Targeting of Fumarate Hydratase from Mycobacterium tuberculosis Using Allosteric Inhibitors with a Dimeric-Binding Mode

Article abstract of DOI:10.1021/acs.jmedchem.9b01203

With the growing worldwide prevalence of antibiotic-resistant strains of tuberculosis (TB), new targets are urgently required for the development of treatments with novel modes of action. Fumarate hydratase (fumarase), a vulnerable component of the citric acid cycle in Mycobacterium tuberculosis (Mtb), is a metabolic target that could satisfy this unmet demand. A key challenge in the targeting of Mtb fumarase is its similarity to the human homolog, which shares an identical active site. A potential solution to this selectivity problem was previously found in a high-throughput screening hit that binds in a nonconserved allosteric site. In this work, a structure-activity relationship study was carried out with the determination of further structural biology on the lead series, affording derivatives with sub-micromolar inhibition. Further, the screening of this series against Mtb in vitro identified compounds with potent minimum inhibitory concentrations.

Full text of DOI:10.1021/acs.jmedchem.9b01203

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Article
Targeting of fumarate hydratase from Mycobacterium tuberculosis
using allosteric inhibitors with a dimeric-binding mode
Andrew J Whitehouse, M. Daben J. Libardo, Monica Kasbekar, Paul D Brear, Gerhard
Fischer, Craig J Thomas, Cliff Barry, Helena I. M. Boshoff, Anthony G Coyne, and Chris Abell
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.9b01203 • Publication Date (Web): 13 Sep 2019
Downloaded from pubs.acs.org on September 14, 2019
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Targeting of fumarate hydratase from
Mycobacterium tuberculosis using allosteric
inhibitors with a dimeric-binding mode
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†
‡
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‖
Andrew J. Whitehouse, M. Daben J. Libardo, Monica Kasbekar, Paul D. Brear, Gerhard
‖,⊥
§
‡
‡
,†
Fischer, Craig J. Thomas, Clifton E. Barry III, Helena I. M. Boshoff, Anthony G. Coyne,*
and Chris Abell*,†
†Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge, CB2 1EW,
UK.
‡Tuberculosis Research Section, Laboratory of Clinical Immunology and Microbiology, National
Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892,
USA.
§National Center for Advancing Translational Sciences, National Institutes of Health, Bethesda,
MD 20850, USA.
‖Department of Biochemistry, University of Cambridge, Tennis Court Road, Cambridge, CB2
1
GA, UK.
⊥Current address: Boehringer Ingelheim RCV, Dr. Boehringer-Gasse 5-11, 1121 Vienna,
Austria.
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KEYWORDS
Mycobacterium tuberculosis; Mtb; tricarboxylic acid cycle; citric acid cycle; Krebs cycle;
fumarate hydratase; fumarase; fumarate; L-malate; high-throughput screening; allosteric;
deconstruction-reconstruction; defragmentation; differential scanning fluorimetry; DSF.
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ABSTRACT
With the growing worldwide prevalence of antibiotic-resistant strains of tuberculosis (TB), new
targets are urgently required for the development of treatments with novel modes of action.
Fumarate hydratase (fumarase), a vulnerable component of the citric acid cycle in
Mycobacterium tuberculosis (Mtb), is a metabolic target that could satisfy this unmet demand. A
key challenge in the targeting of Mtb fumarase is its similarity to the human homolog, which
shares an identical active site. A potential solution to this selectivity problem was previously
found in a high-throughput screening hit that binds in a non-conserved allosteric site. In this
work, a structure-activity relationship study was carried out with the determination of further
structural biology on the lead series, affording derivatives with sub-micromolar inhibition.
Further, the screening of this series against Mtb in vitro identified compounds with potent
minimum inhibitory concentrations (MIC).
INTRODUCTION
Mycobacterium tuberculosis (Mtb), the causative agent of the disease tuberculosis (TB),
infects 10 million people per year and is responsible for the deaths of 1.6 million annually, of
1
which 300,000 are HIV-positive. Further, a quarter of the global population, 1.7 billion
individuals, are thought to be infected with asymptomatic latent TB and hence at risk of active
2
infection. Despite the scope of the disease, treatment remains intensive and long-term with
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multi- and extensively-drug resistant TB strains a growing problem, hence there is a continuing
urgent need for novel improved anti-TB therapeutics.3
4
Mtb fumarate hydratase (fumarase) is a component of the citric acid cycle in Mtb, where it
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catalyzes the reversible interconversion of fumarate and L-malate. When normoxic cells are
rapidly dividing fumarase operates in the forward direction (fumarate to L-malate) and is
involved in energy generation and the synthesis of precursors for biosynthetic pathways. Under
conditions of lowered oxygen availability fumarase operates in the reverse direction allowing
fermentative production and secretion of succinate.5,6 During pathogenesis of tuberculosis anoxia
develops in the center of caseous lesions prior to the development of cavitary lesions, where
sudden reaeration allows rapid bacterial growth to promote bacterial transmission from host-to-
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host. Mtb fumarase is a non-redundant and therefore vulnerable component of the citric acid
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cycle, a situation that is not guaranteed in bacteria. The regulation of fumarase is therefore
central to the realignment of bacterial metabolism that occurs during normal disease progression
in Mtb, which makes it a very high value target for drugs that might allow faster bacterial
clearance. Mtb fumarase has been shown to be essential for Mtb survival with depletion linked to
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impaired growth due to accumulation of fumarate, which can react with cysteine thiols. This
modification adversely impacts the antioxidants catalase and mycothiol, inducing
hypersensitivity to oxidative stress and ultimately Mtb death both in vitro and in vivo.
