Optimization of a Novel Binding Motif to (E)-3-(3,5-Difluoro-4-((1R,3R)-2-(2-fluoro-2-methylpropyl)-3-methyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylic Acid (AZD9496), a Potent and Orally Bioavailable Selective Estrogen Receptor Downregulator and Antagonist

Article abstract of DOI:10.1021/acs.jmedchem.5b00984

The discovery of an orally bioavailable selective estrogen receptor downregulator (SERD) with equivalent potency and preclinical pharmacology to the intramuscular SERD fulvestrant is described. A directed screen identified the 1-aryl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole motif as a novel, druglike ER ligand. Aided by crystal structures of novel ligands bound to an ER construct, medicinal chemistry iterations led to (E)-3-(3,5-difluoro-4-((1R,3R)-2-(2-fluoro-2-methylpropyl)-3-methyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylic acid (30b, AZD9496), a clinical candidate with high oral bioavailability across preclinical species that is currently being evaluated in phase I clinical trials for the treatment of advanced estrogen receptor (ER) positive breast cancer.

Full text of DOI:10.1021/acs.jmedchem.5b00984

Subscriber access provided by NEW YORK MED COLL
Article
Optimization of a novel binding motif to (E)-3-(3,5-difluoro-4-((1R,3R)-2-
(2-fluoro-2-methylpropyl)-3-methyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-
b]indol-1-yl)phenyl)acrylic acid (AZD9496), a potent and orally
bioavailable selective estrogen receptor downregulator and antagonist
Chris De Savi, Robert H. Bradbury, Alfred A. Rabow, Richard A. Norman, Camila de Almeida, David
M. Andrews, Peter Ballard, David Buttar, Rowena Callis, Gordon S Currie, Christopher Davies, Craig
Donald, Lyman Feron, Barry R. Hayter, Syeed Hussain, Galith Karoutchi, Scott Lamont, Philip A. MacFaul,
Thomas A. Moss, Stuart Pearson, Michael Tonge, Graeme Walker, Hazel Weir, and Zena Wilson
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.5b00984 • Publication Date (Web): 25 Sep 2015
Downloaded from http://pubs.acs.org on September 27, 2015
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Journal of Medicinal Chemistry is published by the American Chemical Society. 1155
Sixteenth Street N.W., Washington, DC 20036
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of their duties.

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HO
O
Cl
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F
H
H
N
N
F
N
N
OH
F
ER binding screen hit,
pIC50 = 5.9
AZD9496, orally bioavailable
SERD, pIC50 = 9.9
INTRODUCTION
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Following the pioneering discovery of the estrogen receptor α (ERα) antagonist tamoxifen (Figure 1,
1a) in the 1960s,1,2 identification of the selective estrogen receptor downregulator (SERD) fulvestrant
(2) represented a further step forward in treatment of advanced ERꢀpositive breast cancer.2ꢀ5
Resistance to tamoxifen can develop due to a number of mechanisms that switch the mode of action
from antagonism to agonism, thereby promoting tumour cell reꢀgrowth.6a The pure estrogen
downregulatorꢀantagonist fulvestrant offers the advantage of not only antagonising ERαꢀdriven tumour
cell growth but also degrading ERα via ubiquitinylation and thus diminishing growth due to reduced
ERα content.3b Furthermore, the discovery of ESR1 mutations in metastatic breast cancer patients that
had received prior endocrine therapy, where the mutated receptor is active in the absence of estrogen,
also highlights a further resistance mechanism.6b,c
Fulvestrant has very low oral bioavailability7 and is administered as a monthly intramuscular implant at
a dose of up to 500 mg.8 Clinical efficacy of fulvestrant is believed to be limited by less than 50% ERα
turnover detected in patient samples, compared to complete receptor downregulation seen in in-vitro
breast cell line experiments.9
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3a, R = H
3b, R = OH
3c
1a, R = H
1b, R = OH
HO
O
HO
O
N
O
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R
Cl
R
HN
N
OH
H
2
O
S
F
F
H
H
F
F
HO
F
Figure 1. Chemical structures of tamoxifen (1a), hydroxytamoxifen (1b), fulvestrant (2), GW5638
(3a), GW7604 (3b) and ARNꢀ810 (3c).
A potent, orally bioavailable SERD has potential for significantly higher receptor knockdown
compared to fulvestrant due to higher exposure. A number of SERD agents with low to moderate
rodent oral bioavailability have been reported, including tamoxifen analogs GW5638 (3a) and
GW7604 (3b)10ꢀ12 and various steroidal13,14 and nonꢀsteroidal derivatives.15ꢀ18 Other than ARNꢀ810
(3c)19 no oral SERD has yet entered clinical trials.
Although seldom made explicit in the medicinal chemistry literature,20 achieving oral bioavailability is
a key challenge in the design of ER modulators. Clinical efficacy of tamoxifen reflects formation of
significantly more active hydroxytamoxifen (1b),21,22 in which the phenolic moiety mimics the Aꢀring
phenol group of the endogenous ligand estradiol. Tamoxifen is thus an orally bioavailable prodrug of
more active phenolic metabolites.
Among the efforts of many research groups from the 1980s onwards in designing tissueꢀselective ER
modulators for treatment of hormoneꢀdependent breast cancer and osteoporosis,2,22,23 lasofoxifene20,24
provides a rare example of a phenolic ER modulator with low clearance and high oral bioavailability
across species, a property attributed to conformational effects structurally remote from the phenolic
moiety on the rate of gut wall glucuronidation.20 We however concluded that, exacerbated by high
lipophilicity, the presence of a phenol moiety in ER modulators is more generally associated with high
clearance and/or poor oral absorption.
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Rather than seeking to improve pharmacokinetic properties of known ER motifs, we chose to follow a
less explored strategy of undertaking a directed ER binding screen to identify novel motifs with
druglike properties that could then be optimized for cellular phenotype and potency. In this
publication, we report the discovery of 1ꢀarylꢀ2,3,4,9ꢀtetrahydroꢀ1Hꢀpyrido[3,4ꢀb]indoles as a novel,
orally bioavailable ER binding motif and outline the lead optimization program that led to SERD
clinical candidate AZD9496 (30b).
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RESULTS
Biological data for illustrative compounds selected from several hundred diverse examples prepared
during the course of this work are listed in Table 1, along with reference data for 1a-b, 2 and 3a-b. As
outlined in Schemes 1ꢀ4, central to synthesis of the novel compounds listed in Table 1 was use of the
PictetꢀSpengler cyclisation25ꢀ27 to construct the 1ꢀarylꢀ2,3,4,9ꢀtetrahydroꢀ1Hꢀpyrido[3,4ꢀb]indole
skeleton from appropriate tryptamine and aryl aldehyde precursors.
Racemic screening hit 6 was obtained via alkylation of the readily accessible racemic unsubstituted
amine 5a28 with 2ꢀbromoethanol, and screening hit 7b was similarly obtained by alkylation of 5a with
methyl 3ꢀbromopropionate and subsequent hydrolysis of ester 7a (Scheme 1). Synthesis of trans-1R,
3R compounds containing a methyl group of defined stereochemistry adjacent to the tetrahydro
piperidine nitrogen atom commenced with the appropriate chiral αꢀmethyltryptamine (Schemes 1ꢀ4).
In accord with published work,25ꢀ27 PictetꢀSpengler cyclisation of Nꢀunsubstituted (R)ꢀαꢀ
methyltryptamine 4b with 4ꢀchlorobenzaldehyde gave predominantly the cis isomer as the kinetic
product that could be enriched in trans isomer 5b under acidic conditions. Alkylation of 5b gave 8 and
9a, and the latter hydrolysed to 9b (Scheme 1).
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Scheme 1. Synthesis of compounds 6, 7b, 8, 9ba,b
6
Cl
7
8
H
N
H
(a), (b)
N
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NH2
R1
NH
R1
4a, R1 = H
5a, R1 = H (racemic)
5b, R1 = CH3
4b, R1 = CH3
(c)
Cl
H
N
6, R1 = H, R2 = (CH2)2OH (racemic)
7a, R1 = H, R2 = (CH2)2CO2CH3 (racemic)
7b, R1 = H, R2 = (CH2)2CO2H (racemic)
8, R1 = CH3, R2 = (CH2)2OH
(d)
(d)
N
R2
9a, R1 = CH3, R2 = (CH2)2CO2CH3
9b, R1 = CH3, R2 = (CH2)2CO2H
R1
aCompounds 6 and 7b prepared as racemates; Reagents and conditions: (a) 4ꢀchlorobenzaldehyde,
b
o
o
PhMe, 105 C; (b) AcOH, PhMe, 105 C, 50ꢀ75%; (c) 2ꢀbromoethanol or methyl 3ꢀbromopropionate,
Et3N, MeCN, 140 oC, 72ꢀ76%; (d) NaOH/H2O/THF, 65 oC, 94ꢀ96%.
It is also well established25ꢀ27 that substitution of the αꢀmethyltryptamine nitrogen atom leads
predominantly to the desired trans stereochemistry during the PictetꢀSpengler cyclisation.
Compounds 15 and 16 were thus accessed as shown in Scheme 2. Alkylation of the appropriate αꢀ
methyltryptamines 10 and 4b gave 11 and 12, respectively. PictetꢀSpengler cyclisation with 4ꢀ
chlorobenzaldehyde and subsequent deprotection afforded the desired products 13 and 14.
