Optimization of a Series of Bivalent Triazolopyridazine Based Bromodomain and Extraterminal Inhibitors: The Discovery of (3R)-4-[2-[4-[1-(3-Methoxy-[1,2,4]triazolo[4,3-b]pyridazin-6-yl)-4-piperidyl]phenoxy]ethyl]-1,3-dimethyl-piperazin-2-one (AZD5153)

Article abstract of DOI:10.1021/acs.jmedchem.6b00070

Here we report the discovery and optimization of a series of bivalent bromodomain and extraterminal inhibitors. Starting with the observation of BRD4 activity of compounds from a previous program, the compounds were optimized for BRD4 potency and physical

Full text of DOI:10.1021/acs.jmedchem.6b00070

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Article
Optimisation of a series of bivalent triazolopyridazine based
bromodomain and extraterminal inhibitors: the discovery of
3R)-4-[2-[4-[1-(3-methoxy-[1,2,4]triazolo[4,3-b]pyridazin-6-yl)-4-
(
piperidyl]phenoxy]ethyl]-1,3-dimethyl-piperazin-2-one (AZD5153)
Robert H Bradbury, Rowena Callis, Gregory R. Carr, Huawei Chen, Edwin Clark, Lyman Feron,
Steven C. Glossop, Mark A. Graham, Maureen Hattersley, Chris Jones, Scott G. Lamont, Gilles
Ouvry, Anil Patel, Joe Patel, Alfred A. Rabow, Craig A. Roberts, Stephen Stokes, Natalie Stratton,
Graeme E. Walker, Lara Ward, David Whalley, David Whittaker, Gail Wrigley, and Michael J. Waring
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.6b00070 • Publication Date (Web): 15 Aug 2016
Downloaded from http://pubs.acs.org on August 16, 2016
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Journal of Medicinal Chemistry is published by the American Chemical Society. 1155
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Optimisation of a series of bivalent triazolopyridazine based
bromodomain and extraterminal inhibitors: the discovery of (3R)ꢀ4ꢀ
[
2ꢀ[4ꢀ[1ꢀ(3ꢀmethoxyꢀ[1,2,4]triazolo[4,3ꢀb]pyridazinꢀ6ꢀyl)ꢀ4ꢀ
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piperidyl]phenoxy]ethyl]ꢀ1,3ꢀdimethylꢀpiperazinꢀ2ꢀone (AZD5153)
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1
Robert H. Bradbury , Rowena Callis , Gregory R. Carr , Huawei Chen , Edwin Clark , Lyman Feron ,
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Steve Glossop , Mark A. Graham , Maureen Hattersley , Chris Jones , Scott G. Lamont , Gilles
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Ouvry , Anil Patel , Joe Patel , Alfred A. Rabow , Craig A. Roberts , Stephen Stokes , Natalie
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Stratton , Graeme E. Walker , Lara Ward , David Whalley , David Whittaker , Gail Wrigley and
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Michael J. Waring *.
1
AstraZeneca, Mereside, Alderley Park, Macclesfield, Cheshire, SK10 4TG, UK.
2
AstraZeneca, Gatehouse Park, Waltham, MA 02451, USA.
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AstraZeneca, Chemin du Moulin de Vrilly, 51100 Reims, France.
4
Northern Institute for Cancer Research, School of Chemistry, Newcastle University, Bedson
Building, Newcastle upon Tyne, NE1 7RU, UK.
Abstract
Here we report the discovery and optimization of a series of bivalent bromodomain and extraterminal
inhibitors. Starting with the observation of BRD4 activity of compounds from a previous programme,
the compounds were optimised for BRD4 potency and physical properties. The optimised compound
from this campaign exhibited excellent pharmacokinetic profile and exhibited high potency in vitro
and in vivo effecting cꢀMyc downregulation and tumour growth inhibition in xenograft studies. This
compound was selected as the development candidate AZD5153. The series showed enhanced
potency as a result of bivalent binding and a clear correlation between BRD4 activity and cellular
potency.
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Introduction
The bromodomains (BRD) are a family of proteins responsible for binding acetylated lysine residues
of proteins. The BRDs are rapidly emerging as a target class amenable to pharmacological
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intervention with small molecules . The bromodomain and extraterminal (BET) subꢀfamily are
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perhaps the most widely explored of the class . Initially identified as a result of phenotypic screening
for modulation of ApoA1, subsequent target identification in tandem with optimisation has revealed
multiple small molecule inhibitors of BET bromodomains, which have progressed to preclinical and
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ꢀ5
clinical evaluation (Figure 1) . The BET family consists of four proteins, termed BRD2, BRD3,
BRD4 and BRDT. Each of these proteins contains two separate bromodomains. BRD4 in particular
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has been lauded as a potential drug target although it is worth remembering that there is a high degree
of homology within this class and the majority of compounds, particularly those studied most
extensively in biological elucidation, do not exhibit selectivity within the class.
The BET family regulate gene expression through binding of acetylated lysine residues on histone
proteins followed by activation of transcription elongation driven by RNAꢀPolII. BET inhibitors
inhibit this gene transcription and, as a consequence, exhibit growth inhibition in a number of
haematological and solid tumour models. This action is believed, at least in part, to be due to downꢀ
regulation of critical oncogenes such as cꢀMyc, leading to great interest in BET inhibitors in the
oncology field and a number are currently progressing in early clinical trials.
From the initial ApoA1 screen, pioneering work at GlaxoSmithkline led to the discovery of the
3
,4
triazoloꢀbenzodiazepine 1 (iBETꢀ762, Figure 1) , which has become one of the leading clinical BET
inhibitors. Work carried out by Bradner and the Structural Genomics Consortium identified the
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structurallyꢀrelated compound 2 (JQ1) . 1 and 2 have become key standard compounds in the field
with many studies using these compounds as probes to understand BET biology. More recently,
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OTX015, 3 , the corresponding pꢀhydroxyanilide derivative of 2 has been reported to show
8
encouraging clinical results in AML patients .
We have recently described the discovery that compounds from the androgen receptor (AR)
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downregulator (DR) programme were found to be potent BET inhibitors that are demonstrated to
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act through an inꢀcis bivalent binding mode in which a single inhibitor molecule simultaneously
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engages both bromodomains of a single protein via two acetyl lysine mimicking moieties . This
activity is postulated to explain the effects of the compounds observed in AR and estrogen receptor
(ER) DR assays as well as their effects on cꢀMyc levels. Here we describe the medicinal chemistry
programme that led to the identification of these compounds and their optimization, leading to the
discovery of a clinical candidate bivalent BET inhibitor.
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i-BET762
2 JQ1
3 OTX-015
Figure 1
Results and Discussion
The campaign of optimisation was initiated with the objective of improving the potency of the
previous clinical AR downregulator AZD3514, 4, and the corresponding unsaturated analogue 5
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whilst maintaining their excellent ADMET properties . These compounds exhibited modest cellular
AR DR potencies (pIC50 5.8 and 6.5 for 4 and 5 respectively) and subsequent chemistry was focused
on cellular AR DR potency to guide the chemistry campaign. The BET inhibitory activity of the
compounds and resulting effects on ER and cꢀMyc DR were discovered during the course of this
11
work . Later chemistry focused on the BRD4 potency and ER DR data to develop SAR and was
generated retrospectively on the earlier compounds. The AR DR, ER DR and cꢀMyc DR data
(measured in LNCaP, MCF7 and MM1.S cell lines respectively) are tightly correlated and can be used
reasonably interchangeably to interpret the SAR. The data from these assays are also predictive of
growth inhibitory effects measured in MM1.S cells (see later and Supporting Information for more
detail). In order to assess the effect of the bivalent binding, an assay employing a protein construct
containing both bromodomains was required. Hence, a fluorescence polarisation (FP) assay for full
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tandem domain BRD4 protein (residues 44ꢀ460) was developed using a fluorescently labelled small
molecule probe derived from 2. It should be noted throughout, that whilst data for BRD4 are quoted,
no selectivity of these molecules within the BET family is anticipated due to their close homology.
