Discovery and Optimization of Allosteric Inhibitors of Mutant Isocitrate Dehydrogenase 1 (R132H IDH1) Displaying Activity in Human Acute Myeloid Leukemia Cells

Article abstract of DOI:10.1021/acs.jmedchem.6b01320

A collaborative high throughput screen of 1.35 million compounds against mutant (R132H) isocitrate dehydrogenase IDH1 led to the identification of a novel series of inhibitors. Elucidation of the bound ligand crystal structure showed that the inhibitors exhibited a novel binding mode in a previously identified allosteric site of IDH1 (R132H). This information guided the optimization of the series yielding submicromolar enzyme inhibitors with promising cellular activity. Encouragingly, one compound from this series was found to induce myeloid differentiation in primary human IDH1 R132H AML cells in vitro.

Full text of DOI:10.1021/acs.jmedchem.6b01320

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Article
Discovery and Optimization of Allosteric Inhibitors
of Mutant Isocitrate Dehydrogenase 1 (R132H IDH1)
Displaying Activity in Human Acute Myeloid Leukemia Cells
Stuart Jones, Jonathan Ahmet, Kelly Ayton, Matthew David Ball, Mark Cockerill, Emma E. Fairweather,
Nicola Hamilton, Paul Harper, James R. Hitchin, Allan M. Jordan, Colin W. Levy, Ruth Lopez, Edward
McKenzie, Martin J Packer, Darren Plant, Iain Simpson, Peter Simpson, Ian Sinclair, Tim C. P.
Somervaille, Helen Small, Gary J. Spencer, Graeme J. Thomson, Michael Tonge, Ian D. Waddell,
Jarrod Walsh, Bohdan Waszkowycz, Mark Wigglesworth, Dan Wiseman, and Donald J. Ogilvie
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.6b01320 • Publication Date (Web): 15 Nov 2016
Downloaded from http://pubs.acs.org on November 16, 2016
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Ogilvie, Donald; Cancer Research UK Manchester Institute, Drug Discovery
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Discovery and Optimization of Allosteric Inhibitors
of Mutant Isocitrate Dehydrogenase 1 (R132H
IDH1) Displaying Activity in Human Acute
Myeloid Leukemia Cells
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, ∞
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Stuart Jones,
Jonathan Ahmet, Kelly Ayton, Matthew Ball, Mark Cockerill, Emma
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*,†
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Fairweather, Nicola Hamilton, Paul Harper, James Hitchin, Allan Jordan, Colin Levy,
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Ruth Lopez, Eddie McKenzie, Martin Packer, Darren Plant, Iain Simpson, Peter Simpson,
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‡
┴
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┴
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Ian Sinclair, Tim C. P. Somervaille, Helen Small, Gary J. Spencer, Graeme Thomson,
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Michael Tonge, Ian Waddell, Jarrod Walsh, Bohdan Waszkowycz, Mark Wigglesworth,
┴
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Dan Wiseman and Donald Ogilvie
.
†
Cancer Research UK Manchester Institute, Drug Discovery Unit, University of Manchester,
Wilmslow Road, Withington, Manchester, M20 4BX, UK
┴
Cancer Research UK Manchester Institute, Leukaemia Biology Laboratory, University of
Manchester, Wilmslow Road, Withington, Manchester, M20 4BX, UK
‡
Discovery Sciences, AstraZeneca, Alderley Park, Cheshire, SK10 4TG, UK
§Oncology iMED, AstraZeneca, Alderley Park, Cheshire, SK10 4TG, UK
#
Manchester Institute of Biotechnology, University of Manchester, Princess Street, Manchester,
M1 7DN, UK
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ABSTRACT:
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A collaborative high throughput screen of 1.35 million compounds against mutant (R132H)
isocitrate dehydrogenase IDH1 led to the identification of a novel series of inhibitors.
Elucidation of the bound ligand crystal structure showed that the inhibitors exhibited a novel
binding mode in a previously identified allosteric site of IDH1 (R132H). This information guided
the optimization of the series yielding submicromolar enzyme inhibitors with promising cellular
activity. Encouragingly, one compound from this series was found to induce myeloid
differentiation in primary human IDH1 R132H AML cells in vitro.
