester 8a to give carboxamide tetramate 9a’, which on subsequent
deprotection gave 9b’ in 40 % yield (Scheme 3). The design of carboxamide 9d’ was inspired by
naturally occurring tetramates kibdelomycin20 and amycolamicin,21 and was synthesised by
aminolysis of tetramate 8a with glycosylated amine derivative 12b (Scheme 4). Although it has been
previously reported that the desired glycosylated amine 12b could be directly synthesised from 4-
aminophenol 11a and bromogalactose,22, 23 Fmoc-protected amine 11b was found to be more suitable
for this purpose by Lewis acid-mediated (BF3.OEt2) glycosylation; the equatorial substitution of the
3
aryl group was confirmed by the trans-diaxial JH1-H2 coupling constant (8.0 Hz). Base-mediated
7

Organic & Biomolecular Chemistry
Page 8 of 27
deprotection of the amine gave the desired product 12b which was reacted with tetramate 8a to yield
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carboxamide 9d’ in 60 % yield, and methanolysis under basic conditions afforded 9e’.DOI: 10.1039/C9OB01076A
Table 4: Synthesis of tetramates 9w-g’
Compound
Ar
R
Yield (%)
30
9w
O
O
O
9x
9y
74
75
S
O
S
N
O
O
S
N
9z
72
30
Ph
O
O
O
S
9a’
NH-Fmoc
O
O
S
9b’
40
NH2
8

Page 9 of 27
Organic & Biomolecular Chemistry
p-BrC6H4
O
O
S
N
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DOI: 10.1039/C9OB01076A
9c’
40
60
OAc
OAc
Ph
Ph
O
O
OAc
AcO
9d’
OH
OH
O
O
OH
HO
9e’
9f’
54
30
N
2Cl-4-FC6H3
2Cl-4-FC6H3
9g’
36
N
The carboxamidotetramates 9a-g’, when purified by silica gel column chromatography, gave broad
1
signal resonances in their H NMR spectra, consistent with metal chelation, as has been observed
previously.7 As a result, carboxamides were routinely purified on silica column with 1 % Et3N in the
eluent. Residual Et3N was removed by washing with 5 % citric acid, giving the purified product with
sharp and well-resolved 1H spectra; an example is given for compound 9d (Figure 5). Alternatively, it
was also possible to purify these carboxamides on silica column without any Et3N in the eluent,
provided that they were subsequently washed with 2 M HCl to give the metal-free form.
1
Figure 5. H NMR spectra of tetramate 9d (a) post-column purification, before acid-wash and (b)
post-column purification, after acid-wash.
Since peak broadening was a recurrent observation after column purification, the metal-chelated
tetramate and acid-washed tetramate for compound 9a were analysed by Inductively Coupled Plasma
Mass Spectrometry. The metals Na, Mg, Fe and Zn but especially Ca were found in high abundance in
9

Organic & Biomolecular Chemistry
Page 10 of 27
the metal-chelated tetramate, which were substantially removed by washing with 2M HCl (Table 5
View Article Online
and Table 2, SI). Clearly these tetramates are easily capable of forming metal salts with diverse
DOI: 10.1039/C9OB01076A
cations; the metal chelating behaviour of 3-acyltetramates has been reviewed recently,2, 6, 13, 14, 24 and
chelation both of natural products25-29 especially with antibacterial activity30 and of simpler systems31-
36 has also been reported while in our own work, calcium salt formation (but not other metals) during
the isolation of tetramates had been observed earlier.37
Table 5. Metal content observed in metal-chelated and acid-washed forms of carboxamide 9a.
Metal content (ppm)
Mg
Ca
Fe
Zn
Na
Metal-chelated
Acid-washed
7026.1
15.9
20483.7
24.8
38.2
34.8
71.2
9.9
3385.6
18.8
In an attempt to make use of the C-2 bromoaryl substituent for Suzuki-Miyaura (SM) cross-
coupling reactions for further derivatisation, reaction of both thiazolidines 8b and 9b with 2-
furanylboronic acid was examined, but only unreacted starting material was recovered; this was in
contrast to oxazolidine 13a (Scheme 5) which gave the expected coupled product 13b under the same
conditions. This failure of SM cross-coupling on thiazolidine-derived tetramic acids was attributed to
catalyst deactivation by the cyclic thioether function, and alternative reaction conditions were clearly
necessary. While successful SM cross-coupling on thiophene systems had been documented using
PPh3 and AsPh3,38 no reports on systems containing non-aromatic thioethers such as those of the
thiazolidine-derived tetramates could be found in the literature. However, a more recent report on
Suzuki cross-coupling on biotin-derived systems39 together with a detailed review by Buchwald et
al.40 suggested XPhos as an alternative, and this ligand proved to be successful, with the desired
product 14a being formed along with the return of some unreacted starting material (Scheme 5 and
Table 6). Although it has been reported that the active form of the catalyst for SM cross-coupling
involving dialkylbiaryl phosphine ligands is the monoligated L1Pd(0) species rather than the more
highly coordinated L2Pd(0),40, 41 a ratio of L:Pd=1.5:1 was not sufficient to drive the reaction to
completion even after a reaction time of 48 h. Increasing the L:Pd ratio to 3:1 led to completion of the
reaction and this approach was successfully applied to the synthesis of a range of carboxamide
tetramates 14a-l with elaborated pendant groups at C-2 derived from 9b and 9s (Table 6). The boron
reagents used for the above transformations were commercially available acids or their pinacol esters,
except for 14c where the boron reagent was the glycol ester derived from the corresponding boronic
acid, prepared according to literature procedure.42 Carboxamide tetramate 14k was synthesised as a
mixture of diastereomers from the corresponding commercially available racemic 1-(tetrahydropyran-
10

Page 11 of 27
Organic & Biomolecular Chemistry
2-yl)-1H-pyrazole-5-boronic acid pinacol ester. It would appear the additional nucleophilicity of
XPhos gives a higher electron density at Pd(0) in the monoligated L Pd(0) species, and incrVeieawsAertsiclethOenline
DOI: 10.1039/C9OB01076A
1
rate of oxidative addition leading to overall successful coupling.40
HO
HO
Pd(OAc)2 (5 mol%),
XPhos (7.5 mol%),
aq. Na2CO3, R2B(OH)2
6
6
O
O
HO
O
S
S
O
O
7
N
7
N
R1
R1
O
DME , reflux, 48 h
N
O
O
8b R1 = EtO
9b R1
R2
iPr
Br
iPr
P
13a X = Br
13b X = C4H3O
=
14a-l
X
NH
iPr
9t R1
=
O
NH
XPhos
Scheme 5
Table 6: Suzuki-Miyaura cross-coupling of carboxamide tetramates 9b and 9t.