Mtb fumarase is a 210 kDa class II fumarase,11 characterized by thermostability, iron-
independence and a homotetrameric structure.9,12 Each subunit is defined by an N-terminal
domain (residues 1-137), a central domain (residues 138-393) whose α-helices pack with those
from other subunits to form a central 20-helix bundle that holds together the quaternary structure,
and a conformationally flexible C-terminal domain (residues 394-474). The protein possesses
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four symmetry-related active sites, each located in clefts formed by three subunits and covered
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by an ‘SS’ loop (residues 316-325) that can switch between ‘open’ and ‘closed’ states. The
selective targeting of Mtb fumarase is challenging due to its 53% overall sequence identity with
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the human homolog, which was recently the focus of a study on cancer cell lines.13 In our
previous work, the conservation of active site residues between Mtb and human fumarase was
circumvented through the discovery by high-throughput screening (HTS) of an allosteric
inhibitor 1 of Mtb fumarase (IC50 2.5 ± 1.1 µM) (Figure 1a).14
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Figure 1. a) Allosteric inhibitor 1 of Mtb fumarase. b) X-ray crystal structure of Mtb fumarase
in complex with 1 (PDB code 5F91, subunit A = white, subunit B = green, subunit C = cyan,
subunit D = yellow),14 illustrating relative locations of the allosteric and active sites in the
homotetramer. c) The interactions (yellow dashed lines) of 1 (lilac) in the allosteric site.
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X-ray crystallography showed that inhibitor 1 binds dimerically to Mtb fumarase at the
interface between two adjacent C-terminal domains in an induced pocket (Figure 1b), locking the
nearest active site in an ‘open’ conformation, with the bound molecules interacting with each
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other through π-stacking of their central phenyl rings (Figures 1c and S8a).14 A second
symmetry-related allosteric site exists in the homotetramer however this is unoccupied in the X-
ray crystal structure (Figure 1b), likely due to its participation in a crystal contact. Whilst 1 did
not exhibit bactericidal properties it was able to exert inhibition of Mtb growth in vitro (65%
inhibition at 250 µM) that, in combination with the demonstrated essentiality of Mtb
fumarase,8,10 warranted additional investigation into inhibitor 1. Due to the dimeric-binding
mode of 1, a possible strategy would be to link the molecules together into one inhibitor,
however the geometry of the π-stacking interaction makes this route challenging. One way of
circumventing this problem could be the use of a deconstruction-reconstruction approach,15
where the HTS hit is defragmented into fragment-like molecules allowing the identification of
key binding motifs. This would allow the use of fragment-based methods to more effectively
sample the ligand-binding site and develop a sub-micromolar inhibitor with activity against Mtb
in vitro.16
RESULTS AND DISCUSSION
Defragmentation of HTS hit 1. The defragmentation of HTS hit 1 into ‘fragment-like’
molecules resulted in the assembly of a focused library (Table 1), with members spanning a
range of molecular weights (204 to 326 Da) and incorporating various binding motifs of 1. The
screening of the library with a biochemical assay afforded no measurable inhibition at
concentrations of 100 µM or 1 mM (data not shown). Similarly, the use of differential scanning
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fluorimetry (DSF) showed either negligible change in the melting temperature (ΔT +0.1 °C for
m
1
0) or negative thermal shifts (ΔT
contrast to the thermal shift of +3.9 °C afforded by 1 at 0.625 mM. This includes the higher
molecular weight compounds in the library 4 (ΔT -0.4 °C) and 6 (ΔT -0.6 °C), which retain
m
-1.6 °C for 7) at a ligand concentration of 5 mM (Table 1), in
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the substituted phenyl ring of 1 in addition to either of the sulfonamide or phthalazinone motifs.
These results suggest that the inhibitory capability of 1 for Mtb fumarase is dependent upon all
its binding motifs, with high sensitivity to truncation. The negative impact of defragmentation is
not surprising due to the large number of interactions that doubly-binding 1 makes in the
allosteric site with the two C-terminal domains of Mtb fumarase (in addition to itself through the
π-stacking interaction of its phenyl ring), as well as the induced nature of the site with cryptic
pockets not easily targeted by weak fragments.
Table 1. The change in the melting temperatures (T ) of compounds 2-11.
m
ΔT b
ΔT b
(°C)
Compound
2
m
(°C)
Compound
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-0.6
-1.6
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.625 mM ligand and 2.5 μM Mtb fumarase. 5 mM ligand and 2.5 μM Mtb fumarase.
Structure-activity Relationship Study of HTS hit 1. Due to lack of success in
defragmentation of the HTS hit 1, attention was shifted to a structure-activity relationship study.
Initial modification of the central phenyl ring of 1 through the removal of its methoxy group,
which indirectly engages Leu303, Gly305, Leu306 and His397 through hydrogen bonds with two
ordered water molecules (Figures 2 and S8b), was not tolerated in 12 (16% inhibition at 50 µM)
(Table 2). A similar result was seen upon replacement of the methoxy group with a methyl group
in 13 (<10% inhibition at 50 µM), illustrating the sensitivity of the central phenyl ring of 1 to
alteration. The replacement of the sulfonamide group with an amide, and the resultant
rigidification and rotation of the azepanyl ring was also not tolerated in 14 (14% inhibition at 50
µM).
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Figure 2. X-ray crystal structure of Mtb fumarase in complex with 1 (lilac) (PDB code 5F91,
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subunit A = white, subunit B = green, subunit C = cyan, subunit D = yellow), illustrating the
interactions (yellow dashed lines) of the methoxy group of 1 in the allosteric site.