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4, AZD3514
AR downregulation pIC50 5.8
AR downregulation pIC50 6.5
Figure 2
The synthetic route that could be used to prepare analogues with modifications to the acylpiperazine is
exemplified with the synthesis of 5 (Scheme 1). S Ar addition of 4ꢀ(4ꢀhydroxyphenyl)piperidine to
N
chlorotriazolopyridazine afforded the corresponding phenol (Scheme 1). The phenol was alkylated
with ethylene carbonate to give the ethyl alcohol, which was derivatised as the mesylate and this was
used to alkylate the required amine, in this case acetylpiperazine, to give 5 in 35% overall yield for
the four steps. Compounds 6 and 7 were prepared in an analogous manner.
Scheme 1 – Synthesis of 5. Compounds 6 and 7 were prepared in an analogous manner.
ii
i
iii
iv
5
(
2 3
i) DIPEA, MeCN, rt, 66%. (ii) Ethylene carbonate, K CO , DMF, 80 °C, 72%. (iii) Mesyl chloride,
Et N, DCM, 0 °C, 100%. (iv) Nꢀacetylpiperazine, DIPEA, DMA, 110 °C, 73%.
3
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Alternatively, the amine substituent could be introduced before the triazolopyridazine allowing more
effcient synthesis of compounds with different triazolopyridazine substituents. This is exemplified by
the synthesis of 8 (Scheme 2). BOC protection of 4ꢀ(4ꢀhydroxyphenyl)piperidine was followed by
alkylation of the phenol with 1ꢀbromoꢀ2ꢀchloroethane to give the alkyl halide. This underwent
displacement with Rꢀ1,3ꢀdimethylpiperazinꢀ2ꢀone to afford the BOC protected intermediate.
Removal of the BOC group with ethereal HCl afforded the piperazine, which could be arylated with a
variety of substituted chlorotriazolopyridazines, in this case 6ꢀchloroꢀ3ꢀ(trifluoromethyl)ꢀ
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[
1,2,4]triazolo[4,3ꢀb]pyridazine to give the final compound 8 in five steps and 52% overall yield.
Compounds 9 to 20 were prepared in a similar fashion.
Scheme 2 – Synthesis of 8. Compounds 9 to 20 were prepared in a similar fashion.
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CO
3
, acetone, 90 °C, 97%. (iii)
DIPEA, KI, DMA, 110 °C, 90%. (iv) HCl, Et
2
O, quant. (v) DIPEA, DMA, 80 °C, 68%.
Analogues with variations to the ethoxy linker of the piperazinone were prepared by initial alkylation
of the piperazinone by reductive amination with an appropriate ketone, exemplified with Nꢀ BOC
azetidineꢀ3ꢀone (Scheme 3). Removal of the BOC group with trifluoroacetic acid gave the
intermediate amine. Arylation of 4ꢀ(4ꢀbromophenyl)piperidine with 6ꢀmethoxyꢀ3ꢀ(trifluoromethyl)ꢀ
[1,2,4]triazolo[4,3ꢀb]pyridazine gave the arylbromide, which was coupled with the amine under
BuchwaldꢀHartwig conditions to give the final compound 22 (10% overall yield for the lowest
yielding sequence). Compounds 23 and 24 were prepared in an analogous manner.
Scheme 3 – Synthesis of 22. Compounds 9 to 20 were prepared in a similar fashion.
i
ii
iv
iii
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2
(i) Et
3
3
N, NaBH(OAc) , DCM, 56%. (ii) TFA, DCM, quant. (iii) DIPEA, DMF, 80 °C, 34%.
(iv) Ruphos palladium(II) phenethylamine chloride, dicyclohexyl(2',6'ꢀdiisopropoxyꢀ[1,1'ꢀbiphenyl]ꢀ
ꢀyl)phosphine, Cs CO , toluene, 90 °C, 28%.
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In exploring the structure activity relationships in relation to the potential bromodomain inhibitory
activity, we considered that both the triazolopyridazine and acylpiperazine moieties of the molecule
could act as acetyl lysine mimetics (subsequently, we discovered that in fact both do, resulting in the
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in cis binding mode and accounting for the SAR we observed) . Modifications to both of these
regions were designed with consideration to the overlay of the compounds with acetyl lysine in the
binding site. Changing the acyl piperazine of 5 for Nꢀmethylpiperazinꢀ2ꢀone (6) resulted in retained
potency (AR DR pIC 6.5 for both compounds) (Table 1). The isomeric piperazinꢀ3ꢀone 7 was
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0
slightly less potent (ER DR pIC50 6.0 compared to 6.3 for 6). 3ꢀMethyl substitution of the piperazinꢀ
ꢀone, 8, led to an increase in ER DR potency (pIC50 7.6 compared to 6.3 for 6). In this series, the
2
piperazinones had improved solubility but also increased metabolism, as measured by human
hepatocyte turnover, compared with the acylpiperazine 5. BRD4 assay data reveleaved that
compounds 5 to 7 had weak BRD4 potency (pIC50 range 5.6 ꢀ 6.7) and that introduction of the
piperazinone 3ꢀmethyl substituent led to increased BRD4 activity (pIC50 8.1) consistent with the
increase in the cellular potency.
Table 1 – Effects of structural modifications of the acylpiperazine
Compound
number
R
BRD AR
ER
Myc
MM.
1S
LogD Solubili Hu
7.4
ty / ꢁM heps
4 FP
DR
DR DR
a
i
a
pIC pIC50a pGI50
pK
pIC50
Clint /
a
a
mLmi
5
0
ꢀ
n
1
ꢀ6
×10
cells
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3.3
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a
Measurements reported are the mean of at least three determinations. The standard deviation is not
shown, as all three assays were found to be highly reproducible on repeat testing (see Experimental
Section for more detail).
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Next, the effect of changing the triazolopyridazine trifluoromethyl substituent of 8 was explored
(Table 2). Removal of the substituent (9) led to a loss of BRD4 and cellular potency. Replacement of
the trifluoromethyl group with methyl, chloro, thiomethyl, methoxy and dimethylamino substituents
(10ꢀ14 respectively) resulted in retained activity. Of these compounds, the methoxy derivative 13 had
the lowest logD7.4 value and showed very high solubility and a significantly improved human
hepatocyte stability, resulting in a clear improvement in overall profile. Hence, the methoxy
substituent appeared optimal and was retained in subsequent analogues.