Scheme 2. Synthesis of compounds 15-16a,b
H
H
N
N
(a) or (b)
R1
NHR2
R1
NH2
10, R1 = OCH3
4b, R1 = H
11, R1 = OCH3, R2 = (CH2)2OH
12, R1 = H, R2 = CH2CH(CH3)CO2CH3 (S-epimer)
Cl
Cl
(c), (d) or (e)
(f) or (g)
H
H
N
N
R1
N
R2
R1
N
R2
15, R1 = OH, R2 = (CH2)2OH
16, R1 = H, R2 = CH2CH(CH3)CO2H (S-epimer)
13, R1 = OCH3, R2 = (CH2)2OH
14, R1 = H, R2 = CH2CH(CH3)CO2CH3 (S-epimer)
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aSynthesis of 15 carried out using racemic precursor and enantiomerically pure material obtained by
chiral chromatography at the final step; bReagents and conditions: (a) 2ꢀbromoethanol, K2CO3, MeCN,
80 oC, 53%; (b) (S)ꢀmethyl 2ꢀmethylꢀ3ꢀ(trifluoromethylsulfonyloxy)propanoate (17), iꢀPr2NEt,
o
o
o
dioxane, 80 C, 79%; (c) 4ꢀchlorobenzaldehyde, AcOH, 80 C; (d) NaOH, H2O, MeOH, 60 C, 47%;
o
o
(e) 4ꢀchlorobenzaldehyde, AcOH, PhMe, 90 C, 72%; (f) BBr3, DCM, ꢀ78 C, then RT, 35%; (g)
NaOH, H2O,THF, 65 oC, 93%.
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For synthesis of initial acrylic acid derivatives 22b-24b, a route was developed (Scheme 3) that
enabled variation of the basic nitrogen substituent at a late stage of the synthesis. Reaction of 4b with
aldehyde 18 gave predominantly the expected cis product 19. Alkylation with allyl bromide and
treatment with acid provided a 65:35 mixture of the trans isomer 20 and the corresponding cis isomer,
from which the desired trans intermediate 21 could be isolated pure after removal of the allyl group.
Alkylation and ester hydrolysis then gave the acrylic acid derivatives 22b-24b.
Finally, compounds 27b-30b were obtained by the optimised route shown in Scheme 4, in which 4b
was first alkylated to give 25a-b. PictetꢀSpengler cyclisation under acidic conditions with the
appropriate aldehyde 18 or 26 provided the pure trans products 27a-30a in good yield after
chromatography, and ester hydrolysis then gave 27b-30b.
Scheme 3. Synthesis of compounds 22b-24ba
O
O
O
O
O
OMe
OMe
OR2
OMe
OMe
4b
H
H
H
H
+
N
N
(a)
(d)
N
(b), (c)
N
(e) or (f)
N
NH
N
R1
NH
O
22a, R1 = (CH2)2OH, R2 = CH3
22b, R1 = (CH2)2OH, R2 = H
(g)
21
18
19
20
23a, R1 = CH2CH(CH3)CH2OH, R2 = CH3 (S-epimer)
23b, R1 = CH2CH(CH3)CH2OH, R2 = H (S-epimer)
24a, R1 = CH2CH(CH3)CH2OH, R2 = CH3 (R-epimer)
24b, R1 = CH2CH(CH3)CH2OH, R2 = H (R-epimer)
(g)
(g)
aReagents and conditions: (a) AcOH, 80 oC, 74%; (b) allyl bromide, iꢀPr2NEt, MeCN, 145 oC; (c) TFA,
o
o
DCM, 20 C, 82%; (d) (Ph3P)3RhCl, MeCN, 100 C, 56% ; (e) 2ꢀbromoethanol, iꢀPr2Net, MeCN, 140
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oC, 76%; (f) (R)ꢀ or (S)ꢀ3ꢀbromoꢀ2ꢀmethylꢀ1ꢀpropanol, iꢀPr2Net, MeCN, 135 oC, 64%; (g) NaOH, H2O,
3
4
THF, MeOH, 20 oC, 75ꢀ93%.
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Scheme 4. Synthesis of compounds 27b-30ba
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OR3
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O
OMe
27a, R1 = R2 = H, R3 = CH3
(d)
27b, R1 = R2 = H, R3 = H
28a, R1 = H, R2 = F, R3 = CH3
28b, R1 = H, R2 = F, R3 = H
29a, R1 = F, R2 = H, R3 = CH3
29b, R1 = F, R2 = H, R3 = H
30a, R1 = R2 = F, R3 = CH3
30b, R1 = R2 = F, R3 = H
R2
H
N
H
(a) or (b)
N
(d)
(c)
4b
R2
H
R2
N
(d)
(d)
+
N
R2
R1
O
R1
25a, R1 = H
25b, R1 = F
18, R = H
26, R = F
aReagents and conditions: (a) isobutyraldehyde, Na(OAc)3BH, THF, 0 C, 44%; (b) 2ꢀfluoroꢀ2ꢀ
methylpropyl trifluoromethanesulfonate (31), iꢀPr2NEt, dioxane, 90 oC, 91%; (c) AcOH, PhMe, 80 C,
o
o
68ꢀ74%; (d) NaOH, H2O, THF, MeOH, 20oC, 89ꢀ92%.
Table 1. ER binding, ER downregulation, PR agonism, PR antagonism29 and MCF7
antiproliferation42,43 data for 1-3, 6, 7b, 8, 9b, 15-16, 22b-24b, 27b-30b
a
a
a
a
a
Entry ER binding pIC50 ER downregulation pIC50
PR agonism pIC50
PR antagonism pIC50
MCF7 antiproliferation pIC50
1a
1b
2
7.61
9.51
9.09
8.49
9.84
5.69
4.86
7.4
5.0
<4
6.89
9.1
7.24
9.27
10
5.31
10.2
5.89
8.17
5.93
5.43
7.07
6.11
7.36
7.52
5.88
7.33
7.34
8.68
9.38
9.07
<4
<4
9.68
3a
3b
6
<4
5.4
6.53
NDc
NDc
NDc
4.86
<4.6
<4.5
<4.5
6.09
7.65
NDc
9.11
9.64e
9.49
<4.5
<4
7.92b
5.57
NDd
6.14
5.82
6.04
5.68
5.64
7.31
7.3
7b
8
<4.5
<4
9b
15
16
5.75
7.65
7.15
<4
7.48
7.89
<4
22b 6.16
23b 7.16
24b 7.01
27b 8.19
28b 9.15
29b 9.0
<4
<4
<4
8.44
9.08
8.46
<5.5
<5.5
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30b 9.17
9.86
<5.5
9.55
10.4
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d
an ≥ 3, SEM values are available in the supplementary material; n = 2; cNot determined; Unable to
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determine PR antagonist pIC50 due to inconsistent response across concentration range; n = 1.
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Compounds listed in Table 1 were evaluated in a previouslyꢀdescribed cascade of assays29 designed to
identify compounds with the requisite ER downregulatorꢀantagonist profile in an MCF7 cell line, in
which measurement of progesterone receptor (PR) levels was used as a downstream biomarker of ER
agonism via an ERꢀmediated or indirect mechanism, such as inhibition of histone deacetylase. These
assays were designed specifically to measure ERα downꢀregulation, agonism and antagonism through
simultaneous measurement of ER and PR levels in cells after compound treatment. To confirm the
mechanism of ERα downꢀregulation, assays were performed in the presence of a fixed concentration of
tamoxifen (a representative output in Figure 6b) or cyclohexamide (assay not shown) at 5 hours. If ER
downregulation seen with cyclohexamide treatment is unaltered from the 24 hour value, this indicates
downꢀregulation is not as a result of an alternative pathway that requires further protein synthesis.
Adding a fixed concentration of tamoxifen lowers the IC50 value should a compound and tamoxifen be
competing for binding to the LBD.29
In order to identify novel, drugꢀlike ER binding cores, a directed highꢀthroughput screening set was
generated. A computational approach was used that focused on covering a productive drugꢀlike
subspace with lower complexity compounds. The approach had two main tenets, in firstly choosing a
set of lower complexity compounds as measured by number of heavy atoms, and number of rotatable
bonds, and secondly in using chemical clustering to limit the number of compounds representing each
point in chemical space.
Application of this approach allowed the AstraZeneca collection of 1.5 million compounds to be
covered by a subset of 100K exemplars. This subset of lowerꢀcomplexity compounds was chosen to
represent four different categories of molecules: 7.5% phenol containing compounds, 7.5% compounds
with phenol isosteres, 46% with generalized ER pharmacophores and 39% covering chemotypes not
represented by the preceding categories.
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A hit rate of 4.2 % was obtained30 when the resulting set of 90K nonꢀphenol compounds was evaluated
at a concentration of 10 µM in a directed highꢀthroughput screen that measured affinity for the human
ERα ligand binding domain by fluorescence polarization.29 It is notable that the hit rate of 19.2% was
significantly higher with the 10K phenol compounds tested than that of the nonꢀphenol compounds at
4.2%. As discussed earlier, despite the high hitꢀrate it was not the phenol hits but the nonꢀphenolic
compounds that proved to be the more promising start for identifying drugꢀlike compounds which
possessed low clearance and high oral bioavailability.
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Determination of IC50 values identified the 1ꢀarylꢀ2ꢀ(2ꢀhydroxyethyl)ꢀ2,3,4,9ꢀtetrahydroꢀ1Hꢀ
pyrido[3,4ꢀb]indole derivative 6 as a novel and moderately potent ER binder (pIC50 5.7) that appeared
to be a particularly attractive chemical starting point (MW 317, measured LogD = 3.7). Good
selectivity was seen versus a core panel of nuclear hormone receptor binding assays (pIC50 < 4.5 vs
AR, PR and GR).
Compound 6 showed moderate activity (pIC50 5.9) in the ER downregulation assay, along with
minimal activity in the PR agonism assay.29 The structurally related, more hydrophilic carboxylic acid
7b (measured LogD 1.6) was also detected in the ER binding screen and showed a similar
pharmacological profile to 6. Performing the downregulation assay in the presence of tamoxifen shifted
the IC50 curves of 6 and 7b in a manner consistent with a competitive interaction at the same site in the
ER ligand binding domain. As a measure of ER antagonism, compound 6 also decreased the
downstream estradiolꢀdriven PR response in the MCFꢀ7 cell line.