Carboxamide
Compound
R1
% Yield Compound
Number
R1
% Yield
tetramate (R=) Number
O
O
O
14a
72
74
14g
14h
70
78
O
N
14b
14c
N
O
O
N
80
45
14i
14j
53
70
O
O
N
N
N
14d
14e
N
N
O
N
O
78
14k
60
S
O
N
O
14f
74
30
O
O
14l
Several naturally occurring tetramic acids bearing glycosyl residues are known, including
virgineone,43 kibdelomycin,44 amycolamicin,45 aurantoside46, 47 and streptolydigin.48 While the exact
role of the sugar moiety of these tetramates is not known, it may give improved bioavailability with
increased solubility and transportation, or help modulate toxicity and aid in bacterial target
interaction.49 Thus, investigating the effect of glycosylated tetramates on antibacterial activity was of
11

Organic & Biomolecular Chemistry
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interest, and the systems developed thus far offered the feasibility of C-2 substitution of the tetramate
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ring. In order to demonstrate an alternative process using late-stage ring formation, bicyclic tetramates
DOI: 10.1039/C9OB01076A
with C-2 pendant glycones were derived from β-D-galactose pentaacetate (Scheme 6). Acetobromo-
α-D-galactose 15b was prepared from β-D-galactose pentaacetate 15a according to the literature
3
procedure;50 axial bromination at the anomeric carbon was confirmed from the J(H1-H2)-coupling
constant of H-1 (d, J=4.1 Hz). Solid-liquid phase transfer-catalysed aryl glycosylation of 4-
hydroxybenzaldehyde with acetobromo-α-D-galactose 15b gave the desired aldehyde 15c, according
to a modified literature procedure; BnNBu3Cl proved to be a better phase transfer catalyst than
Bu4NBr while the presence of H2O led to a remarkable reduction in yield.51 The β-anomer was
obtained exclusively, where the equatorial substitution of the phenoxide at the anomeric carbon was
confirmed by 3J-coupling constant of the anomeric C-H (d, J=8.0 Hz). The glycosylated aldehyde 15c
was then condensed with ʟ-cysteine methyl ester hydrochloride to yield the expected thiazolidine in
85 % yield with a cis:trans diastereomeric ratio of 1.6:1, and this material was N-acylated to give the
corresponding malonamide in 52 % yield, which was in turn cyclised to afford the glycosylated
tetramate 16 but only if the crude product was isolated without acidic work-up. Base-catalysed
methanolysis gave tetraol 17 in quantitative yield (Scheme 6); neither hydrolysis nor
transesterification of the ethyl ester at C-7 was observed under these reaction conditions. Aminolysis
of tetramate ester 16 afforded carboxamide tetramates 18 with the acyl groups of the sugar moiety
intact; methanolysis of the former resulted in the very polar carboxamide tetramate 19.
A tetramate with a disaccharide pendant group was derived from commercially available
lactose octaacetate (Scheme 7); this followed a similar approach to the monosaccharide derivative
12

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Organic & Biomolecular Chemistry
above. Commercially available lactose octaacetate 20a (as a 2.9:1 mixture of α- and β- anomers at the
View Article Online
glucopyranosyl end) was subjected to BiBr3-catalysed bromination and afforded 20bDaOsI:t1h0.e10α39-/aCn9OoBm01e0r76A
3
in 92 % yield, confirmed by J-coupling constant of the anomeric C-H. Aryl glycosylation gave
aldehyde 20c, which was condensed with ʟ-cysteine methyl ester hydrochloride, acylated and cyclised
to afford tetramate 21.
Due to the acid-labile nature of phenyl glycosides prepared above and the resulting difficulty
associated with the acidic work-up procedure required to remove chelating metal ions, an alternative
route to the incorporation of glycones via a benzyl alcohol linking group was explored (Scheme 8).
The controlled reduction of terephthalaldehyde with NaBH4 between 0-2°C afforded 4-
(hydroxymethyl)benzaldehyde in 70 % yield according to a modified literature procedure.52 Lewis
acid-catalysed glycosylation with β-D-galactosepentaacetate 15a gave aldehyde 22 as the β-anomer in
71% yield along with the byproduct 4-(acetoxymethyl)benzaldehyde in 28 % yield. Following the
usual synthetic route to access the bicyclic tetramate core, condensation of aldehyde 22 with cysteine
ethyl ester yielded the expected thiazolidine, which was acylated and cyclized under basic conditions
to afford benzyl glycosylated tetramate 23, which proved to be stable to acidic work-up.
In order to permit late stage C-2 substituent manipulation, tetramates 26a and 26b were
derived from tetramates 25a,b by silyl deprotection, which were obtained from the silyl ether of 4-
hydroxybenzaldehyde 24a or 4-(hydroxymethyl)benzaldehyde 24b respectively (Scheme 9).
Conversion of 25a to the corresponding carboxamides 27a-c by aminolysis with the required amine
proceeded using the approach described above, in modest to good yield. The deprotection of silyl
ether 27a to obtain tetramate 28a, however, required some optimisation. While TBAF was a
successful desilylating agent, chromatographic purification was difficult, possibly due to the basicity
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of fluoride ion in an aprotic solvent53 leading to deprotonation of the product tetramic acid. However,
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alternative conditions using KF as a fluoride source were more successful. Two other carboxamide
DOI: 10.1039/C9OB01076A
tetramates 28b and 28c were also synthesised using this approach.
HO
HO
O
O
O
O
O
S
(i), (ii), (iii)
(iv)
S
N
N
O
O
n
TBDMSO
24a n=0 (quant.)
24b n=1 (quant.)
n
25a n=0
25b n=1
O
26a n=0; 30 %
26b n=1; 20 %
n
HO
TBDMS
(v)
HO
O
HO
O
a R =
S
N
R
NH
S
O
N
Ph-
b R =
c R =
NH
O
O
OTBDMS
27a (54%)
27b (27%)
27c (30%)
29a R = CO2Et
30 R = CCMe
R
O
HO
O
(iv)
HMBC correlation data
O
HO
O
S
O
N
HO
O
NH
S
N
R
NH
S
N
(viii)
(vi)
NH
O
O
O
28a (40%)
28b (45%)
28c (45%)
OH
29a (76%)
29b (quant.)
OH
(vii)
30 (30 %)
O
(i) L-Cysteine methyl ester hydrochloride (1.2 eq), Et3N (1.2 eq), Petrol, reflux, 19 h; (ii) Ethyl hydrogen malonate (1.2 eq), DCC (1.2 eq), DMAP (0.1 eq),
CH2Cl2, rt, 15 h; (iii) KOtBu (1.2 eq), THF, reflux, 3h; (iv) for n= 0: KF (2 eq), tetraethyleneglycol/THF, rt, 30 min; for n= 1: KF (2 eq), tetraethyleneglycol/THF, 80
°C, 5 h; (v) RNH2 (1.2 eq), THF/toluene, reflux, 16 h; (vi) ethyl bromoacetate (1.5 eq), K2CO3 (2.1 eq), THF, rt, 18 h; (vii) LiOH.H2O (2.0 eq), THF:H2O (1:1), rt, 2
h; (viii) 1-bromo-2- butyne (1.5 eq), K2CO3 (2.1 eq), THF, rt, 18 h.