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Table 2. The inhibition at 50 µM ligand concentration afforded by compounds 1 and 12-14.
Inhibition (%)
Compound Structure
at 50 µM
1
1
1
> 90
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< 10
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The replacement of the phthalazinone ring of HTS hit 1 with an indole in 15a (27% inhibition
at 50 µM) resulted in a significant drop in inhibition (Table 3), despite X-ray crystallography
showing the maintenance of the cation-π interaction with Arg432 and a hydrogen-bond with the
backbone carbonyl of Leu429 (Figure 3a). The substitution of the ring in 15b (38% inhibition at
50 µM) or extension of the linker in 15c (12% inhibition at 50 µM) did not result in a significant
improvement on 15a. The use of alternative 5-6 fused rings including benzofuran (15d),
benzisoxazole (15e) and benzimidazole (15f) derivatives failed to provide inhibition in the
biochemical assay at 50 µM. In contrast, the screening of quinoline derivative 15g (IC50 4.1 µM)
resulted in a comparable IC50 value to 1 (IC50 2.0 µM), with the X-ray crystal structure of the
Mtb fumarase-15g complex showing the quinoline ring hydrogen-bonding to a water molecule as
opposed to the protein backbone (Figure 3b). Truncation of the quinoline ring of 15g to a
pyridine in 15i eliminated inhibitory activity at 50 µM, in a similar manner to the
defragmentation of 1.
Figure 3. X-ray crystal structures of Mtb fumarase (subunit A = white, subunit B = green,
subunit C = cyan, subunit D = yellow) in complex with a) 15a (blue) (PDB code 6S7U) and b)
1
5g (pink) (PDB code 6S7W), illustrating the interactions (yellow dashed lines) of the ligands in
the allosteric site.
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Table 3. The inhibition at 50 µM ligand concentration or half-maximal inhibitory concentrations
IC50) afforded by compounds 1 and 15a-i.
(
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8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Inhibition (%)
at 50 µM
Inhibition (%)
at 50 µM
Compound
1
R
Compound
15e
R
> 90a
< 10
1
1
5a
5b
27 ± 6
38 ± 5
15f
< 10
15g
86 ± 2b
15c
15d
12 ± 9
< 10
15h
15i
21 ± 17
< 10
a
IC50 2.0 ± 0.1 µM, consistent with previously reported value.14
IC50 4.1 ± 0.3 µM.
b
The truncation of HTS hit 1 to the N-methyl sulfonamide 16a (IC50 57 µM) afforded the lowest
molecular weight derivative (402 Da) to give a measurable IC50 value (Table 4). An X-ray
crystal structure was obtained of the complex of 16a with Mtb fumarase, illustrating a 5 Å
movement in the Arg400 side chain (at Cε) relative to the complex with 1 to form a salt-bridge
with Glu396, significantly reducing the apparent volume for growth around the sulfonamide
(
Figures 4a and S9b). Heterocyclic derivatives of 1 were also produced to explore the volume
defined by His397, Arg400 and Arg432, beginning with the azocanyl and piperidinyl analogues
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1
6b (IC50 4.0 µM) and 16c (IC50 4.4 µM). These 8- and 6-membered derivatives possessed
slightly attenuated inhibition in comparison to 1 (IC50 2.0 µM), however were not sufficiently
different to preclude testing of alternative heterocycles of similar size. X-ray crystallography
showed no significant difference in the binding mode of 16b in Mtb fumarase in comparison to 1
except for Arg400 located above the larger 8-membered ring, which showed evidence of a
second alternative conformation (Figures 4b and S10a). The N-methyl piperazinyl derivative 16d
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(
IC50 38 µM), along with morpholino and thiomorpholine dioxide analogues 16e (IC50 12 µM)
and 16g (IC50 13 µM) afforded IC50 values weaker than 1 or the thiomorpholino derivative 16f
IC50 4.7 µM).
(
Figure 4. X-ray crystal structure of Mtb fumarase (subunit A = white, subunit B = green, subunit
C = cyan, subunit D = yellow) in complex with a) 16a (light pink) (PDB code 6S7K) and b) 16b
(beige) (PDB code 6S43), illustrating the interactions (yellow dashed lines) of the ligands in the
allosteric site.
a)
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8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
b)
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Table 4. The inhibition at 50 µM ligand concentration or half-maximal inhibitory concentrations
IC50) afforded by compounds 16a-n.
(
Compound
R
IC50 (µM)
Compound
16h
R
IC50 (µM)
1
1
6a
6b
57 ± 3
17 ± 3
4.0 ± 0.1
4.4 ± 0.1
16i
16j
NDa
16c
16d
2.2 ± 0.2
38 ± 2
16k
3.4 ± 0.2
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16e
16f
12 ± 1
16l
0.67 ± 0.03
0.67 ± 0.01
NDb
4.7 ± 0.2
13 ± 1
16m
16n
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
6g
a
5
3 ± 3% inhibition at 50 µM concentration.