Summarised in Table 2 are data for selected compounds from a number of routine physical property
and metabolic stability assays. Although the more lipophilic screen hit 6 was highly turned over in rat
and human hepatocytes, the more hydrophilic variant 7b was significantly more stable, and dosing as a
racemate in a rat PK study showed encouragingly low clearance and high oral bioavailability (Table 3).
Further incentive to pursue this novel ER binding motif as a medicinal chemistry start point was
provided by the presence of the 1ꢀarylꢀ2,3,4,9ꢀtetrahydroꢀ1Hꢀpyrido[3,4ꢀb]indole core as a substructure
in the approved PDE5 inhibitor tadalafil33 and in clinical candidates NITDꢀ60934 (antimalarial) and
PTC29935ꢀ37 (VEGFR expression inhibitor), implying that this motif can be regarded as a “privileged
substructure”38ꢀ39 for drug discovery.
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Table 2. LogD,32 aqueous solubility, rat and human protein binding and hepatocyte stability data for 6,
7b, 16, 23b, 27b and 30b
5
6
7
8
a
Entry
6
LogD pH7.4 Solubility µM pH7.4
% Free rat/humanb
4.5/NDd
7.2/NDd
Rat/Human hepatocyte Clint (µl/min/106 cells)c
3.7
1.6
2.8
1.6
2.6
2.8
15 (semiꢀcrystalline)
>1600 (undefined)
300/70
29/19
82/23
21/26
18/40
4.2/32
7b
9
16
1440 (amorphous)
3.2/1.5
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23
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26
27
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30
31
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34
35
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37
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39
40
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44
45
46
47
48
49
50
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52
53
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56
57
58
59
60
23b
27b
30b
>1400 (semiꢀcrystalline)
>1000 (semiꢀcrystalline)
110 (crystalline)
9.1/4.6
0.69/0.51
0.37/0.26
a24 hr thermodynamic solubility of solid sample determined in 0.1M phosphate buffer (parentheses
refer to visual examination of physical form of undissolved sample under a microscope at 24 hr
b
timepoint); Determined from DMSO stock solution by equilibrium dialysis in 10% plasma from Han
Wistar rats or 10% human plasma supplied by Quintiles; cRate of metabolism determined from DMSO
stock solution in isolated rat or human cryopreserved hepatocytes diluted to 1x106 cells/ml; Not
d
determined
.
Varying the nature, position and multiplicity of the pendant or fused aryl ring substituents (halogens,
OMe, CF3, CN) or removal of the Nꢀsubstituent reduced binding potency and downregulation activity
(data not shown). As depicted in Figure 2, a crystal structure of compound 7b bound to a construct of
the ERα ligand binding domain31 could be readily overlaid with the phenol metabolite of tamoxifen
(1b).21,22
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Figure 2. Crystal structure of 7b (yellow, pdb code 5AAU) superimposed on reference structure of 1b
(grey, pdb code 3ERT) bound to the ERα ligand binding domain construct.31 The blue circle indicates
how the ethyl group of 1b occupies a region of the ligand binding domain not accessed by compound
7b termed Pheꢀ404:Pheꢀ425 lipophilic hole. The red circle indicates an additional liphophilic pocket
unoccupied by both 1b and 7b termed the Leuꢀ525:Leu384 lipophilic hole.
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A number of key interactions are evident from the crystal structure of 7b, with the benzenoid ring of
the indole overlaying with the phenolic moiety of 1b, the indole NH picking up a novel interaction by
forming a hydrogen bond to the carbonyl of Leuꢀ346, the carboxylic acid moiety sitting adjacent to
Hisꢀ524 and the pendant aryl substituent orientated towards the Helixꢀ12 region of the ER, an
interaction proposed to be key for ER downregulation.12,40,41
Of particular significance, the overlay of 7b and 1b suggested that introduction of a methyl substituent
adjacent to the ring nitrogen in 7b could boost potency by exploiting the “Pheꢀ404:Pheꢀ425 lipophilic
hole” occupied by the ethyl group of 1b (Figure 2, blue circle). Chiral synthesis of the 4 stereoisomers
containing the additional methyl group showed that activity resided exclusively with the trans-(1R,3R)
enantiomers 8 and 9b, and that these derivatives were significantly more potent ER binders and
downregulators than progenitors 6 and 7b.
Although Figure 2 showed an imperfect overlay of the benzenoid ring of the indole with the phenolic
ring of 1b, we considered it worthwhile to probe the effect of introducing a phenol moiety into 8. In
contrast with 1a-b, no significant increase in binding and downregulation potency was seen for the
resulting phenol 15, and the PR agonism readout revealed that introduction of the phenol moiety had
switched the profile of 15 to an ER agonist.
Structural changes elsewhere also subtly influenced mechanism of action. Further examination of the
crystal structure suggested that introduction of a methyl group on the Nꢀsubstituent of 9b could pick up
additional binding, an observation supported by the increased potency of compound 16, but once again
with adoption of an agonist profile. No clearcut rationale for the change of profile was evident from
the crystal structure of 16 bound to the ERα ligand binding domain. We were encouraged, however, by
the rat oral bioavailability and overall property profile of 16 continuing to represent a viable start point
for further medicinal chemistry (Tables 2 and 3).
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Table 3. Rat,a mouse and dogb pharmacokinetic parameters for 7b, 16, 27b and 30b.
3
4
5
6
7
8
Entry
7b
Species Vdss l/kg
Cl ml/min/kg Bioavailability %
Ratc
Ratc
Ratd
0.6
4
85
16
0.34
14
42
27b
1.1
6.2
21
80
Mousee 3.7
86
Dogf
Ratg
Mouseh 2.2
Dogf
0.40
0.3
0.96
1.0
43
111
63
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60
30b
0.43
128
79
0.28
aDosed to 2 male Han Wistar rats; Dosed to 1 male and 1 female Alderley Park beagle; blood PK
parameters, dosed at 2 ꢁmol/kg i.v. and 5 ꢁmol/kg p.o.; dblood PK parameters, dosed at 5 ꢁmol/kg i.v.
b
c
e
and 13 ꢁmol/kg p.o.; blood PK parameters from 2 male CDꢀ1 mice dosed at 5 ꢁmol/kg i.v. and 13
f
g
ꢁmol/kg p.o.; plasma PK parameters, dosed at 2.5 ꢁmol/kg i.v. and 5 ꢁmol/kg p.o.; plasma PK
h
parameters, dosed at 2.2 ꢁmol/kg i.v. and 4.5 ꢁmol/kg p.o.; blood PK parameters from 2 male SCID
mice dosed at 2.3 ꢁmol/kg i.v. and 5.6 ꢁmol/kg p.o.
Lack of activity of 15 and 16 in an MCF7 breast tumor cell antiproliferation assay used historically to
determine antioestrogenic activity42,43 was consistent with an agonist profile. In contrast with
prototypic downregulators 3a-b, earlier compounds 8 and 9b also showed no effect in the MCF7
antiproliferation assay. On closer examination of the PR agonism data, at the top concentration in the
assay 8, 9b and related compounds showed evidence of weak agonism (typically 15ꢀ30% inhibition at
100 µM), whereas 3a-b gave no detectable inhibition at the highest concentration. We therefore
concluded that whereas 3a-b had a pure downregulatorꢀantagonist profile,12 the lack of antiproliferative
activity of 8 and 9b in the MCF7 cell line could reflect partial agonism.40,41
An overlay of the crystal structures of 3a and the original screen hit 7b bound to the ERα ligand
binding domain (Figure 3) showed the acrylic acid moiety of 3a colocalizing with Aspꢀ351 in the
Helixꢀ12 region of the ER, in an unusual acidꢀacid interaction that has been proposed to be crucial for
achieving a downregulatorꢀantagonist profile.12 Hybridization of compound 8 with 3a thus gave the
moderately potent downregulatorꢀantagonist 22b, with no detectable agonism at the highest test
concentration and ERꢀdownregulation and antagonism activity that translated well into the MCF7
antiproliferative assay.
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Figure 3. Crystal structure of 7b (yellow, pdb code 5AAU) superimposed on reference structure 3a
(grey, pdb code 5AAV) bound to the ERα ligand binding domain construct.31 The blue circle indicates
how the acrylic acid moiety of 3a occupies a region of the ER Helixꢀ12 not accessed by compound 7b.
Potency could be further improved by homologation of the Nꢀsubstituent and, as seen previously, by
introduction of a methyl group to occupy the additional lipophilic pocket (compounds 23b-24b). Due
to the presence of the acrylic acid and alcohol functionalities, these compounds were relatively
hydrophilic (e.g. measured LogD value of 1.6 for 23b) and consequently cellular potency could be
significantly increased by removal of the alcohol moiety. By way of illustration, compounds 27b and
compound 3a share similar profiles in terms of ER downregulation and PR agonism (Figure 4).
Compound 27b showed an encouraging overall profile (Table 2) and excellent PK parameters across
three species (Table 3). Oral exposure scaled well to higher doses in all three species (data not shown).
Based on efficacy seen in an MCF7 mouse tumour xenograft model43 at an oral once daily dose of 5
mg/kg, a preliminary predicted efficacious human dose was derived from an inꢀhouse physiologicallyꢀ
based pharmacokinetic (PBPK) model.44 A predicted once daily dose of 1000 mg prompted us to seek
further potency in order to reduce the predicted dose.
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Figure 4. ER downregulation (black) and PR protein levels (blue) for 3a and 27b in the MCF7 cell
line.
