Design and synthesis of a potent, highly selective, orally bioavailable, retinoic acid receptor alpha agonist

Article abstract of DOI:10.1016/j.bmc.2017.12.015

A ligand-based virtual screening exercise examining likely bioactive conformations of AM 580 (2) and AGN 193836 (3) was used to identify the novel, less lipophilic RARα agonist 4-(3,5-dichloro-4-ethoxybenzamido)benzoic acid 5, which has good selectivity over the RARβ and RARγ receptors. Analysis of the medicinal chemistry parameters of the 3,5-substituents of derivatives of template 5 enabled us to design a class of drug-like molecules with lower intrinsic clearance and higher oral bioavailability which led to the novel RARα agonist 4-(3-chloro-4-ethoxy-5-isopropoxybenzamido)-2-methylbenzoic acid 56 that has high RARα potency and excellent selectivity versus RARβ (2 orders of magnitude) and RARγ (4 orders of magnitude) at both the human and mouse RAR receptors with improved drug-like properties. This RARα specific agonist 56 has high oral bioavailability (>80%) in both mice and dogs with a good PK profile and was shown to be inactive in cytotoxicity and genotoxicity screens.

Full text of DOI:10.1016/j.bmc.2017.12.015

Accepted Manuscript
Design and synthesis of a potent, highly selective, orally bioavailable, retinoic
acid receptor alpha agonist
Earl Clarke, Christopher I. Jarvis, Maria B. Goncalves, S. Barret Kalindjian,
David R. Adams, Jane T. Brown, Jason J. Shiers, David M.A. Taddei, Elodie
Ravier, Stephanie Barlow, Iain Miller, Vanessa Smith, Alan D. Borthwick,
Jonathan P.T. Corcoran
PII:
S0968-0896(17)31909-0
https://doi.org/10.1016/j.bmc.2017.12.015
BMC 14120
DOI:
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
28 September 2017
6 December 2017
8 December 2017
Please cite this article as: Clarke, E., Jarvis, C.I., Goncalves, M.B., Kalindjian, S.B., Adams, D.R., Brown, J.T.,
Shiers, J.J., Taddei, D.M.A., Ravier, E., Barlow, S., Miller, I., Smith, V., Borthwick, A.D., Corcoran, J.P.T., Design
and synthesis of a potent, highly selective, orally bioavailable, retinoic acid receptor alpha agonist, Bioorganic &
Medicinal Chemistry (2017), doi: https://doi.org/10.1016/j.bmc.2017.12.015
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Design and synthesis of a potent, highly selective, orally bioavailable,
retinoic acid receptor alpha agonist.
Earl Clarke,† Christopher I. Jarvis,† Maria B. Goncalves,† S. Barret Kalindjian,† David R.
Adams,‡ Jane T. Brown,‡ Jason J. Shiers,‡ David M. A. Taddei,‡ Elodie Ravier,‡ Stephanie
Barlow,‡ Iain Miller,‡ Vanessa Smith,‡ Alan D. Borthwick,δ* and Jonathan P. T. Corcoran.†*
†Neuroscience Drug Discovery Unit, Wolfson Centre for Age-Related Diseases, Guy’s Campus, King’s College,
London SE1 1UL, UK.
δDrugMolDesign, 15 Temple Grove, London NW11 7UA, UK.
‡Sygnature Discovery Limited, Biocity, Pennyfoot Street, Nottingham NG1 1GF, UK.
ABSTRACT
A ligand-based virtual screening exercise examining likely bioactive conformations of AM 580
(2) and AGN 193836 (3) was used to identify the novel, less lipophilic RARα agonist 4-(3,5-
dichloro-4-ethoxybenzamido)benzoic acid 5, which has good selectivity over the RARβ, and
RARγ receptors. Analysis of the medicinal chemistry parameters of the 3,5-substituents of
derivatives of template 5 enabled us to design a class of drug-like molecules with lower intrinsic
clearance and higher oral bioavailability which led to the novel RARα agonist 4-(3-chloro-4-
ethoxy-5-isopropoxybenzamido)-2-methylbenzoic acid 56 that has high RARα potency and
excellent selectivity versus RARβ (2 orders of magnitude) and RARγ (4 orders of magnitude) at
both the human and mouse RAR receptors with improved drug-like properties. This RARα
specific agonist 56 has high oral bioavailability (>80%) in both mice and dogs with a good PK
profile and was shown to be inactive in cytotoxicity and genotoxicity screens.
1. Introduction
The retinoic acid receptors (RARα, RARβ, and RARγ) are members of the nuclear receptor
superfamily. Compounds which bind to and activate the RARs are termed retinoids and comprise
both natural retinol (Vitamin A) metabolites and synthetic analogs. Retinoids regulate a wide
variety of biological processes such as vertebrate embryonic morphogenesis and organogenesis,
cell growth arrest, differentiation, and apoptosis, as well as their disorders.1
The RARα isoform is found in the majority of tissues and has been implicated in a number of
diseases, most notably acute promyelocytic leukemia (APL). Selective RARα agonists have
been shown to inhibit proliferation and induce apoptosis of mammary tumor oncogenesis in
murine models (MMTV-neu and MMTV-wnt1 transgenic mice) relevant to human cancer,2 and
to inhibit LPS-induced B-lymphocyte proliferation.3 Selective RARα agonists have also been
shown to prevent neuronal cell death caused by amyloid-β and, when administered orally, can
prevent amyloid-β production and Alzheimer’s disease progression in a mouse model.4 It has
been shown5 that selective RARα agonists suppressed allospecific immune response and
significantly prolonged the survival of mouse cardiac allografts and can ameliorate nephritis in
lupus-prone mice, NZB/NZW F1.6 This supports the rationale for using RARα agonists as
immunosuppressants in human organ transplantation. Thus selective RARα agonists have the
1

therapeutic potential for the treatment of cancer, dermatological diseases, Alzheimer’s disease
and immunological disorders.
Synthetic RARα, RARβ, and RARγ agonists have been developed from all-trans-retinoic acid
(ATRA), and usually consist of a lipophilic ring, a linker and a carboxylic acid (Figure 1). There
7,8,9,10
has been an extensive studies on the SAR
of the RARα agonists based essentially on the
bicyclic 5,5,8,8-tetramethyl-5,6,7,8-tetrahydronaphthalene derivatives which evolved from
ATRA. All three agonist, AM 580 (2), AGN 193836 (3), AGN 195183 (4) and antagonist BMS
195614 (1) (Figure 1) which contain an amide linker and a benzoic acid, arose from these
studies.
Figure 1. RARα Agonists and Antagonist.
6
1
4
9
15
18
ATRA
-trams-
all retinoic acid
1
α
Antagonist
BMS 195614
3 X = Br
AGN 193836
2
AM 580
4 X = Cl AGN 195183
5
56
However, although AM 580 (2) and AGN 195183 (4) have moderate and good selectivity
respectively for RARα, over RARβ and RARγ they are quite lipophilic (cLog P 6.3 and 7.2). In
addition AM 580 (2) has been shown to be toxic,11,12 and the more recently discovered compound
AGN 195183 (4)10 which was in Phase I clinical trials for cancer has been discontinued.13 Our
aim was to find a novel, potent, highly selective RARα agonist not based on the bicyclic 5,5,8,8-
tetramethyl-5,6,7,8-tetrahydronaphthalene class that was ligand efficient, orally bioavailable and
without the lipophilic obesity seen with (2), (3) and (4). We outline here how we discovered our
initial hit compound 5 and how this was developed into the orally bioavailable, highly potent and
selective RARα agonist 56 (Figure 1) which exhibits promising drug-like properties.
2

2. Chemistry
The phenyl carboxamido-benzoic acids (Schemes 1, 3, 4 and 5) and phenyl carbamoyl-benzoic
acid 26 (Scheme 2) were prepared by coupling the appropriately substituted aniline with a
substituted benzoic acid using a variety of standard methods for the formation of an amide bond.
The 3,5-dichloro-4-alkoxy compounds 12 – 15 and 17 – 21 (Scheme 1) were prepared by
alkylation of the phenolic group of methyl 3,5-dichloro-4-hydroxybenzoate 6 followed by
hydrolysis of the benzoate ester. Coupling the resultant acid 7 via the acid chloride by reaction
with oxalyl chloride or directly with HATU, with the appropriate methyl 4-aminobenzoate 8
followed by hydrolysis with lithium hydroxide gave the required acids 12 – 15 and 17 – 21.
Scheme 1. 4-(3,5-Dichloro-4-alkoxy-benzamido)benzoic acidsa
9
(vi)
(vii)
(i)
(ii)
7
6
8
(iii)
(iv)
11
10
(v)
(ii)
or (viii)
(ii)
12 R1 = Pr, R2 = H
13 R1 = Bu, R2 = H
14 R1 = iPr, R2 = H
17 R1 = Et, R2 = F
18 R1 = Et, R2 = Me
19 R1 = Et, R2 = OH
20 R1 = Et, R2 = Cl
21 R1 = Et, R2 = CF3
15 R1 =
, R2 = H
16 R1 = tBu, R2 = H
aReagents and conditions: (i) K2CO3, R1Br, DMF, 80 °C, 3h; (ii) LiOH, THF, H2O, room temp, 12h; (iii)
(COCl)2,CH2Cl2, DMF, 0 °C, 1h then methyl 4-amino-2-R2-benzoate, NEt3, room temp, 12h or HATU, DMF,
DIPEA, 5min, then methyl 4-amino-2-R2-benzoate, DMF, room temp, 18h; (iv) R1 =Bn, R2 = Me; BCl3, CH2Cl2, 0
°C then room temp, 12h; (v) 1,1-di-tert-butoxy-N,N-dimethylmethanamine, toluene, 80 °C, 3h, then room temp,
12h; (vi) 1,1-di-tert-butoxy-N,N-dimethylmethanamine, toluene, 80 °C, 3h, then further 1,1-di-tert-butoxy-N,N-
dimethylmethanamine, 2mol, added, 80 °C, 16h. (vii) H2, Pd/C, MeOH, room temp; (viii) R1 =Et, R2 = MeO, R =
tBu; BCl3, CH2Cl2, 0 °C then room temp 2h.
3

For compound 16 the initial alkylation of 6 was carried out with benzyl bromide, and the
resulting benzyloxy compound was hydrolyzed, coupled with the aniline 8 (R2 = H) and the
benzyl group was removed using boron trichloride to result in compound 11 (R2 = H). This
material was then alkylated using 1,1-di-tert-butoxy-N,N-dimethylmethanamine in toluene at 80
°C. A final hydrolysis using lithium hydroxide in a mixture of tetrahydrofuran and water gave
the tertiary butoxy compound 16.
A similar sequence (Scheme 2) coupling the aniline 24 and acid chloride 23 (obtained from acid
22) followed by hydrolysis gave the phenolic acid 25 which upon alkylation with ethyl iodide
followed by hydrolysis gave the reverse amide analog 26.
Scheme 2. 4-(3,5-Dichloro-4-ethoxyphenylcarbamoyl)benzoic acid.a
Cl
Cl
Cl
O
O
(ii)
H
N
HO
O
NH2
HO
+
OMe
O
Cl
OH
23
Cl
24
25
(iii)
Cl
(i)
(iv)
H
N
HO
O
O
EtO
O
O
Cl
OMe
OH
22
26
aReagents and conditions: (i) (COCl)2,CH2Cl2, DMF, 0 °C, then room temp 2h; (ii) DIPEA, CH2Cl2, room temp,
16h, then LiOH, THF, H2O, room temp, 16h; (iii) K2CO3, EtI, DMF, 65 °C, 18h, then futher EtI, 70 °C, 3hr; (iv)
LiOH, THF, H2O, room temp, 5h.
The 3,4,5-trialkoxybenzamido-benzoic acids 31 – 34 were prepared in four or five steps from
methyl 3,4,5-trihydroxybenzoate 27 as illustrated in Scheme 3.
Scheme 3. 4-(3,4,5-Trialkoxybenzamido)benzoic acids.a
(ii)
(iii)
29
28
30
(iv)
(iii)
(ii: R1 = R2)
(i)
(iii)
31 R1 = Et, R2 = R3 = iPr
32 R1 = Et,, R2 = R3 =
33 R1 = R2 = R3 = iPr
34 R1 = R2 = Et, R3 = iPr
27
aReagents and conditions: (i) NaHCO3, R1I, DMF, 30 °C, 72h; (ii) K2CO3, R2Br, DMF, 50 °C, 48h; (iii) LiOH, THF,
H2O, room temp, 16h; (iv) (COCl)2,CH2Cl2, DMF, 0 °C, 1h then methyl 4-amino-benzoate, NEt3, room temp, 12h.
4

