Spirooxindoles as novel 3D-fragment scaffolds: Synthesis and screening against CYP121 from M. tuberculosis

Article abstract of DOI:10.1016/j.bmcl.2016.05.073

The search for new scaffolds to complement current HTS and fragment libraries is an active area of research. The development of novel strategies to synthesise compounds with 3D character in order to expand the diversity of a fragment library was explored. A range of substituted bicyclo[2,2,1]spirooxindoles were synthesised using a Diels–Alder [4+2] cycloaddition reaction. Both diastereoisomers were isolated from the reactions and these 3D fragment scaffolds were screened against the cytochrome P450 enzyme CYP121 from Mycobacterium tuberculosis. A number of hits were identified to bind to CYP121 and were shown to exhibit Type I binding interactions with the heme group.

Full text of DOI:10.1016/j.bmcl.2016.05.073

Bioorganic & Medicinal Chemistry Letters xxx (2016) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Spirooxindoles as novel 3D-fragment scaffolds: Synthesis and
screening against CYP121 from M. tuberculosis
⇑
Holly J. Davis, Madeline E. Kavanagh, Tudor Balan, Chris Abell, Anthony G. Coyne
Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge CB2 1EW, United Kingdom
a r t i c l e i n f o
a b s t r a c t
Article history:
The search for new scaffolds to complement current HTS and fragment libraries is an active area of
research. The development of novel strategies to synthesise compounds with 3D character in order to
expand the diversity of a fragment library was explored. A range of substituted bicyclo[2,2,1]spirooxin-
doles were synthesised using a Diels–Alder [4+2] cycloaddition reaction. Both diastereoisomers were iso-
lated from the reactions and these 3D fragment scaffolds were screened against the cytochrome P450
enzyme CYP121 from Mycobacterium tuberculosis. A number of hits were identified to bind to CYP121
and were shown to exhibit Type I binding interactions with the heme group.
Received 15 April 2016
Revised 23 May 2016
Accepted 25 May 2016
Available online xxxx
Keywords:
Fragment-based drug discovery
CYP121
Ó 2016 Elsevier Ltd. All rights reserved.
Tuberculosis
Fragment-based approaches to drug discovery are now firmly
established in both academia and industry. This methodology has
been applied to a diverse range of target classes, including kinases,
protein–protein interactions and GPCRs, and the scope is ever
increasing.1–3 At the core of this approach is the fragment library,
as it is the composition of these libraries that critically influences
the hit rate and nature of inhibitors that are developed. Proteins
bind substrates and cofactors that typically have 3D character
and it has been argued that fragments or compounds with 3D char-
acter would be preferentially favoured for binding.4 However, frag-
ment and commercial screening libraries typically have a high
proportion of flat heterocycles, and as such there has been a con-
certed effort in recent years to expand the structural diversity of
these libraries. One approach to increasing the structural diversity
of libraries has been to introduce sp3 carbon centres onto the frag-
ment scaffold, thereby increasing the 3D character.4–7 However,
the routes to these compounds are often challenging and require
multiple step syntheses.
A number of research groups have examined various synthetic
methodologies to develop fragments containing a high level of
3D character. Young and co-workers used a diversity oriented syn-
thesis (DOS) approach to synthesise novel bicyclic- and spirocyclic-
pyrrolidine fragments using the proline scaffold as a startpoint.8
Examination of the scaffolds synthesised using cheminformatic
tools showed that they had significantly different 3D character to
commercial compounds found in the ZINC database and main-
tained favourable physicochemical properties. Bull and co-workers
developed a synthetic route to aryl-sulfonyl-oxetanes, which intro-
duce a high degree of 3D functionality into the fragment scaffold
through the non-planar SO2 group and the vectors provided by
the oxetane ring. Ortho-metallation and palladium-catalysed reac-
tions were subsequently used to further expand the structural
diversity of these scaffolds.9 Neither of these papers reported
results from screening these fragments against a biological target.
Only small proportion compounds in of our own in-house frag-
ment library contain 3D character. Analysis of these scaffolds
showed that structural diversity was primarily due to functional
groups such as sulfonamides and biaryl rings, while spirocyclic
containing scaffolds were not represented. In the last few years,
interest in spirocyclic scaffolds has increased because they extend
beyond the chemical space currently represented by drug
molecules.10 The synthesis of spirocycles is generally difficult,
despite a number of single-step routes being available, such as
the Diels–Alder [4+2] and the Huisgen [3+2] cycloaddition reac-
tions. These single step syntheses involve the reaction of a diene
or 1,3-dipole with an exocyclic double bond and can be used to
synthesise spirocycles in high diastereo- and enantioselectivity
by using either metal or organocatalytic methods.11,12 In a recent
report, Carreira and co-workers synthesised a novel thiaazaspiro
[3,4]octane ring using a thiocarbonyl ylide, which was generated
in situ and subsequently reacted with an
a,b-unsaturated ester.