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Table 2 ꢀ Effect of changing the triazolopyridazine trifluoromethyl substituent
Compound
number
R
BRD4 AR
ER
Myc
DR
MM.1S LogD7.4 Solubilit Hu
a
i
a
pK
DR
DR
pGI50
y / ꢁM
heps
a
a
a
pIC50
pIC50
pIC50
Clint /
ꢀ
mLmin
1
ꢀ6
×
10
cells
70
8
9
ꢀCF
3
8.1
7.4
5.8
7.6
<5.5
7.4
7.4
7.5
7.6
8.0
5.9
7.6
7.1
7.7
7.9
8.3
>4.2
1.9
2.4
3.1
2.9
2.3
63
ꢀH
< 6.0
7.8
1000
670
63
180
7.2
24
10
11
12
13
ꢀMe
8.3
7.7
ꢀCl
7.9
7.6
7.6
7.7
ꢀSMe
ꢀOMe
7.9
660
>1600
21
8.3
8.3
<3.5
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14
ꢀNMe
2
7.9
7.6
7.2
7.8
8.5
2.4
>1600
4.4
a
Measurements reported are the mean of at least three determinations. The standard deviation is not
shown, as all three assays were found to be highly reproducible on repeat testing (see Experimental
Section for more detail).
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Further exploration of changes to the piperazinone group (Table 3) showed that the chirality of the 3ꢀ
methyl substituent was important, with the Sꢀenantiomer 15 showing a loss of potency (1.6 log units
relative to 13), suggesting that a specific interaction was formed with the methyl group. The 3,5ꢀ
dimethylꢀ1ꢀacetylpiperazine analogue 16 showed an increase in potency relative to piperazinone 13
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55
56
57
58
59
60
(
0.5 log units vs BRD4 but 1.2 units in ER DR. It is possible that this apparent discrepancy arises due
to the potency limit in the FP assay. Unfortunately, this change also resulted in a decrease in human
hepatocyte stability. Again, the methyl groups appeared to be important for potency, for instance, the
corresponding 2,6ꢀdimethyl isomer 16 showed a loss of potency (1.4 log units in ER DR) relative to
1
5. Further methylation of the piperazinone, for example 18, led to similar potency to 13 but with a
marked decrease in human hepatocyte stability. These data suggested that balancing potency and
metabolic stability in these compounds was very challenging and the (R)ꢀ1,3ꢀdimethylpiperazinꢀ2ꢀone
group appeared to strike the best balance.
Table 3 ꢀ Further exploration of changes to the piperazinone group
Compound
number
R
BRD4 ER
Myc
DR
MM1S LogD7. Solubi Hu
a
i
a
pK
DR
pGI50
4
lity /
ꢁM
heps
a
a
pIC50
pIC50
Clint /
mLmin
ꢀ
1
ꢀ6
×
10
cells
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
13
8.3
6.9
8.8
7.6
6.5
8.8
7.9
8.3
2.3
2.2
2.5
>1600 <3.5
1
5
6
7.1
900
<1.4
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
>8.7
>8.7
>1000 12
17
8.3
8.1
7.4
7.7
3.4
2.3
280
750
48
80
18
7.9
8.2
a
Measurements reported are the mean of at least three determinations. The standard deviation is not
shown, as all three assays were found to be highly reproducible on repeat testing (see Experimental
Section for more detail).
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Modifications to the linker between the piperazinone and the central phenyl ring were also explored
(Table 4). Extending the ethoxyꢀ linker to propoxyꢀ (19) led to increased cellular potency but higher
hepatocyte Clint (Table 4). Further extension to butoxyꢀ (20) led to similar potency but further
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
increased Cl . Deletion of the ether oxygen leading to an ethyl linker (21) resulted in similar potency
int
and Clint to 13. Rigidifcation of the linker by incorporation of an azetidine (22) or piperidine (23)
resulted in some loss of potency, whereas the more flexible azetidinylmethyl linker (24) resulted in an
increase in cellular potency relative to 13 whilst maintaining good solubility and Cl values.
int
Table 4 ꢀ Modifications to the linker between the piperazinone and the central phenyl ring
Compound
number
R
BRD ER
cMyc
DR
MM1S LogD7. Solubil Hu
a
4
DR
pGI50
4
ity /
ꢁM
heps
a
i
a
a
pK
pIC50
pIC50
Clint /
ꢀ
mLmin
1
ꢀ6
×
10
cells
1
3
9
8.3
8.4
7.6
7.7
7.9
8.5
8.3
2.3
2.4
>1600
>1000
<3.5
5.1
O
O
N
N
1
>8.8
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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
20
8.2
7.7
2.8
470
12
2
1
2
8.5
7.2
7.6
6.1
8.0
8.3
2.5
1.7
570
870
<2.2
<1.2
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
23
7.3
8.0
6.8
8.4
2.0
1.7
760
2.7
4.1
24
>8.7
8.8
>1000
a
Measurements reported are the mean of at least three determinations. The standard deviation is not
shown, as all three assays were found to be highly reproducible on repeat testing (see Experimental
Section for more detail).
Solving the Xꢀray crystal structure of 13 in complex with the first bromodomain of BRD4 [BRD4(1)]
1
1
revealed the formation of a dimer as previously observed , in which both the triazolopyridazine and
Nꢀmethylpiperazinone engage the acetyl lysine sites of different bromodomains (Figure 2). In the
first bromodomain, critical hydrogen bonds were observed between the triazolopyridazine nitrogenꢀ1
and Asn140 with the nitrogenꢀ2 and the methoxy substituent acting as the acetyl lysine mimic. In the
second bromodomain, the Nꢀmethylpiperazinone methyl and carbonyl groups acted as the acetyl
lysine mimic with the carbonyl also engaging Asn140. The 3ꢀmethyl group filled a small lipophilic
hole, explaining the increase in potency arising from its introduction. Whilst this structure is
homodimeric, showing the interaction of both ends of the molecule with BRD4(1), given the close
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homology between the first and the second bromodomains, it is reasonable to anticipate that similar
interactions would be made with each bromodomain in an inꢀcis complex with full length BRD4.
This is fully consistent with the observed SAR and explains, for instance, the increase in potency for
16 relative to 13, 17 and 18 since the additional methyl group of 16 is able to occupy a vacant
lipophilic cavity in the binding site. In this model, the diminished potency of 22 and 23 relative to the
other exemplified linkers could be explained by the linear constraints of the linkers of these molecules
requiring an energetically less favoured orientation of the two bromodomains in order to achieve
bivalent binding.
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
Figure 2 – Xꢀray crystal structure of 13 in complex with BRD4(1) showing the formation of a dimeric
structure with two bromodomains. b) Engagement of the triazolopyridazine of 13 with the first
bromodomain (blue ribbon) acetyl lysine binding site. c) Engagement of the Nꢀmethylpiperazinone of
13 with the second bromodomain (red ribbon) acetyl lysine site.