Previous SAR and ER coꢀcrystallisation data suggested limited scope for improving potency through
major structural changes, but from wider medicinal precedent45 we reasoned that relatively small
changes such as introduction of fluorine atom(s) could deliver the requisite increase. Of a number of
variations tried, ortho diꢀsubstitution on the pendant aryl ring (28b) and monofluorination of the Nꢀ
isobutyl substituent (29b) each provided an approximate 10ꢀfold increase in potency across the ER
binding, downregulation, antagonism and antiproliferation assays. Combining these structural changes
gave optimised compound 30b, with minimal increase in LogD relative to 27b and potency comparable
to fulvestrant (2). As shown in Figure 5, an overlay of 30b and 3a bound to the ER construct is
consistent with the earlier overlays depicted in Figures 2 and 3.
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Figure 5. Depiction of 30b (grey, pdb code 5ACC) superimposed on reference structure 3a (yellow,
pdb code 5AAV) bound to the ERα ligand binding domain construct.31 Note the lipophilic holes (Pheꢀ
404:Pheꢀ425 blue circle and Leuꢀ525:Leu384 red circle) are filled with methyl subsitituents increasing
potency for compound 30b.
Detailed pharmacological characterisation of compound 30b, including demonstration of ER and PR
knockdown in cellular assays is described elsewehere,46 but, for the purpose of this report, Figures 6aꢀc
display curves from ER downregulation (Figure 6a), tamoxifen competition (Figure 6b) and
antagonism (Figure 6c) assays that are wholly consistent with the desired ER downregulatorꢀantagonist
profile and comparable to fulvestrant (2) (downregulation curve also shown in Figure 6a).2
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Figure 6a. ER downregulation (black) and PR protein levels (blue) for 30b and 2 in the MCF7 cell
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line, showing no detectable agonism.
Figure 6b. ER downregulation curve (black) for 30b and decrease in potency in the presence of 0.25
µM tamoxifen (red) due to competition at the ER ligand binding site.
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Figure 6c. Doseꢀdependent decrease in estradiolꢀdriven PR response for 30b as a measure of ER
antagonism.
Compound 30b showed an overall property profile (Table 2) broadly comparable to progenitor 27b
while maintaining excellent crossꢀspecies PK (Table 3). The significant degradation of ERα by
Compound 30b in the in cell western assay was confirmed in the Western blot assay with both
Compound 2 (Fulvestrant) and estradiol (E2) as comparator compounds (Figure 7). Selectivity of
compound 30b over other tested nuclear hormone receptors is high: androgen receptor (AR); IC50 30
ꢀM, glucocorticoid receptor (GR); IC50 9.2 ꢀM and progesterone receptor (PR); IC50 0.54 ꢀM (c.f.
estrogen receptor (ERα); IC50 0.0008 ꢀM). When tested in an MCF7 mouse tumour xenograft model,
compound 30b showed doseꢀdependent inhibition of tumour growth (Figure 8). Input of the 24 hour
minimum efficacious concentration into the PBPK model44 gave a predicted once daily human dose of
below 100 mg, a value significantly lower than the maximum absorbable dose (MAD) calculated using
a gastrointestinal simulated model.47 Compound 30b was also tested in a long term estrogen deprived
model (LTED), using the HCCꢀ1428 LTED cell line that grows in the absence of estrogen and is
thought to best represent a model of aromatase inhibition.48 Compound 30b shows significant activity,
with a dose of 5 mg/kg giving tumor regressions in this model (Figure 1 and 2, supporting
information).46
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Figure 7. Degradation of ERα by Compound 30b, Compound 2 and estradiol (E2) by Western blot
analysis.49 MCFꢀ7 cells were incubated with Compound 2, Compound 30b or estradiol at the doses
above. Total protein was extracted after 48 hours incubation with compound and immunoblotting
performed to detect effects on ERα and PR levels.
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Figure 8. Doseꢀdependent antitumor activity of 30b administered orally once daily in an MCF7 mouse
tumor xenograft model.41
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CONCLUSIONS
6
7
8
9
A directed screen identified the 1ꢀarylꢀ2,3,4,9ꢀtetrahydroꢀ1Hꢀpyrido[3,4ꢀb]indole motif as a novel,
druglike ER ligand. Aided by crystal structures of novel ligands bound to an ER construct, medicinal
chemistry iterations led to ER downregulator 30b, an orally bioavailable clinical candidate with
comparable potency and pharmacological profile to the intramuscular SERD fulvestrant. AZD9496 is
currently being evaluated in Phase I clinical trials in patients with advanced estrogen receptor (ER)
positive breast cancer. More inꢀdepth data from antitumour models, including demonstration of ER
and PR knockdown in vivo is reported elsewhere.46
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EXPERIMENTAL SECTION
General synthesis protocols
Operations were carried out at ambient temperature, i.e. in the range 17 to 25oC and under an
atmosphere of an inert gas such as nitrogen, unless otherwise stated. Work up procedures were carried
out after removal of residual solids by filtration. Microwave reactions were carried out using a
Biotage® Initiator unless otherwise stated. Evaporations were carried out under reduced pressure at
below 50oC by rotary evaporation or by using a Biotage v10 evaporator or Genevac equipment. Flash
chromatography was performed on an automated Teledyne Isco CombiFlash® Rf or Teledyne Isco
CombiFlash® Companion®, using prepacked RediSep Rf Gold™ Silica Columns (20ꢀ40 ꢁm,
spherical particles), GraceResolv™ Cartridges (Davisil® silica) or Silicycle cartridges (40 ꢀ 63 ꢁm).
Preparative high performance liquid chromatography (HPLC) was performed on a Gilson instrument
using Waters Xbridge C18 reversedꢀphase silica gel, eluting with a decreasingly polar gradient of water
containing 1% ammonium hydroxide and acetonitrile. Ion exchange purification was performed using
an SCXꢀ2 (Biotage, propylsulfonic acid functionalized silica, manufactured using a trifunctional silane,
o
non endꢀcapped) cartridge. 1H NMR spectra were recorded on Bruker DPX400 instrument at 30 C
unless otherwise stated and are reported as δ values (parts per million) relative to Me4Si as an internal
standard. Mass spectra (MS) were recorded on a Waters ZQ ESCi mass spectrometer scanned over an
1
appropriate mass range. Compound purity was assessed by H NMR and LCMS run on a Waters
Alliance HT (2795) fitted with a Waters ZQ ESCi mass spectrometer and a Phenomenex GeminiꢀNX
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(50x2.1 5µm) column. Accurate masses were determined by high resolution mass spectroscopy
(HRMS) using a Waters Xevo G2 Qtof in ESI +ve ion mode.
5
6
7
8
9
1-(4-Chlorophenyl)-2,3,4,9-tetrahydro-1H-pyrido[3,4b]indole-2-ethanol (6). A solution of 1ꢀ(4ꢀ
chlorophenyl)ꢀ2,3,4,9ꢀtetrahydroꢀ1Hꢀpyrido[3,4ꢀb]indole28 (200 mg, 0.71 mmol), 2ꢀbromoethanol (0.15
mL, 2.12 mmol) and triethylamine (0.30 mL, 2.12 mmol) in MeCN (1.5 mL) was sealed in a
microwave tube. The solution was heated to 140°C for 3 hours in a microwave reactor, then evaporated
to dryness. The residue was purified by reverse phase chromatography, using decreasingly polar
mixtures of water (containing 1% ammonium hydroxide) and MeCN as eluents to give 6 (101 mg,
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1
44%) as a pale yellow solid. H NMR (500 MHz, DMSO d6) 10.33 (1H, s), 7.46 ꢀ 7.48 (1H, m), 7.42 ꢀ
7.46 (2H, m), 7.36 ꢀ 7.4 (2H, m), 7.26 (1H, dt), 7.06 (1H, ddd), 7.00 (1H, ddd), 4.83 (1H, s), 4.43 (1H,
t), 3.60 (1H, ddd), 3.54 (1H, td), 3.15 ꢀ 3.22 (1H, m), 2.74 ꢀ 2.88 (3H, m), 2.65 (1H, dt), 2.57 ꢀ 2.6 (1H,
m). HRMS m/z found 327.1282, C19H20N2OCl requires 327.1264.
Methyl 3-(1-(4-chlorophenyl)-3,4-dihydro-1H-pyrido[3,4-b]indol-2(9H)-yl)propanoate (7a). A
solution of 1ꢀ(4ꢀchlorophenyl)ꢀ2,3,4,9ꢀtetrahydroꢀ1Hꢀpyrido[3,4ꢀb]indole (5a)28 (956 mg, 3.4 mmol),
methyl 3ꢀbromopropanoate (1.1 mL, 10.2 mmol) and triethylamine (1.4 mL, 10.2 mmol) in MeCN
(1.5 mL) was sealed in a microwave tube. The solution was heated at 140 °C for 3 h in a microwave
reactor, then evaporated to dryness and the residue was purified by reverse phase chromatography,
elution gradient 55 to 100% MeCN in water containing 1% ammonium hydroxide, to give 7a (950 mg,
1
76%) as a beige solid. H NMR (400 MHz, DMSO d6) 2.53 ꢀ 2.65 (2H, m), 2.65 ꢀ 2.89 (5H, m), 3.10
(1H, m), 3.57 (3H, s), 4.75 (1H, s), 6.99 (2H, m), 7.22 (1H, d), 7.27 ꢀ 7.33 (2H, m), 7.38 ꢀ 7.46 (3H,
m), 10.30 (1H, s); m/z = 369, 371 [M+H]+.
3-(1-(4-Chlorophenyl)-3,4-dihydro-1H-pyrido[3,4-b]indol-2(9H)-yl)propanoic acid (7b). A 1.25 M
solution of sodium hydroxide (5.2 mL, 6.3 mmol) was added to a stirred solution of 7a (930 mg, 2.5
mmol) in THF (10 mL). The resulting solution was stirred at 65 °C for 2 h, then evaporated to dryness.