Subsequent oxidation of the sulfur to the sulfone using mCPBA
afforded the spirocycles in good yields.12d A drawback of this
method of constructing spirocycles is the limited availability of
sufficiently reactive exocyclic dieno/dipolarophile starting
materials. Steric crowding around the double bond was found to
⇑
Corresponding author. Tel.: +44 (0)1223 330190.
E-mail address: agc40@cam.ac.uk (A.G. Coyne).
significantly decrease the reactivity of these 2p-components.
http://dx.doi.org/10.1016/j.bmcl.2016.05.073
0960-894X/Ó 2016 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Davis, H. J.; et al. Bioorg. Med. Chem. Lett. (2016), http://dx.doi.org/10.1016/j.bmcl.2016.05.073

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H. J. Davis et al. / Bioorg. Med. Chem. Lett. xxx (2016) xxx–xxx
One class of exocyclic dienophiles that has sufficient reactivity
3-alkylidene benzofuranone and benzothiophenone (13a and
13b) scaffolds were also prepared according to literature proce-
dures.18 A number of the substituted 3-alkylidene-2-oxindoles die-
nophiles were reduced with NaBH4 to produce the corresponding
2-oxoindolines 10a–f and 13c, which are themselves interesting
fragment scaffolds that were not represented in our fragment
library.19 These reduced intermediates allowed investigation of
the inherent binding properties of the oxindoline scaffold. The
reaction with NaBH4 was rapid (1–2 min) in converting the highly
coloured 3-alkylidene-2-oxindoles to a clear solution and resulted
in product yields of 28–89%, Scheme 1.
and has recently been studied is based on the 2-oxindoles (isatin)
scaffold.13 These important heterocycles have been incorporated
into a range of drug molecules, such as 1 and 2, and are also found
in natural products such as horsfiline and corulescine 3 (Fig. 1). A
major advantage of the 2-oxindole ring is that it is amenable to
modification at various positions, which would allow scope for
fragment elaboration and optimisation in a medicinal chemistry
program.13c–e We decided to use this scaffold as a start-point to
explore the Diels–Alder [4+2] cycloaddition reaction with the
highly reactive cyclopentadiene 5. The resultant products of the
cycloaddition reactions contained
heptene ring system which has not been previously been
reported as fragment scaffold and has only been briefly
a
novel spirobicyclo[2,2,1]
The next step was to react the substituted 3-alkylidene-2-oxin-
doles 9a–j in a Diels–Alder [4+2] cycloaddition reaction with
cyclopentadiene 5. The reaction proceeded under mild conditions
to give a mixture of two isomers in a ratio of 2–3:1 (Table 1).
The isomers were separable in the majority of cases using flash col-
umn chromatography. The stereochemistry of the isomers was
deduced using 2-D NOESY NMR. An NOE between the C-3 proton
of the bicyclo[2,2,1]heptene ring and one of the bridging methy-
lene protons indicated that the major isomer produced from the
Diels–Alder cycloaddition had the ester and methylene bridge in
the endo conformation (Table 1). No NOE was observed for the
C3-proton of the minor isomer, however NOEs between the methy-
lene bridgehead and the C5-proton of the original 2-oxindoles ring
were identified (Supporting information Fig. S1). The structural
assignments of the major and minor isomers are in agreement with
those previously reported in the literature, which had been deter-
mined by subsequent derivatisation of the products.14
a
mentioned in the literature.14 The aim of this work was to con-
struct a small library of these 3D fragment scaffolds and screen
them against the cytochrome P450 enzyme CYP121 from Mycobac-
terium tuberculosis (Mtb). CYP121 is an essential enzyme, which has
been identified as important drug target for tuberculosis and is a
focus of fragment-based drug discovery efforts within our research
group. We have previously reported the discovery of a number of
fragments and the development of small molecule CYP121 inhibi-
tors with binding affinities in the low micromolar region.15 The
Mtb genome encodes 20 cytochrome P450 enzymes (CYP’s), and
obtaining selectivity for a single CYP isoform is both challenging
and desirable.