Scheme 9
The presence of a phenolic group at the C-2 position provided a platform for further
functionalization of carboxamide tetramates via Williamson ether synthesis. In the presence of
K2CO3, nucleophilic substitution of ethyl bromoacetate and 1-bromo-2-butyne by the phenoxide
derived from 28a gave carboxamide tetramates 29a and 30 respectively (Scheme 9). HMBC
correlation data confirmed the substitution at the phenolic but not at the enolic alcohol group. Ester
hydrolysis of 29a afforded the carboxylic acid bearing tetramate 29b with improved hydrophilicity.
Since the pyroglutamate skeleton is found in antibacterial natural products (e.g.
oxazolomycin54, 55 and pramanicin56), the synthesis of a pyroglutamate-tetramate hybrid system was
also examined. This was achieved by condensing pyroglutaminol 31,57 prepared by the literature
procedure,58, 59 with terephthalaldehyde by reflux in toluene using a Dean-Stark apparatus to give a
mixture of products 32 and 33 in 1:2.4 ratio (Scheme 10); the lactam-functionalized aldehyde 33 was
isolated by column chromatography as a single diastereomer, whose stereochemical assignment was
in agreement with previous reports on N,O-acetal formation of 31 with benzaldehyde.60, 61 Aldehyde
33 was in turn converted to the desired bicyclic tetramate ester 34a in the usual way, which was
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Organic & Biomolecular Chemistry
converted to carboxamide 34b with 1-adamantylamine. The stereochemical assignment of the
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tetramate system was confirmed by NOE experiments (Scheme 10). The relative stereochemistry of
DOI: 10.1039/C9OB01076A
both heterocyclic systems was found to be trans-, in keeping with structures reported in the
literature.62
Of interest is that direct Buchwald-Hartwig amination of bromo-substituted tetramates 8b and
9b using any of XPhos, RuPhos and XantPhos was not successful. Instead, p-bromobenzaldehyde
35a was functionalized via Buchwald amination/amidation to give aldehydes 35b,c and these were
then used to synthesise the tetramate core de novo using a similar route to that described above, giving
esters 36a,b which were converted to carboxamides 37a,b as before (Scheme 11).
In one case, deprotection of the N,S-acetal was examined; in comparison to oxazolidine-based
N,O-acetal systems, thiazolidine-based N,S-acetals have been shown to have higher stability to
acids,63 and their deprotection often requires harsh conditions such as heating under reflux with 5M
HCl for 72 h.64 However, Onoda et al. have shown that Boc-protected thiazolidines can be
deprotected at the N,S-acetal in good yield with 15 eq of TFA in water-saturated CH2Cl2 under
ambient conditions within 2 h.65 Deprotection of carboxamide tetramate 9a was investigated with
TFA in CH2Cl2, but even after a reaction time of 24 h, only starting material remained, and while the
addition of water accelerated the reaction, complete deprotection was not observed in 24 h. However,
deprotection of thiazolidine via the Corey-Reichard protocol66 was successful giving
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carboxamidotetramate 38 in 70 % yield (Scheme 12). This observation highlights the need for
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stronger conditions for the deprotection of the more stable thiazolidine-derived carboxamide
DOI: 10.1039/C9OB01076A
tetramates.
Antibacterial properties
Mode of action of tetramates
Naturally-occurring tetramates, such as kibdelomycin and amycolamycin, are inhibitors of
bacterial topoisomerase IV (topo IV) and DNA gyrase,20, 44, 67 whereas the tetramates streptolydigin
and tirandamycin are inhibitors of RNA polymerase (RNAP).68, 69 Some tetramates have been shown
to provide dual inhibition of both RNAP and undecaprenyl pyrophosphate synthase (UPPS).7 All four
of theses enzymes are essential for viability. The primary function of topo IV is the decatenation of
daughter chromosomes that are multiply-linked, during the final stages of DNA replication,70 and the
role of DNA gyrase is catalysis of negative supercoiling of DNA.71 Bacterial RNAP is an attractive
target in antibacterial chemotherapy as the bacterial RNAP subunit sequences are highly conserved
while being different from eukaryotic RNAP subunit sequences. This permits broad-spectrum activity
and therapeutic selectivity.72 An examination of the mode of action of the tetramates prepared in the
current work was therefore of interest.
Inhibition of possible bacterial target enzymes by some bicyclic tetramates was studied for
Staphylococcus aureus topo IV and RNAP, Escherichia coli RNAP and Mycobacterium tuberculosis
gyrase. A selection of compounds (full data is given in Tables 3-5, SI) initially was tested at a fixed
concentration of 100 µM for the inhibition of topo IV and gyrase, and the percentage of DNA
decatenated or supercoiled in the presence of each test compound was calculated. A lower percentage
of decatenation or supercoiling indicated a higher level of inhibition of the target enzymes. The data
were compared to those for the known topo IV and gyrase inhibitor, ciproflaxacin. Concentrations
resulting in half-maximal inhibition (IC50s) for some of the more potent inhibitors (vide infra) were
determined and are presented in Figure 6(a). Tetramates 9a, 9h and 9m were tested for their
inhibition of S. aureus and E. coli RNAP, 73-75 and the data obtained are presented in Figure 6(b).
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(a)
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DOI: 10.1039/C9OB01076A
(b)
Figure 6.(a) IC50 values of tetramates against M. tuberculosis gyrase and S. aureus topoisomerase IV.
Compounds 9a and 9m were weak inhibitors of M. tuberculosis gyrase and 9d’ was a weak inhibitor
of S. aureus topoisomerase IV. (b) IC50 values of tetramates against S. aureus RNAP and E. coli
RNAP.
Comparison of mimimal inhibitory concentrations (MICs) for antibacterial activity against
methicillin-resistant S. aureus (Table 10, SI; see also Figure 7; vide infra) with IC50s for inhibition of
S. aureus RNAP and topo IV (Figure 6) indicates that compounds exhibiting high antibacterial
activities also exhibit high S. aureus RNAP and topo IV inhibitory activities, consistent with the
possibility that antibacterial activity may be attributable to RNAP and topo IV inhibitory activity.
Compounds 9a, 9h and 9m inhibited both RNAP and S. aureus topo IV, consistent with a possible
dual mode of action for these compounds. Some compounds that showed high efficacy in enzyme
inhibition assays did not show high efficacy in whole-cell assays (e.g 9d’, 19). This potentially may
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be due to low cell permeability or elimination via efflux mechanisms, leading to reduced
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bioavailability in the bacterial cell. In general, E. coli RNAP was less sensitive to tetramates than S.