The residue was purified by reverse phase chromatography, elution gradient 20 to 40% MeCN in water
1
containing 1% ammonium hydroxide, to give 7b (830 mg, 94%) as a pale yellow solid. H NMR (400
MHz, DMSO d6) 1.91 ꢀ 2.04 (1H, m), 2.1 ꢀ 2.25 (1H, m), 2.51 ꢀ 2.63 (1H, m), 2.63 ꢀ 2.86 (4H, m), 3.01
ꢀ 3.13 (1H, m), 4.68 (1H, s), 6.9 ꢀ 7.02 (2H, m), 7.19 (1H, d), 7.29 ꢀ 7.34 (2H, m), 7.34 ꢀ 7.38 (2H, m),
7.40 (1H, d), 10.24 (1H, s). HRMS m/z found 355.1201, C20H19N2O2Cl requires 355.1212.
(1R,3R)-1-(4-Chlorophenyl)-3-methyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole
(5b).
4ꢀ
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Chlorobenzaldehyde (311 mg, 2.2 mmol) was added to a stirred slurry of (R)ꢀ1ꢀ(1Hꢀindolꢀ3ꢀyl)propanꢀ
2ꢀamine (4b) (385 mg, 2.2 mmol) in toluene (7 mL) under nitrogen. The resulting slurry was stirred at
105 °C for 30 minutes, then AcOH (0.70 mL) was added and heating was continued at 105 °C for 2 h.
The reaction mixture was initially purified by ion exchange chromatography, eluting with 7M ammonia
in MeOH. Product containing fractions were evaporated to dryness to give a brown gum, which was
further purified flash chromatography, elution gradient 5 to 90% EtOAc in heptane, to give 5b (330
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mg, 50%) as a pale yellow solid. H NMR (400 MHz, DMSO d6) 1.13 (3H, d), 2.25 ꢀ 2.38 (1H, m),
2.55 ꢀ 2.65 (1H, br s), 2.78 (1H, dd), 2.93 (1H, m), 5.13 (1H, s), 6.93 ꢀ 7 (1H, m), 7 ꢀ 7.06 (1H, m),
7.23 (2H, d), 7.26 (1H, d), 7.34 ꢀ 7.39 (2H, m), 7.42 (1H, d), 10.66 (1H, s); m/z = 295, 297 [MꢀH]ꢀ.
2-((1R,3R)-1-(4-Chlorophenyl)-3-methyl-3,4-dihydro-1H-pyrido[3,4-b]indol-2(9H)-yl)ethanol (8).
A solution of 5b (70 mg, 0.24 mmol), 2ꢀbromoethanol (0.017 mL, 0.24 mmol) and triethylamine (0.033
mL, 0.24 mmol) in MeCN (1.5 mL) was sealed in a microwave tube. The solution was heated at 140
°C for 1.5 h in a microwave reactor, then further quantities of 2ꢀbromoethanol (0.017 mL, 0.24 mmol)
and triethylamine (0.033 mL, 0.24 mmol) were added and heating continued at 140°C for 15 min. The
solution was evaporated to dryness, and the residue was purified by reverse phase chromatography,
elution gradient 45 to 70% MeCN in water containing 1% ammonium hydroxide, to give 8 (61 mg,
1
76%) as a cream colored solid. H NMR (400 MHz, DMSO d6) 1.07 (3H, d), 2.32 ꢀ 2.44 (1H, m), 2.63
ꢀ 2.81 (2H, m), 3.14 (1H, m), 3.27 ꢀ 3.36 (1H, m) 3.46 ꢀ 3.62 (2H, m), 4.38 (1H, br s), 4.93 (1H, s),
6.96 (1H, m), 7.02 (1H, m), 7.25 (1H, d), 7.28 (2H, d), 7.36 (2H, d), 7.42 (1H, d), 10.56 (1H, s);
HRMS m/z found 341.1412, C20H22N2OCl requires 341.1421.
Methyl
3-((1R,3R)-1-(4-chlorophenyl)-3-methyl-3,4-dihydro-1H-pyrido[3,4-b]indol-2(9H)-
yl)propanoate (9a). Obtained from 5b in 72% yield by an analogous method to 7a. 1H NMR (400
MHz, DMSO d6) 1.05 (3H, d), 2.52 ꢀ 2.63 (3H, m), 2.64 ꢀ 2.73 (2H, m), 2.93 ꢀ 3.04 (1H, m), 3.09 (1H,
m), 3.61 (3H, s), 4.88 (1H, s), 6.97 (1H, m), 7.03 (1H, m), 7.22 (2H, d), 7.26 (1H, d), 7.35 ꢀ 7.40 (2H,
m), 7.42 (1H, d), 10.65 (1H, s); m/z = 381, 383 [MꢀH]ꢀ.
3-((1R,3R)-1-(4-Chlorophenyl)-3-methyl-3,4-dihydro-1H-pyrido[3,4-b]indol-2(9H)-yl)propanoic
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acid (9b). Obtained from 9a in 96% yield by an analogous method to 7b. H NMR (400 MHz, DMSO
d6) 1.06 (3H, d), 2.12 (2H, m), 2.55 (1H, m), 2.61 ꢀ 2.73 (2H, m), 2.77 ꢀ 2.92 (1H, m), 3.11 (1H, m),
4.81 (1H, s), 6.67 (1H, br m), 6.73 (1H, br m), 7.12 (1H, m), 7.18 ꢀ 7.31 (3H, m), 7.41 (2H, m),
exchangeable protons not detected; HRMS m/z found 369.1358, C21H22N2O2Cl requires 369.1370.
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2-(1-(5-Methoxy-1H-indol-3-yl)propan-2-ylamino)ethanol (11). A mixture of 1ꢀ(5ꢀmethoxyꢀ1Hꢀ
indolꢀ3ꢀyl)propanꢀ2ꢀamine (1.82 g, 8.9 mmol), 2ꢀbromoethanol (0.63 mL, 8.9 mmol) and potassium
carbonate (1.23 g, 8.9 mmol) in MeCN (6 mL) in a sealed microwave tube was heated to 80 °C for 1 h
in a microwave reactor, then further quantities of 2ꢀbromoethanol (0.63 mL, 8.9 mmol) and potassium
carbonate (1.23 g, 8.9 mmol) were added. Heating was continued at 80°C for 30 min. The mixture
was evaporated to dryness and the residue was purified by reverse phase chromatography, elution
gradient 20 to 40% MeCN in water containing 1% ammonium hydroxide, to give 11 (2.15 g, 53%) as a
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pale yellow gum. H NMR (400 MHz, DMSO d6) 0.97 (3H, d), 2.53 ꢀ 2.71 (4H, m), 2.76 (1H, dd),
2.87 (1H, m), 3.37 ꢀ 3.53 (2H, m), 3.76 (3H, s), 4.37 (1H, t), 6.71 (1H, dd), 6.98 (1H, d), 7.08 (1H, d),
7.22 (1H, d), 10.60 (1H, s); m/z = 249 [M+H]+.
2-(Trans-1-(4-chlorophenyl)-6-methoxy-3-methyl-3,4-dihydro-1H-pyrido[3,4-b]indol-2(9H)-
yl)ethanol (13).
A solution of 11 (1.88 g, 7.6 mmol) and 4ꢀchlorobenzaldehyde (1.06 g, 7.6 mmol) in AcOH (20 mL)
was heated at 80 °C for 7 h under nitrogen. The solution was evaporated to dryness, and the residue
was purified by ion exchange chromatography, eluting with 7M ammonia in MeOH. Product
containing fractions were evaporated to dryness to give the crude acetate derivative as an orange foam,
which was dissolved in MeOH (5 mL), 2M NaOH (5 mL) added and the solution heated at 60oC for 30
min. The solution was evaporated to dryness and the residue purified by reverse phase
chromatography, elution gradient 45 to 70% MeCN in water containing 1% ammonium hydroxide, to
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give 13 (1.33 g, 47%) as a cream solid. H NMR (400 MHz, DMSO d6) 1.06 (3H, d), 2.37 (1H, m),
2.45 ꢀ 2.56 (1H, m), 2.63 ꢀ 2.81 (2H, m), 3.07 ꢀ 3.22 (1H, m), 3.45 ꢀ 3.65 (2H, m), 3.76 (3H, s), 4.38
(1H, s), 4.91 (1H, s), 6.68 (1H, dd), 6.93 (1H, d), 7.14 (1H, d), 7.28 (2H, d), 7.33 ꢀ 7.4 (2H, m), 10.39
(1H, s); m/z = 369, 371 [MꢀH]ꢀ.
(1R,3R)-1-(4-Chlorophenyl)-2-(2-hydroxyethyl)-3-methyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-
b]indol-6-ol (15). A 1 M boron tribromide solution in DCM (4.8 mL, 4.8 mmol) was added dropwise
to a stirred solution of 13 (1.18 g, 3.2 mmol) in DCM (30 mL) at ꢀ78 ºC. The resulting solution was
stirred at ꢀ78 ºC for 30 min then at 20 ºC for 16 h. The solution was cooled to 0 ºC, quenched with
water (10 mL) and diluted with DCM (10 mL). The aqueous phase was separated and passed through
a SCXꢀ2 column. Product containing fractions were combined and evaporated. The crude racemic
product was purified by reverse phase chromatography, elution gradient 30 to 50% MeCN in water
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containing 1% ammonium hydroxide. Product containing fractions were evaporated to dryness to give
racemic 15 (960 mg, 85%) as a cream solid, of which 610 mg was purified by preparative chiral HPLC
on a Lux cellulose 2 column (20 µm silica, 50 mm diameter, 250 mm length), eluting isocratically with
30% EtOH in heptane. Product containing fractions were evaporated to dryness to give the desired
enantiomer as the first eluted product (228 mg). Further purification by reverse phase
chromatography, elution gradient 35 to 60% MeCN in water containing 1% ammonium hydroxide,
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gave pure 15 (107 mg, 35% recovery) as a cream colored solid. H NMR (400 MHz, DMSO d6) 1.05
(3H, d), 2.3 ꢀ 2.48 (2H, m), 2.61 (1H, dd), 2.71 (1H, m), 3.05 ꢀ 3.17 (1H, m), 3.43 ꢀ 3.61 (2H, m), 4.37
(1H, s), 4.88 (1H, s), 6.54 (1H, dd), 6.73 (1H, d), 7.03 (1H, d), 7.28 (2H, d), 7.33 ꢀ 7.39 (2H, m), 8.50
(1H, br s), 10.22 (1H, s); HRMS m/z found 357.1367, C20H22N2O2Cl requires 357.1370.