For the symmetrical tri-alkoxy compound 31, treatment of 27 with sodium hydrogen carbonate
and ethyl iodide gave mainly compound 28 (R1 = Et) where alkylation had only occurred in the
4-position of the substrate. After purification, this compound on treatment with potassium
carbonate and 2-bromopropane gave an intermediate compound where both remaining hydroxyl
groups had reacted with the alkylating reagent. Hydrolysis resulted in the fully alkylated benzoic
acid 29 (R1 = Et, R2 = R3 = iPr) which was coupled via the acid chloride to give the methyl ester
of compound 31. A final hydrolysis using lithium hydroxide yielded compound 31. The other
tri-alkoxy derivatives 32 – 34 were similarly prepared (Scheme 3).
The 3-chloro-4,5-dialkoxybenzamido benzoic acids 39 – 45 and 49 – 59 were prepared as
described in Scheme 4 and Scheme 5. The commercially available 3-chloro-4-hydroxy-5-
methoxybenzoic acid 35 was treated sequentially with boron tribromide and trimethylsilyl
chloride in methanol to leave methyl 3,4-dihydroxy-5-chlorobenzoate 36.
Scheme 4. 4-(3-Chloro-4,5-dialkoxybenzamido)benzoic acids with identical alkoxy groups.a
(iii)
(iv)
(i)
(ii)
35
36
37
(v)
(iv)
38
39 R = iPr, R1 = H
40 R =
43 R = iPr, R1 = F
44 R = iPr, R1 = Me
, R1 = H
45 R = tBu, R1 = Me
41 R = tBu, R1 = H
, R1 = H
42 R =
a Reagents and conditions: (i) BBr3, CH2Cl2, 0 °C, 2h; (ii) TMSCl, MeOH, 50 °C, 16h; (iii) K2CO3, RI, DMF, 70 °C,
46h; (iv) LiOH, THF, H2O, room temp, 18h; (v) (COCl)2, CH2Cl2, DMF, 0 °C, 1h then methyl 4-amino-2-R1-
benzoate, NEt3, room temp, 12h.
For the derivatives 39, 40, 42, 43, and 44, (Scheme 4) where the alkoxy groups are the same,
both hydroxyl groups in 36 were alkylated by using potassium carbonate and the appropriate
alkyl halide in N, N-dimethylformamide heated to 70oC.
Hydrolysis gave rise to the fully substituted benzoic acids 37 (R = iPr), 37 (R = cyclobutyl) and
37 (R = cyclopentyl. These were then coupled to the aniline 38 via the acid chloride generated
by treatment of the benzoic acid with oxalyl chloride.
The di-tert-butoxy derivatives 41 and 45 were synthesized from the acid 37 (R = tBu), which was
prepared by reacting the two hydroxyl groups in 36 with N, N-dimethylformamide di-tert-butyl
acetal followed by hydrolysis, and then coupling the product directly with aniline 38 using
5

HATU. A final treatment of the coupled products with lithium hydroxide in aqueous 1,4-
dioxane gave the required acids.
The non-identical di-alkoxy compounds 49 – 57 and 59 were also prepared via methyl 3,4-
dihydroxy-5-chlorobenzoate 36, while 58 was prepared from 3-chloro-4-hydroxy-5-
methoxybenzoic acid 35 via benzoic acid 48 (R2 = Me, R3 = Et). (Scheme 5).
Scheme 5. 4-(3-Chloro-4,5-dialkoxybenzamido)benzoic acids with non-identical alkoxy
groups.a
(iv)
(iii)
46
47
36
(vii)
(v)
(vi)
(i) (ii)
(ii)
(vi)
(vii)
48
35
38
(viii) (ix)
49 R2 = iPr, R3 = Et, R1 = H
50 R2 =
, R3 = Et, R1= H
51 R2 = iPr, R3 = Me, R1 = H
52 R2 = Et, R3 = iPr, R1 = H
53 R2 = iPr, R3= Me, R1 = F
54 R2 = iPr, R3 = Et, R1 = F
55 R2 = iPr, R3 = Me, R1 = Me
56 R2 = iPr, R3 = Et, R1 = Me
57 R2 = Et, R3 = iPr, R1 = Me
58 R2 = Me, R3 = Et, R1 = Me
59 R2 =
, R3 = Et, R1= Me
a Reagents and conditions: (i) BBr3, CH2Cl2, 0 °C, 2h; (ii) TMSCl, MeOH, 50 °C, 16h; (iii) K2CO3, BnBr, DMF, 60
°C, 0.75h; (iv) K2CO3, R2Br, DMF, 60 °C, 2h; (v) H2, 10% Pd/C, MeOH; (vi) K2CO3, DMF, 60 °C, 10min, then R3I,
40 °C, 3h; (vii) LiOH, THF, H2O, 40 °C, 1h, then room temp, 16h; (viii) T3P, methyl 4-amino-2-R1-benzoate, NEt3,
EtOAc, 60 °C, 4h, then room temp, 16h. (ix) LiOH, THF, H2O, 40 °C, 16h.
On treatment of 36 with potassium carbonate and benzyl bromide, the 4-benzyloxy methyl ester
46 was produced. For 49 this was then alkylated with isopropyl bromide and base to give the 3-
isopropoxy-4-benzyloxy compound 47 (R2 = iPr) which was hydrogenated, alkylated with ethyl
iodide and base and hydrolyzed to give rise to the benzoic acid 48 (R2 = iPr, R3 = Et). This
benzoic acid 48 was then coupled to the aniline 38 (R1 = H) using T3P in ethyl acetate and
triethylamine as a base, followed by hydrolysis with lithium hydroxide to provide the final
6

compound 49. The other non-identical di-alkoxy compounds 50 – 57 and 59 were similarly
prepared via their corresponding benzoic acids 48 (Scheme 5).
3. Results and discussion
A ligand-based virtual screening approach, which ranks compounds by their similarity towards
known active ligands, was adopted in a search for a novel chemical series of small molecule
RARα agonists. The extended electron density representation offered by the Cresset XED force-
field provides a way to characterize the calculated field around a molecule.14 The subsequent
molecular comparison uses four different 3D fields, positive and negative charge, steric shape
and hydrophobicity, and allows a complete 3D conformational analysis of compounds to be
performed.15.16 The crystal structure of the selective RARα antagonist BMS 195614 (1) in the
human RARα active site17 was overlaid with AM 580 (2), the antagonist removed and the
resulting complete assembly minimized to give the putative bioactive conformation of AM 580
(2). This procedure was also performed for AGN 193836 (3) to get its bioactive conformation.
Molecular fields were added to each of these bioactive conformations (Figure 2).
Cresset FieldScreen representation
bioactive conformation of AM580.a
Figure 2.
of
a
Blue field points (spheres) highlight energy minima for a positively charged probe, red for a negative probe.
Yellow spheres represent an attractive van der Waals minima for a neutral probe and orange spheres represent
hydrophobic centroids. Oxygen atoms are shown in red, nitrogen in blue. The size of the points is related to the
strength of the interaction.
These unique molecular field patterns were used to search Cresset’s database of 2.5M
commercially available molecules, and the results ranked in similarity to the initial bioactive
conformations (see Supplementary data for further details).
This methodology identified 3000 commercially available compounds as possible hit
compounds. The 200 compounds that had the highest field overlays, Lipinski likeness, and
synthetic tractability, were purchased. These were tested in transactivation assays at the RARα,
β and γ receptors. Full dose-response curves were generated for each active agonist, and the
potency of each compound was expressed as a ratio of its EC50 compared to that of reference
ATRA EC50 value generated on each 96 well plate. This produced several potent hits, including
the lead compound 5 (Table 1). The 3,5-dichloro-4-ethoxy derivative 5 was considered to be one
of the better starting points for a lead optimization exercise, not only because of its potency as an
RARα agonist but also because of its good selectivity over the RARβ and RARγ receptors, with
7

moderate lipophilicity (cLog P = 4.4) compared to AGN 195183 (4) (cLog P = 7.2). In addition,
it had no systematic Cyp450 liability (inactive at 25µM at Cyp1A, 2C19, 2C9, 2D6 and 3A4
isoforms), and was not cytotoxic in COS-7 cells (i.e. showed <20% cell death @ 50 x EC50 at the
RAR alpha receptors).
Our aim was to increase the RARα potency and selectivity over RARβ while retaining the
excellent selectivity over RARγ shown by 5 and achieve oral bioavailability in the rat. The
target profile was RARα potency (RARα EC50 / ATRA EC50 <10) with a selectivity of 2 orders
of magnitude over RARβ and 3 orders of magnitude over RARγ with an oral bioavailability of >
35% in the rat.
Initial SAR showed that the three aromatic substituents in 5 seemed important for potency as the
disubstituted, 3,5-dichloro derivative 60 was less potent at RARα and also less selective than the
3,5-dichloro-4-ethoxy derivative 5 at RARβ and RARγ. This helped focus our SAR on
derivatives with a 3,4,5 substituted aromatic ring.
3.1. 4-Substituted derivatives
We initially concentrated on the 4-substituent (Table 1). Increasing the length of the 4-alkoxy
substituent to n-propoxy 12 and n-butoxy 13 resulted in a loss of selectivity at RARβ and RARγ.
Table 1. Potency and Selectivity of 3,5-dichloro-4-alkoxy RARα agonists.
Cl
O
RO
26
N
H
CO2H
Cl
Subtype-specific transactivationa
b
Relative EC50
compd
RO
cLogP f
RARα
RARβ
β /α
ratioc
141
RARγ
γ /α
ratioc
867
1564
1917
9836
7.2
4.4
4
5
AGN 195183
11
24
EtO
>300000
79
>12,500
PrO
139
717
1196
1477
4.9
4.6
12
13
15
84
7 d
9.5
8.5
82
18
BuO
iPrO
tBuO
1417
2927
823
4.7
5.1
14
16
205
426
119
909
6250
7
342
4703
5.3
15
10
33
452
-
355
642
>108000
5000
4.4
4.2
3.9
26
60
61
30
12
7
>3600
55
92 d
H
MeO
ATRA
9525
5850
30
318
195
1.0
(1.51 nM)e
1.0
(0.52 nM) e
1.0
(0.22 nM) e
a
Transactivation assays for the RAR alpha, beta and gamma receptors were performed using each of the mouse
RAR ligand binding domains, Subtype-specific activity is expressed in terms of relative EC50 which is the
8

concentration of retinoid required to produce 50% of the maximal observed response, normalised relative to that of
ATRA. .
b
Mean EC50 for each compound divided by the mean EC50 of ATRA. Values were obtained from three separate
experiments. Errors in these assays are approximately 20% of the mean values
c The relative EC50 ratios of α to β and α to γ.
d Compound behaves as a partial agonist relative to the amplitude of the normalizing ATRA output.
e Mean of ATRA EC50 (nM).
f cLog P values were calculated in ChemDraw.
Increasing the bulk of the 4-alkoxy substituent to isopropoxy 14 and tert-butoxy 16 resulted in an
increase in potency at RARα and an increase in selectivity over RARβ but a loss of selectivity at
RARγ. In contrast, the cyclopentoxy compound, 15 was less selective than 5 at both RARβ and
RARγ.
We also explored the reverse amide 26 of 5 which lost significant selectivity against RARβ when
compared to 5 and hence further work on the reverse amides was curtailed.
We next investigated the PK profile of these 3,5-dichloro-4-alkoxy derivatives. We used
intrinsic clearance figures in mouse and human microsomes as a simple in vitro screen to
minimize the risk of Phase 1 metabolism, before progressing to in vivo studies. The PK profile
of the 3,5-dichloro-4-alkoxy series of compounds was poor. The ethoxy 5, tert-butoxy 16 and
cyclopentoxy 15 derivatives all had a high mouse, and moderate human intrinsic clearance and
15 was poorly orally absorbed with very low oral bioavailability in the rat (Table 2).
Table 2. In vitro and in vivo PK.
c
compd aLog D LEb
pH 7.4
intrinsic Clint
rat pK d
AUC po
ng·min
mL−1
Cl
mL/kg/min
F%
ND
mouse
(µL/min/mg
protein)
human
(µL/min/mg
protein)
5
1.7
2.8
2.6
1.7
1.6
2.6
1.7
1.0
0.45
0.41
0.45
0.51
0.36
0.43
0.47
0.44
127
83
91
38
8
18
26
16
14
4
ND
1674
ND
ND
2
0.3
15
16
18
31
39
49
51
ND
1.6
1
ND
12
81
39
-
74396
783782
43569
-
31
41
9
-
10
-
11
12
50940
3
13
a
Measured
LE values were
by octanol/buffer shake flask method at pH 7.4 (see Supplementary data file for details).20
b
calculated by LE = ( RT ln Kd) / N, presuming EC50 ≈ Kd. 18 c Intrinsic clearance Clint data for screening purposes
only: Mouse and Human microsomes were incubated with the test compound at 37 °C in the presence of the co-
factor, NADPH. The data is the mean of 5 separate experiments. Compound disappearance monitored over 45 min
period. SEM is less than 10% of the mean values. d Rat PK (n = 4): AUC (ng·min mL−1) at 10 mg/kg, 8% Ethanol /
92% PEG-400 formulation, Cl in mL min−1 kg−1.24 ND = not determined.
9

