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Development of 5-Hydroxypyrazole Derivatives as Reversible Inhibitors of Lysine Specific
Demethylase 1
Daniel P. Moulda, Ulf Brembergc, Allan M. Jordana, Matthis Geitmannc, Alba Maiques-Diazb, Alison
E. McGonaglea, Helen F. Smalla, Tim C. P. Somervailleb, and Donald Ogilviea
aDrug Discovery Unit, Cancer Research UK Manchester Institute, University of Manchester,
Wilmslow Road, Manchester, M20 4BX, UK
bLeukaemia Biology Group, Cancer Research UK Manchester Institute, University of Manchester,
Wilmslow Road, Manchester, M20 4BX, UK
cBeactica AB, Uppsala Business Park, Virdings allé 2, 75450, Uppsala, SE
ABSTRACT
A series of reversible inhibitors of lysine specific demethylase 1 (LSD1) with a 5-hydroxypyrazole
scaffold have been developed from compound 7, which was identified from the patent literature.
Surface plasmon resonance (SPR) and biochemical analysis showed it to be a reversible LSD1
inhibitor with an IC50 value of 0.23 µM. Optimisation of this compound by rational design afforded
compounds with Kd values of <10 nM. In human THP-1 cells, these compounds were found to
upregulate the expression of the surrogate cellular biomarker CD86. Compound 11p was found to
have moderate oral bioavailability in mice suggesting its potential for use as an in vivo tool
compound.
KEYWORDS:
epigenetics; LSD1; KDM1A; reversible inhibitor; stem cell differentiation; cancer therapy; epigenetic
therapy; acute myeloid leukaemia
1
The many functions of the histone demethylase lysine specific demethylase 1 (LSD1) have unravelled
over the past decade to reveal a complex network of interactions with a number of protein
complexes,1, 2 transcription factors,3-5 and nucleosomes6 in a multitude of cell types. In cancer, Harris
and co-workers found that LSD1 plays a key function in maintaining the oncogenic potential of acute
myeloid leukaemia (AML) cell lines by preventing differentiation.7 Subsequently, research has shown
LSD1 expression to be linked to a number of cancers, most notably small cell lung cancer (SCLC). 8
The development of irreversible inhibitors of LSD1 from the monoamine oxidase (MAO) inhibitor
tranylcypromine (TCP) has been well documented,9 and several compounds are now in clinical trials
for AML and SCLC, either as a monotherapy, or in combination with other pro-differentiation agents
such as all-trans retinoic acid (ATRA).10 Results from a Phase I study of AML patients with resistant
or refractory disease showed that a blast cell differentiation effect was observed in the majority of
treated patients.11
Commercial interest in the development of irreversible TCP LSD1 inhibitors has increased rapidly in
the past decade. Despite this, the rate of optimisation of tranylcypromine derivatives has not been
matched by reversible inhibitors, as evidenced by the paucity of clinical trials involving reversible
agents.12 The large size and polarity of the LSD1 active site presents significant challenges to drug
discovery. However, in the past two years, the patent literature has seen a convergence towards
optimisation of derivatives of GSK-690 (also known as GSK-354),13 a potent reversible LSD1
inhibitor compromised by a significant hERG liability.
2
Figure 1. Structure of GSK-690 (1) and examples of recently disclosed reversible inhibitors of LSD1
from the patent literature (2–7).14-19
Various bicyclic and monocyclic scaffold-hops have been disclosed (Figure 1) in numerous patents.
We became interested in a hydroxypyrazole scaffold template disclosed, but not claimed in a patent
from Quanticel Pharmaceuticals.19 The replacement of the tolyl- (1) or other aryl or heteroaryl- groups
(2–6) with an ether linkage offers a new vector to investigate. Whilst, the patent only disclosed three
pyridylmethyl isomers, the 2-pyridyl isomer (7) was preferred, and was reported to have activity of
<100 nM in biochemical assays. This compound was resynthesised via the disclosed procedure, and
profiled in our biochemical time-resolved fluorescence resonance energy transfer (TR-FRET) assay
against LSD1 where it displayed an IC50 value of 0.23 µM (SD 0.08 µM). Surface plasmon resonance
(SPR) analysis indicated 7 was a reversible inhibitors of LSD1 with a Kd value of 0.042 µM. It also
displayed activity in our previously reported CD86 cellular biomarker assay (EC50 = 2.3 µM).20 These
values suggested 7 was an attractive start point for further development.
3
Scheme 1. Synthesis of compounds 11a–q. Reagents and conditions: (a) oxaloacetic acid, acetic acid,
reflux, 1 h, 93%; (b) COMU, (R)-3-Boc-aminopiperidine, DIPEA, DMF, 30 min, 84%; (c) R-X,
K2CO3, DMF, 100 °C, 1h, then 4M HCl/dioxane, rt, 1 h, 24–71%.