The first step in the preparation of the spirobicyclo[2,2,1]hep-
tene scaffolds was to synthesise the exocyclic dienophile compo-
nent.
A
Wittig reaction between (carbethoxymethylene)
range of commercially available
The low diastereoselectivity of the cycloaddition reaction was
considered advantageous as it allowed us to further expand our
exploration of chemical space. The isomers were readily separable
with the exception of entries 4, 9 and 10, which allowed both the
endo and exo products to be screened independently as fragments.
The scope of the Diels–Alder reaction was investigated with a
range of 5-substituted and N-substituted 3-alkylidene-2-oxindoles
(Table 1, entries 2–7). All substrates yielded the desired cycloaddi-
tion products in moderate to excellent yields (48–99%), and similar
diastereoselectivity (1.8:1 to 3:1). In general, electron-withdraw-
ing groups at the 5-position of the oxindole ring increased the reac-
tion rate, with product formation typically complete within 1–4 h.
In comparison the unsubstituted 3-alkylidene-2-oxindole 9a and
the two N-substituted 3-alkylidene-2-oxindoles 9b and 9g
required reaction times of between 36 and 48 h. When the reaction
was explored with the cyano-substituted 3-alkylidene-2-oxindoles
9h–9j (Table 1, entries 8–10) the reaction rate was further reduced
compared to the carboxyethyl-3-alkylidene-2-oxindoles and addi-
triphenylphosphorane and
a
N- and 5-substituted indoline-2,3-diones 8 was used to synthesise
the substituted 3-alkylidene-2-oxindoles dienophiles 9a–g as
highly coloured crystalline solids in moderate yields (32–65%)
(Scheme 1).16 The structural diversity of the dienophile component
was further expanded by employing Horner–Wadsworth–Emmons
(HWE) methodology to synthesise a range of cyano-substituted
3-alkylidene-2-oxindoles 9h–j in moderate yields (31–58%).17 To
explore the scope of the subsequent Diels–Alder reactions, the
tional equivalents of cyclopentadiene
5 (2 equiv) were also
required for the reactions to go to completion. The diastereoselec-
tivity of the reaction was found to be similar to that with the
carboxyethyl-3-alkylidene-2-oxindoles, however in the case of the
N-methyl 9i (Table 1, entry 9) and 5-trifluoromethoxy substituted
derivative 9j (Table 1, entry 10) the isomers proved difficult to sep-
arate using column chromatography. The benzothiopheneone 13a
and benzofuranone 13b derivatives were also well tolerated by the
Diels–Alder reaction conditions, undergoing reaction with cyclopen-
tadiene 5 (Table 1, entries 11 and 12) to give good yields (up to 76%)
and similar diastereoselectivity to that previously observed. The Fsp3
(Fsp3 = Number of sp3 hybridised carbons/total carbon count) of each
of the compounds isolated was calculated and found to be compara-
ble to that of other 3D like fragments previously reported in the
literature.20 However, as both bicyclo[2,2,1]heptane isomers have
identical Fsp3 values but structurally occupy different 3D space, this
comparison likely underrepresents the structural diversity of the
fragment scaffolds developed here.
Figure 1. (A) Natural products and drugs containing the spirooxindole scaffold. (B)
Synthesis of bicyclo[2,2,1]spirooxindoles using the Diels–Alder [4+2] cycloaddition
methodology. Parent scaffold indicating vectors available for functionalisation.
With the novel bicyclo[2,2,1]heptane scaffolds isolated, we pro-
ceeded to screen this focused library of 3D fragments and reduced
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H. J. Davis et al. / Bioorg. Med. Chem. Lett. xxx (2016) xxx–xxx
3
Scheme 1. Synthesis of dienophile components (9a–j) for the Diels–Alder cycloaddition reaction and synthesis of reduced 3-alkylidene-2-oxindoles (10a–f).
Table 1
Synthesis of bicyclo[2,2,1]heptene scaffolds by the Diels–Alder [4+2] cycloaddition reaction of substituted 3-alkylidene-2-oxindoles with cyclopentadiene
Entry
R1
R2
R3
Time (h)
Yield (%)
Compd No.