To provide further evidence of the bivalent binding mode, compounds were tested against the N140A
and N433A mutant forms of BRD4 in the FP assay and the pK values obtained were compared to the
wild type protein (Table 5). N140 in BD1 and N433 in BD2 of BRD4 have been shown to be
i
1
2
important in the binding of potent BRD ligands . Mutation of these residues was expected to provide
insights into the binding mechanism. Monovalent ligand 1 showed consistent K values across the
i
wild type and both mutant proteins as would be expected. Compounds 5, 6 and 7, which have the
potential for bivalent binding but have an unoptimised second site binding moiety showed a decrease
in affinity for the N140A mutant but an increase in affinity for the N433A mutant in relative to wildꢀ
type. This suggests that the triazolopyridazine moiety binds to BD1 and the acetyl piperazine or
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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
piperazinone group binds to BD2 although this has not been definitively established. The reason for
the increase in affinity for the N433A mutant relative to wildꢀtype for these compounds is unclear. It
may suggest that these the unoptimised second site binding groups employed can interact more
strongly with mutated BD2 than for wild type, indeed, these changes would be expected to disrupt the
critical interaction with the asparagine residue. Compounds 8 – 24, which are all optimised bivalent
compounds consistent show a decrease in affinity of approximately 1 order of magnitude for both
mutant proteins relative to wild type. These structure activity relationships provide further evidence
of the bivalent binding mechanism of the series as a whole.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Table 5 – pK
i
values for BD1 and BD2 mutant proteins compared to wild type
a
Compound
WT FP pKi
N140A
N433A
a
i
a
number
pK
pK
i
1
5
6
7
8
7.3
6.2
6.7
5.6
8.1
< 6.0
7.8
7.9
7.9
8.3
7.9
6.9
8.8
8.3
8.1
7.3
5.5
6.0
5.1
7.2
5.6
6.7
6.5
6.9
6.8
6.5
6.0
7.8
6.8
6.6
7.4
6.6
6.5
6.4
6.4
5.2
6.6
6.7
6.8
6.9
6.5
6.6
7.3
7.3
6.9
9
10
11
12
13
14
15
16
17
18
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19
20
8.4
8.2
8.5
7.2
7.3
8.0
7.2
7.0
6.9
6.4
6.5
7.0
7.1
7.2
7.0
6.7
6.8
6.8
21
22
23
24
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
Measurements reported are the mean of at least three determinations. The standard deviation is not
shown, as all three assays were found to be highly reproducible on repeat testing (see Experimental
Section for more detail).
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8
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
The wild type pK
i
values were shown to accurately predict the cellular potencies such as ER DR
(Figure 3). This provides confidence that the observed pharmacology is BRD4 driven, although we
would expect the compounds to have similar affinities for other members of the BET family due to
their close homology and the lack of selectivity within the BET family for 13 in selectivity screens
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
(
see later). Moreover, the observation of this correlation and lack of drop off between in vitro BRD4
inhibition and cellular potency, in contrast to monovalent inhibitors, which show significantly lower
1
1
cellular potency , suggests that the bivalent mechanism is operative in a cellular environment.
Figure 3 ꢀ Correlation of BRD4 WT pK
i
vs ER Downregulation. Bivalent triazolopyridazine are
2
shown with black dots. Line Fit: ER DR pIC50 = 0.046 + 0.93*[BRD4 WT FP pK
i
], r = 0.84, n=20
The compounds with the best balance of potency and in vitro ADME properties (favorable solubility,
Cacoꢀ2 Papp and metabolic stability) and attractive preꢀclinical pharmacokinetics were selected for
13
further profiling (Table 6). A preliminary human dose prediction was calculated based on achieving
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24 hour free cover above a cellular GI50 (potency in MM.1S cells) from a twiceꢀdaily dosing schedule
(BID, 12 hours apart). The maximum absorbable dose (MAD) and human Fabs were estimated in GIꢀ
14
a
Sim using measured Cacoꢀ2 Papp, solubility and pK data.
Several compounds had low predicted doses (<20 mg/dose) and good margins against the MAD (>
100 fold). Compound 13 was also attractive in that it had its in vivo plasma clearance appeared well
predicted from in vitro data across species, providing greater certainty in the human clearance and
plasma halfꢀlife predictions. 13 also had improved metabolic stability in hepatic microsomal fractions
compared to other examples (data not shown). As a result, 13 was selected for further development
and given the internal identifier AZD5153.
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
Table 6 ꢀ Tabulated PK data. Preclinical PK parameters were derived from plasma concentrationꢀtime
data using nonꢀcompartmental analysis.
Property
13
16
24
21
14
MM.1S GI50
/
µ
M
0.005
2.3 / 4.8
>1650
400
0.002
2.5 / 6.2
>1000
300
0.002
1.7 / 5.9
>1000
460
0.005
2.5 / 6.3
570
0.003
2.4 / ꢀ
>1681
ꢀ
logD / basic pKa
Solubility, pH7.4 / µM
ꢀ1
Cacoꢀ2 Papp / nms
Plasma protein binding
%free) (rat / dog /
280
(
4 / 24 / 23
5 / <1 / <4
6 / 16 / 19
13 / 7 / 12
5 / 27 / 32
2 / 24 / 25
4 / 9 / 16
human)
ꢀ
Hepatocyte CLint / µLmin
1
6
per 10 cells (rat / dog /
<1 / 3 / 4
<1 / <1 / <2 13 / 18 / 4
human)
Rat predicted clearance /
6
4
14
17
2
7
1
3
10
6
ꢀ
1
ꢀ1 a
mLmin kg
Rat in vivo clearance /
ꢀ1
ꢀ1
mLmin kg
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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
ꢀ
1
Rat Vss / Lkg
0.9
81
1.5
40
1.61
57
0.7
72
0.4
42
Rat Foral
%
Dog predicted clearance /
11
17
11
18
5
9
3
ꢀ
ꢀ
ꢀ1
ꢀ1 a
mLmin kg
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
Dog in vivo clearance /
6
ꢀ1
ꢀ1
mLmin kg
ꢀ1
Dog Vss / Lkg
0.4
55
0.6
28
0.7
73
0.9
98
ꢀ
ꢀ
Dog Foral
%
Predicted human
<6.2
9.4
78
8.1
<5.7
<13
6.1
20
ꢀ1
ꢀ1 a
clearance / mLmin kg
Human dose prediction /
<20
<13
b
mg
Predicted maximum
9300
8565
0.93
9935
1.0
8732
0.99
8968
0.92
b
absorbable dose / mg
Predicted human Fabs
0.98
a
Clearance predictions were made from hepatocyte and plasma protein binding data using the wellꢀ
1
5,16 b
stirred model . Preliminary human dose and maximum absorbable dose prediction methods are
described in the Experimental Section.
Compound 13 (10 ꢁM) was tested for activity across 32 bromodomains in the Bromoscan® panel
(
Figure 4). This showed it to be have inherent selectivity for the BET family isolated bromodomains,
the next most potent BRD was TAF1(2) (pK 5.9). This selectivity is further increased when
i
considering the increase in potency due to bivalent binding to fullꢀlength BET proteins, representing a
margin of two orders of magnitude to its full length BRD4 potency as measured in our FP assay and
to cellular potency as determined by cꢀMyc DR, making the assumption that bivalent binding is not
possible for the other BRDs.
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7
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i
Figure 4 ꢀ Selectivity of 13 in the Bromoscan® panel. Figures above the bars indicate pK values.
7.2
7.0
7
.0
7.3
6.9
6
.6
100
6.3
6.4
5.9
90
80
70
60
5
.6
5
.2
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
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60
>5.0
5.6
50
4
0
0
3
20
10
0
a
Inhibition measured at 10 ꢁM compound concentration.
Compound 13 was administered orally to mice bearing MVꢀ4ꢀ11 xenografts and pharmacodynamic
activity (intraturmoural levels of cꢀMyc) were measured at 2, 4, and 8 h post dose (Figure 5). A
considerable decrease in cꢀMyc expression was observed out to 8 h post dose at free plasma levels of
compound <0.2 ꢁM. This decrease in cꢀMyc expression after treatment with 13 is consistent with
17
other published BET inhibitors .