DOI: 10.1039/C9OB01076A
aureus RNAP, and the M. tuberculosis gyrase was less sensitive to tetramates than S. aureus topo IV.
Antibacterial activity: hole-plate method
Whole-cell antibacterial assays were performed using the hole-plate method with Gram-
positive S. aureus DS267 or Gram-negative E. coli X580, using Cephalosporin C as a positive control.
The samples were prepared as 4 mg/mL solutions of 70% DMSO in MeOH, with serial dilution to the
desired concentrations where necessary. The relative potency was estimated by reference to positive
controls prepared with Cephalosporin C.76 Many of the carboxamide analogues 9a-v showed
antibacterial activity against S. aureus, but none showed activity against E. coli (Table 6-9, ESI). The
presence of an adamantyl carboxamide side chain on the tetramate core resulted in a significant
enhancement of antibacterial activity and such tetramates 9a-f were active at a concentration of 1
µg/mL. Tetramates 9h-9k with a 4-cyclohexylphenyl group or 9l-9q with a 4-chloro-2-methylphenyl
group demonstrated good antibacterial activity, although much less potent compared to their
adamantyl analogues, with the 4-cyclohexylphenyl group being the more active. The decrease in
antibacterial activity observed for 9r, 9s and 9w further confirms the requirement for a bulky pendant
group at C-7 of the bicyclic tetramate core.
Since some tetramates have been shown to lose antibacterial activity in the presence of human
serum albumin (HSA),7 further assays were run in the presence of HSA. These data were compared
with the bioactivity of each sample in the absence of HSA and selected examples are given in Figure 7
along with full data in Table 6-9 (ESI). While 9a-9f showed good antibacterial activity at 1 µg/mL in
the absence of HSA, there was a complete loss of activity in the presence of 4 mg/mL of HSA (where
the tetramate:HSA ratio is approximately 1:30, data not shown). These data suggest that carboxamides
are possible ligands for HSA. Nevertheless, of interest was the return of bioactivity for these
tetramates when the concentration of the test compounds was increased to 5 µg/mL (in which
tetratmate:HSA ratio is approximately 1:6). Similarly, tetramates 9h-9q exhibited no antibacterial
activity at their original concentrations when tested in the presence of HSA, but their activity was
regained by increasing the compound concentrations. Interestingly, tetramates 9g,9r,9s and 9w which
were less potent compared to the other tetramates discussed above retained their potency in the
presence of HSA.
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Organic & Biomolecular Chemistry
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DOI: 10.1039/C9OB01076A
Figure 7. Bioactivity of carboxamide tetramates with/without HSA.
The assay samples were prepared in 70% DMSO in MeOH for reasons of solubility, followed
by mixing with albumin solution, and resulted in a final solvent composition of 35:15:50 of
DMSO:MeOH:H2O in the well (35% DMSO). It is known that higher concentrations of DMSO can
lead to protein denaturation,77, 78 and this might have interfered with the above analysis. Carboxamides
9a-9f showed good antibacterial activity at 1 µg/mL, and these samples could be dissolved in a
minimum amount of DMSO at such low concentrations. Thus, the antibacterial activity of
carboxamides 9a-9f in the presence of HSA was tested where the final solvent composition was 2%
DMSO in H2O. The zones of inhibition measured were in agreement with the data obtained in the
presence of 35% DMSO, confirming that the antibacterial activity observed for the tetramates in the
presence of HSA was not a result of the compound unbound to denatured HSA.
Broth dilution method
Broth assays were performed to determine MICs of compounds (Table 10, ESI). The
carboxamido tetramates 9a-g’ showed either weak (MIC= 250 µg/mL) or no activity against Gram-(-)
strains, in agreement with the data obtained from the hole-plate method, while some exhibited
promising antibacterial activity against Gram-(+) strains. Among the C-7 functionalized tetramates,
the most potent carboxamide tetramates 9b-f, h, l-m, q, u-v, z showed an MIC of 0.49 µg/mL against
at least one organism, significantly better than the tetramate ester analogues 8a-g,12 highlighting the
importance of a carboxamide side chain group for whole-cell antibacterial activity. However, the
presence of more polar carboxamides at C-7 led to a loss of activity (see 9d’ and 9e’). Since an MIC
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value of 16-32 µg/mL against a test organism is considered a suitable minimum for optimisation,79
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several tetramates therefore offer suitable starting points for core scaffold elaboration. The selectivity
DOI: 10.1039/C9OB01076A
of these tetramates for prokaryotic cells over mammalian cells was analysed via a primary
cytotoxicity screening against HaCaT cell line (see ESI). Compounds that are worth progressing from
this stage should possess ≥10-fold higher antibacterial activity over cytotoxicity,79 and on this basis,
carboxamido tetramates 9e, 9f, 9g, 9h, 9l, 9m and 9p are of interest.
The tetramates with C-2 functionalization obtained from SM cross-coupling were assayed for
antibacterial activity against Gram-(-) bacterial strains E. coli, K. pneumoniae and P. aeruginosa and
Gram-(+) strains MRSA, Enterococcus and Streptococcus pneumonia but no activity was observed.
There was, however, potent antibacterial activity against Gram-(+) strains MRSA, Enterococcus and
Streptococcus pneumoniae. In particular, compounds 14d, 14h and 14k displayed improved
antibacterial activity against MRSA while 14a, 14d and 14h showed improved activity against
Enterococcus sp. compared to 9a. The glycosylated tetramates were either inactive or only weakly
active (MIC= 250 µg/mL) against Gram-(-) strains tested, but displayed potent antibacterial activity
against Gram-(+) strains MRSA, Enterococcus and Streptococcus pneumoniae. Contrary to the trend
in antibacterial activity observed for the tetramate esters 8a-g,12 tetramate ester 16 proved to be more
active than its adamantyl analogue 18. Increasing polarity at C-2 as in 17 conferred no bioactivity.
Introduction of a disaccharide analogue also led to the complete loss of antibacterial activity of
tetramate ester 21. Further, it was interesting to note that the aglycone analogue 26a was slightly
more potent than the glycosylated tetramate 16. In the case of glycosyl tetramate 23, in which an
acylated galactose is linked to the tetramate via a benzyl ether, improved antibacterial activity was
observed compared to 16. Both 23 and its aglycone analogue 26b displayed similar level of
antibacterial activity. Thus, while the presence of a glycone does not lead to a significant compromise
in bioactivity, it seems that a significant increase in polarity at C-2 leads to a detrimental reduction in
the antibacterial activity (e.g. 17). The exact role of the glycosidic residue is not known, but it may
assist in improving polarity, bioavailability, modulating toxicity and bacterial target interaction.49
Conversion of the tetramate ester 16 to its carboxamide analogues 18 led to a loss in activity.
However, the absence of the glycone moiety in these carboxamide tetramates restored antibacterial
activity as observed for 28a, indicating that the presence of a glycone is not favourable for
antibacterial activity of carboxamide tetramates. This outcome for amides is in contrast to that
observed for tetramate esters.