Fsp3
dr (11:12)
1
2
3
4
5
6
7
8
9
H
H
Cl
NO2
H
COOEt
COOEt
COOEt
COOEt
COOEt
COOEt
COOEt
CN
H
Me
H
H
Ac
H
Me
H
Me
H
48
48
1
1
30
4
70
73
99
48
56
80
87
89
49
45
11a, 12a
11b, 12b
11c, 12c
11d, 12d
11e, 12e
11f, 12f
11g, 12g
11h, 12h
11i, 12i
0.41
0.44
0.41
0.41
0.42
0.41
0.47
0.33
0.37
0.37
65:35
73:27
65:35
75:25a
72:28
70:30
72:28
66:24
72:28a
68:32a
F
OCF3
H
H
4
24
48
48
CN
CN
10
OCF3
11j, 12j
Entry
R1
R2
X
Time (h)
Yield (%)
Compd No.
Fsp3
dr (14:15)
11
12
H
H
COOEt
COOEt
O
S
4
4
74
76
14a, 15a
14b 15b
0.41
0.41
62:38
59:41
a
The two isomers proved inseparable by column chromatography and were isolated as a diastereoisomeric mixture.
3-alkylidene-2-oxindoles against our chosen target CYP121 from
Mtb. Fragments were prepared as 100 mM stock solutions in
DMSO-d6 and diluted to a concentration of 1 mM in aqueous buffer
(50 mM Tris-HCl, pH 7.5, 1 mM EDTA) to assess their solubility
prior to screening. Any compounds which precipitated under these
conditions were not screened to avoid false positive/negative
results (see Supporting information, Table S1).
were in the vicinity of the catalytic heme group. The UV–Vis assay
monitors perturbations in the maximum wavelength (kmax) of the
Soret band of the optical spectrum of heme containing proteins.
The assay is highly sensitive and allows detection of fragments
which bind by either directly ligating the heme iron (Type II), to
cause a red-shift in the Soret band, or which displace the axial
water ligand but do not directly ligate the heme iron (Type I),
which produces a blue-shift in the Soret band. The novel 3D-frag-
ments were screened at 1 mM and the Soret band of CYP121,
A UV–Vis spectrophotometric assay was used to assess whether
fragments bound to CYP121, and specifically if binding interactions
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H. J. Davis et al. / Bioorg. Med. Chem. Lett. xxx (2016) xxx–xxx
which occurs at 416.5 nm in the water-ligated ferric resting state,
was monitored. The previously identified Type II CYP121 ligand 16
was used as a positive control (screened at 1 mM), and was
observed to cause a change in the kmax of the Soret to 421 nm
(+3 nm).15 The X-ray crystal structure of 16 bound to CYP121 has
previously revealed the proximity of the aniline NH2 with the
heme iron which would be consistent with the red-shift in the
Soret band observed in the UV–Vis spectra.15a None of the 3D
fragments or reduced precursors in the present screening library
were identified as Type II hits. This was in sharp contrast to a
previous fragment screening campaign against CYP121, where
the fragment hits produced Type II perturbation’s of the Soret
band.15a However, 7 of the 3D fragments were observed to cause
a blue-shift in the kmax of the Soret band consistent with a Type I
binding mode. Type I, or substrate-like, hits are relatively
uncommon against CYP’s in our experience compared to Type II
ligands, possibly because binding affinity is more dependent on
interactions with diverse active site residues as opposed to
conserved metal-binding interactions. Examining the structures
of the fragment hits revealed that 4 fragments, 12b, 12c, 15a and
15b, were the minor product isomers from the cycloaddition
reactions. There was one example 11a of the major isomer, as
well as a single example of a reduced 2-oxoindoline 10f and
benzofuranone 13b dienophile. The presence of benzofuranone,
benzothiophenone and indolinone containing bicyclo[2,2,1]
heptene scaffolds amongst the fragment hits identified, indicated
that binding affinity of these compounds was unaffected by the
properties of the heterocyclic component itself (Fig. 2).
conducted to determine the apparent KD (KD(app)) of the known
Type II CYP121 ligand 17 (KD = 0.015 M) (Fig. 3A–C), when it
was titrated against CYP121 in the presence of either compound
10f or 12b (Fig. 3D–F). The change in the KD(app) of 17 from the
KD of 17 determined in the absence of the fragments was then used
to calculate the binding affinity of 12b (Fig. 3C–E).21 The KD of com-
l
pound 12b was determined to be 360 lM, which is comparable to
the binding affinities of previous fragments which have been iden-
tified to bind to CYP121 from our standard fragment library
(KD = 400–3000 l
M).15a A KD value could not be determined for
compound 10f, indicating that it bound too weakly to compete
with the type II probe ligand 17. The ligand efficiency of 12b
(LE = 0.21) is significantly lower than that of previously identified
fragments (LE = 0.29–0.39) because of the higher molecular
weight that is required to functionalise the fragment with the
complex spirocycle framework. Murray and Rees from Astex
Pharmaceuticals recently highlighted the importance of balancing
the advantages of structural and stereochemical diversity with
other physicochemical properties.22 They highlighted the need
for new synthetic chemistry efforts to develop lower molecular
weight 3D fragments, which might enable exploration of unchar-
tered areas of chemical space, while preserving the sampling
advantages offered by fragments.