Having demonstrated effects of cꢀMyc inhibition in tumors, the ability of 13 to exert antitumour
activity in the MVꢀ4ꢀ11 xenograft model was assessed (Figure 6). Subcutaneously implanted tumours
3
were allowed to grow to
∼
200 mm before administering 13 daily for 14 days. When dosed at 10
ꢀ1
mgkg , tumour regressions occurred (>100% TGI), with nearly complete regression for the majority
of tumors. Compound 13 was tolerated for 14 days with an average body weight loss of 8%, which is
indicative of acceptable tolerability, further detailed in vivo pharmacology will be communicated in
1
8
subsequent publications .
Figure 5 ꢀ a) Pharmacodynamic data following single oral dose of 10 mg/kg 13 to MVꢀ4ꢀ11 tumorꢀ
bearing mice. Measured levels of cꢀMyc RNA in tumors 2, 4 or 8 hours post dose plotted against free
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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
plasma exposure of 13. cꢀMyc levels for the vehicle group are shown on the plot for comparison at an
arbitrary position on the xꢀaxis. b) Corresponding downregulation of cꢀMyc protein and loading
control measured by Western blot. Each lane represents expression in a tumor from one mouse, n=3
mice per group. Vehicleꢀtreated mice from early and late time points only.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
a)
b)
40
Vehicle
2hr
Vehicle
13
30
4
hr
hr
2h
8h
2h
4h
8h
8
20
c-Myc
GAPDH
1
0 mg/kg
10
QD
0
0
.01
0.1
1
10
Plasma exposure free (uM)
Figure 6 ꢀ Antitumor activity of 13 in the MVꢀ4ꢀ11 xenograft efficacy model. Tumors were allowed
3
to grow to an average size of 200 mm then mice were then treated daily with either vehicle (n=8) or
13 (n=8) for two weeks via oral gavage. Tumor volumes were monitored twice a week for fourteen
days and are plotted as the geometric mean volumes ± standard error (SE) for both vehicle and treated
groups (vehicle group SE bars are too small to see).
Conclusion
Through careful analysis of structure activity relationships, we have been able to elucidate the basis
for secondary pharmacology observed for a series of AR downregulators and ascribe it to BRD4
inhibition. Subsequent optimisation of potency and ADMET properties led to the development of
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potent bivalent BET ligands, culminating in the clinical candidate 13, a potent and selective BET
inhibitor with a novel bivalent binding mode. The in vitro potency of 13 translated directly into cꢀ
Myc modulation and tumour growth inhibition in vivo in xenograft studies at low doses. Further
studies describing the pharmacology of 13 will be the subject of further publications.
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In addition to the discovery of the clinical candidate, this work demonstrates the optimisation of
bivalent inhibitors with maintained drugꢀlike properties. We have previously highlighted the potential
advantages of bivalent binding as a general concept for improved potency and specificity in ligand
1
1
discovery but commonly, this concept has been approached by the linking of two monovalent
ligands with a flexible linker leading to molecules that are large and lipophilic and hence have
18
compromised drugꢀlikeness. Indeed, this approach has been taken for BET inhibitors and it has
been shown that bivalent binding may also be a strategy for overcoming drug resistance, as
19
exemplified with mTOR inhibitors . The concept described here shows that by partially fusing the
two binding elements within a bivalent inhibitor, compounds of clinical candidate quality can be
developed. We anticipate that this will be an attractive strategy for targeting other multidomain
proteins with high potency and specificity in future.
Experimental section
General Experimental
Proton nuclear magnetic resonance spectra were recorded on a Bruker 700 MHz, 500 MHz or 400
1
MHz instrument. All H NMR spectra were measured in parts per million (ppm) relative to the signal
for residual dimethyl sulfoxide (DMSO) in deuterated DMSO (2.49 ppm) or chloroform in deuterated
1
chloroform (7.26 ppm). Data for H NMR spectra were reported as: chemical shift, multiplicity (S =
singlet, d = doublet, t = triplet, q = quartet, h = heptet, m = multiplet), coupling constants, and
integration. Carbon nuclear magnetic resonance spectra were recorded on a Bruker 176 MHz
1
3
instrument. All C NMR spectra were measured in parts per million (ppm) relative to the signal for
residual dimethyl sulfoxide (DMSO) in deuterated DMSO (39.5 ppm) and were obtained with
1
complete H decoupling. Final compound purity was assessed by LSꢀMS analysis and compounds are
≥ 95% unless otherwise stated. All LCꢀMS analysis were performed on a Waters 2795 Separations
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Module, using a GeminiꢀNX 5ꢁ C18 column, with a Waters Micromass detector. High resolution
mass spectra were obtained on either a Thermo LTQꢀFT/Accela/CTC/PDA or Waters Xevo G2 Qtof
instrument in ESI +ve ion mode.
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1-(4-(2-(4-(1-(3-(Trifluoromethyl)-[1,2,4]triazolo[4,3-b]pyridazin-6-yl)piperidin-4-
yl)phenoxy)ethyl)piperazin-1-yl)ethanone (5)
DIPEA (14 mL, 8.2 mmol) was added to 2ꢀ(4ꢀ(1ꢀ(3ꢀ(trifluoromethyl)ꢀ[1,2,4]triazolo[4,3ꢀb]pyridazinꢀ
6ꢀyl)piperidinꢀ4ꢀyl)phenoxy)ethyl methanesulfonate (27) (13 g, 27 mmol) and Nꢀacetylpiperazine (3.9
g, 30 mmol) in DMA (65 mL). The resulting solution was stirred at 110 °C for 1 hour. The reaction
mixture was cooled to room temperature and the solvents evaporated. The residue was diluted with
DCM (250 mL) and washed with water (250 mL) (emulsion formed). The aqueous was reꢀextracted
with DCM (250 mL) and the combined organics were washed with brine (250 mL), dried over
4
MgSO , filtered and evaporated to give crude product. The crude product was purified by flash silica
chromatography, elution gradient 0 to 3% MeOH in DCM then 5% MeOH in DCM. Pure fractions
were evaporated to give 1ꢀ(4ꢀ(2ꢀ(4ꢀ(1ꢀ(3ꢀ(trifluoromethyl)ꢀ[1,2,4]triazolo[4,3ꢀb]pyridazinꢀ6ꢀ
yl)piperidinꢀ4ꢀyl)phenoxy)ethyl)piperazinꢀ1ꢀyl)ethanone (5) (10 g, 73%) as a white solid. 1H NMR
(
(
700 MHz, DMSO) δ 1.64 (qd, J = 4.0, 12.8 Hz, 2H), 1.82 – 1.89 (m, 2H), 1.96 (s, 3H), 2.38 – 2.42
m, 2H), 2.45 – 2.48 (m, 2H), 2.69 (t, J = 5.8 Hz, 2H), 2.78 (tt, J = 3.4, 12.0 Hz, 1H), 3.08 (td, J = 2.0,
1
3.1 Hz, 2H), 3.40 (dt, J = 5.2, 10.4 Hz, 3H), 4.04 (t, J = 5.8 Hz, 2H), 4.38 (d, J = 13.4 Hz, 2H), 6.85
d, J = 8.7 Hz, 2H), 7.15 (d, J = 8.7 Hz, 2H), 7.63 (d, J = 10.3 Hz, 1H), 8.21 (d, J = 10.3 Hz, 1H). 13C
NMR (176 MHz, DMSO, 30 °C) δ 21.03, 32.31, 40.51, 40.75, 45.57, 45.82, 52.66, 53.15, 56.40,
(
65.38, 114.34, 116.78, 118.61 (q, J = 269.2 Hz), 124.05, 127.53, 137.15 (q, J = 39.6 Hz), 137.49,
+
+
1
44.10, 155.43, 156.78, 167.96. Purity 100% by LCꢀMS, m/z: ES [M+H] 518. HRMS (ESI) Calcd.