Etherification of 28a to 29a resulted in a 64-fold reduction in antibacterial activity. Ethyl ester
hydrolysis of 29a to give carboxylic acid 29b resulted in a further 8-fold reduction in potency (MIC of
29b against MRSA was 250 µg/mL). However, etherification of 28a with 1-bromo-2-butyne afforded
30 without much compromise in antibacterial activity. It was also interesting to note that replacement
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of the adamantyl side chain with either phenyl (28b) or tetrahydropyranyl (28c) groups led to a
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reduction in potency, with no antibacterial activity being observed for the latter. An evaluation of
DOI: 10.1039/C9OB01076A
adamantyl carboxamide tetramates with aliphatic heterocycles 34b, 37a and 37b appended at C-2
showed good antibacterial activity when compared to the corresponding tetramate esters. This
observation is in agreement with the general trend observed for tetramate esters and their carboxamide
analogues, highlighting the need for a bulky, hydrophobic C-7 pendant group. In a separate
comparison of C-7 adamantyl carboxamides bearing morpholine, pyroglutamate and β-lactam
moieties at C-2, morpholine substituted tetramate 37a was the most active while pyroglutamate
substituted tetramate 34b was the least potent.
In the absence of a C-2 substituent, antibacterial activity of tetramate 38 against Gram-positive
S. aureus is significantly reduced, and to obtain a similar zone of inhibition to that of 9a, a 1000-fold
increase in concentration of 38 was necessary. However, upon deprotection, activity against Gram-
negative E. coli could be observed; thus, at a concentration of 4 mg/mL, 38 showed activity, while 9a
was completely inactive. It is noteworthy that deprotected tetramate 38 does not exhibit significant
binding to albumin as its antibacterial activity is retained when treated with HSA. This improvement
in activity observed for E. coli could be due at least in part to its increased polarity, which in turn
leads to increased cell membrane permeability.
O'Shea and Moser have analysed the major antibacterial classes of compounds and have
shown that mean values for physicochemical properties define distinct classes;80 antibacterial efficacy
does not, however, necessarily correspond to oral bioavailability codified in the Lipinski Rule of 5,81
primarily as a result of bacterial cell wall penetration.82, 83 A correlation of calculated physicochemical
properties with the antibacterial activity of some known tetramates was undertaken (using Marvin
(16.4.18.0), 2016, ChemAxon (http://www.chemaxon.com)) (Table 11, SI), and their physicochemical
properties show general resemblance to fluoroquinolones, with relative PSA values of 13-25% and a
MW range of 300-600Da. A similar comparison of physicochemical properties (MW, clogD7.4 and
rel. PSA) and antibacterial activity (where 'activity' was defined as MIC of ≤ 8 µg/mL80 against at
least one bacterial strain) of the tetramate compound library (Figure 8 and Table 11, SI) showed
compounds that are active largely occupy physicochemical space with MW of 300-600, clogD7.4 of -
2.5 to 4 and rel. PSA of 11-22; in this, they also generally resemble fluoroquinolones.80 The 'active'
tetramate esters 16 and 23 with glycone pendants appear as outliers due to their higher MW in the
region of 650-670. Compounds that are more hydrophilic (that is, with lower clogD7.4 and higher
relative PSA) did not exhibit antibacterial activity against Gram-positive strains, even though the
mean MW of known Gram-positive antibacterial agents is around 800.80 However, not all polar
tetramates that fit in to the optimal chemical space of antibacterials with Gram-negative activity
showed the expected activity, although the antibacterial activity of N,S-acetal deprotected tetramate 38
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against E. coli suggests suitable polarity improves Gram-negative activity of tetramates, since
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uptake/penetration through the cell membrane might be better.70 Tetramates with antibacterial activity
DOI: 10.1039/C9OB01076A
possessed desired physicochemical properties required by Lipinsky's Ro5, and therefore have
potential to be developed as oral drug candidates, but these highly hydropbobic compounds are likely
to be penetrative of the blood brain barrier on the basis of calculation of log BB values using a
published algorithm.84 Furthermore, the Fsp3 value (Fsp3 = sp3 hybridized carbon count/ total carbon
count85) of the tetramate scaffold 8 is 0.43; Lovering and co-workers have demonstrated that
complexity as measured by Fsp3 increased for molecules in clinical progression and that drugs have an
average Fsp3 of 0.47.85 This places the tetramate scaffold in an ideal region of chemical space for
development. Moreover, increased complexity appears to reduce toxicity of drug candidates,86 and
this was consistent with our observations: for the antibacterially active tetramates for which
cytotoxicity against HaCaT mammalian cell line was determined, the mean Fsp3 calculated for
tetramates with low toxicity (Fsp3 = 0.41), defined as those that possess ≥10-fold antibacterial activity
(against at least one bacterial strain) over cytotoxicity, was higher than those with higher toxicity
levels (Fsp3 = 0.38).86 No reaction of tetramate 9f with benzylamine and cysteine under ambient
conditions was observed, consistent with the lack of toxicity observed in these systems.
Figure 8. Correlation of physicochemical properties (MW, clogD7.4, and rel. PSA) with antibacterial
activity.
Experimental
General procedure: Esterification of ʟ-serine and ᴅʟ-cysteine.12
To a suspension of the amino acid (1.0 eq) in MeOH (2 mL/mmol) at 0 °C , SOCl2 (1.2 eq) was added
drop-wise under continuous stirring and warmed to rt, then heated at reflux for 1-3 h. The reaction
mixture was concentrated in vacuo to obtain the respective amino ester.
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General procedure: Synthesis of N-acylated thiazolidines 6,7.12
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DOI: 10.1039/C9OB01076A
Step 1: To ʟ-cysteine methyl ester hydrochloride (1.0 eq) in petrol (25 mL/1 g), Et3N (1.2 eq) and
aldehyde (1.2 eq) were added. The mixture was heated at reflux, with continuous removal of water
using a Dean-Stark apparatus, for 19 h. It was then filtered and washed with Et2O. The combined
filtrates were concentrated in vacuo and residue was purified by silica gel flash column
chromatography (eluent: EtOAc/petrol) to give the required thiazolidine.
Step 2: A solution of ethyl hydrogen malonate (1.2 eq) in CH2Cl2 (2.5 mL/mmol) was added to a
stirred solution of thiazolidine (1.0 eq), DCC (1.2 eq) and DMAP (0.1 eq) in CH2Cl2 (5 mL/mmol) at
°
0°C. The mixture was stirred at 0 C for 15 min and then at rt for 15 h. The reaction mixture was
filtered to remove dicyclohexylurea and the residue was washed with CH2Cl2. The combined filtrates
were concentrated in vacuo and purified by silica gel flash column chromatography (eluent:
EtOAc/petrol) to give N-acylated thiazolidines 6,7.