Introduction
The enzyme isocitrate dehydrogenase 1 (IDH1) is a Nicotinamide adenine dinucleotide
+
phosphate (NADP )ꢀdependent dehydrogenase which catalyzes the conversion of isocitrate to α
ketoglutarate (αꢀKG). Mutations have been found in both IDH1 (R132H) and IDH2 (R140Q,
R172K, R172M) in several cancer types including up to 70% of low grade and secondary glioma
1ꢀ4
cases and up to 10% of acute myeloid leukemia cases. IDH1 has been found to be frequently
mutated in its active site at residue arginine 132 to histidine (R132H). This mutation leads to a
loss of the normal enzymatic activity of IDH1 and the acquisition of a neomorphic activity, the
5ꢀ7
conversion of α ꢀketoglutarate to 2ꢀhydroxyglutarate (2ꢀHG) . 2ꢀHG is considered to be an
“oncometabolite” which accumulates to high levels and competitively inhibits αꢀketoglutarateꢀ
dependent enzymes, such as histone demethylases and TET2, which mediates DNA
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demethylation. There is an increasing body of research to investigate the effect of inhibition of
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IDH1 R132H on the cancer disease state
1
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A number of inhibitors of IDH1 R132H have been reported,
(Figure 1). Crystal structures of
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IDH1 R132H containing isocitrate (ICT) have provided some understanding of the change of
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function of the enzyme. Of the reported IDH1 R132H inhibitors, a number have associated
crystallographic data and these may be separated into two distinct classes – those that occupy the
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active site of the protein
such as 2 and 4 (Figure 1) and those that occupy a remote
allosteric site which then renders the protein inactive through conformational change such as 3
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and 6.
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Figure 1. Examples of chemotypes reported as inhibitors of IDH1 (R132H) AGI 5198 1
,
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Pyridone 2
, Sanofi Bisimidazole 3 , Thiohydantoin 4 , Novartis Inhibitor 5 , GSK321 6 ,
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Bay 1436032 7 .
As part of the AstraZeneca (AZ) Open Innovation initiative, the Cancer Research UK
Manchester Institute (CRUKꢀMI) and AZ entered into a collaboration to screen IDH1 (R132H)
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against the AZ compound collection. A highꢀthroughput screen (HTS) of approximately 1.35
million compounds was undertaken against the R132H mutant form of IDH1 in order to identify
novel chemical matter for development as inhibitors.
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The results of HTS screening (see Figure 2 for screening cascade) are described below.
Figure 2. Screening cascade used to identify hit series from 1.35M compounds
The primary HTS performed well with a mean Z’ of 0.58 and generated an initial hit rate of 2.1%
using a cut off of 50% inhibition at 30 µM compound concentration in the IDH1 R132H
biochemical assay. Compounds were then retested at a lower concentration (10 µM) resulting in
61.3% of hits at 30 µM reconfirming (17126 compounds). False positives (e.g. those that
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interfered with the assay detection system), were identified using an orthogonal mass
spectrometry assay leaving 50.5% confirmed as active. Prior to IC determination confirmed
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actives were subjected to cluster analysis and virtual chemical filtering (to remove potential
reactive and assay interference compounds) resulting in the selection of 5976 compounds
alongside 2748 ‘near neighbors’ for 10 point dose response determination. In order to further
improve the quality of the hit output, compounds were tested in a ratio assay, i.e. testing at 1×
and 10× enzyme concentration to identify any additional assay interference compounds.
Compounds showing a greater than 0.5 log10 shift in IC were removed from further testing, the
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majority of these showing a decrease in potency under the 10x standard enzyme concentration
conditions, potentially due to redox cycling or colloidal aggregation.
After these measures, further clustering by substructural analysis and Tanimoto similarity gave
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85 compounds of interest, with IC values ranging from 0.3 to 30 µM. These compounds were
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tested against wildꢀtype IDH1 in a biochemical assay to determine whether the compounds were
selective for the mutant isoform. None of the compounds tested showed significant activity
against wildꢀtype IDH1.
In parallel, compounds were also examined in a surface plasmon resonance (SPR) assay to
confirm direct binding to the IDH R132H enzyme. Compounds that bound with a 1:1
stoichiometry were prioritized for further exploration (data not shown).
The overall result from the described screening cascade and triaging process was that compounds
belonging to 3 distinct chemical series were identified for further evaluation. Previously we had
found that the IDH1 (R132H) biochemical assay was sensitive to a number of metal
contaminants including palladium, potentially giving rise to false positives. Several other hits,
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which had used palladium in a late stage of their synthesis, were reꢀsynthesized with particular
care taken to eliminate any potential contamination of the products with residual palladium by
the use of a palladium scavenger (Quadrapure® MPA) and purification of reaction products by
both normal and reverse phase chromatography. However, one hit series did not use palladium in
the synthetic route and examples were resynthesized inꢀhouse without these additional
precautions.
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On testing the inꢀhouse prepared compounds in the IDH1 R132H biochemical assay only this
palladiumꢀfree series reconfirmed strongly suggesting that contamination of the original HTS
samples (potentially by palladium) was responsible for the false positive response. The results
for the one remaining series are shown in Table 1.
