Stereoselective synthesis of allele-specific BET inhibitors

Article abstract of DOI:10.1039/d0ob01165g

Developing stereoselective synthetic routes that are efficient and cost-effective allows easy access to biologically active molecules. Our previous syntheses of allele-selective bumped inhibitors of the Bromo and Extra-Terminal (BET) domain proteins, Brd2

Full text of DOI:10.1039/d0ob01165g

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Stereoselective synthesis of allele-specific BET
inhibitors†
Cite this: DOI: 10.1039/d0ob01165g
Adam G. Bond,
Andrea Testa ‡ and Alessio Ciulli
*
Received 5th June 2020,
Accepted 9th July 2020
DOI: 10.1039/d0ob01165g
rsc.li/obc
Developing stereoselective synthetic routes that are efficient and develop stereoselective routes and processes which yield solely
cost-effective allows easy access to biologically active molecules. the desired biologically active molecule.2–5
Our previous syntheses of allele-selective bumped inhibitors of the
The four Bromo and Extra-Terminal (BET) proteins, Brd2,
Bromo and Extra-Terminal (BET) domain proteins, Brd2, Brd3, Brd4 Brd3, Brd4 and BrdT, play a crucial role in transcriptional regu-
and BrdT, required a wasteful, late-stage alkylation step and expen- lation and other processes such as cell proliferation and cell
sive chiral separation. To circumvent these limitations, we devel- cycle progression.6–8 BET proteins have become attractive
oped a route based on stereocontrolled alkylation of an N-Pf pro- therapeutic targets as their misregulation has been linked to
tected aspartic acid derivative that was used in a divergent, racemi- diseases such as cancer, neurological disorders and
sation-free protocol to yield structurally diverse and enantiopure inflammation.9,10 Association to disease has fuelled great
triazolodiazepines. With this approach, we synthesized bumped interest in the field to develop small molecule BET inhibitors,
thienodiazepine-based BET inhibitor, ET-JQ1-OMe, in five steps many of which are in the clinic.11–14 Many BET inhibitors
and 99% ee without the need for chiral chromatography. Exquisite include a triazolodiazepine scaffold, including JQ1 (ref. 15)
selectivity of ET-JQ1-OMe for Leu-Ala and Leu-Val mutants over and I-BET762 (ref. 16) (Fig. 1). Due to the high conservation of
wild-type bromodomain was established by isothermal titration BET bromodomains at the acetyl-lysine binding pocket, these
calorimetry and X-ray crystallography. Our new approach provides inhibitors are pan-selective so do not significantly discrimi-
unambiguous chemical evidence for the absolute stereochemistry nate between the bromodomains within and across the BET
of the active, allele-specific BET inhibitors and a viable route that
will open wider access to this compound class.
Chemical biology and therapeutic development rely on the
design or discovery of biologically active compounds that typi-
cally contain one or more stereocenters. Amongst the different
stereoisomers for a given compound, it is often the case that
only one (so-called eutomer) exhibits the desired biological
activity, while the other(s) (distomers) may be inactive or have
toxic and off-target effects.1 Studying and testing diastereo-
meric or racemic mixtures has limitations and could lead to
unwanted or artefactual results, it is therefore important to
Division of Biological Chemistry and Drug Discovery, School of Life Sciences,
University of Dundee, James Black Centre, Dow Street, Dundee DD1 5EH, UK.
E-mail: a.ciulli@dundee.ac.uk
†Electronic supplementary information (ESI) available: Supplementary results
(Fig. S1–S5 and Table S1); Materials and methods section and Scheme S1; sup-
plementary references and NMR spectra. Atomic coordinates and structure
factors for the crystal structure of Brd2(BD2)-L383V mutant in complex with
ET-JQ1-OMe has been deposited in the protein databank (PDB) under accession
number 6YTM. See DOI: 10.1039/d0ob01165g
Fig. 1 Pan and allele-selective BET inhibitors. Pan-selective inhibitors
(+)-JQ1 and I-BET762 (top) and allele selective probes ME, ET, 9-ME-1
and 9-ET-1 (bottom).
‡Present address: Andrea Testa: Amphista Therapeutics Ltd, Bo’Ness Road,
Newhouse, ML1 5UH, United Kingdom.
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family.17,18 More recently, compounds have been reported to products are racemic and require chiral separation to isolate
show selective binding to the first bromodomains (BD1), or to the eutomers.24–26 Separation of the enantiomers can be
the second bromodomains (BD2), of the BET family.19–22 costly (up to £1000 for <150 mg of racemate) and leads to loss
Although these compounds can discriminate between BD1 of material during the separation.
and BD2 within a given BET protein, they still cannot discrimi-
nate across the four BET proteins.