A series of docking studies were used to ascertain how the frag-
ment hits bound to CYP121 and to explore the preference of
CYP121 for the minor product isomer. A range of different binding
scenarios were modelled, employing positional constraints to
either restrict binding interactions to the vicinity of the heme
cofactor, or to enforce hydrogen-bonding interactions between
fragments and the axial heme water ligand. In one scenario this
resulted in a conserved binding orientation for the minor product
isomers. The fragments docked to form H-bonding interactions
between the indolinone carbonyl group and Arg386, which
Attempts were made to determine the binding affinities (KD val-
ues) of the most potent hits, 10f and 12b, using both direct isother-
mal titration calorimetry (ITC) and optical titrations. However,
titration curves could not be saturated within the solubility limits
of the fragments. As a result, an indirect optical titration was
Figure 2. Results from UV–Vis fragment screening assay showing the distribution of compounds plotted against the change in wavelength (Dkmax) of the Soret band of
CYP121.
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H. J. Davis et al. / Bioorg. Med. Chem. Lett. xxx (2016) xxx–xxx
5
Figure 3. (a) UV–Vis spectra of CYP121 (5
17 induced absorbance change plotted against the concentration of compound 17. (d) UV–Vis spectra of CYP121 (5
12b (500 M). (e) Difference spectra generated from the competition titration of CYP121 with compound 17 in the presence of fragment 12b. (f) Compound 17 induced
absorbance change plotted against the concentration of compound 17 when titrated against CYP121 in the presence of fragment 12b.
l
M) titrated with compound 17. (b) Difference spectra of generated from the titration of CYP121 with compound 17. (c) Compound
lM) titrated with compound in the presence of compound
l
positioned the indole N to point towards the heme iron or axial
water ligand. In contrast, the major product isomers docked in a
range of conformations. Either displacement of the axial water
ligand or weakening the coordination of the axial water ligand to
the heme iron through forming H-bonding interactions with frag-
ments would be consistent with the Type I shift in the Soret band
that was observed in UV–Vis assays. This binding mode has not
been previously observed for fragments identified using our previ-
ous fragment-based approaches, in which all the hits coordinated
directly to the heme iron using an aniline group (Fig. 4).
and benzothiophenone precursors were also explored. The yields
obtained for the reactions were good to excellent and the
diastereoselectivity was typically in the range of 2:1, which
enabled the isolation of both isomers for use in fragment screen-
ing. The novel 3D fragments were screened against CYP121 from
Mtb using a UV–Vis spectrophotometric assay. Seven Type I hits
were identified and using indirect titration compound 12b was
identified to have an apparent KD of 360 lM. Co-crystallisation
of a selection of these hits with CYP121 is currently in progress.
Owing to the success of this methodology for identifying novel
ligand scaffolds, a selection of these 3D fragments will be incor-
porated into our main fragment library and screened against a
range of other protein targets.
In conclusion we have explored the Diels–Alder [4+2] cycload-
dition reaction of substituted oxindoles with cyclopentadiene to
synthesise a series of novel spirocycles. Alternative benzofuranone
Figure 4. (a) Overlay of X-ray crystal structures of previously identified fragments which bind to CYP121 (pdb codes 4G44 (Blue), 4G47 (Green), 4G45 (Yellow)). (b) Docking
of 12a, 12b and 12f (minor isomers) into CYP121 showing conformations relative to the positive control 16 (pdb code 4G1X, the protein was removed to make viewing the
binding poses easier to observe).
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H. J. Davis et al. / Bioorg. Med. Chem. Lett. xxx (2016) xxx–xxx
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Supplementary data
Supplementary data (synthesis and characterization of organic
molecules, UV–Vis assays and docking conditions can be found in
the online version at http://dx.doi.org/10.1016/j.bmcl.2016.05.
073. Additionally, NMR spectra related to this publication are also
available at the University of Cambridge data repository (www.
repository.cam.ac.uk/handle/1810/254133).
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Please cite this article in press as: Davis, H. J.; et al. Bioorg. Med. Chem. Lett. (2016), http://dx.doi.org/10.1016/j.bmcl.2016.05.073