General procedure: Synthesis of bicyclic tetramates 8.12
KOtBu (1.2 eq) was added to a solution of the N-acylated thiazolidine in THF and heated at reflux for
3 h. It was then cooled to rt, concentrated in vacuo and partitioned between Et2O and water. The
aqueous layer was extracted and acidified with 2M HCl (to pH 1) and extracted with EtOAc. The
combined EtOAc extracts were washed with brine, dried over Na2SO4, filtered and concentrated in
vacuo. The residue was purified by flash column chromatography (with 1% Et3N) to give the desired
product. The product was then dissolved in CH2Cl2 and washed with 5% citric acid. The organic
fractions were dried over Na2SO4, filtered and concentrated in vacuo to yield the desired bicyclic
tetramates 8a-g.
General procedure: Synthesis of carboxamide tetramates 9a-g’
To tetramic acid (1.0 eq) dissolved in THF/toluene or DMSO/toluene (1:9, 10 mL/mmol), amine (1.5
eq) was added. The solution was heated at reflux for 16 h, cooled to rt and concentrated in vacuo. The
residue was purified by silica gel flash column chromatography (eluent: EtOAc/MeOH/1 % Et3N).
The product was dissolved in CH2Cl2 and washed with 5% citric acid. The organic fractions were
dried over Na2SO4, filtered and concentrated in vacuo to yield the bicyclic carboxamide tetramate.
Conclusion
Thiazolidine derived tetramate esters were readily elaborated to carboxamido tetramates by
direct uncatalyzed ester to amide exchange, and conditions were identified which permit skeletal
elaboration by Suzuki-Miyaura coupling despite the presence of a cyclic thioether function. While
tetramate esters were inactive against both Gram-positive and Gram-negative bacterial strains, the
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presence of a carboxamide pendant group at C-7 of the tetramate core conferred antibacterial activity
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against Gram-positive strains; however, the introduction of more polar substituents led to reduced or
DOI: 10.1039/C9OB01076A
no activity as observed for 17 and 29b. The antibacterial activity spectrum observed for various C-2
functionalized tetramates demonstrated the ability to accommodate a range of substituents while
retaining antibacterial activity, providing scope for physicochemical property optimization, and an
analysis of physicochemical properties indicates that bioactive tetramates generally occupy
physicochemical space with MW of 300-600, clogD7.4 of -2.5 to 4 and rel. PSA of 11-22%. With
regards to cytotoxicity, compounds with low toxicity had a higher Fsp3 value compared to those with
high toxicity. Evaluation of antibacterial activity of tetramates in the presence of HSA suggests
possible binding of tetramates to albumin may occur. Although the major tautomeric pair of these
species has been identified in solution, it should not be assumed that this is also the binding tautomer
at its enzyme target site, since a number of tautomeric forms may be in free equilibrium.
More generally, the identification of novel, non-planar, synthetically accessible heterocyclic
systems has become of interest in drug discovery.85, 87, 88 The work described here shows that highly
functionalised heterocyclic skeletons with several points of diversity, modelled on bioactive natural
products, are available in a short synthetic sequence, and that further functional group elaboration may
give structures which exhibit similar bioactivity profiles to that of the parent natural product, in this
case antibacterial activity. Importantly, these compounds provide well-defined 3D templates which
minimise aromatic ring count,88 but maintain acceptable starting values of MW, PSA, numbers of
rotatable bonds, H-bond acceptors and H-bond donors, while leaving ample scope for lead
optimisation in the drug discovery process. They therefore offer suitable skeletons for application in
fragment-based drug design89, 90 which allow escape “from flatland”.85
Conflicts of interest
There are no conflicts of interest to declare.
Acknowledgements
This work was supported by MRC Confidence in Concept grant to M.G.M. and National Institutes of
Health Grants GM041376 and AI109713-1 to R.H.E.
References
1.
2.
3.
4.
A. Stefanucci, E. Novellino, R. Costante and A. Mollica, Heterocycles, 2014, 89, 1801 - 1825.
R. Schobert, Naturwissenschaften, 2007, 94, 1-11.
B. J. L. Royles, Chem. Rev., 1995, 95, 1981-2001.
H. G. Henning and A. Gelbin, in Adv. Heterocycl. Chem., ed. A. R. Katritzky, 1993, 57, 139-
185.
5.
6.
X. H. Mo, Q. L. Li and J. H. Ju, RSC Advances, 2014, 4, 50566-50593.
R. Schobert and A. Schlenk, Bioorg. Med. Chem., 2008, 16, 4203–4221.
24

Page 25 of 27
Organic & Biomolecular Chemistry
7.
Y.-C. Jeong, M. Anwar, M. G. Moloney, Z. Bikadi and E. Hazai, Chem. Sci., 2013, 4, 1008-
View Article Online
DOI: 10.1039/C9OB01076A
1015.
8.
M. Anwar and M. G. Moloney, Tetrahedron Lett., 2007, 48, 7259–7262.
9.
10.
11.
L. Josa-Cullere, M. G. Moloney and A. L. Thompson, Synlett, 2016, 27, 1677-1681.
D. Matiadis, Catalysts, 2019, 9.
Y.-C. Jeong, M. Anwar, T. M. Nguyen, B. S. W. Tan, C. L. L. Chai and M. G. Moloney, Org.
Biomol. Chem., 2011, 9, 6663-6669.
12.
T. D. Panduwawala, S. Iqbal, R. Tirfoin and M. G. Moloney, Org. Biomol. Chem., 2016, 14,
4464-4478.
13.
14.
15.
16.
17.
M. Petermichl and R. Schobert, Synlett, 2017, 28, 654-663.
M. Zaghouani and B. Nay, Natural Product Reports, 2016, 33, 540-548.
Y.-C. Jeong and M. G. Moloney, J. Org. Chem., 2011, 76, 1342-1354.
Y.-C. Jeong and M. G. Moloney, Beilstein J. Org. Chem., 2013, 9, 1899-1906.
Low temperature,91 single crystal X-ray diffraction data were collected on a representative
crystal of 9f using a (Rigaku) Oxford Diffraction Supernova A diffractometer (λ = 1.54180 Å).
Raw frame data were reduced using CrysAlisPro and solved using Superflip92 prior to
refinement with CRYSTALS.93 Full details can be found in the ESI (CIF); Crystallographic
data have also been deposited with the Cambridge Crystallographic Data Centre (CCDC
1904326) and copies of these data can be obtained free of charge via
http://www.ccdc.cam.ac.uk/data_request/cif.
18.
19.
20.
M. J. Nolte, P. S. Steyn and P. L. Wessels, J. Chem. Soc., Perkin Trans. 1, 1980, 1057 - 1065.
M. D. Coleman, Br. J. Dermatol., 1993, 129, 507–513.
J. Phillips, M. Goetz, S. Smith, D. Zink, J. Polishook, R. Onishi, S. S, J. Wiltsie, J. Allocco, J.
Sigmund, K. Dorso, S. Lee, S. Skwish, M. de la Cruz, J. Martín, F. Vicente, O. Genilloud, J.