4 ± 5% inhibition at 50 µM concentration.
b
4
The replacement of the methyl group of 16a with a phenyl ring in 16h (IC50 17 µM) (Table 4)
partially restored the conformation of the Arg400 side chain, with observation of a cation-π
interaction by X-ray crystallography (Figures S9a and S10b). The addition of more flexibility to
16h in 16i through insertion of a methylene linker had a detrimental impact on inhibition (53%
inhibition at 50 µM). The rigidification of 16i through incorporation of its benzyl group into a
tetrahydroisoquinolyl ring system in 16j (IC50 2.2 µM) was more successful, with comparable
inhibition to 1 (IC50 2.0 µM). X-ray crystallography showed the tetrahydroisoquinolyl ring of 16j
lying between Arg400 and Arg432, with the side chain of Arg400 maintaining the conformation
exhibited in the Mtb fumarase-16h structure through a cation-π interaction with the phenyl
portion of the ring (Figure 5a). Ring expansion of 16j to improve the interaction with Arg432 in
16l (IC50 0.67 µM) resulted in a sub-micromolar IC50 value, 3-fold lower than that of 1 (IC50 2.0
µM) (Figure 5b). Addition of a methoxy group in 16m (IC50 0.67 µM) maintained the sub-
micromolar inhibitory profile, however further modification of 16l through addition of a
methylene bridge to the tetrahydrobenzoazepanyl ring in 16n was not tolerated (44% inhibition
at 50 µM), revealing the limits of occupancy of this pocket in the allosteric site.
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Figure 5. X-ray crystal structure of Mtb fumarase (subunit A = white, subunit B = green, subunit
C = cyan, subunit D = yellow) in complex with a) 16j (light green) (PDB code 6S7Z) and b) 16l
(light blue) (PDB code 6S88), illustrating the interactions (yellow dashed lines) of the ligands in
the allosteric site.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
a)
b)
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Screening against H37Rv Mycobacterium tuberculosis. As previously reported,14 no
bactericidal activity was observed for HTS hit 1 (Table 5). This was also found with derivatives
15g-i, 16c-h and 16j-m (data not shown). However, activity was observed with 15a-d and 16b
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(
Table 5). The balance of metabolic intermediates in Mtb can be programmed by feeding the
bacterium various carbon sources. As such, Mtb growing under glucose as the sole carbon source
relies on glycolysis while bacteria that grow on dipalmitoyl phosphatidylcholine (DPPC) will
rely heavily on β-oxidation to supply intermediates for the central carbon metabolism. Both
glucose and DPPC are carbon sources predicted to be relevant during the in vivo pathogenesis of
17
Mtb. Because pyruvate from glycolysis will go on to feed several other anabolic pathways, on
balance bacteria get more acetyl-CoA equivalence under growth in DPPC. These acetyl-CoA
feed directly into the citric acid cycle (among other pathways) in which fumarase lies. Therefore,
the flux of intermediates in the citric acid cycle is greater under growth in DPPC and Mtb are
more reliant on the activity of enzymes in this pathway for growth. Hence, it is reassuring that
compounds 15a-d and 16b were highly active in DPPC-containing medium. In particular, the
benzofuran analogue 15d gave the lowest MIC value against Mtb at 6.3 µM in DPPC-containing
medium. Of these active compounds 16b (IC50 4.0 µM, MIC 19 µM in 7H9/DPPC) was the most
potent against purified Mtb fumarase, and all were more lipophilic than 1 (cLogP 1.6) with the
majority possessing a cLogP between 3.6 and 4.1. The poor activity in 7H9 medium
supplemented with ADC was likely due to high protein binding since this medium contains 0.4%
bovine serum albumin. This suggests overcoming protein binding will be an important
component of future optimization.
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2
3
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6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Table 5. The minimum inhibitory concentrations (MIC) against H37Rv Mycobacterium
tuberculosis (Mtb) afforded by compounds 1, 15a-d and 16b.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Compound R1
R2
cLogPa H37Rv Mtb MIC (µM)
GAST- 7H9/ 7H9/
7H9/
ADC glucose DPPC
Fe
1
1.6
3.6
3.6
> 100
> 100 > 100
> 100 100
> 100
9.4
1
1
5a
5b
> 100
> 100
> 100 > 100
12.5
1
1
5c
5d
4.1
4.1
> 100
> 100
> 100 25
> 100 9.4
9.4
6.3
1
6b
2.1
> 100
> 100 25
19
a
Calculated using ChemDraw (PerkinElmer, Waltham MA).
Synthetic Chemistry. The synthesis of 1 was achieved in a convergent manner with the T3P®-
mediated amide coupling of carboxylic acid 3 and aniline 5 (60% yield) (Scheme 1), each
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14
produced as previously reported. Synthesis of aniline 5 involved the trifluoroacetyl-protection
of the amino group of o-anisidine 19 as 20 (99% yield), followed by S Ar of a sulfonyl chloride
E
group by treatment with chlorosulfonic acid (77% yield). Reaction of 21 with
hexamethyleneimine and sodium hydride converted the sulfonyl chloride to a sulfonamide, with
the resultant intermediate heated under reflux in an aqueous mixture of ethanol and HCl to
remove the trifluoroacetyl protecting group, affording 5 (83% yield). Carboxylic acid 3 was
obtained by a route beginning with the Horner-Wadsworth-Emmons reaction of phthalic
anhydride 17 and (carbethoxymethylene)triphenylphosphorane to give 18 (67% yield), followed
by heating under reflux with hydrazine in ethanol to afford the phthalazinone ester 2 (99% yield).
Hydrolysis of 2 produced acid 3 (44% yield), which was also used in the synthesis of 4 through
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
®
T3P -mediated amide coupling with o-anisidine 19 (82% yield) (Scheme 1). The application of
acetic anhydride and pyridine to aniline 5 also allowed the production of 6 (79% yield).
Scheme 1. Synthesis of Compounds 1-6 and 21.