3.2. 3,5-Disubstituted derivatives
To overcome these difficulties we turned our attention to the 3,5-sustituents in 5. The patent
analysis in this class of compounds showed that non-alkyl substituents in the 3,4,5-substituted
aromatic ring of 5 appeared novel. With this in mind we analysed the medicinal chemistry
parameters of the 3,5-substituents of our initial 4-OEt derivatives containing non-alkyl 3,5-
substituents 5, 62, 63 and 3,5-dialkyl substituents 64 (Table 3). Ranking these four derivatives in
terms of RARα potency against the properties of the 3,5 substituents in the second aromatic ring,
such as size (MR), lipophilicity (π) and electronic resonance (σ) (Table 3), shows that potency
only increases with the lipophilicity π of the 3,5-sustituents (and not with the size or resonance
effects of these substituents).
Table 3. 3,5-Disubstituted-4-ethoxy derivatives.
R1
O
EtO
CO2H
N
H
R2
πb
R1+R2 R1+R2 rel EC50
σc
RARα
d
compd
R1
R2
MRa
R1+R2
EtO
Cl
EtO
Cl
25
0.76
1.42
1.72
3.96
1.70
0.2
0.74
0.78
-0.20
0.20
370
24
62
5
12.06
17.76
39.24
34.12
Br
Br
5
63
64
31
tBu
iPrO
tBu
iPrO
0.2
26e
a Sum of size (MR) of meta substituents R1
and R2.
b Sum of lipophilicity (π) of substituents R1 and R2.
c Sum of electronic resonance effect (σ) of meta substituents R1 and R2.
For parameters see ref 19.
d relative EC50 see a,b Table 1
e partial agonist see d Table 1
A search of possible aromatic substituents showed that the isopropoxy group has a similar
lipophilicity to a chlorine / bromine atom found in 5 / 63 and a similar size to a tert-butyl found
in the more potent derivative 64. This suggested that the 3,5-diisopropoxy derivative 31 should
be at least as active as the chloro and bromo derivatives 5 and 63, and why the 3,5-diethoxy
analog 62 which is the least lipophilic, is the least active.
3.3. 3,4,5-Trialkoxy and 3,4,-Dialkoxy derivatives
Encouragingly 31 proved to have good RARα potency (Table 3). In addition, 31 has high
selectivity over RARβ and RARγ (Table 4), and low mouse and human intrinsic clearance with
excellent oral absorption and bioavailability (81%) in the rat (Table 2), although it was shown to
be only a partial RARα agonist. The close profile of 5 and 31 in terms of RARα potency, as well
10

as RARβ and RARγ selectivity, shows that in this case, the iPrO group is a good bioisostere of
the Cl group. This led the project away from the 3,5-dichloro template and enabled exploration
of the alkoxy derivatives at these positions which give a lipophilic surface without the high
lipophilicity of the similar sized tertiary butyl group seen in 64, making the template more drug-
like. Further analogs of this trialkoxy template 31 were investigated in an attempt to increase its
alpha potency while maintaining the excellent beta and gamma selectivity as well as its good PK
profile. Increasing the size of the 3,5-substituents in 31 to give the di-cyclopentoxy derivative 32
or increasing the size of the 4-substituents to give 33 maintained the good RARα potency and
RARβ selectivity but lost selectivity against RARγ (Table 4). Decreasing the size of both the 3-
and 5- isopropoxy groups to give the 3,4,5-triethoxy derivative 62, resulted in a substantial loss
of RARα potency (Table 3). In addition 31, 32 and 33 all exhibited some partial agonist activity
at RARα. However a close analog the 3,4-diethoxy-5-isopropoxy derivative 34 showed that it
was possible to have full RARα agonist properties with trialkoxy derivatives (Table 4).
Table 4. Potency and Selectivity of 3,4,5-Trialkoxy and 3,4,-Dialkoxy RARα agonists.
R1
O
O
2
O
O
R
N
H
R3
OH
Subtype-specific transactivationa
b
Relative EC50
compd
R1O
R2O
R3
RARα
RARβ
β /α
ratioc
174
RARγ
γ /α
ratioc
16
EtO
iPrO
EtO
EtO
EtO
EtO
iPrO
64148
4560
4200
5882
56900
550
62
31
32
368
26 d
29 d
175
145
2190
19
O
O
iPrO
iPrO
iPrO
2600
225
33
27d
96
8
iPrO
iPrO
EtO
EtO
EtO
Cl
2450
103
960
34
49
29
0.7 d
84
34
8083
150
11721
EtO
iPrO
Cl
115
1706
50
1.0 d
115
1706
iPrO
Cl
Cl
89
53
1386
1059
39
40
1.7
54
22
838
447
2.4 d
tBuO
tBuO
Cl
Cl
38
55
162
571
41
42
0.9
44
32
189
336
1.7 d
iPrO
EtO
MeO
iPrO
Cl
Cl
1500
7.1
10833
1202
1.0
51
52
5.3
2.1
1.0
283
3.1
2043
570
ATRA
1.0
(1.51 nM)e (0.52 nM) e
(0.22 nM) e
aTransactivation assays were performed using the RAR alpha, beta and gamma receptors containing each of the
mouse RAR ligand binding domains. Subtype-specific activity is expressed in terms of relative EC50 which is the
concentration of retinoid required to produce 50% of the maximal observed response, normalised relative to that of
ATRA.
11

b The relative EC50 is the mean EC50 for each compound divided by the mean EC50 of ATRA. Values were obtained
from three separate experiments. Errors in these assays are approximately 20% of the mean values
c The relative EC50 ratios of α to β and α to γ.
d Compound behaves as a partial agonist relative to the amplitude of the normalizing ATRA output.
e Mean of ATRA EC50 (nM).
This unsymmetrical derivative was further exploited by the investigation of a series of 3,4-
alkoxy derivatives (Table 4). Replacing one of the isopropoxy groups in the lead 31 with a
chloro atom gave the chloro-dialkoxy derivative 49 which had increased potency at RARα and
also maintained the excellent selectivity at RARβ and RARγ. However, this compound was also
only a partial agonist at RARα.
Increasing the size of the 3-isopropoxy in 49 to 3-cyclobutyl in 50 gave no change in profile.
However, increasing the 4-ethoxy group in 49 to the 4-isopropoxy in 39 gave a similar level of
potency at RARα as a full agonist. The molecule was also an order of magnitude more potent
than 31 at RARα while maintaining excellent selectivity at RARγ with moderate selectivity at
RARβ. In addition, the di-isopropoxy derivative 39 was orally well absorbed in the rat with a
bioavailability of 39% (Table 2). Thus 39 satisfied our target profile except for selectivity at
RARβ. Increasing the size of the alkoxy groups to the di-cyclobutyl in 40, di-tert-butyl in 41 and
di-cyclopentyl in 42 maintained potency at RARα, but decreased selectivity at RARβ and RARγ.
Interestingly reducing the size of the 4-ethoxy in 49 to 4-methoxy in 51 gave a full agonist with
good RARα potency and selectivity at RARβ and RARγ. However, it had a low oral
bioavailability (13%) in the rat (Table 2).
3.4. Substitution of the benzoic acid ring.
Ortho-fluoro substitution in the benzoic acid ring of the bicyclic 5,5,8,8-tetramethyl-5,6,7,8-
tetrahydronaphthalene analogs which lead to AGN 195183 (4)10 has been shown to increase
RARα binding potency and increase selectivity over RARβ and RARγ in the transactivation
assay.
Table 5. Ortho-substituted benzoic acid derivatives of 5.
Cl
O
EtO
N
H
CO2H
Cl
z
Compd
Z
RARα
rel EC50
β /α
ratiob
γ /α
ratiob
a
H
F
24
25
80
>12,500
>80
5
80
82
37
64
1.5
17
18
19
20
21
CH3
OH
Cl
0.9
151
33c
151
1.7c
149
>50
CF3
11
a,b and c see Table 1.
12

Based on this precedent, a series of ortho-substituted benzoic acid derivatives of our initial lead
template 5 were prepared (Table 5). While the ortho-fluoro substitution product 17 maintained
potency and selectivity, the ortho-methyl substitution product, 18 improved RARα potency 20-
fold and maintained good RARβ and RARγ selectivity. In addition, 18 had a lower mouse and
human intrinsic clearance, as well as a somewhat improved bioavailability (12%) in the rat
(Table 2), compared to the unsubstituted benzoic acid derivative 5. Compounds 19, 20 and 21
with larger substituent groups, either lost RARα potency or RARβ / RARγ selectivity compared
to 5. As a result of these findings, a series ortho-methyl and ortho-fluoro substituted benzoic
acid derivatives of the 3,4-dialkoxy-5-chloro template were prepared (Table 6). The initial trend
from the 5 series (Table 5) was also seen in the 3,4-dialkoxy-5-chloro series (Table 6).
The ortho-fluoro substituted derivatives 53, 54 and 43 (Table 6) maintained RARα potency and
selectivity compared to their corresponding unsubstituted derivatives 51, 49 and 39 and, in
addition, had a lower mouse and human intrinsic clearance with the latter being in single figures.
However, although both 53 and 54 met our target profile in terms of high RARα potency with a
selectivity of 2 orders of magnitude over RARβ and 3 orders of magnitude over RARγ, they both
had low oral bioavailability (12% and 15% respectively) in the rat.
Table 6. Ortho-Fluoro and ortho-Methyl (3-chloro-4,5-dialkoxybenzamido)benzoic acids.
R1
O
O
2
O
O
R
N
H
Cl
OH
R3
e
compd
R1O
R2O
R3
LogD
d
pH 7.4
intrinsic Clint
rat PKf
AUC Cl
po
RARα
rel EC50
β /α
ratiob
γ /α
ratiob
a
mouse human
F
%
15 15
12 12
iPrO
iPrO
iPrO
iPrO
iPrO
iPrO
EtO
MeO
EtO
F
F
F
1.0
1.4
2.0
1.3
2.3
1.8
1.9
-
11
0.3
3.3
6.9
1.5
15
10871
53
54
43
55
44
56
57
58
6.7
1.1
2.7
4.7c
0.97
1.6
7
609
487
34
17600
6169
300
17.9
18.8
10.6
26.2
37.6
25.7
18.4
15376
-
iPrO
-
-
-
-
MeO Me
-
77670
70765
241
45
509
iPrO
EtO
iPrO
EtO
Me
Me
Me
Me
5.4 66
2947
11000
1675
>250
5.3
8.7
-
7
-
40
-
200
286
210
-
-
MeO
-
-
33
EtO
Me
-
42.3
59
2.6
58
38461
-
-
-
-
-
-
tBuO
tBuO Me
2.9
36.2
28.7
45
0.64
13200
128
-
a relative EC50, brelative EC50 ratios and cpartial agonist see Table 1
d Measured by octanol/buffer shake flask method at pH 7.4 see Table 2.
e Clint (µL/min/mg protein) and fAUC (ng·min mL−1), Cl (mL min−1 kg−1) see Table 2.
The ortho-methyl substituted derivative 55 had a similar mouse and lower human intrinsic
clearance (Table 6) compared to the unsubstituted derivative 51 (Table 2). However, it had only
partial RARα agonist activity. Both the ortho-methyl derivatives 56 and 44 had good
bioavailability (≥40%) in the rat and lower mouse intrinsic clearance (Table 6) compared to the
13

unsubstituted derivatives 49 and 39 (Table 2), with 56 having the lowest (single figure) human
intrinsic clearance of these four derivatives.
While both derivatives 56 and 44 had high RARα potency and good bioavailability 56 was
superior in terms of selectivity at RARβ (2 orders of magnitude) and RARγ (4 orders of
magnitude) and possessed a better overall potency, selectivity and PK profile than the other
analogues 45, 57– 59 shown in Table 6.
The 3-OEt, 4-OiPr geometrical isomer 57 was less potent and less selective at RARγ than 56
which is analogous to the trend seen with compounds 51 and 49 in the unsubstituted benzoic acid
series. This emphasizes the need for a more lipophilic group than OEt in the 3-and 5-position in
this template which was initially seen in Table 3. Thus the 3-OiPr, 4-OEt derivative 56 reached
our target profile in terms of potency, selectivity, and oral bioavailability.
The excellent RARα potency, good RARβ and RARγ selectivity and PK profile of the full
agonist 56 suggested further investigations to see if it had sufficient drug-like properties to be an
orally bioavailable, highly potent and selective RARα agonist with therapeutic potential.
3.5. Predevelopment studies of 4-(3-chloro-4-ethoxy-5-isopropoxybenzamido)-2-
methylbenzoic acid 56.
3.5.1 ADME profiling.
Predevelopment ADME studies revealed that 56 has a good Cyp 450 profile with no significant
inhibition IC50 > 25µM against five Cyp 450 isozymes (1A2, 2C9,2C19, 2D6, 3A4), and has a
human and mouse plasma protein binding of 93% and 91% respectively.20
Compound 56 has also been examined by CEREP in a panel of 120 other receptors, channels and
enzymes. The compound at 10 µM demonstrated no significant interactions with any of the sites
examined leaving a window of some 4 orders of magnitude between its actions at RAR and any
non-RAR site.21 The highest inhibition of 25% was found for the 5HT2B site. To exclude
potential cardiovascular side effects, compound 56 was tested in vitro on the cardiac hERG
channel and did not show any significant binding to hERG up to the concentration of 10 µM.20
3.5.2 Hepatocyte stability.
We initially used a microsomes assay as a screen to rank order compounds of interest in terms of
their metabolic stability. As microsomes only contain phase I metabolising enzymes it was of
interest to screen our lead compound 56 in a secondary screen using hepatocytes which contain
the full complement of drug metabolising enzymes present in the liver.
The metabolic stability of compound 56 was tested at two concentrations (1 µM and 30 µM) in
mouse, rat, dog, Cynomolgus monkey and human cryopreserved hepatocytes. The compound
was shown to be stable, with a long t½ and low clearance in all species (Table 7), which
correlates with the available PK data (Table 8)
Table 7. Stability of 56 in Hepatocytes
14