(S)-Methyl 2-methyl-3-(trifluoromethylsulfonyloxy)propanoate (17). To a stirred solution of (S)ꢀ
methyl 3ꢀhydroxyꢀ2ꢀmethylpropanoate (4.0 g, 34 mmol) in DCM (15 mL) at 0°C was added dropwise
trifluoromethanesulfonic anhydride (6.84 mL, 41 mmol), followed by 2,6ꢀlutidine (4.35 g, 41 mmol)
dropwise over 5 min (caution large exotherm). The reaction mixture was stirred at 0 °C for 15 min,
then diluted with DCM (50 mL). The solution was washed with 2M HCl (2 x 100 mL) and brine (100
mL). The organic layer was dried over Na2SO4 and concentrated in vacuo to give 17 (8.46 g, 100%) as
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a pink liquid which was used without further purification. H NMR (400 MHz, CDCl3) 1.30 (3H, d),
2.86 ꢀ 3.07 (1H, m), 3.75 (3H, s), 4.56 (1H, dd), 4.69 (1H, dd).
(S)-Methyl 3-((R)-1-(1H-indol-3-yl)propan-2-ylamino)-2-methylpropanoate (12). A solution of 17
(607 mg, 2.4 mmol), 4b (423 mg, 2.4 mmol) and N,Nꢀdiisopropylethylamine (0.93 mL, 5.3 mmol) in
dioxane (10 mL) was purged with nitrogen for 5 min and then sealed in a microwave tube. The solution
was heated at 80 °C for 40 min in a microwave reactor, cooled to RT and diluted with DCM (10 mL).
The solution was washed sequentially with water (5 mL) and brine (5 mL), dried over MgSO4 and
evaporated to dryness. The residue was purified by flash silica chromatography, elution gradient 0 to
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30% MeOH in DCM, to give 12 (525 mg, 79 %) as a pale yellow gum. H NMR (400 MHz, DMSO
d6) 0.97 (3H, d), 1.03 (3H, d), 2.53 ꢀ 2.72 (3H, m), 2.78 (2H, dt), 2.83 ꢀ 2.93 (1H, m), 3.54 (3H, s), 6.94
ꢀ 7 (1H, m), 7.02 ꢀ 7.09 (1H, m), 7.13 (1H, d), 7.34 (1H, d), 7.51 (1H, d), 10.79 (1H, s), exchangeable
amine N proton not detected; m/z = 275 [M+H]+.
(S)-Methyl 3-((1R,3R)-1-(4-chlorophenyl)-3-methyl-3,4-dihydro-1H-pyrido[3,4-b]indol-2(9H)-yl)-
2-methylpropanoate (14). A solution of 12 (140 mg, 0.51 mmol) and 4ꢀchlorobenzaldehyde (72 mg,
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0.51 mmol) in toluene (4 mL) containing molecular sieves was stirred at 90 °C for 1 h. AcOH (1 mL)
was added and heating continued for 5 h. The solution was evaporated to dryness and the residue was
initially purified by ion exchange chromatography, eluting with 7M ammonia in MeOH. Product
containing fractions were evaporated to dryness, and the residue further purified by reverse phase
chromatography, elution gradient 60 to 100% MeCN in water containing 1% ammonium hydroxide, to
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give 14 (146 mg, 72%) as a pale yellow solid. H NMR (400 MHz, DMSO d6) 1.03 (6H, d), 2.3 ꢀ 2.39
(1H, m), 2.52 (1H, m), 2.63 (1H, dd), 2.81 ꢀ 2.96 (2H, m), 3.00 (1H, m), 3.67 (3H, s), 4.94 (1H, s), 6.95
ꢀ 7.02 (1H, m), 7.03 ꢀ 7.1 (1H, m), 7.25 (2H, d), 7.30 (1H, d), 7.38 (2H, d), 7.44 (1H, d), 10.71 (1H, s);
m/z = 395, 397[MꢀH]ꢀ.
(S)-3-((1R,3R)-1-(4-Chlorophenyl)-3-methyl-3,4-dihydro-1H-pyrido[3,4-b]indol-2(9H)-yl)-2-
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methylpropanoic acid (16). Obtained from 14 in 93% yield by an analogous method to 7b. H NMR
(400 MHz, DMSO d6) 0.86 (3H, d), 1.01 (3H, d), 2.13 (1H, m), 2.23 (1H, m), 2.51 (1H, m), 2.77 (2H,
m), 3.21 (1H, m), 4.79 (1H, s), 6.91 ꢀ 6.97 (1H, m), 6.97 ꢀ 7.03 (1H, m), 7.22 (1H, d), 7.31 (2H, d),
7.37 (2H, d), 7.40 (1H, d), 10.44 (1H, s), exchangeable carboxylic acid proton not detected; HRMS m/z
found 383.1502, C22H24N2O2Cl requires 383.1526.
(E)-Methyl 3-(4-formylphenyl)acrylate (18). 4ꢀBromobenzaldehyde (30 g, 162 mmol) and methyl
acrylate (20.9 g, 243 mmol) were taken up in well degassed DMA (300 mL). Triꢀoꢀtolylphosphine (4.9
g, 16.2 mmol), palladium(II) acetate (1.8 g, 8.1 mmol) and triethylamine (45.2 mL, 324 mmol) were
added and the mixture was heated at 110 °C for 16 h. The mixture was poured into water (4 L) and the
resulting precipitate was filtered off and dried. The solid was purified by flash chromatography, elution
gradient 0 to 30% EtOAc in heptane, to give a solid which was triturated with heptane to give 18 (25.6
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g, 83%) as a yellow crystalline solid. H NMR (400 MHz, DMSO d6) 3.75 (3H, s), 6.79 (1H, d), 7.72
(1H, d), 7.93 (4H, s), 10.03 (1H, s). No mass ion observed in LCMS.
(E)-Methyl 3-(4-((1S,3R)-3-methyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylate
(19). 18 (18.6 g, 97.6 mmol) was added to a stirred solution of 4b (17 g, 97.6 mmol) in AcOH (250
mL) under nitrogen. The resulting solution was stirred at 80 °C for 2 h. The reaction mixture was
evaporated to dryness, redissolved in DCM (500 mL), and the solution was washed sequentially with
saturated NaHCO3 (2 x 300 mL), 2 M NaOH (300 mL), water (300 mL), and saturated brine (300 mL).
The organic layer was dried over MgSO4 and evaporated to dryness. The residue was purified by flash
chromatography, elution gradient 1 to 7% MeOH in DCM, to give 19 (25.1 g, 74%) as a beige foam,
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which contained 12% of the inseparable trans (1R,3R) epimer. H NMR (400 MHz, DMSO d6) 1.25
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(3H, d), 2.37 ꢀ 2.48 (1H, m), 2.74 (1H, d), 3.12 (1H, s), 3.73 (3H, s), 5.18 (1H, s), 6.64 (1H, d), 6.97
(2H, dd), 7.19 (1H, d), 7.36 ꢀ 7.46 (3H, m), 7.64 ꢀ 7.75 (3H, m), 10.19 (1H, s), no NH observed; m/z =
347 [M+H]+.
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yl)phenyl)acrylate (20). 19 (35 g, 101 mmol), allyl bromide (9.6 mL, 111 mmol) and N,Nꢀ
diisopropylethylamine (19 mL, 111 mmol) were suspended in MeCN (160 mL). The mixture was
purged with nitrogen for 5 min and sealed in a microwave tube. The reaction was heated at 140 °C for
3.5 h in a microwave reactor, then cooled to room temperature. The reaction mixture was evaporated to
dryness, the residue was redissolved in DCM (100 mL) and the solution was washed sequentially with
1 M citric acid (100 mL), water (100 mL) and brine (100 mL). The organic layer was dried over
MgSO4 and evaporated to dryness. The residue was purified by flash chromatography, elution gradient
0 to 20% EtOAc in heptane. Pure fractions were evaporated to dryness to afford a 50:50 mixture of 20
and the corresponding cis (1S,3R) epimer (10 g, 25 mmol, 26%) as a pale yellow solid. The epimeric
mixture was dissolved in DCM (100 mL), TFA (5.6 mL, 76 mmol) was added, and the solution was
stirred at 20 °C for 3 days under nitrogen. The reaction mixture was diluted cautiously with saturated
NaHCO3 solution (250 mL), and the organic layer was washed sequentially with water (250 mL) and
brine (250 mL). The organic layer was dried over MgSO4 and evaporated to dryness. The residue was
purified by flash column chromatography, elution gradient 10 to 20% EtOAc in heptane, to give a
65:35 mixture of 20 and the corresponding cis (1S,3R) epimer (8.0 g, 82% recovery) as a pale yellow
solid, which was used in the next stage without further separation. m/z = 387 [M+H]+.