Table 1. Activity of original and resynthesized hits
a
Compound
Structure
Biochemical IC50 µM
Library sample
Resynthesized
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3.0 (n/a)
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IC s are mean values of a minimum of 2 replicates. Standard deviations are listed in
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parentheses.
Compounds from this chemical series have previously been reported by AZ as inhibitors of 11βꢀ
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hydroxysteroid dehydrogenase Type 1 (11βꢀHSD1) offering us the opportunity to rapidly
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examine additional near neighbors of the original hits, sourced from the AZ compound
collection.
The preliminary SAR obtained in this manner suggested that the molecule could be simplified.
Thus, the hydroxyadamantylꢀamide group was replaced with cyclohexylꢀamide and the sulphur
linkage replaced by an ether linkage with no loss of enzyme potency, e.g. compound 12 (Figure
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Figure 3 IDH1 (R132H) IC (µM) data for selected near neighbor of hit compound 8.
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As an aid to guide optimization, attempts were made early in the project to determine the binding
mode of the initial hits using Xꢀray crystallography. The crystal structure of compound 9
complexed with IDH1 (R132H) homodimer in the presence of NADPH (PDB submission code
5
L58) was obtained at 3.0 Å resolution (Figure 4). Although several loop regions proved to be
highly disordered at this resolution, the crystal structure revealed that the unit cell contained a
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symmetrical dimer of (R132H) IDH1 in an “open” conformation , with electron density for
compound 9 and the NADP cofactor found in both protomer subunits.
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Figure 4. (A) Symmetrical dimer of (R132H) IDH1 in an “open” conformation , with compound
(green carbons) and the NADP cofactor (blue carbons) bound in both subunits (PDB accession
9
code 5L58) (B) Crystal structure of compound 9 bound at the dimer interface of IDH1 (R132H),
with the two protomers distinguished by ribbon colour. (C) Protein:ligand interaction diagram
hydrogen bonds indicated by magenta arrows, aromatic stacking interactions by green vectors).
(
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The inhibitor occupies a binding site at the dimer interface in a similar region to that described
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previously for other allosteric inhibitors.
. This binding site is distinct from the substrate and
+
coꢀfactor binding sites, with the closest distance between 9 and NADP being approximately 14
Å. Notably, the packing of the two protomers differs significantly between our crystal structure
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and the structure of (R132H) IDH bound to αꢀketoglutarate (PDB 3INM ), with considerable
disruption of the helical dimer interface, in particular with the helix N271 to Y285 being
completely disordered.
The binding interactions around 9 are mainly lipophilic in nature, with the only direct protein
hydrogen bonds arising from an ionic interaction between the carboxylic acid of compound 9 and
Arg119. The central pyridine ring is piꢀstacked with the side chain of Tyr285. The pyridyl 2ꢀ
cyclohexylthio group occupies a discrete lipophilic pocket bounded by the residues, Trp124 and
Trp 267 of chain A, and Val255 and Met259 of chains A and B. The ꢀNHꢀ of the pyridine 3ꢀ
carboxamide potentially forms a hydrogen bond with the 2ꢀpyridyl sulfur atom, which may serve
to constrain the ligand conformation. While the sidechain of R132H is located close to the
substrate binding site in the αꢀketoglutarateꢀbound crystal structure 3INM (where it presumably
modulates the neomorphic catalytic activity), in our structure it has rotated approximately 5Å
away from the substrate site and towards compound 9. Although the 3ꢀcarboxamide carbonyl
group of 9 points towards R132H, it appears too distant for direct hydrogen bonding. However,
the recruitment of R132H to form part of the binding cavity, away from the catalytic site, offers
some explanation as to the loss of neomorphic activity in the presence of these compounds. The
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pendant hydroxy adamantyl group is sited in an area which is largely unresolved (the residues
Tyr272ꢀVal281 are missing in the electron density) giving only partial structural information.
The adamantyl moiety appears loosely bound although it makes several lipophilic contacts with
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A
B
B
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Ile251 , Ile251 , Val255 and Trp267 . The hydroxyl group is positioned close to the sidechain
of Asn271 but does not appear to make any direct hydrogen bonds.
Examination of the Xꢀray crystal structure suggested a number of avenues for the rapid
exploration of the SAR (Figure 5).