To explore the SAR around the ether functionality, we synthesised hydroxypyrazole 9 from 4-
cyanophenylhydrazine (8) via reaction with oxaloacetic acid (Scheme 1). The amide was then
installed under standard coupling conditions to give Boc-protected amide 10. The hydroxypyrazole
could be alkylated under basic conditions with a variety of alkylating agents, and then deprotected
under acidic conditions to afford compounds 11a–q. From the limited SAR disclosed in the patent,
we envisaged that the pyridyl nitrogen may be acting as a H-bond acceptor, hence we focused on
replacement of the pyridyl with groups containing functionalities which have hydrogen-bonding
groups analogous to the pyridyl nitrogen (Table 1). While the primary amide 11a had only modest
affinity for LSD1 by SPR, methylation at either carbon (11b) or nitrogen (11c) resulted in a
significant improvement in potency in both assay formats, with compound 11c achieving sub-
micromolar affinity by SPR. Extending the dimethylamide further to a morpholine (11d) improved
potency as well as resulting in decreased lipophilicity. More promising were alcohols 11g–i.
Compound 11h achieved a biochemical IC50 of 50 nM and a Kd value of 6 nM (Figure 2), a significant
improvement over 7, and one of the most potent reversible inhibitors of LSD1 reported to date. The
acidifying effect of the trifluoromethyl group appears important, giving a 32-fold improvement in
potency over 11g. These compounds demonstrated that a variety of alcohol side chains could be
tolerated. Finally, we incorporated a number of different 5-membered heterocyclic groups to
investigate how the presence of different heteroatoms in varying positions would affect the activity
4
(11i–q). Most notably, methylisoxazole 11o displayed excellent affinity for LSD1 by SPR, with the
extension of the methyl group to ethyl (11p) and isopropyl (11q) also well tolerated.
Table 1a
Compound R IC50 (µM) SPR Kd
(µM)
11a >150 17.7
11b 16.0 (0.9) 3.9
11c 2.8 (0.15) 0.76
11d 1.5 (0.35) 0.21
11e 4.3 (1.2) 0.45
11f 6.3 (0.7) 1.7
11g 1.6 (0.2) 0.13
11h
CF3
OH0.050 (0.01) 0.006
11i 0.27 (0.06) 0.022
11j 0.41 (0.02) 0.066
11k 2.6 (0.54) 0.32
11l 0.75 (0.13) 0.12
5
NN
N
O
N
OH2N
R
NH2
O
NH2
O
N
O
N
O
O
HN
O
OH
OH
NN
N
NN
N
N N
11m 0.19 (0.03) 0.034
11n 1.2 (0.2) 0.33
11o 0.15 (0.09) 0.007
11p 0.079 (0.024) 0.021
11q 0.083 (0.034) 0.017
aIC50 and Kd values for selected compounds against the LSD1 enzyme in biochemical and biophysical
assays. Standard deviation given in parentheses. IC50 determined from 10 point concentration/effect
experiments. Geometric mean of at least two independent experimental determinations given.
Figure 2. Surface plasmon resonance sensorgrams of the interaction between LSD1 and selected
reversible inhibitors in two-fold dilution series (highest concentration indicated in the graph).
6
N
NO
O
N N
O N
O N
O N
In an attempt to account for the observed SAR, we performed docking studies using the previously
reported structure of LSD1 bound to the reversible ligand tetrahydrofolate (PDB accession code
4KUM).21 Protein preparation and docking were performed in Glide (Schrödinger, New York,
USA).22 Previous disclosure of the crystal structure of compound 1 bound to LSD1 has shown that the
nitrile displaces a bridging water molecule between K661 and FAD, and that the basic centre is
directed towards a pair of adjacent aspartate residues, Asp555 and Asp556. Docking studies of
compound 7 identified a binding mode in which the pyridyl moiety makes an additional hydrogen
bonding interaction with Asn535 (Figure 3). Another credible binding pose suggested that the
flexibility of the ether link allows this group to be directed towards Gln358, achieving a similar
hydrogen bonding interaction as the first binding mode. Compounds 11h and 11o displayed similar
predicted binding modes, with the possibility of additional hydrogen bonding interactions from the
ether oxygen to His564.
7
Figure 3. Predicted binding modes of compounds 7 (green carbons), 11h (yellow carbons) and 11o
(magenta carbons) in the LSD1 active site (4KUM). Visualised in PyMOL. FAD truncated for clarity.