We sought to address these problems by developing a new
stereoselective synthetic strategy. We hypothesised that incor-
To aid individual intra-BET selectivity, we previously devel- porating the “bump” earlier in the synthesis via a diastereo-
oped a chemical genetics approach to engineer orthogonal selective alkylation of an aspartic acid derivative would circum-
protein/ligand pairs between the BET proteins and selective vent the limitations of the original route (Fig. 2B). Here, we
inhibitors.23 Our “bump & hole” approach involved the intro- describe a new synthetic route that allows the preparation of
duction of a single point mutation to the target BET bromo- both novel and previously described bumped BET inhibitors
domain by replacing a leucine residue that is conserved stereoselectively in 99% ee. We also provide unambiguous
across all BET bromodomains, with a smaller residue (e.g. chemical evidence to the absolute stereochemistry of the active
alanine) to generate a “hole”.24 Simultaneously, an alkyl allele-specific ligand, previously only assumed based on co-
“bump” is incorporated at a diastereotopic, methylene group crystal structures.25
on the parent scaffold based on the I-BET762 inhibitor,
Our first efforts to alkylate (+)-JQ1 directly proved unsuc-
aimed to both complement the size of the “hole” and provide cessful, likely due to the steric hindrance caused by the tert-
a steric clash to the wild-type protein. This approach led to butyl ester. Conversion from the tert-butyl to a methyl ester
the generation of allele specific chemical probes ET (ref. 24) was required to allow for the introduction of the bump.
and 9-ME-1 (ref. 25) (Fig. 1) targeting the leucine to alanine Alkylation with KHMDS and alkyl iodides proceeded with
(L/A) or the less disruptive, leucine to valine (L/V) mutation, undesired diastereoselectivity towards the (S,S) diastereomer
respectively. We used this new system to dissect individual over the (S,R) diastereomer in overall alkylation yields of
roles of BD1 vs. BD2 in Brd4 and the other BET proteins and approx. 20%. Epimerisation of the major (S,S) isomer with
showed that while the BD1 is necessary and sufficient for sodium methoxide allows access to the desired (S,R) isomer in
chromatin binding, the BD2 plays a role in transcriptional a 1 : 1 ratio with the starting (S,S) isomer. However, the use of
regulation.24,25
strong bases during alkylation and additional epimerisation
Our previous approaches for incorporation of an alkyl steps may lead to complete racemisation of both stereocentres
“bump” into the I-BET scaffold involved alkylation of a pot- as previously reported,24–26 (see Scheme S1 in ESI†). With
assium enolate to afford bumped I-BET derivatives with unde- enantiopure (+)-JQ1 costing >$750 per gram, and its derivatives
sired diastereoselectivity and in low yields (Fig. 2A). e.g. (+)-JQ1 carboxylic acid being even more expensive, this
Epimerisation was required to enhance the amount of wasteful approach is not a viable strategy for the preparation of
desired diastereomer, and further separation by high per- enantiopure bumped JQ1.
formance liquid chromatography (HPLC) to isolate the
To efficiently prepare the desired bumped JQ1 derivatives
desired isomer. Due to the use of potassium hexamethyl- as single enantiomers, we sought to stereoselectively introduce
disilazide (KHMDS) for the enolization, and the need of an the “bump” earlier in the synthesis. Our synthetic method-
epimerisation step at such a late stage of the synthesis, the ology was based on previously reported stereoselective alkyl-
ation of L-aspartic acid diester derivatives.27–30 We so hypoth-
esized that protection of the amino group with both 9-phenyl-
9-fluorenyl (Pf) and benzyl (Bn) groups could drive diastereo-
selective alkylation, with the Pf group providing strong steric
hindrance of the β-carbon to the nitrogen while also protecting
the α-proton from epimerisation.
L-Aspartic acid derived diester 1 was first treated with benz-
aldehyde in dichloromethane (DCM) and the formation of the
intermediate imine was monitored by 1H-NMR. Reduction of
the imine with sodium borohydride yielded the mono-benzyl
protected amine 2. Amine 2 was treated with 9-phenyl-9-fluore-
nyl bromide, lead(II) nitrate and tribasic potassium phosphate
in acetonitrile to form the N-diprotected diester 3.
Deprotonation of diester 3 with lithium hexamethyldisilazide
(LHMDS) at −78 °C in tetrahydrofuran (THF) afforded the
desired E-lithium enolate, which was reacted with methyl
iodide at −40 °C over 16 h. This yielded methylated diastereo-
mers 4a (S,R) and 4b (S,S) in a 6 : 1 ratio respectively. Ethylated
Fig. 2 Synthetic routes to bumped BET inhibitors. (A) Previous synthetic
compounds 5a and 5b were prepared in a similar way by
deprotonation of diester 3 with LHMDS at −78 °C in THF fol-
strategy for bumped I-BET762 derivatives. (B) Retrosynthetic analysis for
enantiopure bumped JQ1 derivatives.