…
……………………………………………………………………………………………………
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6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Reagents and Conditions: (a) (carbethoxymethylene)triphenylphosphorane, CHCl , reflux, 3 h;
3
°
(
b) N
2
H
4
°
·H
2
O, EtOH, 50 C, 2 h; (c) NaOH (10% w/v), THF, reflux, 1 h; (d) TFAA, pyridine,
°
DCM, 0 C to rt, 3 d; (e) HSO
3
Cl, DCM, 0 C to rt, 16 h; (f) (i) hexamethyleneimine, NaH, DMF,
°
®
0 C to rt, 3 h (ii) EtOH, HCl (37.5% w/v), H
2
O, reflux, 20 h; (g) T3P (50 wt. % in DMF),
°
®
°
DIPEA, DMF, 70 C, 1 h; (h) 19, T3P (50 wt. % in DMF), DIPEA, DMF, 40 C, 2 h; (i) Ac O,
2
pyridine, DCM, 90 min.
Fragment 7 was synthesized in three steps, beginning with the treatment of phenol 22 with
chlorosulfonic acid in a similar manner to 20 (78% yield) (Scheme 2). The resultant sulfonyl
chloride 23 was reacted with hexamethyleneimine in the presence of DIPEA to afford
sulfonamide 24 (64% yield), whose nitro group was reduced by a mixture of sodium borohydride
and nickel(II) chloride (62% yield). The production of fragment 8 and the corresponding methyl
analogue 29 involved the functionalization of commercial sulfonyl chlorides 25 and 26 with
hexamethyleneimine and sodium hydride, affording sulfonamides 27 and 28 (42-72% yield)
(
Scheme 2). The nitro groups of 27 and 28 were reduced in the same manner as 24 to the
corresponding anilines 8 and 29 (82-88% yield). Aniline 8 was also acetylated to fragment 9 in
the same manner as 5 (86% yield). Anisole 30 was used to make fragment 10 through stirring
with chlorosulfonic acid, with the resultant sulfonyl chloride taken forwards crude for reaction
with hexamethyleneimine and sodium hydride (49% yield overall) (Scheme 2). The sulfonyl
chloride 21 was converted to N-methyl sulfonamide 31a by heating under reflux with
methylamine in THF (82% yield), with 31a also acetylated in the same manner as 5 to afford
fragment 11 (56% yield).
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Scheme 2. Synthesis of Compounds 7-11, 29 and 31a.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
…
……………………………………………………………………………………………………
…
……………………………………………………………………………………………………
Reagents and Conditions: (a) HSO
DIPEA, DCM, 15 h; (c) NaBH , NiCl
NaH, DMF, 0 C to rt, 1 h to 90 min; (e) NaBH
pyridine, DCM, 5 h; (g) (i) HSO Cl, CHCl , 0 °C to rt, 30 min (ii) hexamethyleneimine, NaH,
DMF, 0 °C to rt, 1 h; (h) (i) methylamine (2 M in THF), THF, reflux, 90 min (ii) EtOH, HCl
(37.5% w/v), H O, reflux, 3 h 30 min; (i) Ac O, pyridine, DCM, 2 d.
3
Cl, CHCl
3
, 0 °C to reflux, 90 min; (b) hexamethyleneimine,
4
2
·6H O, MeOH, 0 °C to rt, 2 h; (d) hexamethyleneimine,
2
°
°
4
, NiCl
2
, MeOH, 0 C to rt, 45 min; (f) Ac O,
2
3
3
2
2
Compounds 12 and 13 were synthesized using anilines 8 and 29 (Scheme 2), which were
®
coupled with carboxylic acid 3 using T3P as with the synthesis of 1 (45-50% yield) (Scheme 3).
®
In contrast, the route for 14 required the initial T3P -mediated amide coupling of carboxylic acid
32 with hexamethyleneimine, affording phenol 33 (24% yield) that was methylated with methyl
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9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
iodide (77% yield) (Scheme 3). The nitro group of 34 was reduced in the same manner as 27 and
2
8 (64% yield), with the resultant aniline 35 coupled with acid 3 to afford 14 (45% yield).
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Scheme 3. Synthesis of Compounds 12-14.
…
……………………………………………………………………………………………………
®
°
Reagents and Conditions: (a) T3P (50 wt. % in EtOAc), DIPEA, DMF, 70 C, 2 h; (b)
®
hexamethyleneimine, T3P (50% in DMF), DIPEA, DMF, 1 d; (c) Me
2
SO
4
, K
2
CO , acetone,
3
°
®
reflux, 2 h; (d) NaBH
DIPEA, DMF, 70 C, 2 h.
4
, NiCl
2
·6H
2
O, MeOH, 0 C to rt, 90 min; (e) 3, T3P (50 wt. % in EtOAc),
°
®
Compounds 15a-f and 15h were synthesized in one step from aniline 5 via T3P -mediated
amide coupling with commercially available carboxylic acids (21-67% yield), whilst 15i required
alternative amide-coupling conditions with the use of EDC (64% yield) (Scheme 4). In the
production of 15g, 4-methylquinoline 36 was converted using LDA and carbon dioxide to 4-
quinolylacetic acid, which was taken forwards crude for reaction with aniline 5 (5% yield
®
overall). The synthesis of compounds 16a-n involved the T3P -mediated amide coupling of
anilines 31a-n with carboxylic acid 3 as with the synthesis of 1 (10-43% yield) (Scheme 4).
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Anilines 31b-f and 31h-l were produced by the reaction of sulfonyl chloride 21 with
commercially available amines in the presence of sodium hydride, followed by trifluoroacetyl
deprotection via heating under reflux with ethanol and HCl as in 5 (37-81% yield). In
comparison, whilst the trifluoroacetyl deprotection step in the synthesis of anilines 31g and 31m-
n was the same as 31b-f and 31h-l, the prior reaction of sulfonyl chloride 21 with an amine was
carried out with triethylamine and DMAP (65-80% yield overall).