Figure 5. Potential opportunities for SAR exploration
Thus for Region 1 (R1), the potential for hydrogen bonding between the pyridyl carbonyl and the
protein could be explored, alongside attempts to increase potency by finding additional contacts
in the large pocket currently occupied by the hydroxy adamantyl group. The crystal structure
shows that the pocket occupied by the ꢀSꢀcyclohexyl moiety, Region 2 (R2), is a discrete
lipophilic pocket and is likely to respond to the precise nature and shape of any ‘small’
substituents. The effects of changes to the linkage could also be explored. Region 3 (R3) is
dominated by the charged interaction between the carboxylic acid and Arg119. However the
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linkage between the carboxylic acid and the pyridyl ring offers scope for exploration. It was
imagined that modification of each of the areas in the molecule in turn would give an
understanding of the component SAR which may allow a rationale for the design of molecules
with acceptable in vitro and in vivo DMPK properties, alongside improvements in selectivity
against 11βꢀHSD1.
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CHEMISTRY
A series of compounds allowing simple substitution at the 2ꢀposition of the central pyridine (R2)
were prepared with the 3ꢀcarboxamide function held as cyclohexyl and the 6ꢀcarboxylic acid
function was maintained as (S)ꢀ2ꢀ(piperidinꢀ3ꢀyl)acetic acid using the methods shown in Scheme
1.
Scheme 1. Route to R2 substituted compounds
Reagents and conditions: i. Cyclohexylamine, DIPEA, DCM, RT. ii. RXH, NaH/DMF RT, 22ꢀ99%, or
potassium tꢀbutoxide /THF RT, 38ꢀ99%. iii. Ethyl (S)ꢀ2ꢀ(piperidinꢀ3ꢀyl)acetate, DIPEA/PrCN, 110 °C,
23ꢀ99%. iv. NaOH/MeOH rtꢀ50 °C, 4ꢀ99%
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,6ꢀDichloronicotinoyl chloride was condensed with cyclohexylamine in dichloromethane
(DCM) in the presence of diisopropylethylamine (DIPEA) to give 2,6ꢀdichloroꢀNꢀ
cyclohexylnicotinamide 13 as a key intermediate. Displacement of the 2ꢀchloro group was
accomplished using the appropriate alcohols and either sodium hydride in DMF or potassium
tertꢀbutoxide in THF to give the 2ꢀOꢀsubstituted 6ꢀchloroꢀNꢀcyclohexyl nicotinamides 14aꢀaz.
The regiochemistry of this reaction was confirmed for R = ꢀOꢀisopentyl by removal of the
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
1
remaining chlorine atom by hydrogenation and examination of the H NMR aryl region for
characteristic coupling patterns. The 6ꢀchloro group was then displaced by ethyl (S)ꢀ2ꢀ(piperidinꢀ
3ꢀyl)acetate using potassium carbonate in butyronitrile at 110 °C to give the intermediate esters
15aꢀaz which were hydrolysed to the acid using aqueous sodium hydroxide in methanol to give
the carboxylic acids 16aꢀaz. The final ester hydrolysis step was frequently carried out on
unpurified isolated esters allowing expedient production of final compounds.
Compounds designed to explore the SAR around the 3ꢀcarboxamide group, R1, were prepared
either by the sequential route shown in Scheme 1 above (although steps were ‘telescoped’ by
taking crude isolated products into the next reaction and using preparative HPLC to isolate the
final product in a suitable purity) or using an alternative route which was developed to be more
efficient, as shown in Scheme 2.
Scheme 2. Route to R1 substituted compounds
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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: i. Sodium hydride 60% in mineral oil, THF/DCM, 3ꢀmethyl butanꢀ2ꢀol, RT,
73%. ii. DIPEA/PrCN 110 °C, 42ꢀ75%. iii. Aq NaOH, methanol or dioxane.
6ꢀChloroꢀ2ꢀ(isopentyloxy)nicotinic acid 17 was prepared by reaction of 2,6ꢀdichloronicotinic
acid with isopentanol, (confirmation of the regiochemistry of the alkoxide addition was again
obtained by removal of the remaining chlorine atom by hydrogenolysis and examination of the
1
H NMR aryl region for characteristic coupling patterns). Condensation of the aminoꢀester ethyl
(1R,5S,6R)ꢀ3ꢀazabicyclo[3.1.0]hexaneꢀ6ꢀcarboxylate (an alternative aminoꢀacid function that
had been identified in the original hit set) using conditions previously described followed by
hydrolysis of the ester with sodium hydroxide gave the target compounds 20aꢀae. The latter two
steps were combined without purification of the intermediate ester product.
Compounds bearing alternative carboxylic acid groups were prepared either using similar
methods as described above (21 – 23) or by using Suzuki or Buchwald–Hartwig chemistry (24 –
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2
3
4
5
6
7
8
9
2
8). (For these examples particular care was taken to remove residual palladium from final
compounds to avoid apparent activity due to metal contamination).