+
for C25
H
30
F
3
N
7
O
2
[M+H] 518.2486; Found 518.2490.
The following compounds were synthesised using analogous procedures:
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1-Methyl-4-(2-(4-(1-(3-(trifluoromethyl)-[1,2,4]triazolo[4,3-b]pyridazin-6-yl)piperidin-4-
yl)phenoxy)ethyl)piperazin-2-one (6)
1H NMR (700 MHz, DMSO) δ 1.64 (qd, J = 3.9, 12.8 Hz, 2H), 1.86 (d, J = 11.5 Hz, 2H), 2.73 (dd, J
= 4.9, 5.6 Hz, 4H), 2.75 – 2.79 (m, 1H), 2.80 (s, 3H), 3.04 – 3.1 (m, 4H), 3.22 – 3.26 (m, 2H), 4.05 (t,
J = 5.6 Hz, 2H), 4.38 (d, J = 13.3 Hz, 2H), 6.85 (d, J = 8.7 Hz, 2H), 7.15 (d, J = 8.7 Hz, 2H), 7.63 (d,
J = 10.3 Hz, 1H), 8.21 (d, J = 10.3 Hz, 1H). 13C NMR (176 MHz, DMSO, 30 °C) δ 32.31, 32.88,
10
11
12
13
14
15
16
17
18
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22
23
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40.52, 45.81, 47.78, 49.50, 55.29, 57.13, 65.42, 114.33, 116.77, 118.61 (q, J = 269.2), 124.04, 127.54,
+
137.15 (q, J = 39.6), 137.54, 144.09, 155.42, 156.72, 165.74. Purity 99% by LCꢀMS, m/z: ES
+
+
[
28 3 7 2
M+H] 504. HRMS (ESI) Calcd. for C24H F N O [M+H] 504.2329; Found 504.2329.
4-Methyl-1-(2-(4-(1-(3-(trifluoromethyl)-[1,2,4]triazolo[4,3-b]pyridazin-6-yl)piperidin-4-
yl)phenoxy)ethyl)piperazin-2-one (7)
1H NMR (700 MHz, DMSO) δ 1.64 (qd, J = 3.9, 12.8 Hz, 2H), 1.86 (d, J = 12.4 Hz, 2H), 2.18 (s,
3H), 2.51 – 2.54 (m, 2H), 2.78 (tt, J = 3.3, 12.0 Hz, 1H), 2.90 (s, 2H), 3.07 (td, J = 2.0, 13.1 Hz, 2H),
3.39 – 3.42 (m, 2H), 3.61 (t, J = 5.7 Hz, 2H), 4.06 (t, J = 5.7 Hz, 2H), 4.38 (d, J = 13.4 Hz, 2H), 6.85
(
d, J = 8.7 Hz, 2H), 7.15 (d, J = 8.7 Hz, 2H), 7.63 (d, J = 10.3 Hz, 1H), 8.21 (d, J = 10.3 Hz, 1H). 13C
NMR (176 MHz, DMSO, 30 °C) δ 32.86, 41.08, 44.92, 45.75, 46.37, 47.95, 51.71, 59.28, 65.74,
114.89, 117.33, 119.17 (q, J = 269.3), 124.60, 128.14, 137.71 (q, J = 39.6), 138.27, 144.66, 155.98,
+
+
1
57.18, 166.64. Purity 100% by LCꢀMS, m/z: ES [M+H] 504. HRMS (ESI) Calcd. for
+
C
24
H
28
F
3
N
7
O
2
[M+H] 504.2329; Found 504.2327.
(
R)-1,3-Dimethyl-4-(2-(4-(1-(3-(trifluoromethyl)-[1,2,4]triazolo[4,3-b]pyridazin-6-yl)piperidin-4-
yl)phenoxy)ethyl)piperazin-2-one (8)
DIPEA (0.23 mL, 1.4 mmol) was added to (R)ꢀ1,3ꢀdimethylꢀ4ꢀ(2ꢀ(4ꢀ(piperidinꢀ4ꢀ
yl)phenoxy)ethyl)piperazinꢀ2ꢀone (31) (0.14 g, 0.34 mmol) and 6ꢀchloroꢀ3ꢀ(trifluoromethyl)ꢀ
[1,2,4]triazolo[4,3ꢀb]pyridazine (75 mg, 0.34 mmol) in DMA (2.0 mL) at 20 °C under nitrogen. The
resulting solution was stirred at 80 °C for 1 hour then cooled to room temperature. The cooled
reaction mixture was then partitioned between DCM (50 mL) and water (100 mL) and extracted. The
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aqueous was extracted a further twice with DCM (2 × 20 mL). The combined organic layers were
passed through a phase separating cartridge and evaporated to give a yellow gum. The crude product
was purified by preparative HPLC (Waters XBridge Prep C18 OBD column, 5 µ silica, 19 mm
diameter, 100 mm length), using decreasingly polar mixtures of water (containing 1% NH
MeCN as eluents. Fractions containing the desired compound were evaporated to dryness to afford
R)ꢀ1,3ꢀdimethylꢀ4ꢀ(2ꢀ(4ꢀ(1ꢀ(3ꢀ(trifluoromethyl)ꢀ[1,2,4]triazolo[4,3ꢀb]pyridazinꢀ6ꢀyl)piperidinꢀ4ꢀ
yl)phenoxy)ethyl)piperazinꢀ2ꢀone (8) (0.12 g, 68%) as a brown solid. 1H NMR (700 MHz, DMSO) δ
3
) and
0
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2
3
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9
0
1
2
3
4
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8
9
0
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(
1
7
2
6
1
6
1
.22 (d, J = 6.8 Hz, 2H), 1.64 (qd, J = 3.7, 12.8 Hz, 2H), 1.86 (d, J = 12.4 Hz, 2H), 2.67 (ddd, J = 4.8,
.0, 12.1 Hz, 1H), 2.72 – 2.78 (m, 1H), 2.79 (s, 3H), 2.90 (dt, J = 5.9, 13.5 Hz, 1H), 3.02 – 3.1 (m,
H), 3.13 (q, J = 6.8 Hz, 1H), 3.19 – 3.26 (m, 2H), 4.03 (t, J = 5.8 Hz, 1H), 4.38 (d, J = 13.4 Hz, 2H),
.85 (d, J = 8.6 Hz, 2H), 7.15 (d, J = 8.6 Hz, 2H), 7.63 (d, J = 10.3 Hz, 1H), 8.21 (d, J = 10.3 Hz, 1H).
3C NMR (176 MHz, DMSO, 30 °C) δ 14.97, 32.32, 33.62, 40.52, 45.81, 45.84, 47.39, 52.01, 59.61,
5.87, 114.34, 116.77, 118.61 (q, J = 269.3), 124.04, 127.53, 137.16 (q, J = 39.7), 137.51, 144.10,
+
+
55.42, 156.78, 169.23. Purity 100% by LCꢀMS, m/z: ES [M+H] 518. HRMS (ESI) Calcd. for
+
C
25
H
30
F
3
N
7
O
2
[M+H] 518.2486; Found 518.2488.