Lu, R. Painter, K. Young, K. Overbye, R. Donald and S. Singh, Chem Biol., 2011, 18, 955-
965.
21.
R. Sawa, Y. Takahashi, H. Hashizume, K. Sasaki, Y. Ishizaki, M. Umekita, M. Hatano, H.
Abe, T. Watanabe, N. Kinoshita, Y. Homma, C. Hayashi, K. Inoue, S. Ohba, T. Masuda, M.
Arakawa, Y. Kobayashi, M. Hamada, M. Igarashi, H. Adachi, Y. Nishimura and Y. Akamatsu,
Chem. - Eur. J., 2012, 18, 15772-15781.
22.
23.
C. D. Spicer and B. G. Davis, Chem. Commun. , 2013, 49, 2747–2749.
E. Smits, J. B. F. N. Engberts, R. M. Kellogg and H. A. van Doren, J. Chem. Soc., Perkin
Trans. 1 1996, 2873–2877.
24.
25.
26.
27.
G. Athanasellis, O. Igglessi-Markopoulou and J. Markopoulos, Bioinorg. Chem. Appl., 2010,
315056.
P. T. Cherian, A. Deshpande, M. N. Cheramie, D. F. Bruhn, J. G. Hurdle and R. E. Lee, J.
Antibiot., 2017, 70, 65–72.
Z. Shang, L. Li, B. P. Espósito, A. A. Salim, Z. G. Khalil, M. Quezada, P. V. Bernhardt and R.
J. Capon, Org. Biomol. Chem., 2015, 13, 7795-7802.
B. Biersack, R. Diestel, C. Jagusch, F. Sasse and R. Schobert, J. Inorg. Biochem., 2009, 103,
72-76.
28.
29.
30.
P.-K. Chang, K. C. Ehrlich and I. Fujii, Toxins, 2009, 1, 74-99.
P. S. Steyn and C. J. Rabie, Phytochemistry, 1976, 15, 1977-1979.
P. Dandawate, S. Padhye, R. Schobert and B. Biersack, Expert Opin. Drug Discovery, 2019,
14, 563-576.
31.
32.
33.
34.
E. Gavrielatos, G. Athanasellis, B. T. Heaton, A. Steiner, J. F. Bickley, O. Igglessi-
Markopoulou and J. Markopoulos, Inorg. Chim. Acta, 2003, 351, 21-26.
E. Gavrielatos, C. Mitsos, G. Athanasellis, B. T. Heaton, A. Steiner, J. F. Bickley, O. Igglessi-
Markopoulou and J. Markopoulos, J. Chem. Soc., Dalton Trans., 2001, 639–644.
B. T. Heaton, C. Jacob, J. Markopoulos, O. Markopoulou, J. Nahring, C.-K. Skylaris and A. K.
Smith, J. Chem. Soc., Dalton Trans., 1996, 1701-1706.
O. Tietze, G. Reck, B. Schulz and A. Zschunke, J. Prakt. Chem./Chem.-Ztg., 1996, 338, 642-
646.
25

Organic & Biomolecular Chemistry
Page 26 of 27
35.
36.
37.
J. V. Barkley, J. Markopoulos and O. Markopoulou, J. Chem. Soc., Perkin Trans. 2, 1994,
View Article Online
DOI: 10.1039/C9OB01076A
1271-1275.
M.-H. Lebrun, P. Duvert, F. Gaudemer, A. Gaudemer, C. Deballon and P. Boucly, J. Inorg.
Biochem., 1985, 24, 167–181.
L. Josa-Culleré, C. Towers, F. Willenbrock, A. L. Thompson and M. G. Moloney, Eur. J. Org.
Chem., 2018, 7055-7059.
38.
39.
A. Suzuki, Angew. Chem. Int. Ed. Engl., 2011, 50, 6722–6737.
M. Fairhead, D. Shen, L. K. M. Chan, E. D. Lowe, T. J. Donohoe and M. Howarth, Bioorg.
Med. Chem., 2014, 22, 5476–5486.
40.
41.
R. Martin and S. L. Buchwald, Acc. Chem. Res., 2008, 41, 1461–1473.
K. C. K. Swamy, N. N. B. Kumar, E. Balaraman and K. V. P. P. Kumar, Chem. Rev., 2009,
109, 2551–2651.
42.
43.
Y. Iwai, K. M. Gligorich and M. S. Sigman, Angew. Chem. Int. Ed. Engl., 2008, 47, 3219–
3222.
M. Figueroa, H. Raja, J. O. Falkinham, A. F. Adcock, D. J. Kroll, M. C. Wani, C. J. Pearce
and N. H. Oberlies, J. Nat. Prod. , 2013, 76, 1007–1015.
44.
45.
S. B. Singh, Bioorg. Med. Chem., 2016, 24, 6291–6297.
R. Sawa, Y. Takahashi, H. Hashizume, K. Sasaki, Y. Ishizaki, M. Umekita, M. Hatano, H.
Abe, T. Watanabe, N. Kinoshita, Y. Homma, C. Hayashi, K. Inoue, S. Ohba, T. Masuda, M.
Arakawa, Y. Kobayashi, M. Hamada, M. Igarashi, H. Adachi, Y. Nishimura and Y. Akamatsu,
Chem. - Eur. J., 2012, 18, 15772–15781.
46.
47.
48.
F. Reinscheid and U. M. Reinscheid, J. Mol. Struct., 2017, 1147, 96-102.
M. Petermichl, S. Loscher and R. Schobert, Angew. Chem. Int. Ed., 2016, 55, 10122 –10125.
S. V. Pronin, A. Martinez, K. Kuznedelov, K. Severinov, H. A. Shuman and S. A. Kozmin, J.
Am. Chem. Soc., 2011, 133, 12172-12184.
49.
50.
V. Křen and T. Řezanka, FEMS Microbiol Rev, 2008, 32, 858 - 889.
J.-L. Montero, J.-Y. Winum, A. Leydet, M. Kamal, A. A. Pavia and J.-P. Roque, Carbohydr.
Res., 1997, 297, 175–180.
51.
52.
53.
54.
55.
M. Hongu, K. Saito and K. Tsujihara, Synth. Commun., 1999, 29, 2775–2781.
N. M. Loim and E. S. Kelbyscheva, Russ. Chem. Bull., 2004, 53, 2080–2085.
J. H. Clark, Chem. Rev., 1980, 80, 429–452.
J. Ishihara and S. Hatakeyama, The Chemical Record, 2014, 14, 663–677.
M. G. Moloney, P. C. Trippier, M. Yaqoob and Z. Wang, Curr. Drug Discovery Technol.,
2004, 1, 181-199.
56.