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
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Scheme 4. Synthesis of Compounds 15-16.
…
……………………………………………………………………………………………………
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®
°
Reagents and Conditions: (a) RCH
2
CO
2
H, T3P (50 wt. % in EtOAc), DIPEA, DMF, 70 C, 1
to 4 h; (b) 4-pyridylacetic acid HCl, EDC.HCl, DIPEA, DMAP, DCM, 90 min; (c) (i) CO
LDA (2 M in THF/heptane/ethylbenzene), THF, -78 C to rt, 1 h (ii) 5, T3P (50 wt. % in
EtOAc), DIPEA, DMF, 70 C, 40 min; (d) (i) RH, NaH, DMF, 0 C to rt, 30 min to 20 h (ii)
EtOH, HCl (37.5% w/v), H O, reflux, 9 h to 2 d; (e) (i) RH, NEt , DMAP, DCM, 30 min to 1 h
2
(s),
°
®
°
°
2
3
®
(ii) EtOH, HCl (37.5% w/v), H
DMF, 70 C, 45 min to 5 h.
2
O, reflux, 3 to 16 h; (f) T3P (50 wt. % in EtOAc), DIPEA,
°
0
1
2
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9
0
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0
1
2
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8
9
0
CONCLUSIONS
The application of a deconstruction-reconstruction approach with the previously identified
HTS hit 1 did not lead to the development of improved inhibitors, with defragmentation failing
to afford fragments with either measurable inhibition in the biochemical assay or a positive result
by DSF. This points to the essentiality of all components of 1 for its unique dimeric, induced-fit
binding mode in the allosteric site of Mtb fumarase. A subsequent SAR study of 1 showed that
modifications including replacement of both the phthalazinone and azepanyl ring systems were
possible, with benzoazepanyl derivatives 16l-m affording sub-micromolar IC50 values (0.67 µM)
three-fold stronger than that of 1. Derivatives of 1 maintained the dimeric-binding mode in the
allosteric site of Mtb fumarase under X-ray crystallography soaking conditions, including in the
case of attenuated inhibition as in 16a (IC50 57 µM). In contrast to 1, a subset of the derivatives
afforded measurable MIC values against Mtb in vitro. These derivatives ranged from
conservative expansion of the azepanyl ring of 1 with a subsequent rise in lipophilicity in 16b
(
MIC 19 µM in 7H9/DPPC) to complete replacement of the phthalazinone ring with alternative
heterocyclic systems in 15a-d (MIC 6.3-12.5 µM in 7H9/DPPC). These results represent a
significant improvement on the original HTS hit 1 and encourage further work on Mtb fumarase
as a target for the development of compounds with bactericidal activity.
EXPERIMENTAL SECTION
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General Chemistry. All reactions were carried out in oven-dried glassware under a positive
pressure of dry nitrogen atmosphere. Temperatures of 0 and -78 °C were obtained by submerging
the reaction vessel in a bath containing either ice or a mixture of solid CO pellets and acetone
2
1
1
1
1
1
1
1
1
1
1
2
2
2
2
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1
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1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
respectively. The solvents DCM, ethyl acetate, acetonitrile, methanol, petroleum ether and
toluene were distilled over calcium hydride under a dry nitrogen atmosphere prior to use, with
THF distilled over a mixture of lithium aluminium hydride, calcium hydride and
triphenylphosphine. DMF was purchased as anhydrous from commercial suppliers, with ethanol
obtained in the absolute form. All purchased chemicals were used as received. Solutions of
Na
2
CO and NaCl (brine) were aqueous and saturated.
3
®
Flash column chromatography was performed using automated Biotage Isolera™ Spektra
®
purification systems with appropriately sized Biotage SNAP cartridges, containing either KP 50
µm silica in ‘normal phase’ purification or HP-sphere 25 µm C18 silica in ‘reverse phase’
purification. Analytical thin layer chromatography (TLC) was performed using Merck glass-
backed silica plates, with visualization by 254 or 365 nm ultraviolet light.
®
Liquid chromatography mass spectrometry (LCMS) was carried out using a Waters Acquity
®
UPLC H-Class system, with samples run on a solvent gradient from 0 to 95% acetonitrile in
water (+ 0.1% formic acid) over 4 minutes. Peaks corresponding to desired product are
described, including the retention time (rt) and % purity by integration. High-resolution mass
spectrometry (HRMS) was mainly performed using ThermoFinnigan Orbitrap Classic, Waters®
LCT PremierTM or Waters Vion IMS QTof systems. A Perkin-Elmer Spectrum One FT-IR
®
TM
®
spectrometer fitted with a universal attenuated total reflectance accessory was used to record
-1
infrared spectra, with wavelengths of maximum absorbance (νmax) quoted in wavenumbers (cm )
for signals outside of the fingerprint region (br = broad). Only peaks corresponding to key
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functional groups were characterized. Nuclear magnetic resonance (NMR) spectra were recorded
in the indicated deuterated solvents with AvanceTM III HD (400 MHz), QNP Cryoprobe (400
1
MHz) or DCH Cryoprobe (500 MHz) Bruker spectrometers. H NMR data are presented in the
0
1
2
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following order: chemical shift (in ppm on a δ scale relative to the residual solvent resonance
peak), integration, multiplicity (s = singlet, d = doublet, t = triplet, q = quartet, quin = quintet,
1
3
sep = septet, m = multiplet) and coupling constant (J, in Hz). C NMR spectra were proton-
decoupled, with chemical shifts presented.