Reverse carboxamide compounds 39a – 39c were prepared from the appropriately substituted
aminopyridine compound 37 as shown in Scheme 3 using cyclohexane carboxylic acid chloride,
cyclohexylisocyanate and cyclohexane sulfonyl chloride as electrophiles.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Scheme 3. Route to 3ꢀamino substituted compounds
Reagents: i. 3ꢀMethyl butanol, NaH, THF, rt, 93%. ii. Amine, DIPEA, butyronitrile, 100 °C, 75%. iii. Fe,
NH Cl, aq. ethanol, reflux, 74%. iv. Acyl chloride, sulfonyl chloride or isocyanate, rt, 73ꢀ86%. v. Aq
4
NaOH in methanol, rt, 38ꢀ85%.
Thus, sequential displacements of halogen from 2,5ꢀdichloroꢀ3ꢀnitropyridine by isopentoxide
and ethyl (1R,5S,6r)ꢀ3ꢀazabicyclo[3.1.0]hexaneꢀ6ꢀcarboxylate followed by reduction to the
amine gave intermediate 37 which was then substituted further before hydrolysis to the
carboxylic acid.
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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
Sulfonamide replacement compound 43 was prepared from 2,6ꢀdichloropyridineꢀ3ꢀsulfonyl
chloride as shown in Scheme 4.
Scheme 4. Route to 3ꢀsulfonamide compound 43.
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: i. Amine, DIPEA, THF/DCM 39%. ii. 3ꢀmethyl butanol, potassium tꢀbutoxide, THF, 85%. iii.
Amine, DIPEA, butyronitrile, 70%. iv. Aq NaOH in methanol, 18%.
Reaction of the commercially available 2,6ꢀdichloropyridineꢀ3ꢀsulfonyl chloride with 4ꢀ
aminoadamantanꢀ1ꢀol to give 40 followed by sequential displacements of halogen by 3ꢀmethyl
butoxide and ethyl (1R,5S,6R)ꢀ3ꢀazabicyclo[3.1.0]hexaneꢀ6ꢀcarboxylate followed by hydrolysis
to the carboxylic acid gave compound 43.
The desꢀcarbonyl compound 47 was prepared by the route shown in Scheme 5 below.
Scheme 5 Synthesis of ‘desꢀcarbonyl’ compound 47.
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9
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16
17
18
19
20
21
22
23
24
25
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27
28
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30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Reagents: i. Boraneꢀtetrahydrofuran complex 1M in THF 80%. ii DessꢀMartin periodinane, 15 wt.% in
DCM, 65%. iii. Amine, DIPEA, butyronitrile, 80%. iv. a. 4ꢀAminoꢀ1ꢀadamantanol, sodium borohydride,
methanol. b. NaOH, aq. Methanol, 4%.
Compound 17 was reduced to alcohol 44 and then oxidised to give aldehyde 45 which was
reacted with ethyl (1R,5S,6R)ꢀ3ꢀazabicyclo[3.1.0]hexaneꢀ6ꢀcarboxylate followed by addition of
the hydroxyadamantyl amine by reductive amination and then hydrolysis of the ester to the
carboxylic acid to give compound 47, albeit with poor recovery after preparative HPLC
purification.
Results.
Region R2. Compounds 16a ꢀ 16az with differing substituents in the core pyridine 2ꢀposition,
R2, were intended to explore the topology and the nature of the small lipophilic pocket identified
in the crystal structure of compound 3. These compounds were screened in the IDH1 (R132H)
biochemical assay, the results of which are shown in Table 2.
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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 2: Biochemical screening data for R2 aliphatic substituents.
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
a
IC µM
IC µM
5
0
50
2
2
R
R
(SD)
(SD)
1
2
6.4 (1.9)
8.0 (0.95)
7.6 (2.4)
16m
16n
16o
16 (4.4)
26 (12.7)
14 (7.5)
1
6a
16b
6c
6d
6e
16f
1
9.7 (4.4)
7.2 (3.2)
16p
16q
20 (6.9)
30 (3.3)
1
1
12 (2.1)
12 (2.7)
12 (3.1)
15 (0.47)
12 (5.1)
16r
16s
16t
20 (12.8)
>50
1
1
6g
6h
>50
16u
16v
>50
1
1
6i
6j
>50
14 (2.7)
14 (4.7)
12 (5.9)
16w
16x
16y
>50
>50
>50
16k
16l
a
IC s are mean values of a minimum of 3 replicates. Standard deviations are listed in
5
0
parentheses.