Conc Species
(µM)
Mouse
Half-life
(minutes)a million cells)
Clint (µl/min/
224
357
12
4
Rat
Dog
1
>450
>450
>450
>300
>450
>450
>450
>450
<3
<3
<3
<9
<3
<3
<3
<3
Cynomolgus monkey
Human
Mouse
Rat
30
Dog
Cynomolgus monkey
Human
aData are expressed as mean values (n=2). For assay details see
Supplementary data file.
3.5.3 PK profile in Mice and Dogs.
The PK profile of 56 was also studied in mice and dogs (Table 8). Compound 56 showed low
plasma clearance (Cl) and low volume of distribution (Vss), resulting in sustained plasma half-
lives in each species (iv t1/2: mice, 1.9 h; dog, 9.2 h). In addition, oral administration of 56
exhibited high bioavailabilities > 80% in both mice and dogs. These results encouraged us to
investigate 56 further as a predevelopment candidate.
Table 8. Pharmacokinetic Profiles of 56 in Mice and Dogs a
iv
Vss (mL/kg)
po
Tmax (h) F (%)
Cl (mL/h/kg)
t1/2 (h)
Cmax (ng/mL)
2007
Species
mice b
dog c
4.7
2.3
0.3
1.9
9.2
0.25
84
2050
0.5
83
0.66
aAdministered at a dose of 1mg/kg by both iv and po routes in mice. Administered at a dose of 0.5 mg/kg, iv,
1mg/kg, po, in dogs.Vehicle = 2% DMSO in 0.05M phosphate buffered saline, pH 7.4. Data are expressed as mean
values (mice, n=3. dogs n=3).
b C57 mice. For assay details see Supplementary data file
c Beagle dogs. For assay details see Supplementary data file.
3.5.4 Human RAR alpha receptor.
As we planned to perform PK and further in vivo evaluation in rodents, we initially used the
corresponding in vitro transcriptional transactivation assays with gal4 fusion receptor constructs,
created using each of the mouse RAR ligand-binding domains. Although the percentage identity
of amino acid sequences between the mouse and human RAR ligand-binding domains of all
three RAR types (α,β or γ) is 99-100%,23 we thought it prudent to confirm the activity and
selectivity of our lead compound 56 against the human RAR ligand-binding domains in a
transcriptional transactivation assay before further predevelopment studies were investigated.
We also tested an earlier less active analog 15 from the 3,5-dichloro template, and AM 580 (2)
for comparison (Table 9).
15

Table 9. Human and Mouse RARα Potency plus β and γ Selectivity.
56
15
AM 580 (2)
ATRA
human
property
56
human b
15
mousea
AM580(2)
mousea
mousea
human b
human b
mouse
RARα
rel EC50
1.0
1.0
1.6
0.59
10.4
33-fold
452-fold
8.1
10.4
8.1
(1.51nM)c (1.01nM)d
Selectivity
β /α ratio
0.33-fold
0.11-fold
200-fold
290-fold
289-fold
33-fold
289-fold
0.34-fold
0.15-fold
Selectivity
11000-fold >13000-fold
2322-fold
452-fold 2322-fold
γ /α ratio
a see Table 1.
bTransactivation RAR human assay. For details see Supplementary data file.
c Mean of ATRA EC50 (nM) mouse assay RARα
d Mean of ATRA EC50 (nM). human assay RARα
There is a good correspondence for RARα potency between human vs mouse for 56 and 15 with
the human being slightly more potent, in contrast to the RARα potency for AM 580 (2) where
the human is less potent than the mouse (Table 9). Similarly, the α vs β selectivity comparison
for 56 and 15, shows that the human is more selective than the mouse, while for AM 580 (2) the
human is less selective than the mouse. Also, α vs γ selectivity for 56 is 4 orders of magnitude
compared to AM 580 (2) where it is only 2 orders of magnitude for both human and mouse.
3.5.5 In vitro toxicology.
In common with most of the other compounds in the series, the lead compound 56 showed no
cytotoxicity in COS-7 cells at a 50-fold multiple of its EC50.20 When examined in a high content
cell toxicity screen in HEPG2 cells (Cyprotex), 56 was found to have no effect at concentrations
up to 50 µM on cell or mitochondrial viability markers.20 This is in contrast to the more
lipophilic molecule AM 580 (2) which caused a significant increase in cell membrane
permeability and a significant decrease in mitochondrial membrane potential at concentrations
between 10 and 30 µM.
When 56 was examined for genetic toxicity, it was negative in bacterial cytotoxicity tests up to
100 µM, negative in an Ames test in three bacterial strains and in an in vitro micronucleus test in
CHO-K1 cells, in all cases in both the presence and absence of S9.21 In the absence of S9 it
should be noted that AM 580 (2), the reference RARα agonist has been shown by others to be a
mutagen in vitro.11,12
16

3.5.6 Ease of Synthesis.
The 4-(3-chloro-4-ethoxy-5-isopropoxybenzamido)-2-methylbenzoic acid 56 can be synthesized
in 9 high yielding reaction steps from 3-chloro-4-hydroxy-5-methoxybenzoic acid (35) (Scheme
5). It is available as a stable highly crystalline, non-hygroscopic, white powder with a melting
point of 186 °C, and with a solubility of >5 mg/mL, as the sodium salt in water at 35 °C.
3.5.7 Profile of lead compound 56.
The 3-OiPr, 4-OEt, 5-Cl ortho methyl benzoic acid derivative 56 met our target profile in terms
of high RARα agonist potency with a high degree of selectivity over RARβ (of 2 orders of
magnitude) and excellent selectivity over RARγ (4 orders of magnitude) at both the mouse and
human receptors. It has high levels of potency in the RARα binding assay (IC50) showing that
the transactivation activity observed was being mediated through the alpha receptor (Table 10).
As expected 56 was also selective vs RXR (IC50 > 10µM in human RXR α and β binding
assays).22 It also possesses good drug-like properties, a low human intrinsic clearance (5.3
µL/min/mg protein) in microsomes and a measured Log D =1.8, which resulted in good oral
exposure with low clearance and good bioavailability (40%) in the rat (Table 10). In contrast,
both 15 and 2 have human intrinsic clearance in double figures and a higher Log D =2.8, which
resulted in low oral exposure in the rat with low bioavailability (0.3%) for 15. Compound 56 was
also shown to be metabolically stable to hepatocytes with a long t½ and low clearance in human
and 4 animal species (Table 7) together with a high bioavailability (>80%) in both mice and dogs
with low plasma clearance (CL) and a sustained plasma half-live (iv t1/2: mice, 1.9 h; dog, 9.2 h)
(Table 8). In addition 56 has a solubility of >5 mg/mL as the sodium salt, no systematic Cyp 450
liability against five isoforms (1A2, 2C9,2C19, 2D6, 3A4) and demonstrated no inhibition (at 10
µM) in a binding assay for hERG channels. It was not cytotoxic in COS-7 cells and was
negative for genetic toxicity in the Ames test and micronucleus test in CHO-K1 cells.
Table 10. Comparison of the RARα Agonist Potency, selectivity versus the RARβ and RARγ
Human and Mouse Receptors, Human Intrinsic Clearance and Pharmacokinetic Profile in Rat for
56 and 15.
intrinsic
d
Clint
RARα
rel IC50
RARα
rel EC50
β / α
ratio
γ / α
ratio
rat PK e
f Log D
pH 7.4
a
compd
bm/ chu
1.6 / 0.6
10.4 / 8.1
bm/ chu
200 / 298
33 / 289
bm/ chu
11000 / >13000
452 / 2322
human
5.3
AUC po Cl F%
3.6
115
9
70765
1674
-
7
2
-
40
0.3
-
1.8
2.8
2.8
56
15
26
AM 580(2)
0.02 / 0.13 1130/ 162
826 / 505
15.6
a
RARα binding assay. The relative IC50 is the mean IC50 for each compound divided by the mean IC50 of ATRA
(IC50 = 0.6 nM). Values were obtained from three separate experiments.
b m = mouse receptor, see Table 1.
c hu = human receptor, see Table 9.
d Human microsomes Clint (µL/min/mg protein),
eAUC po ng·min mL−1, Cl mL/kg/min
f Log D see Table 2.
4. Conclusions
17

We have used a ligand-based virtual screening exercise based on the bioactive conformation of
AM 580 (2) and AGN 193836 (3) to identify the novel, less lipophilic RARα agonist 4-(3,5-
dichloro-4-ethoxybenzamido) benzoic acid 5, which has good selectivity over the RARβ, and
RARγ receptors. Analysis of the medicinal chemistry parameters of the 3,5-substituents of
derivatives of template 5 showed that RARα potency is driven by the lipophilicity of these
substituents. It showed that the iPrO group is a good bioisostere of the Cl group in this case and
that the 4’-(3,5-diisopropoxy-4-ethoxybenzamido)benzoic acid derivative 31 has a close profile
to 5 in terms of RARα potency as well as RARβ and RARγ selectivity. The low mouse and
human intrinsic clearance with excellent oral absorption and bioavailability (81%) in the rat
shown by 31 led to the exploration of the more drug-like branched dialkoxy derivatives, the best
of which was the 4-(3-chloro-4,5-diisopropoxybenzamido)benzoic acid derivative 39 which was
an order of magnitude more potent than 31 at RARα, while maintaining excellent selectivity over
RARγ with moderate selectivity at RARβ and was orally well absorbed in the rat with a
bioavailability of 39%. Substitution at the ortho-position of benzoic acid 5, with a range of
groups, has shown that methyl groups are the best at increasing potency while maintaining good
RARβ and RARγ selectivity. Methyl substitution at the ortho-position of the 4’-benzoic acid
ring of a series of 4’-(3-chloro-4,5-dialkoxybenzamido)benzoic acid derivatives gave the novel
RARα agonist 4-(3-chloro-4-ethoxy-5-isopropoxybenzamido)-2-methylbenzoic acid 56 as the
best in terms of RARα agonist potency and selectivity versus RARβ (2 orders of magnitude)
and RARγ (4 orders of magnitude) at both the human and mouse RAR receptors. This potent
RARα-specific agonist with improved physicochemical properties also has high bioavailability
(>80%) in both mice and dogs with a good PK profile and drug-like properties and was shown to
be negative in the cytotoxicity and genotoxicity screens warranting further consideration as a
potential therapeutic agent.
5. Experimental Procedures.
All starting materials and solvents, as well as compounds 5, 60, 61 and 62, were obtained from
commercial sources. Hydrogenations were performed either on a Thales H-cube flow reactor or
with a suspension of the catalyst under a balloon of hydrogen. Microwave reactions were carried
out on a Personal Chemistry Smith Synthesizer Workstation with a 300 W single mode
microwave cavity. Ion exchange chromatography was performed using strong cation exchange
resin (SCX) cartridges purchased from Sigma-Aldrich and washed with methanol prior to use.
The reaction mixture to be purified was first dissolved in methanol and then loaded directly onto
the SCX and washed with methanol. The desired material was then eluted by washing with 1%
NH3 in methanol. Silica gel column chromatography was performed using Silicycle pre-packed
silica (230-400 mesh, 40-63 µM) cartridges. Preparative HPLC was carried out using a Gilson
HPLC and an Agilent 5 µm Prep-C18 21.2 x 50 mm column. Detection was achieved using a
UV detector at 254 nm. Mobile phase A: 0.1% aqueous formic acid, Mobile phase B: 0.1%
formic acid in methanol. A flow rate of 40 mL/min was used and a gradient employed as
follows; 0.0-0.8 min 5% B; 0.8-7.3 min 5-95% B; 7.3-8.3 min 95% B; 8.3-8.4 min 95-5% B.
Analytical LCMS was performed using an Agilent 1200 HPLC and mass spectrometer system
with a Scalar 5 µm C18 4.6 x 50 mm column and peaks detected by positive or negative ion
electrospray ionization and a UV detector at 254 nm. All tested compounds were found to be of
1
13
≥95% purity using analytical LCMS. H and C NMR spectra were recorded using a Bruker
TM
Avance III 400 spectrometer at 400 and 110 MHz respectively, using either residual non-
deuterated solvent or tetramethylsilane as a reference in the various solvents specified. All
18