11o 11h 11p
Caco2 A–B mean Papp (10-6
cm/s); (efflux ratio) 0.73 (12) <0.1 0.415 (111)
Hu Mic CLint (μL/min/106 cells) 6.91 5.6 5.6
Hu Mic T½ (min) 201 250 250
Mo Hepatocytes T½ (min) 68 ND 121
Table 2. In vitro DMPK properties for selected compounds. ND – not determined
In vitro DMPK experiments (Table 2) identified a potential liability in the Caco-2 membrane
permeability assay, with high levels of efflux observed, or in the case of compound 11f, levels of
permeability below detectable levels. This has also been previously observed in series of
hydroxypyrazole compounds.23
To investigate if we could improve the DMPK properties, we looked to alter the basic centre while
retaining the methyl-isoxazole functional group. These compounds were synthesised in four steps
from hydrazine 8 (Scheme 2). Cycloaddition of 8 with diethyl acetylenedicarboxylate afforded
hydroxypyrazole 12. This was alkylated with 5-(bromomethyl)-3-methylisoxazole under basic
conditions to afford ether 13. Ester hydrolysis with lithium hydroxide afforded acid 14, which was
reacted with various di-amines (Boc-protected where necessary) under standard amide coupling
conditions, then deprotected where necessary to afford compounds 15a–m (Table 3).
The stereoisomer of compound 11o (15a) was roughly equipotent by biochemical assay. Methylation
of the basic centre was slightly deleterious to activity. Interestingly, the two enantiomers of the 3-
aminopyrollidine derivatives 15i and 15j showed a significant disparity in activity, with the S-
enantiomer strongly favoured. To see how modification of the pKa of the amine would affect the
activity, we synthesised 15k with a fluorine beta- to the basic centre, however this was poorly
8
tolerated. Overall, there appeared to be little scope to optimise the basic centre to further improve
potency.
Scheme 2. Reagents and conditions: (a) diethyl acetylenedicarboxylate, K2CO3, ethanol, reflux, 16 h,
79%, (b) 5-(bromomethyl)-3-methylisoxazole, K2CO3, DMF, 80 °C, 4 h, 65%; (c) LiOH, MeOH,
H2O, 50 °C, 30 min, 80%; (d) COMU, appropriate amine, DIPEA, DMF, 1 h, rt, then 4M
HCl/dioxane (if required), 20–74%.
Table 3a
Compound R IC50 (µM) SPR KD
(µM)
15a 0.20 (0.04) 0.032
15b 0.34 (0.03) 0.042
15c 0.65 (0.21) 0.087
9
15d 0.67 (0.08) 0.099
15e 0.72 (0.08) 0.14
15f 1.1 (0.3) 0.24
15g 1.2 (0.2) 0.23
15h 0.22 (0.04) 0.037
15i 0.17 (0.04) 0.013
15j 1.4 (0.1) 0.17
15k 2.6 (0.4) 0.11
15l 0.51 (0.07) 0.097
15m 0.71 (0) 0.24
aIC50 and Kd values for selected compounds against the LSD1 enzyme in biochemical and biophysical assays. Standard deviation given in parentheses. IC50 determined from 10 point concentration/effect experiments. Geometric mean of at least two independent experimental determinations given.
Compound CD86 (µM)
7 2.3 (0.41)
11o 1.9 (0.18)
11m 0.42 (0.07)
11h 0.48 (0.08)
11p 0.52 (0.09)
Table 4. IC50 values for selected compounds in CD86 expression based cellular assays. IC50
determined from 10 point concentration/effect experiments. Geometric mean of at least three independent experimental determinations given.
10
The most active compounds were taken forwards into previously described cellular assays.12
Upregulation of the cell surface protein CD86 can be used as a reliable surrogate cellular biomarker of
LSD1 inhibition, and is superior to other cell assays such as global histone methylation levels, which
should not be trusted as an indication of LSD1 inhibition in a cellular context. Here, the optimised
derivatives showed improved activity over compound 7, with the biochemical activity correlating
well, albeit with a significant, but consistent, ~5-fold drop-off in activity in the cellular assay.
Figure 4. In vivo DMPK properties of compound 11p.
Compound 11p was subjected to in vivo pharmacokinetic assessment using serial microsampling,24, 25
to determine whether the high efflux ratio and low permeability observed would transfer into low
bioavailability in mice. We were relieved to find that 11p was moderately bioavailable and only
moderately cleared with a half-life of around 1 h in both the i.v. and p.o. dosed mice (Figure 4).
Unfortunately, when tested against the hERG ion channel in a patch clamp assay this compound was
found to have an IC50 of ~5 µM. While this was an improvement over compound 1 (IC50 = 3 µM), it
still represented a significant liability that must be overcome when developing LSD1 inhibitors that
contain basic moieties adjacent to an aryl or heteroaryl core, especially when developing a rigid
aromatic scaffold such as hydroxypyrazoles or other recently patented mono- and bicyclic scaffold
modifications of 1.