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The introduction of a heteroatom (i.e. N or O) to the substituent was poorly tolerated, as
exemplified by the methyl ether compound 16s, pyranyl compound 16t and dimethylamine
compound 16w, which were all inactive (IC > 50 µM). These results were consistent with the
5
0
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Xꢀray crystallographic data from the original hit, which showed that the alkyl group at the 2ꢀ
position occupies a lipophilic pocket devoid of any potential polar interactions. Reducing the
length of the Oꢀlinked alkyl chain at the 2ꢀposition to an isobutyl 16n, ethyl 16u or methyl group
16v all led to a 3ꢀfold to >7ꢀfold drop off in potency. Given the potencies observed for similarly
sized moieties at this position, these data (and the data for 16x and 16y) are rather surprising and
remain unexplained.
2
Attempts to reduce the conformational flexibility of the 2ꢀsubstituent by introducing sp
character or a small ring, compounds 16a, 16d, 16j, 16p and 16q, all led to a decrease in the
potency compared with compound 12. The introduction of a 1,1,1ꢀtrifluorobutoxy group 16h
resulted in a 2ꢀfold decrease in the activity towards IDH1 (R132H) compared with an isopentyl
group, roughly equipotent with the butoxyl compound 16g. All this provided confirmation that
the binding pocket of the IDH1 (R132H) enzyme preferred lipophilic, nonꢀpolar groups at the 2ꢀ
position of the pyridine ring and that from the examples prepared, the isopentyl moiety was
optimal.
The introduction of a variety of different substituted Oꢀlinked benzyl groups at this position
bearing electronꢀdonating or electronꢀwithdrawing substituents are shown in Table 3.
Unsubstituted benzyl compound 16z gave an IC of 8.6 µM, representing a marginal decrease in
5
0
the potency compared with the isopentyl group at this position, 12.
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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
The addition of an electronꢀdonating methyl group to the phenyl ring of the benzyl group had a
significant impact on the activity of these compounds. For example, the orthoꢀmethyl, compound
16aa and metaꢀsubstituted benzyl, compound 16ab, compounds led to a twoꢀfold increase in the
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
potency compared with the unsubstituted system compound 9z. However, the corresponding
paraꢀsubstituted compound 16ac was inactive against IDH1 (R132H). Methoxyꢀsubstituted
benzyl systems, however, gave a marginal loss in potency for orthoꢀ and metaꢀsubstituted benzyl
groups, compounds 16ad and 16ae while the paraꢀmethoxy substituted benzyl compound 16af
was inactive. Interestingly, 16ad and 16ae contrast strongly with 16s and 16t, where an oxygen
atom in a similar spatial location is clearly not tolerated, perhaps highlighting a requirement for
addionla steric bulk or lipophilic interactions. The introduction of electronꢀwithdrawing
substituents onto the phenyl ring of the benzyl group was also investigated. In contrast to the
results observed for the electronꢀdonating substituents, the introduction of a fluoro or chloro
substituent was tolerated in all positions of the phenyl ring showing only a marginal increase in
potency for the ortho fluoro compound 16ag and equivalent potency for fluorine in the mꢀ and pꢀ
positions and chlorine in all positions.
Table 3. IDH1 (R132H) enzyme assay data for R2 substitutions.
a
a
IC µM
IC µM
50
50
R
R
(SD)
(SD)
1
2
6.4 (1.9)
8.6 (3.7)
16ai
16aj
8.8 (3.6)
7.0 (1.8)
16z
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a
a
IC µM
IC µM
5
0
50
R
R
(SD)
(SD)
1
6aa
4.5 (1.3)
16ak
16al
9.9 (11)
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
16ab
4.6 (0.08)
7.7 (0.49)
1
6ac
>50
16am
16an
43 (4.7)
16ad
16ae
13 (0.37)
5.9 (0.87)
12 (0.65)
>50
16ao
16ap
11 (1.1)
5.8 (1.0)
1
6af
16ag
5.6 (0.32)
10 (3.1)
16aq
38 (7.1)
16ah
a
IC s are mean values of a minimum of 3 replicates. Standard deviations are listed in
5
0
parentheses.
The introduction of a methyl group at the αꢀbenzylic position of the unsubstituted benzyl was
investigated, with the (R)ꢀenantiomer, compound 16an, showing a small increase in potency
compared with the unsubstituted system; conversely the (S)ꢀenantiomer, compound 16ao showed
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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
a small loss in potency. Extending from benzyl to phenethyl (16am) gave a significant decrease
in the potency, consistent with the small size of the binding pocket.
Taken together, these data show that the introduction of an orthoꢀmethyl substituted benzyl
system at the 2ꢀposition of the pyridine ring was the optimal benyl moiety, albeit delivering only
a marginal increase in potency at best, compared with the isopentyl substituent (4.5 vs. 6.4 µM).
Combination of the orthoꢀmethyl group and an αꢀmethyl group compound 16ap showed no
advantage over the individual substitutions whilst combination of the orthoꢀmethyl group and a
paraꢀchloro group compounds 16aq was detrimental.