animal studies were ethically reviewed and carried out in accordance with the United Kingdom
Animals (Scientific Procedures) Act 1986 by CXR Biosciences Ltd, James Lindsay Place,
Dundee Technopole, Dundee DD 5JJ.
5.1. Chemistry
5.1.1. 4-(3,5-Dichloro-4-(cyclopentyloxy)benzamido)benzoic acid (15).
Step (i): Methyl 3,5-dichloro-4-(cyclopentyloxy)benzoate Methyl 3,5-dichloro-4-
hydroxybenzoate 6 (1.00 g, 4.52 mmol) was dissolved in N, N-dimethylformamide (8 mL) and
treated with bromocyclopentane (534 µL, 4.98 mmol), followed by potassium carbonate (937
mg, 6.79 mmol). The mixture was stirred at 80 °C for 3 h and then partitioned between ethyl
acetate (100 mL) and water (100 mL). The aqueous phase was extracted with ethyl acetate (50
mL) and the combined organic phases washed successively with water (5 x 50 mL) and brine (50
mL), then dried over magnesium sulfate and filtered. The solvent was removed in vacuo to
afford methyl 3,5-dichloro-4-(cyclopentyloxy) benzoate (1.10 g, 84% yield). 1H NMR (400MHz,
CDCl3) δ 7.97 (2H, s), 5.04 (1H, m), 3.90 (3H, s), 2.04 – 1.91 (4H, m), 1.82 – 1.75 (2H, m), 1.69
– 1.60 (2H, m).
Step (ii): 3,5-Dichloro-4-(cyclopentyloxy)benzoic acid (7: R1 = cyclopentyloxy). Methyl
3,5-dichloro-4-(cyclopentyloxy)benzoate (1.05 g, 3.63 mmol) and lithium hydroxide (174 mg,
7.26 mmol) were combined in tetrahydrofuran (10 mL), and water (1.5 mL) was added dropwise
until a solution formed. The resultant mixture was stirred at room temperature for 12 h. The
tetrahydrofuran was removed in vacuo and the residue acidified using aqueous 1M hydrochloric
acid. The resultant precipitate was filtered to afford 3,5-dichloro-4-(cyclopentyloxy)benzoic acid
1
(7: R1 = cyclopentyloxy), (820 mg, 82% yield). H NMR (400MHz, DMSO-d6) δ 8.10 (2H, s),
5.03 (1H, m), 2.04 – 1.91 (4H, m), 1.82 – 1.75 (2H, m), 1.69 – 1.60 (2H, m).
Step (iii): Methyl 4-[3,5-dichloro-4-(cyclopentyloxy)benzamido]benzoate (10: R1
=
cyclopentyloxy, R2 = H, R = Me). A solution of (7: R1 = cyclopentyloxy) (100 mg, 363 µmol)
in dichloromethane (5 mL), cooled to 0 °C, was treated with oxalyl chloride (63.6 µL, 727 µmol),
followed by a drop of N, N-dimethylformamide. The resultant mixture was stirred for 1 h at
room temperature. The solvent was evaporated in vacuo and the residue dissolved in
dichloromethane (5 mL) and then treated with a solution of methyl 4-aminobenzoate (8: R2 = H,
R = Me) (54.9 mg, 363 µmol) and di-isopropylethylamine (190 µL, 1.09 mmol) in
dichloromethane (5 mL). The reaction mixture was stirred for12 h at room temperature and then
partitioned between dichloromethane (20 mL) and aqueous 1M hydrochloric acid (20 mL). The
phases were separated, and the organic phase was washed successively with water (2 x 20 mL),
and brine (20 mL), dried over magnesium sulfate, filtered and then the solvent was removed in
vacuo. The residue was purified by silica gel chromatography (12 g, 0-100% ethyl acetate /
isohexane) to afford methyl 4-[3,5-dichloro-4-(cyclopentyloxy)benzamido]benzoate (10: R1 =
cyclopentyloxy, R2 = H, R = Me), (30 mg, 20% yield). 1H NMR (400MHz, CDCl3) δ 8.06 (2H,
d, J = 8.8 Hz,), 7.85 (1H, br s), 7.82 (2H, s), 7.71 (2H, d, J = 8.8 Hz ), 5.07 – 5.03 (1H, m), 3.92
(3H, s), 2.10 – 1.90 (4H, m), 1.85 – 1.70 (2H, m), 1.70 – 1.60 (2H, m).
Step (iv): 4-[3,5-Dichloro-4-(cyclopentyloxy)benzamido]benzoic acid (15). Compound (10:
R1 = cyclopentyloxy, R2 = H, R = Me), (30.0 mg, 73 µmol) and lithium hydroxide (3.5 mg, 0.147
mmol) were combined in tetrahydrofuran (3 mL) and water was added dropwise until a solution
formed. The resultant mixture was stirred at room temperature for 16 h. The tetrahydrofuran
was removed in vacuo and the residue acidified using aqueous 1M hydrochloric acid. The
19

resultant precipitate was filtered to afford 4-[3,5-dichloro-4-(cyclopentyloxy)benzamido]benzoic
1
acid 15 (15.0 mg, 51% yield) as a white solid. H NMR (400MHz, DMSO-d6) δ 12.77 (1H, s),
10.58 (1H, s), 8.07 (2H, s), 7.93 (2H, d, J = 8.8 Hz,), 7.88 (2H, d, J = 8.8 Hz,), 5.06 – 5.01 (1H,
m), 1.90 – 1.60 (8H, m). m/z 392 (M-H)- (ES-).
The compounds 12 – 14, 17, 20, 21, 63 and 64 were similarly prepared as 15: see
Supplementary data for experimental and spectroscopic details.
5.1.2. 4-(4-(tert-Butoxy)-3,5-dichlorobenzamido)benzoic acid (16).
Step (i): Methyl 4-(benzyloxy)-3,5-dichlorobenzoate. Crude methyl 4-(benzyloxy)-3,5-
dichlorobenzoate (16.9 g) was prepared from methyl 3,5-dichloro-4-hydroxybenzoate (6) (10 g,
45.2 mmol) and benzyl bromide (15.5 g, 90 mmol) using a procedure essentially the same as in
step (i) for 15, except that the mixture was stirred at room temperature for 18 h. The crude
product was partially purified by silica gel chromatography (330 g, 0-10% EtOAc/isohexane) to
afford a white solid. The material was used in the next step without further purification.
Step (ii): 4-(Benzyloxy)-3,5-dichlorobenzoic acid (7: R1 = CH2Ph). 4-(Benzyloxy)-3,5-
dichlorobenzoic acid (7: R1 = CH2Ph) (12.8 g, 96% over 2 steps) was prepared from crude 4-
(benzyloxy)-3,5-dichlorobenzoate (16.9 g) using a procedure essentially the same as in step (iv)
for 15: 1H NMR (400 MHz, DMSO-d6) δ 7.88 (2H, s), 7.56 – 7.48 (2H, m), 7.44 – 7.37 (3H, m),
5.05 (2H, s). m/z 295 (M-H)- (ES-).
Step (iii): Methyl 4-(4-(benzyloxy)-3,5-dichlorobenzamido)benzoate (10: R1 = CH2Ph, R2
= H, R = Me). Methyl 4-(4-(benzyloxy)-3,5-dichlorobenzamido)benzoate (10: R1 = CH2Ph, R2 =
H, R = Me) (9.81 g, 51%) was prepared from 4-(benzyloxy)-3,5-dichlorobenzoic acid (7: R1 =
CH2Ph) (12.8 g, 43.2 mmol) using a procedure essentially the same as in step (iii) for 15, except
the crude product was crystallized from isohexane/EtOAc to afford the product as a white solid.
1H NMR (400 MHz, CDCl3) δ 8.07 (2H, d, J = 8.8 Hz), 7.84 (2H, s), 7.73 (2H, d, J = 8.8 Hz),
7.59 – 7.52 (2H, m), 7.44 – 7.36 (3H, m), 5.13 (2H, s), 3.92 (3H, s). m/z 428 (M-H)- (ES-).
Step (iv): Methyl 4-(3,5-dichloro-4-hydroxybenzamido)benzoate (11: R2 = H). A solution
of methyl 4-(4-(benzyloxy)-3,5-dichlorobenzamido)benzoate (10: R1 = CH2Ph, R2 = H, R = Me)
(8.8 g, 20.5 mmol) in DCM (500 mL) was cooled to 0 °C and treated dropwise with boron
trichloride (20.5 mL, 20.5 mmol, 1 M in DCM). The mixture was then allowed to stir at room
temperature for 12 h. The mixture was cooled in an ice bath then quenched by addition of water
(150 mL). The resultant mixture was partitioned between EtOAc (200 mL) and H2O (100 mL).
The aqueous phase was extracted with EtOAc (2 x 75 mL) and the combined organic phases
washed successively with water (50 mL) and brine (50 mL), then dried over MgSO4 and filtered.
The solvent was removed in vacuo. The residue was crystallized from isohexane/EtOAc to
1
afford methyl 4-(3,5-dichloro-4-hydroxybenzamido)benzoate (11: R2 = H) (5.81 g, 84%): H
NMR (400 MHz, DMSO-d6) δ 11.06 (1H, s), 10.52 (1H, s), 8.06 (2H, s), 8.04 – 7.91 (4H, m),
3.88 (3H, s). m/z 338 (M-H)- (ES-).
Step (v): Methyl 4-(4-(tert-butoxy)-3,5-dichlorobenzamido)benzoate. A stirred suspension
of methyl 4-(3,5-dichloro-4-hydroxybenzamido)benzoate (11: R2 = H) (100 mg, 294 µmol) in
toluene (2 mL) was heated at 80 °C until homogenous. The resultant solution was treated with
1,1-di-tert-butoxy-N,N-dimethylmethanamine (141 µL, 588 µmol) and the mixture heated at 80
°C for 3 h, and then at room temperature for 18 h. Additional 1,1-di-tert-butoxy-N,N-
dimethylmethanamine (141 µL, 588 µmol) was added and the mixture was heated at 80 °C for 5
h. The reaction mixture was cooled to room temperature and the solvent was removed in vacuo.
The residue was diluted with water and extracted with Et2O. The organic layer was dried over
MgSO4, filtered and concentrated in vacuo. The residue was partially purified by silica gel
20

chromatography (12 g, 0-50% EtOAc in isohexane) to afford methyl 4-(4-(tert-butoxy)-3,5-
dichlorobenzamido)benzoate (82 mg, 71%). The material was used in the next step without
further purification.
Step (vi): 4-(4-(tert-Butoxy)-3,5-dichlorobenzamido)benzoic acid (16). 4-(4-(tert-Butoxy)-
3,5-dichlorobenzamido)benzoic acid 16 (39 mg, 51%) was prepared as a white solid from methyl
4-(4-(tert-butoxy)-3,5-dichlorobenzamido)benzoate (82 mg, 294 µmol) using a procedure
essentially the same as in step (ii) for 15: 1H NMR (400 MHz, DMSO-d6) δ 12.79 (1H, s), 10.60
(1H, s), 8.06 (2H, s), 7.94 (2H, d, J = 8.1 Hz), 7.87 (2H d, J = 8.1 Hz), 1.49 (9H, s). m/z 380 [M-
H]- (ES-).
5.1.3. 4-(3,5-Dichloro-4-ethoxybenzamido)-2-methylbenzoic acid (18).
Step (iii): Methyl 4-(3,5-dichloro-4-ethoxybenzamido)-2-methylbenzoate (10: R1 = Et, R2
= Me ). A solution of 3,5-dichloro-4-ethoxybenzoic acid (7: R1 = Et) (285 mg, 1.21 mmol) and
DIPEA (1.05 mL, 6.05 mmol) in DMF (2.5 mL) was added to HATU (690 mg, 1.82 mmol) and
the orange mixture was stirred for 5 min prior to the addition of methyl 4-amino-2-
methylbenzoate (8: R = R2 = Me) (200 mg, 1.21 mmol) in DMF (1 mL). The resulting dark
orange solution was stirred for 18 h. 2 M HCl (10 mL) was added and stirring continued for 10
min, and then the mixture was extracted with diethyl ether. The organic layer was washed with
water (3 x 15 mL), dried over MgSO4, filtered and the solvent was evaporated in vacuo. The
yellow residue was purified by silica gel chromatography (40 g, 0–100% EtOAc in isohexane) to
afford methyl 4-(3,5-dichloro-4-ethoxybenzamido)-2-methylbenzoate (10: R1 = Et, R = R2 = Me)
(267 mg, 56%): 1H NMR (400 MHz, CDCl3) δ 7.97 (d, J = 8.5 Hz, 1H), 7.81 (2H, s), 7.83 – 7.77
(1H, m), 7.59 – 7.48 (2H, m), 4.18 (2H, q, J = 7.0 Hz), 3.89 (3H, s), 2.63 (3H, s), 1.49 (3H, t, J =
7.0 Hz). m/z 380 (M-H)- (ES-).
Step (ii): 4-(3,5-Dichloro-4-ethoxybenzamido)-2-methylbenzoic acid (18).
Lithium
hydroxide (60 mg, 2.51 mmol) in water (1 mL) was added dropwise to a stirring solution of
methyl 4-(3,5-dichloro-4-ethoxybenzamido)-2-methylbenzoate (10: R1 = Et, R = R2 = Me) (267
mg, 56%) (240 mg, 0.628 mmol) in THF (5 mL) and the resulting yellow solution was stirred for
5 days at room temperature. The solvent was evaporated in vacuo and dissolved in water (5
mL), then acidified with 2 M HCl. The resultant mixture was extracted with EtOAc. The
organic layer was washed with water, dried over MgSO4 and filtered and pre-adsorbed on silica.
Silica gel chromatography (40 g, 0-10% IPA in DCM) provided 4-(3,5-dichloro-4-
ethoxybenzamido)-2-methylbenzoic acid 18 (52 mg, 22%) as a white solid: 1H NMR (400 MHz,
DMSO-d6) δ 12.66 (1H, s), 10.49 (1H, s), 8.08 (2H, s), 7.94 – 7.84 (1H, m), 7.75 – 7.65 (2H, m),
4.14 (2H, q, J = 7.0 Hz), 2.54 (3H, s), 1.40 (3H, t, J = 7.0 Hz). m/z 366 (M-H)- (ES-).
The compound 58 was similarly prepared as 18: see Supplementary data for experimental and
spectroscopic details.
5.1.4. 4-(3,5-Dichloro-4-ethoxybenzamido)-2-hydroxybenzoic acid (19).
t
Steps (vi) and (vii): tert-Butyl 4-amino-2-methoxybenzoate (8: R = Bu, R2 = OMe). 1,1-di-
tert-Butoxy-N,N-dimethylmethanamine (608 µL, 2.54 mmol) was added dropwise to a solution
of 2-methoxy-4-nitrobenzoic acid 9 (250 mg, 1.27 mmol) in toluene (7.5 mL) at 80 °C. The
reaction mixture was heated at 80 °C for 3 h, then a further quantity of 1,1-di-tert-butoxy-N,N-
dimethylmethanamine (608 µL, 2.54 mmol) was added. The reaction mixture was heated at 80
°C for 16 h, then diluted with water (10 mL) and extracted with Et2O (3 x 10 mL). The combined
organic phases were washed with brine (30 mL), dried over MgSO4, filtered and then
21