R. E. Schwartz, G. L. Helms, E. A. Bolessa, K. E. Wilson, R. A. Giacobbe, J. S. Tkacz, G. F.
Bills, J. M. Liesch, D. L. Zink, J. E. Curotto, B. Pramanik and J. C. Onishi, Tetrahedron, 1994,
50, 1675-1686.
57.
58.
J. Dyer, S. Keeling, A. King and M. G. Moloney, J. Chem. Soc., Perkin Trans. 1, 2000, 2793-
2804.
J. H. Bailey, D. T. Cherry, K. M. Crapnell, M. G. Moloney, S. B. Shim, M. Bamford and R. B.
Lamont, Tetrahedron, 1997, 53, 11731-11744.
59.
60.
R. B. Silverman and M. A. Levy, J. Org. Chem., 1980, 45, 815-818.
M. J. Beard, J. H. Bailey, D. T. Cherry, M. G. Moloney, S. B. Shim, K. Statham, M. Bamford
and R. B. Lamont, Tetrahedron, 1996, 52, 3719-3740 and corrigenda 1997, 3753, 1177.
J. K. Thottathil, J. M. Moniot, R. H. Mueller, M. K. Y. Wong and T. P. Kissick, J. Org.
Chem., 1986, 51, 3140-3143.
61.
62.
A. R. Cowley, T. J. Hill, P. Kocis, M. G. Moloney, R. D. Stevenson and A. L. Thompson,
Org. Biomol. Chem., 2011, 9, 7042–7056.
63.
64.
65.
66.
T. M. Postma and F. Albericio, Org. Lett. , 2014, 16, 1772–1775.
G. Pattenden, S. M. Thom and M. F. Jones, Tetrahedron, 1993, 49, 2131–2138.
T. Onoda, R. Shirai, Y. Koiso and S. Iwasaki, Tetrahedron, 1996, 52, 14543–14562.
E. J. Corey and G. A. Reichard, J. Am. Chem. Soc., 1992, 114, 10677-10678.
26

Page 27 of 27
Organic & Biomolecular Chemistry
67.
68.
S. B. Singh, M. A. Goetz, S. K. Smith, D. L. Zink, J. Polishook, R. Onishi, S. Salowe, J.
View Article Online
Wiltsie, J. Allocco, J. Sigmund, K. Dorso, M. d. l. Cruz, J. Martín, F. Vicente, O. Genilloud,
DOI: 10.1039/C9OB01076A
R. G. K. Donald and J. W. Phillips, Biorg. Med. Chem. Lett., 2012, 22, 7127–7130.
S. Tuske, S. G. Sarafianos, X. Wang, B. Hudson, E. Sineva, J. Mukhopadhyay, J. J. Birktoft,
O. Leroy, S. Ismail, A. D. Clark, C. Dharia, A. Napoli, O. Laptenko, J. Lee, S. Borukhov, R.
H. Ebright and E. Arnold, Cell, 2005, 122, 541–552.
69.
70.
F. Reusser, Antimicrob Agents Chemother., 1976, 10, 618-622.
S. Alt, L. A. Mitchenall, A. Maxwell and L. Heide, Antimicrob Agents Chemother., 2011, 66,
2061-2069.
71.
72.
73.
A. Aubry, X.-S. Pan, L. M. Fisher, V. Jarlier and E. Cambau, Antimicrob Agents Chemother.,
2004, 48, 1281-1288.
J. Mukhopadhyay, K. Das, S. Ismail, D. Koppstein, M. Jang, B. Hudson, S. Sarafianos, S.
Tuske, J. Patel, R. Jansen, H. Irschik, E. Arnold and R. H. Ebright, Cell, 2008, 135, 295-307.
S. I. Maffioli, Y. Zhang, D. Degen, T. Carzaniga, G. Del Gatto, S. Serina, P. Monciardini, C.
Mazzetti, P. Guglierame, G. Candiani, A. I. Chiriac, G. Facchetti, P. Kaltofen, H.-G. Sahl, G.
Dehò, S. Donadio and R. H. Ebright, Cell, 2017, 169, 1240-1248.e1223.
Y. Zhang, D. Degen, M. X. Ho, E. Sineva, K. Y. Ebright, Y. W. Ebright, V. Mekler, H.
Vahedian-Movahed, Y. Feng, R. Yin, S. Tuske, H. Irschik, R. Jansen, S. Maffioli, S. Donadio,
E. Arnold and R. H. Ebright, eLife, 2014, 3, e02450-e02450.
74.
75.
Liu C-Y. 2007. "The use of single-molecule nanomanipulation to study transcription kinetics",
Ph.D. thesis, Rutgers University.
76.
77.
78.
79.
80.
81.
J. E. Baldwin, A. J. Pratt and M. G. Moloney, Tetrahedron, 1987, 43, 2565-2575.
K. R. Grigoryan, J. Phys. Chem. A, 2009, 83, 2368-2370.
T. Arakawa, Y. Kita and S. N. Timasheff, Biophys. Chem., 2007, 131, 62-70.
A. J. O’Neill and I. Chopra, Expert Opin. Drug Discov., 2004, 13, 1045-1063.
R. O’Shea and H. E. Moser, J. Med. Chem., 2008, 51, 2871-2878.
C. A. Lipinski, F. Lombardo, B. W. Dominy and P. J. Feeney, Adv. Drug Delivery Rev., 2001,
46, 3-26.
82.
83.
84.
G. Mugumbate and J. P. Overington, Bioorg. Med. Chem., 2015, 23, 5218–5224.
M. P. Gleeson, J. Med. Chem., 2008, 51, 817–834.
Y. Fan, R. Unwalla, R. A. Denny, L. Di, E. H. Kerns, D. J. Diller and C. Humblet, J. Chem.
Inf. Model., 2010, 50, 1123-1133.
85.
86.
87.
88.
89.
90.
91.
92.
93.
F. Lovering, J. Bikker and C. Humblet, J. Med. Chem., 2009, 52, 6752–6756.
F. Lovering, MedChemComm, 2013, 4, 515-519.
W. R. Pitt, D. M. Parry, B. G. Perry and C. R. Groom, J. Med. Chem., 2009, 52, 2952–2963.
T. J. Ritchie and S. J. F. MacDonald, Drug Discov. Today, 2009, 14, 1011-1020.
M. Congreve, G. Chessari, D. Tisi and A. J. Woodhead, J. Med. Chem., 2008, 51, 3661-3680.
P. J. Hajduk, J. Med. Chem., 2006, 49, 6972-6976.
J. Cosier and A. M. Glazer, J. Appl. Cryst., 1986, 19, 105-107.
L. Palatinus and G. Chapuis, J. Appl. Cryst. , 2007, 40, 786–790.
P. W. Betteridge, J. R. Carruthers, R. I. Cooper, K. Prout and D. J. Watkin, J. Appl. Cryst.,
2003, 36, 1487-1487.
27