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
In order to understand the influence of linkers other than oxygen on the SAR, a small set of
matched pairs were prepared in a similar manner to that shown in Scheme 1, using the
appropriate starting materials with O, N and S linker atoms. Full details of synthesis are given in
the experimental section.
Table 4. IDH1 (R132H) enzyme assay data IC (µM) for R2 and X substitutions
5
0
X
O
S
N
IC50
a
a
IC50 µM
IC50 µM
(SD)
a
µM
(SD)
(SD)
12
6.4 (1.9)
16ar
10 (1.3)
16av
>50
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6
7
8
9
16e
12 (2.1)
9.7 (4.4)
16aw
16ax
>50
>50
1
1
6c
6z
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
8.6 (3.7)
4.5 (1.3)
16as
16at
39 (9.8)
>50
16ay
16az
>50
>50
16aa
16ag 5.6 (0.32) 16au
21 (5.4)
a
IC s are mean values of a minimum of 3 replicates. Standard deviations are listed in
50
parentheses.
Comparison of the IDH1 (R132H) activities of the Oꢀisopentyl compound 12 and Sꢀisopentyl
compound 16ar showed only a small difference in potencies (Table 4). However, it was apparent
that in all other examples that the Sꢀlinkage was inferior to Oꢀlinkage and that the Nꢀlinkage was
not tolerated at all, perhaps as a result of the change from an Hꢀbond acceptor to an Hꢀbond
donor.
Summarising SAR in this region, having investigated a wide variety of small lipophilic groups in
the R2 position, ꢀOꢀisopentyl and ꢀOꢀorthoꢀmethylbenzyl were found to be the preferred
substituents for this vector.
Region R1. Compounds 20a – 20af with differing substituents on the pyridine 3ꢀcarboxamide,
R1, were intended to explore topology and the nature of the more open and less well defined
pocket identified in the crystal structure of compound 9. The results of the investigation can be
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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
seen in Table 5, all new compounds were compared against the R1 cyclohexyl carboxamide
compound 20ab, enzyme IC 7.9µM.
50
Aliphatic and cycloaliphatic groups, with or without the addition of hetero atoms or single bridge
structures, failed to offer any increase in potency against IDH1 (R132H) except for 20l. Direct
aryl attachments gave a range of inhibitory activities from unsubstituted phenyl (>50 µM) (not
shown in Table 5) to 20f (2.9 µM), compounds with a hydroxyl pendent group (direct or
indirect) being the most potent suggesting the possibility of a positive interaction between the
hydroxyl group and the protein structure.
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
Benzyl carboxamides gave a range of activities. The baseline benzyl substituent group 20y was
marginally improved upon by simple monosubstitutions and modest (up to 2ꢀfold) improvements
were obtained from the Rꢀαꢀmethyl compound 20t and the Sꢀαꢀmethyl compound 20k.
Extending the aryl group further with R1 as phenethyl, 20i, gave an additional small increase in
enzyme potency.
Table 5. IDH1 (R132H) enzyme assay data (µM) for R1 substitutions.
a
a
IC µM
IC µM
5
0
50
R1
R1
(SD)
(SD)
12
6.4 (1.9)
20p
20q
5.0 (0.74)
20a
0.27 (0.22)
12 (13)
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7
8
9
20b
19a
>50
20r
20s
5.6 (0.95)
6.0 (0.94)
13.5 (11)
2
R = Et
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
2
0c
0d
0e
0.27 (0.14)
0.23 (0.02)
20t
5.4 (1.9)
6.2 (1.1)
2
20u
2
0.86 (0.33)
20v
6.3 (1.3)
20f
20g
20h
20i
2.9 (0.09)
3.1 (0.44)
3.0 (0.46)
20w
20x
20y
6.5 (2.0)
6.6 (1.8)
9.4 (1.6)
3.9 (0.70)
3.2 (0.67)
4.4 (0.51)
20z
20aa
20ab
8.2 (2.2)
7.4 (3.4)
7.9 (3.5)
2
0j
0k
0l
2
2
3.9 (0.69)
4.6 (0.54)
20ac
20ad
17 (4.5)
14 (8.6)
20m
2
2
0n
0o
5.6 (1.7)
20ae
20af
5.6 (0.95)
17 (11)
5.4 (0.26)
a
IC s are mean values of a minimum of 3 replicates. Standard deviations are listed in
5
0
2
parentheses. R =H in all cases except where stated otherwise (20??)