(
R)-4-(2-(4-(1-([1,2,4]Triazolo[4,3-b]pyridazin-6-yl)piperidin-4-yl)phenoxy)ethyl)-1,3-
dimethylpiperazin-2-one (9)
Zinc powder (65 mg, 0.99 mmol) was added to (R)ꢀ4ꢀ(2ꢀ(4ꢀ(1ꢀ(3ꢀbromoꢀ[1,2,4]triazolo[4,3ꢀ
b]pyridazinꢀ6ꢀyl)piperidinꢀ4ꢀyl)phenoxy)ethyl)ꢀ1,3ꢀdimethylpiperazinꢀ2ꢀone (32) (0.11 g, 0.20 mmol)
in AcOH (4.0 mL). The resulting mixture was stirred for 20 minutes. The reaction mixture was
evaporated to dryness and redissolved in EtOAc (25 mL), and washed sequentially with saturated
NaHCO
MgSO , filtered and evaporated to afford crude product. This was purified by preparative HPLC
(Waters XBridge Prep C18 OBD column, 5µ silica, 19 mm diameter, 100 mm length), using
decreasingly polar mixtures of water (containing 1% NH ) and MeCN as eluents. Fractions containing
3
(2 × 25 mL), water (25 mL), and saturated brine (25 mL). The organic layer was dried over
4
3
the desired compound were evaporated to dryness to afford (R)ꢀ4ꢀ(2ꢀ(4ꢀ(1ꢀ(3ꢀbromoꢀ
[1,2,4]triazolo[4,3ꢀb]pyridazinꢀ6ꢀyl)piperidinꢀ4ꢀyl)phenoxy)ethyl)ꢀ1,3ꢀdimethylpiperazinꢀ2ꢀone
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(25.00 mg, 23.81%) as a white solid and (R)ꢀ4ꢀ(2ꢀ(4ꢀ(1ꢀ([1,2,4]triazolo[4,3ꢀb]pyridazinꢀ6ꢀ
yl)piperidinꢀ4ꢀyl)phenoxy)ethyl)ꢀ1,3ꢀdimethylpiperazinꢀ2ꢀone (9) (27 mg, 30%) as a white solid. 1H
NMR (700 MHz, DMSO) δ 1.22 (d, J = 6.8 Hz, 3H), 1.64 (qd, J = 3.9, 12.8 Hz, 2H), 1.83 (d, J = 12.8
Hz, 2H), 2.67 (ddd, J = 4.8, 7.0, 12.1 Hz, 1H), 2.73 – 2.79 (m, 2H), 2.79 (s, 3H), 2.90 (dt, J = 5.9,
13.6 Hz, 1H), 3 – 3.06 (m, 3H), 3.13 (q, J = 6.8 Hz, 1H), 3.2 – 3.26 (m, 2H), 4.03 (t, J = 5.8 Hz, 2H),
10
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4
8
3
1
.33 (d, J = 13.3 Hz, 2H), 6.85 (d, J = 8.7 Hz, 2H), 7.15 (d, J = 8.7 Hz, 2H), 7.40 (d, J = 10.2 Hz, 1H),
.02 – 8.06 (m, 1H), 9.17 (d, J = 0.7 Hz, 1H). 13C NMR (176 MHz, DMSO, 30 °C) δ 14.98, 32.42,
3.63, 40.65, 45.83, 46.10, 47.39, 52.01, 59.60, 65.87, 114.34, 115.17, 123.90, 127.53, 137.66,
+
+
38.21, 141.56, 155.08, 156.75, 169.24. Purity 100% by LCꢀMS, m/z: ES [M+H] 450. HRMS (ESI)
+
Calcd. for C24
H
31
N
7
O
2
[M+H] 450.2612; Found 450.2612.
The following compounds were synthesised using analogous procedures:
(
R)-1,3-Dimethyl-4-(2-(4-(1-(3-methyl-[1,2,4]triazolo[4,3-b]pyridazin-6-yl)piperidin-4-
yl)phenoxy)ethyl)piperazin-2-one (10)
1H NMR (700 MHz, DMSO) δ 1.22 (d, J = 6.8 Hz, 3H), 1.64 (qd, J = 3.9, 12.8 Hz, 2H), 1.84 (d, J =
12.5 Hz, 2H), 2.54 (s, 3H), 2.67 (ddd, J = 4.8, 7.1, 12.1 Hz, 1H), 2.72 – 2.78 (m, 2H), 2.79 (s, 3H),
2.90 (dt, J = 5.9, 13.6 Hz, 1H), 2.98 – 3.06 (m, 3H), 3.13 (q, J = 6.8 Hz, 1H), 3.19 – 3.26 (m, 2H),
4.01 – 4.04 (m, 2H), 4.35 (d, J = 13.3 Hz, 2H), 6.85 (d, J = 8.7 Hz, 2H), 7.15 (d, J = 8.7 Hz, 2H), 7.35
(
d, J = 10.2 Hz, 1H), 7.97 (d, J = 10.2 Hz, 1H). 13C NMR (176 MHz, DMSO, 30 °C) δ 9.14, 14.97,
32.42, 33.62, 40.63, 45.84, 46.04, 47.39, 52.01, 59.61, 65.87, 114.00, 114.33, 124.01, 127.52, 137.67,
+
+
141.76, 145.16, 154.73, 156.75, 169.23. Purity 100% by LCꢀMS, m/z: ES [M+H] 464. HRMS (ESI)
+
Calcd. for C25
H
33
N
7
O
2
[M+H] 464.2769; Found 464.2766.
(R)-4-(2-(4-(1-(3-Chloro-[1,2,4]triazolo[4,3-b]pyridazin-6-yl)piperidin-4-yl)phenoxy)ethyl)-1,3-
dimethylpiperazin-2-one (11)
1H NMR (700 MHz, DMSO) δ 1.22 (d, J = 6.8 Hz, 3H), 1.64 (qd, J = 3.7, 12.7, 12.8, 12.8 Hz, 2H),
1.85 (d, J = 12.5 Hz, 2H), 2.67 (ddd, J = 4.8, 6.9, 12.1 Hz, 1H), 2.72 – 2.78 (m, 2H), 2.79 (s, 3H),
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2.87 – 2.93 (m, 1H), 3.01 – 3.08 (m, 3H), 3.13 (q, J = 6.8, 6.8, 6.8 Hz, 1H), 3.19 – 3.26 (m, 2H), 4.03
(t, J = 5.7, 5.7 Hz, 2H), 4.39 (d, J = 13.3 Hz, 2H), 6.85 (d, J = 8.6 Hz, 2H), 7.15 (d, J = 8.6 Hz, 2H),
7.48 (d, J = 10.2 Hz, 1H), 8.06 (d, J = 10.2 Hz, 1H). 13C NMR (176 MHz, DMSO, 30 °C) δ 14.97,
32.40, 33.62, 40.57, 45.83, 45.88, 47.39, 52.00, 59.60, 65.87, 114.33, 115.49, 124.19, 127.52, 134.33,
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
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7
8
9
0
1
2
3
4
5
6
7
8
9
0
+
+
137.56, 143.31, 155.22, 156.76, 169.22. Purity 100% by LCꢀMS, m/z: ES [M+H] 484. HRMS (ESI)
3
5
+
3 2
Calcd. for C24H30ClN O [ M+H] 484.2222; Found 484.2226.