concentrated in vacuo to afford tert-butyl 2-methoxy-4-nitrobenzoate (271 mg, 78%) as a pale
yellow solid. The material was used in the next step without further purification. tert-Butyl 2-
methoxy-4-nitrobenzoate (271 mg, 1.07 mmol) was dissolved in MeOH (270 mL) and passed
through a Thales ‘H-cube’ cartridge (10% Pd/C) at a flow rate of 1 mL/min at 25 °C under full
H2 mode. The solvent was removed in vacuo to afford tert-butyl 4-amino-2-methoxybenzoate (8:
R= tBu, R2 = OMe) (234 mg, 92%) as a pale yellow solid: 1H-NMR (400 MHz, DMSO-d6) δ 7.41
(1H, d, J = 8.5 Hz), 6.16 (1H, d, J = 2.0 Hz), 6.09 (1H, dd, J = 8.5, 2.0 Hz), 5.82 (2H, br s), 3.68
(3H, s), 1.45 (9H, s). m/z 222 [M-H]- (ES-).
Step (iii): tert-Butyl 4-(3,5-dichloro-4-ethoxybenzamido)-2-methoxybenzoate (10: R1 = Et,
1
t
R2 = OMe, R = Bu ). 3,5-Dichloro-4-ethoxybenzoic acid (7: R = Et) (75 mg, 0.32 mmol) in
DCM (5 mL) was treated with oxalyl chloride (56 µL, 0.64 mmol) dropwise, followed by a drop
of DMF. The reaction mixture was stirred at room temperature for 1 h, and then the solvent was
removed in vacuo. The residue was dissolved in DCM (5 mL) and TEA (133 µL, 957 µmol) was
added. The mixture was added to tert-butyl 4-amino-2-methoxybenzoate (8 : R= tBu, R2 = OMe)
(71 mg, 0.32 mmol) and stirred at room temperature for 16 h. The mixture was sequentially
washed with sat. aq. NaHCO3 (5 mL) and 1 M HCl (5 mL), and the organic phase was
concentrated in vacuo. The residue was purified by silica gel chromatography (12 g, 0–100%
EtOAc in isohexane) to afford tert-butyl 4-(3,5-dichloro-4-ethoxybenzamido)-2-
methoxybenzoate (10: R1 = Et, R2 = OMe, R= tBu ) (59 mg, 42%) as a white solid: 1H-NMR (400
MHz, DMSO-d6) δ 10.51 (1H, s), 8.09 (2H, s), 7.63 (1H, d, J = 8.5 Hz), 7.60 (1H, d, J = 1.9 Hz),
7.43 (1 H, dd, J = 8.5, 1.9 Hz), 4.15 (2H, q, J = 7.0 Hz), 3.81 (3H, s), 1.51 (9H, s), 1.41 (3H, t, J
= 7.0 Hz). m/z 384 [M-tBu+2H]+ (ES+).
Step (viii): 4-(3,5-Dichloro-4-ethoxybenzamido)-2-hydroxybenzoic acid (19). A solution of
tert-butyl 4-(3,5-dichloro-4-ethoxybenzamido)-2-methoxybenzoate (10: R1 = Et, R2 = OMe, R =
tBu ) (55 mg, 0.13 mmol) in DCM (5 mL) was cooled to 0 °C and treated dropwise with a
solution of 1 M boron trichloride in DCM (349 µL, 349 µmol). The reaction mixture was stirred
at 0 °C for 1 h and then at room temperature for 2 h. The reaction mixture was cooled to 0 °C
and water (0.5 mL) and sat. aq. NaHCO3 (2 mL) were added. The resulting white precipitate was
collected by filtration and washed with water (2 mL). The solid was dried, then purified by
capture and release on SAX, eluting with 5% AcOH in THF to afford 4-(3,5-dichloro-4-
1
ethoxybenzamido)-2-hydroxybenzoic acid 19 (11 mg, 24%) as a white solid: H-NMR (400
MHz, DMSO-d6) δ 10.51 (1H, s), 8.06 (2H, s), 7.76 (1H, d, J = 8.7 Hz), 7.48 (1H, d, J = 2.0 Hz),
7.32 – 7.25 (1H, m), 4.14 (2H, q, J = 7.0 Hz), 1.91 (1H, s), 1.40 (3H, t, J = 7.0 Hz), 1.35 (1H, s).
m/z 370 [M+H]+ (ES+), 368 [M-H]- (ES-).
5.1.5. 4-(3,5-Dichloro-4-ethoxyphenylcarbamoyl)benzoic acid (26).
Steps (i) and (ii): 4-(3,5-Dichloro-4-hydroxyphenylcarbamoyl)benzoic acid (25). A mixture
of 4-(chlorocarbonyl)benzoic acid methyl ester 23 (600 mg, ca. 3.02 mmol) contaminated with
4-(methoxycarbonyl)benzoic acid 22 was suspended in DCM (5 mL) and cooled to 0 °C. The
mixture was treated with oxalyl chloride (529 µL, 6.04 mmol) and DMF (1 drop). The resultant
mixture was warmed to room temperature, stirred for 2 h, and then concentrated in vacuo. The
residue was dissolved in DCM (3 mL) and a suspension of 4-amino-2,6-dichlorophenol 24 (511
mg, 2.9 mmol) in DCM (18 mL) was added. The resultant suspension was treated with DIPEA
(1.58 mL, 9.06 mmol) and was stirred at room temperature for 16 h. The solvent was removed in
vacuo and the residue partitioned between EtOAc/DCM and aqueous HCl (1 M). The layers
22

were separated and the organic layer was washed with water and brine. The organic layer was
dried over MgSO4, filtered and then the solvent evaporated in vacuo to afford a pale brown solid
(930 mg), which was triturated in hot acetonitrile/methanol (9:1) and filtered. The precipitate
and filtrate were recombined, the solvent was evaporated in vacuo and then the residue was
dissolved in THF (40 mL). Water (10 mL) was added and the mixture treated with lithium
hydroxide (340 mg, 14.2 mmol). The mixture was stirred for 16 h and then partitioned between
EtOAc and aqueous HCl (1 M). The organic layer was washed successively with water (2 x 50
mL), brine, dried over MgSO4, filtered and then concentrated in vacuo to afford crude 4-(3,5-
dichloro-4-hydroxyphenylcarbamoyl)benzoic acid 25 as a pale brown solid. This material was
used in the subsequent reaction step without purification.
Step (iii): Ethyl 4-(3,5-dichloro-4-ethoxyphenylcarbamoyl)benzoate.
Crude 4-(3,5-
dichloro-4-hydroxyphenylcarbamoyl)benzoic acid 25 (450 mg) was dissolved in DMF (15 mL)
and treated with potassium carbonate (829 mg, 6.00 mmol) and iodoethane (436 µL, 5.4 mmol).
The mixture was stirred at 65 °C for 16 h. Iodoethane (200 µL, 2.48 mmol) was added and the
reaction mixture stirred at 70 °C for 3 h. The mixture was partitioned between EtOAc (150 mL)
and aqueous HCl (100 mL, 1 M). The layers were separated and the organic layer was washed
successively with saturated aqueous NaHCO3 and water. The organic layer was dried over
MgSO4, filtered and the solvent evaporated in vacuo. The residue was purified by silica gel
chromatography
(10-25%
EtOAc/isohexane)
to
afford
ethyl
4-(3,5-dichloro-4-
ethoxyphenylcarbamoyl)benzoate (500 mg, 75% over 2 steps) as a pale pink solid: m/z 380 (M-
H)+ (ES-).
Step (iv): 4-(3,5-Dichloro-4-ethoxyphenylcarbamoyl)benzoic acid (26). Ethyl 4-(3,5-
dichloro-4-ethoxyphenylcarbamoyl)benzoate (109 mg, 285 µmol) in THF (5 mL) was treated
with aqueous lithium hydroxide (1.43 mL, 1 M, 1.43 mmol) and the mixture was stirred at room
temperature for 5 h. The reaction mixture was partitioned between EtOAc and aqueous HCl
(1 M). The organic layer was separated and washed successively with water and brine. The
organic layer was dried over MgSO4, filtered and then concentrated in vacuo to afford 4-(3,5-
1
dichloro-4-ethoxyphenylcarbamoyl)benzoic acid 26 (89 mg, 88%) as a pale lilac solid: H NMR
(400 MHz, DMSO-d6) δ 13.30 (1H, s), 10.58 (1H, s), 8.13 – 7.99 (4H, m), 7.94 (2H, s), 4.04 (2H,
q, J = 7.0 Hz), 1.37 (3H, t, J = 7.0 Hz). m/z 352 [M-H]- (ES-).
5.1.6. 4-(4-Ethoxy-3,5-diisopropoxybenzamido)benzoic acid (31).
Step (i): Methyl 3,5-dihydroxy-4-ethoxybenzoate (28: R1 = Et). A mixture of methyl 3,4,5-
trihydroxybenzoate 27 (5g, 27.2 mmol), iodoethane (2.194 mL, 27.2 mmol) and sodium
hydrogen carbonate (9.12 g, 109 mmol) was stirred in N,N-dimethylformamide (50 mL) at 30 °C
for 72 h. Water (50 mL) was added and the mixture was extracted with ethyl acetate (2 x 50
mL). The organic layer was then washed with water (50 mL), brine (50 mL), dried over
magnesium sulphate, filtered and concentrated in vacuo. The product was then purified by silica
gel chromatography (80 g, 0-20% hexane/ethyl acetate) to leave 28 (2.90g, 50% yield). 1H NMR
(400MHz, CDCl3) δ 7.24 (2H, s), 5.68 (2H, s), 4.21 (2H, q, J = 7.1 Hz), 3.89 (3H, s), 1.42 (3H, t,
J = 7.0 Hz).
Step (ii): 3,5-Diisopropoxy-4-ethoxybenzoic acid (29: R1 = Et, R2 = R3 = iPr). Compound
28 (500 mg, 2.36 mmol) was combined with 2-bromopropane (885 µL, 9.43 mmol) and
potassium carbonate (651 mg, 4.71 mmol) in N,N-dimethylformamide (5 mL). The resulting
suspension was stirred at 50 °C for 48 h. Water (5 mL) was added and the mixture was extracted
with ethyl acetate (2 x 5 mL). The organic layer was then washed with water (5 mL), brine (5
23