(E)-Methyl
3-(4-((1R,3R)-3-methyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-
yl)phenyl)acrylate (21). Seven separate batches of the foregoing 65:35 mixture of 20 and the
corresponding cis (1S,3R) epimer (2.0 g, 5.2 mmol) were each suspended with
chlorotris(triphenylphosphine)rhodium(I) (Wilkinson's catalyst) (2.35 g, 2.5 mmol) in MeCN (12 mL)
and water (2.4 mL). The mixtures were purged with nitrogen for 5 min, sealed in a microwave tube
and heated to 100 °C for 60 min in the microwave reactor. The reaction mixtures were combined,
evaporated to dryness and partitioned between DCM (200 mL) and saturated aqueous NaHCO3 (200
mL). The organic layer was separated, washed sequentially with water (200 mL) and brine (200 mL),
dried over MgSO4, and evaporated to dryness. The residue was purified by flash chromatography,
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elution gradient 0 to 5% methanol in DCM, to give 21 (7.0 g, 56%) as a pale yellow solid. H NMR
(400 MHz, DMSO d6) 1.14 (3H, d), 2.27 ꢀ 2.4 (1H, m), 2.81 (1H, dd), 2.94 ꢀ 3.05 (1H, m), 3.72 (3H, s),
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5.19 (1H, s), 6.60 (1H, d), 6.94 ꢀ 7 (1H, m), 7.01 ꢀ 7.09 (1H, m), 7.26 (3H, d), 7.43 (1H, d), 7.59 ꢀ 7.68
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(3H, m), 10.70 (1H, s), NH not observed; m/z = 347 [M+H]+.
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(E)-Methyl
3-(4-((1R,3R)-2-((S)-3-hydroxy-2-methylpropyl)-3-methyl-2,3,4,9-tetrahydro-1H-
pyrido[3,4-b]indol-1-yl)phenyl)acrylate (23a). 21 (1.0 g, 2.9 mmol), (R)ꢀ3ꢀbromoꢀ2ꢀmethylꢀ1ꢀ
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propanol (0.45 mL, 4.3 mmol), N,Nꢀdiisopropylethylamine (1.51 mL, 8.7 mmol), and acetonitrile (12
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mL) were degassed with nitrogen and sealed in a microwave tube. The mixture was heated at 135 C
overnight in a microwave reactor, cooled to room temperature, and evaporated to dryness. The residue
was dissolved in DCM and filtered through a pad of silica gel, eluting with 0ꢀ50% ether in DCM to
give 23a (770 mg, 64%) as an offꢀwhite foam. 1H NMR (400 MHz, DMSO d6) 0.80 (3H, d), 1.07 (3H,
d), 1.88 (1H, m), 2.14 (1H, dd), 2.54 ꢀ 274 (3H, m), 3.14 (1H, m), 3.32 (1H, m), 3.54 (1H, m), 3.72
(3H, s), 4.59 (1H, tr), 4.89 (1H, s), 6.60 (1H, d), 6.96 (1H, m), 7.02 (1H, m), 7.29 (3H, m), 7.42 (1H,
d), 7.67 (3H, m), 10.60 (1H, s). m/z = 419 [M+H]+.
(E)-3-(4-((1R,3R)-2-((S)-3-Hydroxy-2-methylpropyl)-3-methyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-
b]indol-1-yl)phenyl)acrylic acid (23b). A solution of 2 M sodium hydroxide (1.31 mL, 9.8 mmol)
was added to a solution of 23a (412 mg, 0.98 mmol) in THF (10 mL), MeOH (2 mL) and water (2
mL). The mixture was stirred at room temperature for 4 h under nitrogen and the organics were
removed by evaporation. The aqueous residue was applied to a SCXꢀ2 column loaded with MeOH.
Elution with 7 M ammonia in MeOH gave crude product, which was further purified by reverse phase
chromatography, elution gradient 20 to 40% MeCN in water containing 1% ammonium hydroxide, to
give 23b (305 mg, 75%) as a solid. 1H NMR (400 MHz, DMSO d6) 0.80 (3H, d), 1.07 (3H, d), 1.89
(1H, m), 2.16 (1H, dd), 2.52 ꢀ 2.72 (3H, m), 3.18 (1H, m), 3.32 (1H, m), 3.54 (1H, m), 4.89 (1H, s),
6.49 (1H, d), 6.97 (1H, m), 7.03 (1H, m), 7.28 (3H, m), 7.45 (2H, m), 7.59 (2H, m), 10.60 (1H, s),
carboxylic acid and hydroxyl protons seen in broad hump between 6 and 4 ppm. HRMS m/z found
405.2175, C25H29N2O3 requires 405.2178.
(E)-3-(4-((1R,3R)-2-(2-Hydroxyethyl)-3-methyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-
yl)phenyl)acrylic acid (22b) was obtained by an analogous method to 23b by alkylation of 21 with 2ꢀ
bromoethanol to followed by hydrolysis of ester 22a (see Supporting Information for 22a
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characterization data). H NMR (400 MHz, DMSO d6) 1.07 (3H, d), 2.36 ꢀ 2.43 (1H, m), 2.52 ꢀ 2.54
(1H, m), 2.55 (1H, dt), 2.75 (2H, ddd), 3.13 ꢀ 3.2 (1H, m), 3.48 ꢀ 3.6 (2H, m), 4.38 (1H, s), 4.96 (1H,
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s), 6.48 (1H, d), 6.97 (1H, td), 7.03 (1H, ddd), 7.22 ꢀ 7.28 (1H, m), 7.31 (2H, d), 7.43 (1H, d), 7.54 (1H,
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d), 7.61 (2H, d), 10.58 (1H, s). HRMS m/z found 377.1870, C23H25N2O3 requires 377.1865.
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(E)-3-(4-((1R,3R)-2-((R)-3-hydroxy-2-methylpropyl)-3-methyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-
b]indol-1-yl)phenyl)acrylic acid (24b) was obtained by an analogous method to 23b by alkylation of
21 with (S)ꢀ3ꢀbromoꢀ2ꢀmethylpropanꢀ1ꢀol followed by hydrolysis of ester 24a (see Supporting
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Information for 24a characterization data). H NMR (400 MHz, DMSO d6) 0.93 (3H, d), 1.05 (3H, d),
1.84 (1H, dt), 2.34 (2H, q), 2.48 (1H, m), 2.64 (1H, dd), 3.08 ꢀ 3.23 (2H, m), 3.28 (1H, dd), 4.80 (1H,
s), 6.43 (1H, d), 6.92 ꢀ 6.98 (1H, m), 6.98 ꢀ 7.05 (1H, m), 7.17 ꢀ 7.25 (3H, m), 7.27 (1H, d), 7.40 (1H,
d), 7.48 (2H, d), 10.67 (1H, s), exchangeable protons not detected. HRMS m/z found 405.2178,
C25H29N2O3 requires 405.2178.
(R)-N-(1-(1H-Indol-3-yl)propan-2-yl)-2-methylpropan-1-amine
(25a).
Sodium
triacetoxyborohydride (42.6 g, 201 mmol) was added in one portion to 4b (25 g, 143 mmol) and
isobutyraldehyde (14.4 mL, 158 mmol) in THF (500 mL) at 0 °C under nitrogen. The resulting
mixture was stirred at 0 °C for 30 min, then quenched with saturated aqueous sodium bicarbonate (50
mL) and extracted with EtOAc (2 x 500 mL). The organic layer was dried over MgSO4 and evaporated
to dryness. The residue was purified by flash chromatography, elution gradient 0 to 5% 7M ammonia
1
in MeOH in DCM, to give 25a (14.6 g, 44.3 %) as a waxy yellow gum. H NMR (400 MHz, DMSO
d6) 0.82 (6H, d), 0.98 (3H, d), 1.58 (1H, dt), 2.38 (2H, qd), 2.61 (1H, dd), 2.83 (2H, ddd), 6.93 ꢀ 7.01
(1H, m), 7.02 ꢀ 7.09 (1H, m), 7.13 (1H, d), 7.34 (1H, d), 7.52 (1H, d), 10.79 (1H, s); m/z = 231
[M+H]+.
2-Fluoro-2-methylpropan-1-ol. Lithium aluminium hydride (3.37 g, 88.6 mmol) was added
portionwise over 15 min to a solution of ethyl 2ꢀfluoroꢀ2ꢀmethylpropanoate (9.9 g, 73.8 mmol) in
diethyl ether (185 mL) at 0°C. The mixture was stirred for 1 h, then water (3.3 mL), 15% NaOH
solution (3.3 mL) and water (6.7 mL) were added sequentially. The suspension was stirred for 15 min,
then filtered, and the solids were washed with diethyl ether. The filtrate was evaporated to give 2ꢀ
fluoroꢀ2ꢀmethylpropanꢀ1ꢀol (5.90 g, 87 %) as a colourless oil which was used without purification in
1
the next step. H NMR (400 MHz, CDCl3) 1.37 (6H, d), 3.56 (2H, d), OH not observed.
2-Fluoro-2-methylpropyl trifluoromethanesulfonate (31). Trifluoromethanesulfonic anhydride (12.1
mL, 71.2 mmol) and 2,6ꢀlutidine (11.4 mL, 81.4 mmol) were added to a solution of 2ꢀfluoroꢀ2ꢀ
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methylpropanꢀ1ꢀol (6.25 g, 67.8 mmol) in DCM (150 mL) at ꢀ10 °C. The solution was stirred for 1 h,
washed with 2M HCl (2 x 100 mL) and saturated aqueous NaHCO3 (2 x 100 mL), dried over Na2SO4
and concentrated to give 31 (12.9 g, 85%) as a red oil which was used without purification in the next
step. 1H NMR (400 MHz, CDCl3) 1.46 (6H, d), 4.41 (2H, d).