(
R)-1,3-Dimethyl-4-(2-(4-(1-(3-(methylthio)-[1,2,4]triazolo[4,3-b]pyridazin-6-yl)piperidin-4-
yl)phenoxy)ethyl)piperazin-2-one (12)
1
1
1
2
1
4
1
H NMR (700 MHz, DMSO) δ 1.22 (d, J = 6.8 Hz, 3H), 1.64 (qd, J = 3.8, 12.8 Hz, 2H), 1.85 (d, J =
2.6 Hz, 2H), 2.53 (s, 3H), 2.65 – 2.69 (m, 2H), 2.73 – 2.78 (m, 1H), 2.79 (s, 3H), 2.91 (dt, J = 5.9,
3.6 Hz, 1H), 3.01 – 3.06 (m, 3H), 3.13 (q, J = 6.8 Hz, 1H), 3.19 – 3.26 (m, 2H), 4.03 (t, J = 6.2 Hz,
H), 4.35 (d, J = 13.2 Hz, 2H), 6.85 (d, J = 8.7 Hz, 2H), 7.16 (d, J = 8.7 Hz, 2H), 7.39 (d, J = 10.2 Hz,
H), 8.01 (d, J = 10.2 Hz, 1H). 13C NMR (176 MHz, DMSO, 30 °C) δ 13.41, 14.99, 32.39, 33.63,
0.41, 40.61, 45.84, 45.99, 47.40, 52.01, 59.61, 65.88, 114.35, 114.61, 124.10, 127.54, 137.65,
+
+
43.41, 144.16, 154.79, 156.76, 169.24. Purity 97% by LCꢀMS, m/z: ES [M+H] 496. HRMS (ESI)
+
Calcd. for C25
H
33
N
7
O
2
S [M+H] 496.2489; Found 496.2488.
(
R)-4-(2-(4-(1-(3-Methoxy-[1,2,4]triazolo[4,3-b]pyridazin-6-yl)piperidin-4-yl)phenoxy)ethyl)-1,3-
dimethylpiperazin-2-one (13)
1H NMR (700 MHz, DMSO) δ 1.22 (d, J = 6.8 Hz, 3H), 1.62 (qd, J = 3.9, 12.8 Hz, 2H), 1.82 (d, J =
11.3 Hz, 2H), 2.67 (ddd, J = 4.7, 7.1, 12.1 Hz, 1H), 2.7 – 2.78 (m, 2H), 2.79 (s, 3H), 2.90 (dt, J = 5.9,
13.6 Hz, 1H), 2.97 (td, J = 2.1, 13.0 Hz, 2H), 3.04 (dt, J = 5.0, 12.4 Hz, 1H), 3.13 (q, J = 6.8 Hz, 1H),
3.19 – 3.26 (m, 2H), 4.01 – 4.04 (m, 2H), 4.17 (s, 3H), 4.28 (d, J = 13.3 Hz, 2H), 6.85 (d, J = 8.7 Hz,
2H), 7.14 (d, J = 8.7 Hz, 2H), 7.27 (d, J = 10.3 Hz, 1H), 7.84 (d, J = 10.3 Hz, 1H). 13C NMR (176
MHz, DMSO, 30 °C) δ 14.97, 32.42, 33.62, 40.63, 45.84, 46.09, 47.39, 52.01, 57.67, 59.61, 65.87,
114.33, 114.38, 124.31, 127.50, 137.66, 140.39, 153.61, 154.43, 156.74, 169.22. Purity 100% by LCꢀ
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Page 29 of 60
Journal of Medicinal Chemistry
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MS, m/z: ES [M+H] 480. Enantiomeric purity >98% as measured by chiral HPLC. HRMS (ESI)
+
Calcd. for C H N O [M+H] 480.2718; Found 480.2718.
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(R)-4-(2-(4-(1-(3-(Dimethylamino)-[1,2,4]triazolo[4,3-b]pyridazin-6-yl)piperidin-4-
yl)phenoxy)ethyl)-1,3-dimethylpiperazin-2-one (14)
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A solution of (R)ꢀ1,3ꢀdimethylꢀ4ꢀ(2ꢀ(4ꢀ(piperidinꢀ4ꢀyl)phenoxy)ethyl)piperazinꢀ2ꢀone (31) (0.21 g,
0.63 mmol) and 3,6ꢀdichloroꢀ[1,2,4]triazolo[4,3ꢀb]pyridazine (0.12 g, 0.63 mmol) dissolved in DMF
(
3.0 mL) was treated with NꢀethylꢀNꢀisopropylpropanꢀ2ꢀamine (0.17 mL, 0.95 mmol) under nitrogen.
The resulting mixture was stirred at 20 °C for 16 hours. The mixture was treated with dimethylamine
in THF (2.0 M, 1.3 mL, 2.5 mmol) and heated in a sealed tube in microwave at 140 °C for 72 hours.
The reaction mixture was evaporated to afford crude product. The crude product was purified by
preparative HPLC (Waters XBridge Prep C18 OBD column, 5 µ silica, 50 mm diameter, 150 mm
length), using decreasingly polar mixtures of water (containing 1% NH
Fractions containing the desired compound were evaporated to dryness to afford (R)ꢀ4ꢀ(2ꢀ(4ꢀ(1ꢀ(3ꢀ
dimethylamino)ꢀ[1,2,4]triazolo[4,3ꢀb]pyridazinꢀ6ꢀyl)piperidinꢀ4ꢀyl)phenoxy)ethyl)ꢀ1,3ꢀ
dimethylpiperazinꢀ2ꢀone (14) (0.15 g, 48%) as a pale yellow solid. 1H NMR (700 MHz, DMSO) 1.22
d, J = 6.8 Hz, 3H), 1.64 (qd, J = 3.8, 12.7 Hz, 2H), 1.83 (d, J = 12.1 Hz, 2H), 2.67 (ddd, J = 4.8, 7.0,
3
) and MeCN as eluents.
(
(
1
2.0 Hz, 1H), 2.71 – 2.78 (m, 2H), 2.79 (s, 3H), 2.90 (dt, J = 5.9, 13.6 Hz, 1H), 2.99 (t, J = 11.9 Hz,
H), 3.01 – 3.06 (m, 1H), 3.04 (s, 6H), 3.13 (q, J = 6.8 Hz, 1H), 3.19 – 3.26 (m, 2H), 4.02 (t, J = 5.7
2
Hz, 2H), 4.25 (d, J = 13.2 Hz, 2H), 6.85 (d, J = 8.7 Hz, 2H), 7.15 (d, J = 8.7 Hz, 2H), 7.21 (d, J = 10.2
Hz, 1H), 7.82 (d, J = 10.2 Hz, 1H). 13C NMR (176 MHz, DMSO, 30 °C) 14.98, 32.36, 33.62, 39.58,
40.41, 40.63, 45.84, 46.26, 47.39, 52.01, 59.61, 65.87, 113.09, 114.34, 124.44, 127.51, 137.69,
+
+
1
40.85, 152.01, 153.75, 156.74, 169.23. Purity 100% by LCꢀMS, m/z: ES [M+H] 493. HRMS (ESI)
+
Calcd. for C26
H
36
N
8
O
2
[M+H] 493.3034; Found 493.3032.
The following compounds were synthesised using analogous procedures:
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