A combination of TLC and LCMS analysis was used to monitor reactions. All tested
compounds possessed a purity of at least 95% as determined by LCMS analysis.
N-(5-(Azepan-1-ylsulfonyl)-2-methoxyphenyl)-2-(4-oxo-3,4-dihydrophthalazin-1-
®
yl)acetamide (1). T3P (50 wt. % in DMF, 1.3 mL, 2.2 mmol) and N,N-diisopropylethylamine
(
0.64 mL, 3.7 mmol) were added to a solution of 2-(4-oxo-3,4-dihydrophthalazin-1-yl)acetic acid
3 (0.150 g, 0.735 mmol) and 5-(azepan-1-ylsulfonyl)-2-methoxyaniline 5 (0.209 g, 0.735 mmol)
in DMF (2 mL). The reaction mixture was heated to 70 °C over 1 hour. The reaction mixture was
diluted with water (15 mL), adjusted to pH 1 and extracted into ethyl acetate (3 x 20 mL). The
combined organic extracts were dried (MgSO
4
) and concentrated in vacuo. Purification by flash
column chromatography (0 – 20% methanol in DCM) afforded 1 (0.206 g, 60% yield). LCMS
+
-
1
(
ESI+): m/z 471.3 [M + H] , (ESI-): m/z 469.1 [M - H] , rt 1.92 minutes, >99%; H NMR (400
MHz, (CD SO) 12.62 (1H, s), 9.86 (1H, s), 8.45 (1H, d, J = 2.1 Hz), 8.27 (1H, d, J = 7.9 Hz),
3 2
)
7.99-7.91 (2H, m), 7.90-7.82 (1H, m), 7.49 (1H, dd, J = 8.5, 2.2 Hz), 7.24 (1H, d, J = 8.8 Hz),
13
4
.21 (2H, s), 3.96 (3H, s), 3.12 (4H, t, J = 5.9 Hz), 1.64-1.51 (4H, m), 1.50-1.39 (4H, m); C
NMR (125 MHz, (CD
3
)
2
SO) 168.4, 159.5, 152.0, 142.1, 133.5, 131.6, 130.4, 129.8, 127.7, 127.6,
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25.9, 125.5, 123.4, 119.1, 111.2, 56.3, 47.6, 28.5, 26.3 (1 peak missing); spectroscopic data
consistent with literature.14
Ethyl 2-(4-oxo-3,4-dihydrophthalazin-1-yl)acetate (2).14,18 Hydrazine monohydrate (0.47
mL, 9.0 mmol) was added dropwise to a solution of ethyl (E)-2-(3-oxoisobenzofuran-1(3H)-
ylidene)acetate 18 (1.97 g, 9.03 mmol) in ethanol (10 mL). The reaction mixture was stirred at
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
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5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
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8
9
0
1
2
3
4
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9
0
5
0 °C over 2 hours. The reaction mixture was cooled to room temperature and left to stand for 12
hours. The resulting precipitate was obtained by vacuum filtration and washed with ethanol (10
+
mL) to afford 2 (2.21 g, 99% yield). LCMS (ESI+): m/z 233.2 [M + H] , rt 1.53 minutes, >99%;
1H NMR (400 MHz, (CD
3
2
) SO) 12.63 (1H, s), 8.30-8.24 (1H, m), 7.98-7.91 (1H, m), 7.90-7.83
1
3
(2H, m), 4.11 (2H, q, J = 7.1 Hz), 4.05 (2H, s), 1.17 (3H, t, J = 7.1 Hz); C NMR (100 MHz,
1
(
CD
3
)
2
SO) 169.8, 159.4, 140.9, 133.6, 131.7, 129.4, 127.5, 125.9, 125.5, 60.7, 37.9, 14.0; H
NMR spectroscopic data consistent with literature.18
2-(4-Oxo-3,4-dihydrophthalazin-1-yl)acetic
acid
(3).14
Ethyl
2-(4-oxo-3,4-
dihydrophthalazin-1-yl)acetate 2 (0.260 g, 1.12 mmol) was dissolved in a mixture of aqueous
NaOH (10% w/v, 10 mL) and THF (10 mL). The reaction mixture was heated under reflux for 1
hour. The reaction mixture was adjusted to pH 1 by the addition of aqueous HCl (2 M) at 0 °C,
then extracted into diethyl ether (3 x 100 mL). The combined organic extracts were washed
(
(
brine), dried (MgSO
4
) and concentrated in vacuo. Purification by flash column chromatography
1
0 – 10% methanol in DCM) afforded 3 (0.101 g, 44% yield). H NMR (400 MHz, (CD
3
2
) SO)
12.61 (1H, s), 8.26 (1H, d, J = 7.7 Hz), 7.94 (1H, ddd, J = 8.3, 6.9, 1.4 Hz), 7.90-7.82 (2H, m),
13
3
1
.95 (2H, s); C NMR (100 MHz, (CD
3
2
) SO) 171.4, 159.5, 141.5, 133.5, 131.6, 129.6, 127.5,
25.9, 125.6, 38.2; H NMR spectroscopic data consistent with literature.18
1
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®
N-(2-Methoxyphenyl)-2-(4-oxo-3,4-dihydrophthalazin-1-yl)acetamide (4). T3P (50 wt. %
in DMF, 0.35 mL, 0.59 mmol) and N,N-diisopropylethylamine (0.10 mL, 0.59 mmol) were
added to a solution of 2-(4-oxo-3,4-dihydrophthalazin-1-yl)acetic acid 3 (40 mg, 0.20 mmol) and
o-anisidine 19 (33 µL, 0.29 mmol) in DMF (1 mL). The reaction mixture was heated to 40 °C
over 2 hours. The reaction mixture was diluted with water (15 mL) and extracted into DCM (3 x
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
0 mL). The combined organic extracts were dried (MgSO ) and concentrated in vacuo. The
4
residue was dissolved in toluene (10 mL) and concentrated in vacuo. Purification by flash
column chromatography (50 – 100% ethyl acetate in petroleum ether, 0 – 20% methanol in
+
DCM) afforded 4 (50 mg, 82% yield). LCMS (ESI+): m/z 332.1 [M + Na] , rt 1.64 minutes,
1
>99%; H NMR (500 MHz, (CD
3
)
2
SO) 12.61 (1H, s), 9.56 (1H, s), 8.27 (1H, d, J = 7.9 Hz),
13
8
.01-7.79 (4H, m), 7.11-7.00 (2H, m), 6.92-6.83 (1H, m), 4.16 (2H, s), 3.86 (3H, s); C NMR
(
125 MHz, (CD
3
) SO) 167.6, 159.5, 149.6, 142.3, 133.5, 131.5, 129.8, 127.6, 127.2, 125.8,
2
-
1
125.6, 124.6, 121.8, 120.2, 111.2, 55.7 (1 peak missing); νmax/cm 2905, 1665 (C=O), 1646,
+
1
3
597, 1539; HRMS (ESI)+: m/z calculated for [C17
10.1175.