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(R)-N-(1-(1H-Indol-3-yl)propan-2-yl)-2-fluoro-2-methylpropan-1-amine (25b). 31 (8.04 g, 35.9
mmol) was added to a solution of 4b (5.0 g, 28.7 mmol) and N,Nꢀdiisopropylethylamine (7.4 mL, 43
mmol) in dioxane (50 mL). The solution was heated to 90 °C for 3 h, cooled to room temperature,
diluted with EtOAc (200 mL) and washed with saturated aqueous NaHCO3 (2 x 100 mL). The aqueous
phase was extracted with EtOAc (150 mL), and the combined organics were dried over MgSO4 and
concentrated. The residue was purified by flash chromatography, eluting with EtOAc, to give 25b (6.49
1
g, 91 %) as a brown oil. H NMR (400 MHz, CDCl3) 1.14 (3H, d), 1.31 (3H, d), 1.37 (3H, d), 1.94
(1H, s), 2.63 ꢀ 2.87 (3H, m), 2.92 (1H, dd), 3.07 (1H, h), 7.07 (1H, d), 7.08 ꢀ 7.15 (1H, m), 7.16 ꢀ 7.24
(1H, m), 7.37 (1H, d), 7.62 (1H, d), 8.04 (1H, s); m/z = 249 [M+H]+.
(E)-Methyl 3-(3,5-difluoro-4-formylphenyl)acrylate (26). 4ꢀBromoꢀ2,6ꢀdifluorobenzaldehyde (10 g,
45.2 mmol) and methyl acrylate (6.1 mL, 67.8 mmol) were taken up in well degassed DMA (100 mL)
and triꢀoꢀtolylphosphine (1.38 g, 4.5 mmol), palladium(II) acetate (0.51 g, 2.26 mmol) and
triethylamine (12.6 mL, 90.4 mmol) were added. The mixture was stirred and heated at 80 °C for 6 h,
then filtered through a layer of celite and washed with MeOH (50 mL). The filtrate was preꢀabsorbed
onto silica and purified by suction chromatography, eluting with 0ꢀ10% ether in DCM. Fractions
containing the desired product were evaporated, and the residue was triturated successively with ether
(50 mL) and water (50 mL) before being dried under high vacuum at 50 °C to give 26 (8.85 g, 87%) as
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a yellow solid. H NMR (400 MHz, DMSO d6) 3.75 (3H, s), 6.93 (1H, d), 7.52 ꢀ 7.81 (3H, m), 10.18
(1H, s). No mass ion observed in LCMS.
(E)-Methyl 3-(3,5-difluoro-4-((1R,3R)-2-(2-fluoro-2-methylpropyl)-3-methyl-2,3,4,9-tetrahydro-
1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylate (30a). 26 was added to a suspension of 25b (6.02 g,
24.2 mmol) in toluene (50 mL) and AcOH (2.8 mL, 48.5 mmol). The mixture was heated to 80 °C for 5
h and then loaded onto an SCXꢀ2 column, eluting with 7 M ammonia in MeOH. Product containing
fractions were evaporated to dryness and the residual solid purified by flash chromatography, elution
gradient 0 to 30% EtOAc in heptane, to give 30a (7.52 g, 68%) as a yellow solid. 1H NMR (400 MHz,
DMSO d6, 100°C) 1.10 (3H, d), 1.12 ꢀ 1.31 (6H, m), 2.28 ꢀ 2.72 (2H, m), 2.84 ꢀ 3.09 (2H, m), 3.52 ꢀ
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3.69 (1H, m), 3.76 (3H, s), 5.30 (1H, s), 6.64 (1H, d), 6.9 ꢀ 7.11 (2H, m), 7.21 (1H, d), 7.32 (2H, d),
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7.42 (1H, d), 7.58 (1H, d), 10.14 (1H, s); m/z = 457 [M+H]+.
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(E)-3-(3,5-Difluoro-4-((1R,3R)-2-(2-fluoro-2-methylpropyl)-3-methyl-2,3,4,9-tetrahydro-1H-
pyrido[3,4-b]indol-1-yl)phenyl)acrylic acid (30b). A 7.5 M solution of sodium hydroxide (32.9 mL,
247 mmol) was added to a solution of 30a (11.28 g, 24.7 mmol) in THF (143 mL) and MeOH (71 mL).
The mixture was stirred at room temperature for 4 h. The pH was adjusted to 6.5 by addition of 2 M
HCl solution, then the solution was extracted with diethyl ether (3 x 150 mL). The combined organics
were dried over Na2SO4 and concentrated. The residue was purified by flash chromatography, elution
gradient 0 to 20% MeOH in DCM to give a yellow solid. Attempted trituration with acetone/heptane
failed due to higher than expected solubility. The solvents were removed to give a yellow solid which
was triturated in isohexane (50 mL) with a few drops of ether, and the resulting solid was filtered off
and dried to give a yellow powder (11.1 g). The solid was dissolved in EtOH (100 mL) under nitrogen
and in the dark. The solution was evaporated to 5 mbar using a vacuum pump at 62 ˚C in the dark.
This procedure was repeated twice and the resulting yellow glass scratched with a spatula into a fine
powder and subjected to 5 mbar using a vacuum pump at 62 ˚C for 60 min to give a yellow powder.
The powder was then left in a vacuum oven over P2O5 at 62 ˚C at 300 mbar overnight to give 30b (9.77
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g, 89%) as a pale yellow solid. H NMR (400 MHz, DMSO d6) 1.07 ꢀ 1.16 (3H, m), 1.18 ꢀ 1.29 (6H,
m), 2.39 (1H, dd), 2.62 (1H, dd), 2.92 (2H, dd), 3.56 (1H, d), 5.26 (1H, s), 6.70 (1H, d), 7.02 (2H, dd),
7.22 (1H, d), 7.47 (3H, dd), 7.58 (1H, d), 10.60 (1H, s), 12.60 (1H, s); HRMS m/z found 443.19394,
C20H17ON4 requires 443.19409.
Compounds 27b-29b were obtained by hydrolysis of 27a-29a, in turn obtained by analogous routes to
30b (see Supporting Information for characterization of intermediates 27a-29a).
(E)-3-(4-((1R,3R)-2-isobutyl-3-methyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-
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yl)phenyl)acrylic acid (27b): H NMR (400 MHz, CDCl3) 0.80 (3H, d), 0.93 (3H, d), 1.08 (3H, d),
1.80 (1H, dt), 2.14 (1H, dd), 2.36 (1H, dd), 2.61 (1H, dd), 2.88 (1H, dd), 3.33 (1H, q), 4.72 (1H, s),
6.41 (1H, d), 7.12 (2H, pd), 7.24 (1H, d), 7.35 (2H, d), 7.46 (2H, t), 7.5 ꢀ 7.56 (1H, m), 7.76 (1H, d),
exchangeable protons not observed; HRMS m/z found 389.2248, C25H29N2O2 requires 389.2229.
(E)-3-(3,5-Difluoro-4-((1R,3R)-2-isobutyl-3-methyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-
yl)phenyl)acrylic acid (28b): 1H NMR (400 MHz, DMSO d6) 0.68 (3H, d), 0.82 (3H, d), 1.04 (3H, d),
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1.64 ꢀ 1.76 (1H, m), 2.05 (1H, dd), 2.43 (1H, dd), 2.58 (1H, dd), 2.88 (1H, dd), 3.40 (1H, h), 5.10 (1H,
s), 6.68 (1H, d), 6.95 (1H, td), 7.00 (1H, td), 7.20 (1H, d), 7.39 ꢀ 7.45 (3H, m), 7.49 (1H, d), 10.55 (1H,
s). HRMS m/z found 425.2052, C25H27N2O2F2 requires 425.2041.
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(E)-3-(4-((1R,3R)-2-(2-Fluoro-2-methylpropyl)-3-methyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-
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b]indol-1-yl)phenyl)acrylic acid (29b): H NMR (400 MHz, DMSO d6) 1.06 (3H, d), 1.30 (3H, d),
1.47 (3H, d), 2.53 ꢀ 2.64 (2H, m), 2.79 (2H, s), 3.10 (1H, d), 5.08 (1H, s), 6.47 (1H, d), 6.98 (1H, t),
7.06 (1H, t), 7.19 ꢀ 7.37 (3H, m), 7.44 (1H, d), 7.56 (1H, d), 7.63 (2H, d), 10.81 (1H, s), 12.30 (1H, s).
HRMS m/z found 407.2144, C25H28N2O2F requires 407.2135.
ACKNOWLEDGEMENTS
We thank Calum R. Cook, Julie Demeritt, Sam Groombridge, Lorraine Hassall, Karen Jones, Anil
Patel, Karen Roberts and Paul Turner for technical assistance.
SUPPORTING INFORMATION AVAILABLE
SEM values for data in Table 1 and characterization data for precursors 22a, 24a, 27a-29a (Table 1),
selected preꢀclinical comparator data between compounds 30b and 3c (Table 2), and in vivo activity of
compound 30b in a long term estrogen deprived (LTED) model are available (Figure 1 and Figure 2,
supporting information) free of charge via the Internet at http://pubs.acs.org.
REFERENCES AND NOTES
1. Jordan, V. C. Tamoxifen: a most unlikely pioneering medicine. Nature Rev. Drug Discovery 2003,
2, 205ꢀ213.
2. Ariazi, E. A.; Jordan, V. C. Estrogen receptors as therapeutic targets in breast cancer. In Nuclear
Receptors as drug targets. Eckhard, O.; Weinmann, H.; Eds. WileyꢀVCH Verlag GmbH: Weinheim,
Germany, 2008; pp 127ꢀ199.
3. (a) Morris, C.; Wakeling, A. E. Fulvestrant (“Faslodex”) – a new treatment option for patients
progressing on prior endocrine therapy. Endocrine-Related Cancer 2002, 267ꢀ276. (b) Nephew, K. P.;
Long, X. Fulvestrant (ICI 182,780)ꢀdependent interacting proteins mediate immobilization and
degradation of estrogen receptorꢀα. J. Biol. Chem. 2006, 14, 9607ꢀ9615
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