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lowed by addition of ethyl iodide and stirring at −78 °C for which led to the formation of the triazole ring in the final
16 h. This was left for a further 24 h at −23 °C to yield diaster- compounds 15 (ME-JQ1-OMe) and 16 (ET-JQ1-OMe) with 99%
eomers 5a (S,R) and 5b (S,S) in a 2 : 1 ratio respectively. The ee determined with supercritical fluid chiral chromatography
choice of LHMDS over the respective potassium base, KHMDS, (Scheme 2, see ESI† for analytical details). Overall, we were
was motivated by prior findings that switching between these able to achieve ∼40 mg of enantiopure product in just five,
bases can reverse the diastereoselectivity on a similar aspartate yield limiting, steps from <£100 worth of starting materials. In
derived diester to compound 3.27 Using the potassium base comparison, our previous approach required six steps, includ-
leads to a chelate controlled enolate-ester intermediate which ing expensive chiral purification (∼£1000) and 1 g of JQ1
has the opposite geometry to the non-chelated, ‘open’ lithium ($750) to achieve the same amount of pure product.
enolate intermediate and influences facial selectivity to attack
by the electrophile (alkyl iodide).
Quantitative conversion to carboxylic acids 17 (ME-JQ1-OH)
and 18 (ET-JQ1-OH) was achieved by treating esters 15 and 16
Removal of both Pf and Bn groups were performed by in a 4 : 1 mixture of THF to either a 0.54 M or 0.65 M lithium
hydrogenation of alkylated diesters 4a and 5a with a suspen- hydroxide (LiOH) solution respectfully. Heating to 45 °C was
sion of 10% palladium on carbon (Pd/C) in acetic acid to give required for ethyl bumped compound 16 due to the conversion
free amines 6 and 7 in high yields. The resulting free amines being much slower in comparison to the methyl bumped com-
were dissolved in a 1 : 1 mixture of trifluoroacetic acid (TFA) pound 15. These very mild conditions were essential to avoid
and dichloromethane to achieve the tert-butyl ester de- epimerisation of the alkylated stereocenter adjacent to the car-
protection and leave the free amino acid as a TFA salt. The bonyl group. Using higher concentrations of LiOH and/or
TFA salts were then dissolved in 2 M HCl and freeze-dried to higher temperatures resulted in an increased rate of hydrolysis
obtain the amino acids 8 and 9 as HCl salts. Conversion of but led to substantial epimerisation to the undesired (S,S) dia-
salts proved crucial for the next step as any TFA present led to stereomer. Access to these free acids allows the possibility for
the formation of trifluoroacetamide by-products. Amino acids further functionalisation (e.g. via amide or ester bond
8 and 9 (HCl salts) were treated with triphosgene in THF over formation).
16 h to yield the key N-carboxyanhydrides (NCAs) 10 and 11, as
Having achieved the novel bumped JQ1 derivatives, we next
precursors of the alkylated sidechain fragment. These were sought to demonstrate the versatility and scope of our new
used in the next steps without the need for further purification route by attempting to synthesise the I-BET-based bumped
(Scheme 1).
probes, 9-ME-1 and 9-ET-1.25 By using NCA precursors 10 and
Next, thienodiazepines 13 and 14 were formed in a conden- 11, and treating them with aminobenzophenone 19 in the
sation reaction between NCAs 10 and 11 and amino ketone 12 same condensation reaction as described previously, yielded
by heating in the presence of TFA and subsequently triethyl- benzodiazepines 20 and 21. Subsequent triazole ring for-
amine (TEA) in toluene as reported by Fier et al.31 The use of mation via
a similar phosphorylimidate intermediate as
this methodology for the benzodiazepine ring formation was described above yielded the final ligands, 22 (9-ME-1) and 23
crucial in our synthetic strategy as it was found to retain the (9-ET-1), with 99% ee determined with supercritical fluid
stereochemistry of the amino acid derived NCA. Deprotonation chiral chromatography (see ESI† for analytical details). We
of the lactam in both 13 and 14 with potassium tert-butoxide have also demonstrated the accessibility for the (S,S) diastereo-
and addition of diethyl chlorophosphate, as described,32 gave mer (reported as 16* in the ESI†) of 16 using the minor ethy-
the activated phosphorylimidate intermediate which was not lated diastereomer 5b as the starting point. Stereoselective
isolated. This was subsequently reacted with acetylhydrazine access to ‘inactive’ stereoisomers provides important negative
Scheme 1 Stereoselective synthesis of N-carboxyanhydrides 10 and 11. Conditions: (a) (i) Na2CO3, EtOAc, H2O, (ii) PhCHO, DCM, 2 h, r.t., (iii)
NaBH4, MeOH, 1 h, 0 °C–r.t. (66%); (b) PfBr, K3PO4, Pb(NO3)2, MeCN, r.t., 4 h (98%); (c) (i) LHMDS, THF, −78 °C, 1 h, (ii) MeI/EtI, −78 °C to −23 °C,
48 h/96 h (65%/48%); (d) H2, Pd/C, AcOH, r.t., 24 h (quant.); (e) (i) TFA, DCM, r.t., 2 h, (ii) 2 M HCl, freeze dry (quant.); (f) triphosgene, THF, r.t., 16 h
(quant.).