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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
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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
The most significant improvements in IDH1 (R132H) inhibition came from the use of bulkier
structures. Although the adamantyl carboxamide 20b was essentially inactive (>50 µM),
substitution of the adamantyl group in the 1ꢀposition with hydroxyl 20a or methoxy 20c gave
compounds with subꢀmicromolar activity. Extending the hydroxyadamantyl group further from
the carboxamide link with a methyl substituted methylene 20d gave a similar subꢀmicromolar
level of activity whilst the addition of a difluoromethoxy group 20e gave a noticeable decrease in
activity, albeit still at a submicromolar level.
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
Region R3. Simple replacement of the carboxylic acid group of 20b by ethyl ester (19a)
dramatically reduced activity, as might be expected by elimination of the key interaction with
Arg119. Although a number of alternative carboxylic acid scaffolds were tolerated, it was noted
that the scaffolds in 20b, 21 and 22 were the most effective (Table 6). One exception to this trend
was the thiazole carboxylic acid 26 which displayed submicromolar potency, although this is still
some threeꢀ fold less active than the most potent compound 20a.
Table 6. IDH1 (R132H) enzyme assay data (µM) for R3 substitutions.
3
a
R
IC µM
50
(SD)
2
0a
0.27 (0.22)
1.3 (0.33)
2
1
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8
9
22
1.2 (1.3)
2
2
2
3
4
5
3.4 (1.1)
3.0 (6.5)
11 (2.4)
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
2
2
6
7
21 (6.7)
0.81 (0.32)
28
>50
a
IC s are mean values of a minimum of 3 replicates. Standard deviations are listed in
5
0
parentheses.
With this information to hand, a set of compounds were then synthesized combining the more
potent R2 and R3 components with the hydroxyadamantyl carboxamide R1 group to examine the
additive nature of the group SAR.
Table 7. Matched pair combinations of R2 and R3 substitutions, IDH1 R132H IC µM.
5
0
R3
a
a
IC µM (SD)
IC µM (SD)
5
0
50
2
0a 0.27 (0.22)
21 1.2 (0.33)
32 2.3 (0.79)
2
9 27 (15)
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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
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5
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5
5
5
5
5
6
30 4.5 (4.7)
1 3.2 (2.0)
IC s are mean values of a minimum of 3 replicates. Standard deviations are listed in
33 0.69 (0.30)
3
34 2.3 (0.39)
a
5
0
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
parentheses.
It is evident from the results in Table 7 that the component SAR is not truly additive and that the
overall SAR is dominated by the R3 carboxylic acid group. It is logical to suggest that the rigid
nature of the azaꢀbicyclohexane acid, which gave rise to the most potent inhibitors with a small
subset of R1 and R2 groups, would be less tolerant of alternative R2 substitutions than the more
flexible (and accommodating) piperidine acetic acid.
The Xꢀray crystal structure obtained for compound 9 indicated that there may be some potential
for hydrogen bonding between the pyridine carboxamide ꢀNHꢀ with Tyr285 and ꢀCOꢀ with
His132, in this latter case via water mediation. In order to understand further the importance of
the role of the 3ꢀcarboxamido group, compounds were prepared modifying the nature of the 3ꢀ
carboxamide group by substitution, by reversal of the carboxamide or by replacement with other
groups.
A comparison of benzyl carboxamide compound 20y and cyclohexyl carboxamide compound
2
0ab with the Nꢀmethyl analogues, 20aa and 20ac shows either no change or a small loss of
potency on replacement of the carboxamide –NH– by –NMe–, suggesting that either any
potential internal hydrogen bond to the pyridyl 2ꢀoxygen atom added little to the potency of the
compounds or the increased rotational freedom of the amide bond is beneficial.
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9
Replacement of the pyridine carboxamide with the reversed carboxamide, 39a or urea 39b
resulted in complete loss of activity, as did direct replacement of the carbonyl group with a
sulfonamide 43 (Figure 6). Further, reduction of the carbonyl group completely to yield 47,
resulted in a complete loss of activity suggesting that the carbonyl group has a role in hydrogen
bonding and/or rotational stability. Only the reverse sulfonamide 39c (8.6 µM) was found to
have a similar potency when compared to 20ab. Here, perhaps, the differing geometry allows a
strong hydrogen bond with His132 or reorientates the R1 lipophilic group improving contact
with the enzyme, presenting an opportunity for further study.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
a
Figure 6. IDH1 IC results for carboxamide replacements. IC s are mean values of a minimum
5
0
50
of 3 replicates. Standard deviations are listed in parentheses.
Having established that compound 20a was a potent compound against IDH1 (R132H) in this
series we chose to reꢀexamine selectivity against other related enzymes (Table 8).
Whilst retaining some activity against an alternative mutant IDH1 (R132C), (0.47 ꢁM),
compound 20a was inactive against both wild type IDH1 and IDH2 and glucoseꢀ6ꢀphosphate
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