H
15
N
3
O
3
+ H] = 310.1186, observed
5
-(Azepan-1-ylsulfonyl)-2-methoxyaniline (5).14 Hexamethyleneimine (0.212 mL, 1.89
mmol) was added dropwise at 0 °C to a suspension of sodium hydride (60% in mineral oil, 0.189
g, 4.72 mmol) in DMF (2 mL). The reaction mixture was stirred at 0 °C over 30 minutes. A
solution of 4-methoxy-3-(2,2,2-trifluoroacetamido)benzenesulfonyl chloride 21 (0.500 g, 1.57
mmol) in DMF (3 mL) was added dropwise at 0 °C to the reaction mixture. The reaction mixture
was warmed to room temperature and stirred over 3 hours. Ethanol (10 mL) was added dropwise
at 0 °C to the reaction mixture, followed by water (10 mL) and aqueous HCl (37.5% w/v, 10
mL). The reaction mixture was heated under reflux for 20 hours. The reaction mixture was
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concentrated in vacuo to remove ethanol, then adjusted to pH 9 by the dropwise addition of
aqueous NaOH (10% w/v). The mixture was diluted with ethyl acetate (50 mL), and the resultant
aqueous layer discarded. The organic layer was washed with water (3 x 50 mL) and brine (50
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
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3
3
3
3
3
3
4
4
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4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
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1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
mL), dried (MgSO
4
) and concentrated in vacuo. Purification by flash column chromatography
(
20 – 50% ethyl acetate in petroleum ether) afforded 5 (0.373 g, 83% yield). LCMS (ESI+): m/z
+
1
2
85.2 [M + H] , rt 1.85 minutes, >99%; H NMR (400 MHz, CDCl ) 7.17 (1H, dd, J = 8.4, 2.3
3
Hz), 7.09 (1H, d, J = 2.2 Hz), 6.81 (1H, d, J = 8.4 Hz), 3.98 (2H, br s), 3.90 (3H, s), 3.23 (4H, t, J
13
=
5.9 Hz), 1.75-1.64 (4H, m), 1.62-1.53 (4H, m); C NMR (100 MHz, CDCl
3
) 150.1, 136.7,
-1
1
31.6, 118.1, 112.7, 109.7, 55.8, 48.3, 29.3, 27.1; νmax/cm 3484 (N-H), 3379 (N-H), 2932,
+
2849, 1610, 1577, 1513; HRMS (ESI)+: m/z calculated for [C13
observed 323.0835.
H
20
N
2
O
3
S + K] = 323.0826,
N-(5-(Azepan-1-ylsulfonyl)-2-methoxyphenyl)acetamide (6). Acetic anhydride (10 µL, 0.11
mmol) was added to a solution of 5-(azepan-1-ylsulfonyl)-2-methoxyaniline 5 (15 mg, 0.053
mmol) and pyridine (8.5 µL, 0.11 mmol) in DCM (2 mL). The reaction mixture was stirred over
9
1
0 minutes. The reaction mixture was diluted with water (10 mL) and extracted into DCM (3 x
5 mL). The combined organic extracts were dried (MgSO ) and concentrated in vacuo.
4
Purification by flash column chromatography (50 – 80% ethyl acetate in petroleum ether)
+
-
afforded 6 (14 mg, 79% yield). LCMS (ESI+): m/z 327.3 [M + H] , (ESI-): m/z 325.2 [M - H] ,
1
rt 1.90 minutes, 96%; H NMR (400 MHz, CDCl
3
) 8.78 (1H, d, J = 1.8 Hz), 7.76 (1H, br s), 7.53
(1H, dd, J = 8.5, 2.3 Hz), 6.91 (1H, d, J = 8.6 Hz), 3.94 (3H, s), 3.30 (4H, t, J = 6.0 Hz), 2.22
13
(
3H, s), 1.80-1.67 (4H, m), 1.65-1.53 (4H, m); C NMR (125 MHz, CDCl
3
) 168.4, 150.4, 132.1,
-1
1
28.2, 123.5, 118.0, 109.5, 56.2, 48.5, 29.3, 27.1, 25.0; νmax/cm 3349, 2930, 2855, 1673 (C=O),
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