mL), dried over magnesium sulphate, filtered and concentrated in vacuo. The crude product was
then purified by silica gel chromatography (40 g, 0-50% hexane/ethyl acetate) to leave methyl
1
3,5-diisopropoxy-4-ethoxybenzoate (530 mg, 76% yield). H NMR (400MHz, CDCl3) δ 7.27
(2H, s), 4.61 – 4.55 (2H, m), 4.10 (2H, q, J = 7.1 Hz), 3.88 (3H, s), 1.38–1.33 (15H, m).
Step (iii): Methyl 3,5-diisopropoxy-4-ethoxybenzoate was converted to compound (29: R1 =
Et, R2 = R3 = iPr) in 57% yield using lithium hydroxide in the procedure described for
1
compound 15. H NMR (400MHz, CDCl3) δ 7.34 (2H, s), 4.61 – 4.55 (2H, m), 4.10 (2H, q, J =
7.1 Hz), 1.40–1.33 (15H, m).
Step (iv): 4-(4-Ethoxy-3,5-diisopropoxybenzamido)benzoic acid (31). Compound (29: R1 =
Et, R2 = R3 = iPr) and 30 were coupled and hydrolyzed to the title compound 31 (288 mg, 57%
for final step) as a white solid, using the procedures described for the preparation of 15. 1H NMR
(400 MHz, CDCl3) δ 8.02 (2H, d, J = 8.7 Hz), 7.85 (1H, br s), 7.75 (2H, d, J = 8.8 Hz), 7.09 (2H,
s), 4.66 – 4.58 (2H, m), 4.10 (2H, q, J = 7.1 Hz), 1.39 – 1.33 (15H, m). m/z 400 (M-H)- (ES-),
402 (M+H)+ (ES+).
The compounds 32 – 34 were similarly prepared as 31: see Supplementary data for experimental
and spectroscopic details.
5.1.7. 4-[3-Chloro-4,5-bis(cyclopentyloxy)benzamido]benzoic acid (42).
Step (i): Methyl 3-chloro-4,5-dihydroxybenzoate (36). Tribromoborane (7.86 mL, 82 mmol)
was added dropwise to a stirring mixture of 3-chloro-4-hydroxy-5-methoxybenzoic acid 35 (6.61
g, 32.6 mmol) in dichloromethane (50 mL) under nitrogen at 0 °C. The resulting orange mixture
was stirred at the same temperature for 2 h then poured portion wise onto ice/brine (250 mL).
The aqueous phase was extracted with ethyl acetate (2 x 150 mL) and the combined organic
extracts were dried over magnesium sulfate and filtered. The solvent was removed in vacuo to
1
give 3-chloro-4,5-dihydroxybenzoic acid (5.11g, 79% yield). H NMR (400MHz, DMSO-d6) δ
12.69 (1H, br s), 10.14 (2H, br s), 7.35 (1H, d, J = 2.0 Hz), 7.32 (1H, d, J = 2.0 Hz). m/z 187 [M-
H]- (ES).
Step (ii): A solution of 3-chloro-4,5-dihydroxybenzoic acid (3.16 g, 16.76 mmol) and
chlorotrimethylsilane (6.36 mL, 50.3 mmol) in methanol (50 mL) was stirred at 50 °C, for 16 h,
under an atmosphere of nitrogen. The solvent was removed in vacuo and the residue was
partitioned between brine (75 mL) and ethyl acetate (75 mL). The organic layer was washed with
brine (75 mL), dried over magnesium sulfate and filtered. The solvent was removed in vacuo to
give methyl 3-chloro-4,5-dihydroxybenzoate 36 (3.26 g, 82 % yield). 1H NMR (400 MHz,
DMSO-d6) δ 10.17 (2H, br s), 7.38 (1H, d, J = 2.0 Hz ), 7.35 (1H, d, J = 2.0 Hz), 3.78 (3H, s).
m/z 201 [M-H]- (ES-).
Step (iii): 3-Chloro-4,5-bis(cyclopentyloxy)benzoic acid (37, R = cyclopentyl). A mixture of
methyl 3-chloro-4,5-dihydroxybenzoate 36 (300 mg, 1.48 mmol), iodocyclopentane (558 µL,
4.44 mmol) and potassium carbonate (614 mg, 4.44 mmol) in DMF (10 mL) was stirred at 70 °C
for 46 h. The reaction mixture was cooled to room temperature and then partitioned between 1M
hydrochloric acid (75 mL) and ethyl acetate (100 mL). The phases were separated and the
organic phase was washed with brine (2 x 75 mL) then dried over magnesium sulfate and
filtered. The solvent was removed in vacuo and the residue was purified by silica gel
chromatography (40 g, 0–100% EtOAc and isohexane) to give methyl 3-chloro-4,5-
bis(cyclopentyloxy)benzoate (427 mg, 85 % yield). 1H NMR (400 MHz, CDCl3) δ 7.66 (1H, d, J
= 2.0 Hz), 7.45 (1H, d, J = 2.0 Hz), 5.05 – 4.98 (1H, m), 4.87 – 4.83 (1H, m), 3.89 (3H, s), 1.95–
1.55 (16H, m). m/z 339 [M+H]+ (ES+).
24

Step (iv): Methyl 3-chloro-4,5-bis(cyclopentyloxy)benzoate (400 mg, 1.18 mmol) was dissolved
in a mixture of 1,4-dioxane (10 mL) and water (5 mL) and lithium hydroxide (226 mg, 9.44
mmol) was added. After stirring for 18 h at room temperature, the mixture was partitioned
between 1M hydrochloric acid (20 mL) and ethyl acetate (25 mL). The phases were separated
and the organic phase was washed with water (20 mL) then dried over magnesium sulfate and
filtered. The solvent was removed in vacuo to give the title compound (37: R = cyclopentyl) (380
1
mg, 99% yield). H NMR (400 MHz, DMSO-d6) δ 13.07 (1H, br s), 7.52 (1H, d, J = 2.0 Hz),
7.45 (1H, d, J = 2.0 Hz), 4.97 – 4.91 (2H, m), 1.99 – 1.90 (2H, m), 1.70–1.57 (14H, m). m/z 323
[M-H]- (ES-).
Step (v) and (iv): 4-[3-Chloro-4,5-bis(cyclopentyloxy)benzamido]benzoic acid (42).
mixture of 3-chloro-4,5-bis(cyclopentyloxy)benzoic acid (37: R = cyclopentyl) and methyl 4-
aminobenzoate (38: R1
H) was converted to the methyl 4-[3-chloro-4,5-
A
=
bis(cyclopentyloxy)benzamido]benzoate in 49% yield using the procedure in step (iii) described
1
for compound 15 H NMR (400 MHz, CDCl3) δ 8.06 (2H, d, J = 8.8 H), 7.84 (1H, br s), 7.72
(2H, d, J = 8.8 Hz), 7.42 – 7.35 (2H, m), 5.08 – 4.98 (1H, m), 4.90 – 4.86 (1H, m), 3.92 (3H, s),
1.95 – 1.63 (16H, m). m/z 458 [M+H]+ (ES+), 456 [M-H]- (ES-). Hydrolysis of methyl-4-[3-
chloro-4,5-bis(cyclopentyloxy)benzamido]benzoate using the procedure in step (iv) described in
the preparation of compound (37:
R
=
cyclopentyl) gave 4-[3-Chloro-4,5-
1
bis(cyclopentyloxy)benzamido]benzoic acid 42 in 72% yield as white solid. H NMR (400 MHz,
DMSO-d6) δ 12.74 (1H, br s), 10.45 (1H, s), 7.98 – 7.84 (4H, m), 7.69 (1H, d, J = 2.0 Hz), 7.52
(1H, d, J = 2.0 Hz), 5.01 – 4.95 (2H, m), 1.99–1.93 (2H, m), 1.73–1.48 (14H, m). m/z 442 [M-
H]- (ES-).
The compounds 39, 40, 43, 44 were similarly prepared as 42: see Supplementary data for
experimental and spectroscopic details.
5.1.8. 4-(3,4-Di-tert-butoxy-5-chlorobenzamido)benzoic acid (41).
t
Step (ix): 3,4-Di-tert-butoxy-5-chlorobenzoic acid (37: R = Bu). N,N-Dimethylformamide
di-tert-butyl acetal (5.92 mL, 24.7 mmol) was added to a solution of methyl 3-chloro-4,5-
dihydroxybenzoate 36 (500 mg, 2.47 mmol) in toluene (10 mL) and the reaction mixture was
stirred at RT under nitrogen for 21 h. The solvent was removed in vacuo and the residue was
purified by silica gel chromatography (40 g, 0 – 20% EtOAc in iso-hexane) to give the bis-
alkylated intermediate, which was dissolved in 1,4-dioxane/water (20 mL, 1:1) and treated with
lithium hydroxide (591 mg, 24.7 mmol). The mixture was stirred 18 h at room temperature. The
mixture was poured into 10% aqueous citric acid (100 mL) and the precipitate was collected by
filtration. The solid was washed with water and dried to give 3,4-di-tert-butoxy-5-chlorobenzoic
acid (37: R = tBu) (534 mg, 70%). 1H NMR (400 MHz, DMSO-d6) δ 13.13 (1H, br s), 7.67 (1H,
s), 7.53 (1H, s), 1.39 (9H, s), 1.32 (9H, s). m/z 299 [M-H]- (ES-).
Step (x): 4-(3,4-Di-tert-butoxy-5-chlorobenzamido)benzoic acid (41).
A mixture of 3,4-di-tert-butoxy-5-chlorobenzoic acid (37: R = Bu) (250 mg, 0.831 mmol) and
t
methyl 4-aminobenzoate (38: R1 = H) was converted to the methyl 4-(3,4-di-tert-butoxy-5-
chlorobenzamido)benzoate (185 mg, 50%) using the procedure described for compound 18. H
1
NMR (400 MHz, DMSO-d6) δ:10.54 (1H, s), 7.96 (2H, d), 7.91 (2H, d), 7.86 (1H, d), 7.60 (1H,
d), 3.84 (3H, s), 1.41 (9H, s), 1.32 (9H, s). m/z 432 [M-H]- (ES-). Hydrolysis of methyl 4-(3,4-
di-tert-butoxy-5-chlorobenzamido)benzoate (175 mg, 0.403 mmol) using the procedure
described in the preparation of compound (37: R = cyclopentyl) step (vii). gave 4-(3,4-di-tert-
butoxy-5-chlorobenzamido)benzoic acid 41 (110 mg, 64%) as a white solid: 1H NMR (400 MHz,
25

DMSO-d6) δ 12.78 (1H, br s). 10.49 (1H, s), 7.97 – 7.86 (3H, m), 7.85 (2H, d, J = 2.3 Hz), 7.59
(1H, d, J = 2.2 Hz), 1.40 (9H, s), 1.34 (9H, s). m/z 418 [M-H]- (ES-).
The compound 45 was similarly prepared as 41: see Supplementary data for experimental
and spectroscopic details.
5.1.9. 4-(3-Chloro-4-ethoxy-5-isopropoxybenzamido)-2-methylbenzoic acid (56).
Step (iii): Methyl 4-benzyloxy-3-chloro-5-hydroxybenzoate (46). Methyl 3-chloro-4,5-
dihydroxybenzoate 36 (14.19 g, 70 mmol) was dissolved in N,N-dimethylformamide (210 mL)
and treated with potassium carbonate (8.71g, 63 mmol). After stirring for 5 min, benzyl bromide
(8.32 mL, 70 mmol) was added and the mixture was heated to 60o C for 0.75h. The reaction
mixture was diluted with diethyl ether (500 mL) and washed successively with 1M hydrochloric
acid (500 mL) and with brine (2 x 500 mL). The aqueous phase was re-extracted with diethyl
ether (500 mL) and the combined organic layers were washed with brine (2 x 500 mL) and dried
with magnesium sulfate. Filtration and evaporation left the crude product which was purified by
silica gel chromatography (330g, 0-100% ethyl acetate / isohexane) to leave methyl 4-
(benzyloxy)-3-chloro-5-hydroxybenzoate 46 as an off-white solid (9.84 g, 48% yield). 1H NMR
(400 MHz, DMSO-d6) δ 10.50 (1H, s), 7.57 – 7.53 (2H, m), 7.52 (1H, d, J = 2.1 Hz), 7.47 (1H, d,
J = 2.1 Hz), 7.46 – 7.37 (3H, m), 5.14 (2H, s), 3.82 (3H, s). (m/z 293.3 [M+H]+ (ES+), 291.2 [M-
H]- (ES-).
Step (iv): Methyl 3-chloro-4-benzyloxy-5-isopropoxybenzoate (47: R2 = iPr). Methyl 4-
(benzyloxy)-3-chloro-5-hydroxybenzoate 46 (7.5 g, 25.6 mmol) was combined with potassium
carbonate (7.08 g, 51.2 mmol) in N,N-dimethylformamide (25 mL). The mixture was stirred at
RT for 5 min. 2-Bromopropane (4.81 mL, 51.2 mmol) was added and the mixture stirred at 60oC
for 2 h. Water (25 mL) was added and the mixture was extracted with ethyl acetate (3 x 50 mL).
The combined organic phase was washed with brine (2 x 50 mL) and then dried over magnesium
sulfate, filtered and concentrated in vacuo to leave a crude mixture which was purified by silica
gel chromatography (120g, 0-100% ethyl acetate / isohexane) to afford methyl 3-chloro-4-
benzyloxy-5-isopropoxybenzoate (47: R2 = iPr), as a clear oil (4.91 g, 57% yield). 1H NMR (400
MHz, DMSO-d6) δ 7.68 (1H, d, J = 2.0 Hz), 7.56 – 7.47 (3H, m), 7.43 – 7.28 (3H, m), 5.12 (2H,
s), 4.77 – 4.72 (1H, m), 3.90 (3H, s), 1.38 (6H, d, J = 6.1 Hz). m/z 335 [M+H]+ (ES+).
3-Chloro-4-ethoxy 5-isopropoxybenzoic acid (48: R2 = iPr, R3 = Et). Steps (v), (vi) and (vii).
Step (v): Methyl 3-chloro-4-hydroxy-5-isopropoxybenzoate.
Methyl 3-chloro-4-
(benzyloxy)-5-isopropoxybenzoate (47: R2 = iPr) (4.91 g, 14.7 mmol) was dissolved in a mixture
of methanol (160 mL), dichloromethane (16 mL) and acetic acid (0.16 mL) and the solution was
passed through a Thales ‘H-cube’ cartridge (10% Pd/C) at a flow rate of 1 mL/min at 25 °C
under an atmosphere of hydrogen (full H2 mode). The solvents were removed in vacuo to afford
methyl 3-chloro-4-hydroxy-5-isopropoxybenzoate (3.42 g, 85%). 1H NMR (400 MHz,
DMSO-d6) δ 10.07 (1H, s), 7.53 (1H, d, J = 2.0 Hz), 7.43 (1H, d, J = 2.0 Hz), 4.70 – 4.63 (1H,
m), 3.82 (3H, s), 1.30 (6H, d, J = 6.0 Hz). m/z 245 [M+H]+ (ES+), 243 [M-H]- (ES-).
Step (vi): Methyl 3-chloro-4-ethoxy-5-isopropoxybenzoate. Methyl 3-chloro-4-hydroxy-5-
isopropoxybenzoate (3.42 g, 14 mmol) was combined with potassium carbonate (3.86 g, 28
mmol) in N,N-dimethylformamide (5 mL) and the mixture heated at 60oC for 10 min.
Iodoethane (2.26 mL, 28 mmol) was added dropwise whereupon the mixture was stirred at 40oC
for 3 h. A further aliquot of iodoethane was added and heating and stirring was continued for 16
h. Water (50 mL) was added and the mixture was extracted with ethyl acetate (3 x 100 mL).
The combined organic phase was washed with brine (3 x 50 mL) and then dried over magnesium
sulfate, filtered and concentrated in vacuo to leave a crude mixture which was purified by silica
26