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Scheme 2 Formation of thienodiazepines 15–18 and benzodiazepine derivatives 22 and 23. Conditions: (a) (i) TFA, toluene, 60 °C, 0.5 h, (ii) TEA,
80 °C, 16 h (30–51%); (b) (i) KOtBu, THF, −78 °C to −10 °C, 0.5 h, (ii) (OEt)2P(O)Cl, −78 °C to −10 °C, 0.75 h, (iii) AcNHNH2, r.t., 1 h, (iv) n-butanol,
90 °C, 1 h (8–39%); (c) LiOH, THF : H2O 4 : 1, R = Me; r.t., 1 to 3 days; R = Et: 45 °C, 1 week (quant.).
controls to increase validity and robustness of findings in both
biophysical and biological assays.
To further characterise our novel bumped compound, we
studied the binding of 16 to Brd4(2) L387A, L387V and wild-
type using isothermal titration calorimetry (ITC). We found
undetectable/no binding of 16 to the wild-type protein
(Fig. 3A, see also ESI Fig. S5†), consistent with ethyl bumped
compounds reported previously.24,25 Crucially, 16 demon-
strated very high binding affinity to both L/A and L/V mutants
with equipotent Kd values of 65 nM (Fig. 3A, see also ESI
Fig. S3 and S4†). To validate the binding mode, we solved a
high resolution (1.56 Å) X-ray structure of Brd2(2)L383V co-crys-
tallised with 16 (Fig. 3B, see also ESI Fig. S2 and Table S1†).
We found that 16 adopts a similar binding mode to 9-ME-1
and 9-ET-1 (see ESI Fig. S1†), positioning the ethyl “bump”
towards the “hole” formed by the L/V mutation.
In summary, we describe
a versatile stereoselective
approach to successfully synthesise bumped BET inhibitors.
We demonstrate scope by synthesizing both novel JQ1 deriva-
tives and previously described I-BET762 derivatives, all in 99%
ee. Compared to the previous method, our new route achieves
enantiopure products in one less step, from widely available
and relatively inexpensive starting materials, while avoiding
wasteful, late-stage alkylation steps and chiral separation. We
qualified the remarkable allele-selectivity of ET-JQ1-OMe over
wild-type and provided unambiguous chemical evidence to the
Fig. 3 Biophysical and structural characterization of novel JQ1-based
absolute stereochemistry of the eutomer. Access to carboxylic bumped inhibitor. (A) ITC titrations of ET-JQ1-OMe (16) against wild-
type (W.T.) (maroon), L387A (blue) and L387V (red) constructs of Brd4(2).
Table shows values taken as a mean and standard deviation from three
replicates. (B) Co-crystal structure of 16 (pink carbons) with Fo-Fc omit
map (grey mesh, contour: 3σ) bound to Brd2(2)L383V mutant (green,
acid derivatives retaining enantiomeric purity enables functio-
nalisation into conjugates such as biotinylated and fluorescent
probes and PROTACs,23 which will further expand the scope
and utility of this new synthetic strategy for chemical biology
investigation.
cartoon representation; green carbons, binding site side chains) (PDB
code: 6YTM). Bump and hole residue Val 383 is highlighted.
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with ITC and crystallography; Darryl McConnell (Boehringer
Ingelheim) for discussions; Reach Separations for chiral ana-
lysis; and staff at Diamond Light Source for synchrotron
access.
Abbreviations
AcNHNH2 Acetyl hydrazine
AcOH
BD1
Acetic acid
First bromodomain
BD2
BET
Brd
Second bromodomain
Bromo and extra-terminal
Bromodomain
References
DCM
ITC
KHMDS
LHMDS
NCA
Dichloromethane
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Medical Research Scotland (PhD studentship 1170-2017 to
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Conflicts of interest
The authors declare the following competing financial interest
(s): The Ciulli laboratory receives or has received sponsored
research support from Amphista Therapeutics, Boehringer
Ingelheim, Eisai, Nurix, and Ono Pharmaceuticals.
A.C. is a scientific founder, shareholder, non-executive
director and consultant of Amphista Therapeutics, a company
that is developing targeted protein degradation therapeutic
platforms. The remaining authors report no competing
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