In summary, we have described herein our development of a series of reversible 5-hydroxypyrazole
based inhibitors of LSD1. These compounds are highly potent by biochemical assay and SPR, and
have been shown to induce differentiation of THP-1 cells at sub-micromolar concentrations in cellular
11
assays. Compound 11p confounded in vitro DMPK experiments that indicated low levels of
permeability and high efflux by displaying moderate levels of bioavailability and reasonable half-life
in mice. These compounds are some of the most potent cell-active LSD1 inhibitors described to date
with the potential for oral bioavailability, and should enable further research in the area.
Acknowledgments
This work was supported by Cancer Research UK (Grants C5759/A12328, C480/A11411,
C5759/A17098 and C5759/A02901). Additional support was provided to DPM by the Society of
Chemical Industry through a Messel Scholarship. We are thankful to Bohdan Waszkowycz for his
assistance with Glide Docking. In vitro pharmacokinetic and hERG data was provided by Cyprotex
Discovery (Macclesfield, UK). Images of protein-ligand docking were captured within the PyMOL
Molecular Graphics System, Version 1.7.6.2. (Schrödinger, LLC, New York).
References and Notes
Tim Somervaille has ongoing research collaborations with Oryzon Genomics and consults for Imago
Biosciences. The other authors declare no competing financial interest.
1. Shi Y-J, Matson C, Lan F, Iwase S, Baba T, Shi Y. Mol Cell. 2005;19(6): 857-864.
2. Metzger E, Wissmann M, Yin N, et al. Nature. 2005;437(7057): 436-439.
3. Liu L, Souto J, Liao W, et al, J. Bio. Chem. 2013;288(48): 34719–34728.
4. Thambyrajah R, Mazan M, Patel R, et al. Nat. Cell. Biol. 2016;18(1): 21-32.
5. Saleque S, Kim J, Rooke HM, Orkin SH. Mol. Cell. 2007;27(4): 562-572.
6. Pilotto S, Speranzini V, Tortorici M, et al. Proc. Natl. Acad. Sci. U.S.A. 2015;112(9): 2752–
2757.
12
7. Harris WJ, Huang X, Lynch JT, et al. Cancer Cell. 2012;21(4): 473–487.
8. Mohammad HP, Smitheman KN, Kamat CD, et al. Cancer Cell. 2015;28(1): 57-69.
9. Hojfeldt JW, Agger K, Helin K. Nat. Rev. Drug Discov. 2013;12(12): 917-930.
10. Schenk T, Chen WC, Gollner S, et al. Nat. Med. 2012;18(4): 605-611.
11. Somervaille T, Salamero O, Montesinos P, et al. Blood. 2016;128(22): 4060.
12. Mould DP, McGonagle AE, Wiseman DH, Williams EL, Jordan AM. Med. Res. Rev.
2015;35(3): 586-618.
13. Hitchin JR, Blagg J, Burke R, et al. Development and evaluation of selective, reversible
LSD1 inhibitors derived from fragments. MedChemComm. 2013;4(11): 1513-1522.
14. Chen YK, Kanouni T, Nie Z, Stafford JA, Veal JM, Sung LM. WO2016004105. Cellgene
Quanticel Research Inc.; 2016.
15. Chen YK, Kanouni T, Nie Z, Stafford JA, Veal JM, Sung LM. WO2016003917. Cellgene
Quanticel Research Inc.; 2016.
16. Liangxing W, Xiaozhao W, Wenqing Y, Zhang C. US20160009711. Incyte Corporation;
2016.
17. Liangxing W, Konkol LC, Lajkiewicz N, et al. US20160009712. Incyte Corporation; 2016.
18. Liangxing W, Courter JR, Chunhong H, et al. US20160009720. Incyte Corporation; 2016.
19. Chen YK, Kanouni T, Kaldor SW, Stafford JA, Veal JM. Cellgene Quanticel Research Inc.;
WO2015089192.
20. Lynch JT, Cockerill MJ, Hitchin JR, Wiseman DH, Somervaille TCP. Anal. Biochem.
2013;442(1): 104-106.
13
21. Luka Z, Pakhomova S, Loukachevitch LV, Calcutt MW, Newcomer ME, Wagner C. Protein
Sci. 2014;23(7): 993-998.
22. Friesner RA, Banks JL, Murphy RB, et al. J. Med. Chem. 2004;47(7): 1739-1749.
23. Cadieux JA, Zhang Z, Mattice M, et al. Bioorg. Med. Chem. Lett. 2012;22(1): 90-95.
24. Li C, Liu B, Chang J, et al.. Drug Discov. Today. 2013;18(1–2): 71-78.
25. Liu B, Chang J, Gordon WP, Isbell J, Zhou Y, Tuntland T. Drug Discov. Today. 2008;13(7–
8): 360-367.
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