gel chromatography (120g, 0-100% ethyl acetate / isohexane) to afford methyl 3-chloro-4-
ethoxy-5-isopropoxybenzoate as a white solid (3.17 g, 82% yield). H NMR (400 MHz,
1
DMSO-d6) δ 7.67 (1H, d, J = 2.0 Hz), 7.48 (1H, d, J = 2.5 Hz), 4.66 – 4.59 (1H, m), 4.16 (2H, q,
J = 7.1 Hz), 3.90 (3H, s), 1.40 (3H, t, J = 7.0 Hz), 1.37 (6H, d, J = 6.0 Hz). m/z 245 [M+H]+
(ES+), 243 [M-H]- (ES-).
Step (vii): 3-Chloro-4-ethoxy 5-isopropoxybenzoic acid (48: R2 = iPr, R3 = Et). Methyl 3-
chloro-4-ethoxy-5-isopropoxybenzoate (3.17 g, 11.6 mmol) was dissolved in tetrahydrofuran
(226 mL) and treated with 1M aqueous lithium hydroxide solution (23.25 mL, 23.25 mmol).
Methanol (5 mL) was added so that a solution formed and this was heated at 40oC for 1h. After
stirring for a further 16h at room temperature, the reaction mixture was acidified with 1M
hydrochloric acid and extracted with diethyl ether (3 x 100 mL). The organic layer was dried
over magnesium sulfate, filtered and concentrated in vacuo to leave 3-chloro-4-ethoxy 5-
1
isopropoxybenzoic acid (48: R2 = iPr, R3 = Et) as a white solid (2.83 g, 94% yield). H NMR
(400 MHz, DMSO-d6) δ 13.21 (1H, s), 7.54 (1H, d, J = 1.9 Hz), 7.48 (1H, d J = 1.9 Hz,), 4.74 –
4.78 (1H, m), 4.11 (2H, q, J = 7.0 Hz), 1.37 – 1.21 (9H, m). m/z 257 [M-H]- (ES-).
4-(3-Chloro-4-ethoxy-5-isopropoxybenzamido)-2-methylbenzoic acid (56).
Step (viii): Methyl 4-(3-chloro-4-ethoxy-5-isopropoxybenzamido)-2-methylbenzoate.
A
suspension of 3-chloro-4-ethoxy 5-isopropoxybenzoic acid (48: R2 = iPr, R3 = Et) (2.82 g, 10.9
mmol) and methyl 4-amino-2-methylbenzoate (38, R1 = Me) (2.16 g, 13.1mmol) in ethyl acetate
(33 mL) was treated with triethylamine (4.56 mL, 32.7 mmol) followed by T3P (50 wt% in ethyl
acetate) (17.34 mL, 27.3 mmol) and the mixture was heated at 60°C for 4 h and allowed to cool
to room temperature for 16 h. The reaction mixture was stirred vigorously with an aqueous
solution of sodium hydrogencarbonate (50 mL) for 10 min and separated. The aqueous layer
was extracted with dichloromethane (3 x 100 mL) and the combined organic phases were dried
(magnesium sulphate), filtered concentrated in vacuo and the residue purified by silica gel
chromatography (40 g, 0:50:50 to 20:40:40 ethyl acetate:dichloromethane:isohexane) to produce
methyl 4-(3-chloro-4-ethoxy-5-isopropoxybenzamido)-2-methylbenzoate as a beige solid (3.24
1
g, 70% yield). H NMR (400 MHz, CDCl3) δ 8.0 (1H, d, J = 8.4 Hz), 7.8 (1H, s), 7.59 – 7.50
(2H, m), 7.43 – 7.36 (2H, m), 4.69 – 4.73 (1H, m), 4.18 (2H, q, J = 7.1 Hz), 3.92 (3H, s), 2.6
(3H, s), 1.46 – 1.37 (9 H, m). m/z 406 [M+H]+ (ES+), 404 [M- H]- (ES-).
Step (ix): 1M Lithium hydroxide solution (15.97 mL, 15.97 mmol) was added to a solution of
methyl 4-(3-chloro-4-ethoxy-5-isopropoxybenzamido)-2-methylbenzoate (3.24 g, 7.98 mmol) in
tetrahydrofuran (32 mL). Methanol (5 mL) was added and the mixture stirred at 40oC for 16h.
A further aliquot of lithium hydroxide solution (7.98 mL, 7.98 mmol) in methanol (5 mL) was
added and stirring at 40oC was continued for 3h. The reaction mixture was partitioned between
water (50 mL) and diethyl ether (100 mL). The layers were separated and the aqueous layer was
acidified with 1M hydrochloric acid solution. A precipitate evolved which was filtered and
washed with water (3 x 10 mL) and diethyl ether (3 x 10 mL). After drying, this left 4-(3-chloro-
4-ethoxy-5-isopropoxybenzamido)-2-methylbenzoic acid 56 as a white solid (2.55 g, 81%).
1
Recrystallisation from dioxane/water (82:18) gave white crystals mp 186 °C. H NMR (400
MHz, DMSO-d6) δ 12.64 (1H, br s), 10.34 (1H, s), 7.86 (1H, d, J = 8.5 Hz,), 7.74 – 7.63 (3H,
m), 7.55 (1H, d, J = 2.0 Hz), 4.79 – 4.73 (1H, m), 4.10 (2H, q, J = 7.1 Hz), 2.52 (3H, s), 1.37–
1.26 (9H, m). 13C NMR (101 MHz, DMSO-d6) δ 168.5, 164.4, 151.9, 148.0, 142.4, 140.9,
132.0, 130.9, 127.9, 125.5, 123.0, 121.4, 117.7, 114.2, 71.6, 69.3, 22.3, 22.2, 15.9. m/z 392
[M+H]+ (ES+), 390 [M-H]- (ES-). HRMS: C20H23ClNO5 requires (M+H)+ 392.1265, found
392.1249 (error -4.0 ppm).
27

The compounds 49 – 55, 57 and 59 were similarly prepared as 56: see Supplementary data for
experimental and spectroscopic details.
5.2. Biological and ADME assays
5.2.1. Transactivation assays for mouse RAR alpha, beta and gamma receptors
Transcriptional transactivation assays have been performed with gal4 fusion receptor constructs,
created using each of the mouse RAR ligand-binding domains, co-transfected with the pFR-luc
(Stratagene) reporter construct in COS-7 cells. Thus, transfected cells will constitutively express
the gal4-RAR fusion protein which in turn may be transactivated by ATRA to induce the
expression of the luciferase that is driven by a gal4UAS. Briefly, on day one, 96 well plates
were seeded with 8000 cells per well then left to recover overnight. On day two, the cells were
co-transfected with 100ng of reporter plasmid and 10ng of the appropriate receptor plasmid per
well using lipofectamine (Invitrogen). On day three, the lipofectamine containing media was
replaced by a DMEM without phenol red, followed by the addition of novel compounds
dissolved in 1µl of DMSO to each well’s 100µl total volume. Finally, on day four, the cells
were lysed and their luciferase substrate was provided by the BrightGlo reagent (Promega), the
plates were then read on the MicroBeta TriLux (Perkin Elmer). In each experiment, an 8 point
dose response curve of ATRA was run in duplicate, and the various compounds tested were
compared to these values.
5.2.2. FlashPlate® Scintillation Proximity Binding Assay (SPA)
In the FlashPlate® Scintillation Proximity Assay (SPA) wells of a 96-well plate are coated with
scintillant and capture antibody (or similar) for tagged proteins. This requires just 100ng of RAR
proteins and 2nM [3H]-retinoic acid per well. This enables competition of specifically bound
[3H]-retinoic acid by unlabelled retinoid compounds. As only radioligand specifically bound to
the captured protein is sufficiently close to the scintillant to produce a signal, separation of bound
and free radioactivity is not required. Binding of the tritiated retinoid to biotinylated RARα is
specific, saturable, time dependent and reversible. We have successfully applied our assay to a
screen of known retinoid standards and novel compounds and it is both rapid and reproducible
(see Supplementary data file for details).
5.2.3. Intrinsic clearance Clint
In this in vitro model of hepatic clearance mouse or human liver microsomes were incubated
with the test compound at 37 °C in the presence of the co-factor, NADPH, which initiates the
reaction. The reaction is terminated by the addition of methanol. Following centrifugation, the
supernatant is analyzed on the LC-MS/MS. The disappearance of the test compound is monitored
over a 45 minute time period. The data is the mean on 5 separate experiments. SEM is less than
10% of the mean values.
The ln peak area ratio (compound peak area/ internal standard peak area) is plotted against time
and the gradient of the line determined.
The elimination rate constant (k) = (- gradient), the Half life (t1/2)(min) = 0.693/k and V(µL/mg)
= volume of incubation (µL) / protein in the incubation (mg).
Intrinsic Clearance = (CLint)(µL/min/mg/protein) = V x 0.693 / k. (see Supplementary data file
for details).
28

5.2.4. PK studies in rats
Test compounds were administered orally and intravenously to groups of 4 male Sprague-
Dawley rats. Oral dosing solutions of each Test Item were prepared at a concentration of
1mg/mL in 8% ethanol and 92% PEG-400. The Test Items were orally administered at a dose of
10mg/kg and a dosing volume of 10mL/kg. Intravenous dosing solutions of each Test Item were
prepared at a concentration of 0.25mg/mL in 8% ethanol, 92% PEG-400. The Test Items were
intravenously administered at a dose of 0.5mg/kg and a dosing volume of 2mL/kg.
Approximately eight blood samples were collected from each animal at appropriate intervals up
to 6 hours post dosing for the iv groups and up to 24h for the oral groups. Whole blood
concentrations of the Test Items were measured using LC-MS/MS and selected pharmacokinetic
parameters calculated using Pharsight WinNonLin software. For furthers details see
Supplementary data file.
Acknowledgements
We thank the Wellcome Trust for their financial support (Grant 084286).
Supplementary data
Supplementary data including experimental and spectroscopic details for similarly prepared
compounds 12 – 14, 17, 20, 21, 32 – 34, 39, 40, 43 – 45, 49 – 55, 57 – 59, 63 and 64. ADME,
biological assays, and virtual screening details associated with this article can be found, in the
online version, at http://dx.doi.org/
References and notes
1. P. Dollé and K. Neiderreither.,Eds. The Retinoids: Biology, Biochemistry, and Disease, John Wiley & Sons, Inc,
Hoboken, NJ, 2015.
2. Lu Y, Bertran S, Samuels TA, Mira-y-Lopez R, Farias EF. Mechanism of inhibition of MMTV-neu and MMTV-
wnt1 induced mammary oncogenesis by RAR[alpha] agonist AM 580. Oncogene, 2010;29:3665–3676.
3. Yoshimura H, Kikuchi K, Hibi S, Tagami K, Satoh T, Yamauchi T, Inshibahi A, Tai K, Hida T, Tokuhara N,
Nagai M. Discovery of novel and potent retinoic acid receptor α agonists: synthesis and evaluation of benzofuranyl-
pyrrole and benzothiophenyl-pyrrole derivatives. J Med Chem. 2000;43:2929–2937.
4. Jarvis CI, Goncalves MB, Clarke E, Dogruel M, Kalindjian SB, Thomas SA, Maden M, Corcoran JPT. Retinoic
acid receptor-α signalling antagonizes both intracellular and extracellular amyloid-β production and prevents
neuronal cell death caused by amyloid-β. Eur. J. Neurosci. 2010;32:1246–1255
5. Seino KI, Yamauchi T, Shikata K, Kobayashi S, Nagai M, Taniguchi M, Fukao K. Prevention of acute and
chronic allograft rejection by a novel retinoic acid receptor-α-selective agonist Int. Immunol. 2004;16:665–673.
6. Yamauchi T, Ishibashi A, Shikata K, Tokuhara N, Seino KI, Kobayashi S, Nagai M. Effect of E6060 [4-{5-[7-
fluoro-4-(trifluoromethyl)benzo[b]furan-2-yl]-1H- 2-pyrrolyl}benzoic acid], a novel subtype-selective retinoid, on
lupus-like nephritis in female (NZBxNZW)F1 mice. J. Pharmacol. Exp. Ther. 2005;312:938–944.
7. Shudo K, Kagechika H. Structural Evolution of Retinoids. Adv Drug Res. 1993;24:81–119.
8. Teng M, Duong TT, Klein ES, Pino ME, Chandraratna RA. Identification of a retinoic acid receptor α subtype
specific agonist. J Med Chem. 1996;39:3035–3038.
9. Kagechika H, Kawachi E, Hashimoto Y, Himi T, Shudo K. Retinobenzoic Acids. 1. Structure-Activity
Relationships of Aromatic Amides with Retinoidal Activity. J Med Chem. 1988;31:2182–2192.
29