Development and Characterization of Novel and Selective Inhibitors of Cytochrome P450 CYP26A1, the Human Liver Retinoic Acid Hydroxylase

Article abstract of DOI:10.1021/acs.jmedchem.5b01780

Cytochrome P450 CYP26 enzymes are responsible for all-trans-retinoic acid (atRA) clearance. Inhibition of CYP26 enzymes will increase endogenous atRA concentrations and is an attractive therapeutic target. However, the selectivity and potency of the exist

Full text of DOI:10.1021/acs.jmedchem.5b01780

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Article
Development and characterization of novel and selective inhibitors of
cytochrome P450 CYP26A1, the human liver retinoic acid hydroxylase
Philippe Diaz, Weize Huang, Charles M. Keyari, Brian Buttrick, Lauren Price, Nicolas Guilloteau,
Sasmita Tripathy, Vanessa G. Sperandio, Frank R Fronczek, Fanny Astruc-Diaz, and Nina Isoherranen
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.5b01780 • Publication Date (Web): 26 Feb 2016
Downloaded from http://pubs.acs.org on March 11, 2016
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Journal of Medicinal Chemistry
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Development and characterization of novel and selective
inhibitors of cytochrome P450 CYP26A1, the human
liver retinoic acid hydroxylase
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*,§,δ
±
§
±
±
Philippe Diaz , Weize Huang , Charles M. Keyari , Brian Buttrick , Lauren Price , Nicolas
δ
±
§
€
δ
Guilloteau , Sasmita Tripathy , Vanessa G. Sperandio , Frank R. Fronczek , Fanny AstrucꢀDiaz , Nina
,
±
Isoherranen*
±
§
Department of Pharmaceutics, University of Washington, Seattle, WA98195
Department of Biomedical and Pharmaceutical Sciences, The University of Montana, 32 Campus
Drive, Missoula, MT 59812, USA
€
Chemistry Department, Louisiana State University, 612 Choppin Hall, Baton Rouge, LA 70803, USA
δ
DermaXon LLC, 32 Campus Drive, Missoula, MT 59812, USA
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ABSTRACT
Cytochrome P450 CYP26 enzymes are responsible for allꢀtransꢀretinoic acid (atRA) clearance.
Inhibition of CYP26 enzymes will increase endogenous atRA concentrations and is an attractive
therapeutic target. However, the selectivity and potency of the existing atRA metabolism inhibitors
towards CYP26A1 and CYP26B1 is unknown, and no selective CYP26A1 or CYP26B1 inhibitors have
been developed. Here the synthesis and potent inhibitory activity of the first CYP26A1 selective
inhibitors is reported. A series of nonꢀazole CYP26A1 selective inhibitors was identified with low nM
potency.
‐{4‐[2‐(5,5,8,8‐tetramethyl‐5,6,7,8‐tetrahydronaphthalen‐2‐yl)‐1,3‐dioxolan‐2‐yl] phenyl}4 propanoic
acid (24) had 43ꢀfold selectivity towards CYP26A1 with an IC of 340 nM. Compound 24 and its two
The
lead
compound
3
5
0
structural analogs also inhibited atRA metabolism in HepG2 cells resulting in increased potency of
atRA towards RAR activation. The identified compounds have potential to become novel treatments
aiming to elevate endogenous atRA concentrations and may be useful as cotreatment with atRA to
combat therapy resistance.
KEYWORDS: Cytochrome P450, retinoic acid, CYP26, retinoic acid receptor
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INTRODUCTION
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R
etinoic acid (RA) is the endogenous active metabolite of vitamin A (retinol). It is an essential regulator of
1
,2
cell growth, cell cycle and differentiation. RA exists in several isomeric forms, allꢀtrans retinoic acid
(atRA), 13ꢀcisꢀretinoic acid (13ꢀcisRA), 9,13ꢀdicisꢀretinoic acid (9,13ꢀdicisRA), and 9ꢀcisꢀretinoic acid
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(9ꢀcisRA). Of these isomers, atRA is believed to be the main biologically active form exhibiting its
activity through binding predominantly to nuclear retinoic acid receptors (RARs). Through its binding
to nuclear receptors and regulating gene transcription, atRA plays a critical role in many biological
processes including reproduction, maintenance of skin and epithelia, regulation of the immune system
4–6
and Tꢀ and Bꢀcell function, and in fetal development. In addition, imbalance in vitamin A and RA
homeostasis has been implied in development and progression of several human diseases such as acne,
1
psoriasis and ichthyosis, type II diabetes, neurodegenerative diseases and some cancers. The potential
of RA in the treatment of human diseases has been manifested in the success of 13ꢀcisRA (Accutane) in
treatment of acne and neuroblastoma, and the effectiveness of atRA in the treatment of acute
promyelocytic leukemia (APL). However, the use and long term efficacy of atRA has been limited in all
of the therapeutic areas by its high clearance that limits oral administration of this agent, and the
7
resistance that develops towards atRA due to autoinduction of its metabolism. Additionally 13cisꢀRA
via isomerization to atRA, and atRA both cause broad activation of RARs that is believed to be
responsible for some of the untoward side effects of these agents. Due to these shortcomings, the full
clinical and therapeutic potential of retinoids has not been reached.
The clearance of atRA is predominantly mediated by cytochrome P450 family 26 enzymes
8
,9
(CYP26). The CYP26 family has three isoforms: CYP26A1, CYP26B1 and CYP26C1 which are
expressed in a tissue specific manner. In the human liver CYP26A1 is the predominant CYP26 enzyme
10
responsible for majority of the hepatic clearance of atRA. CYP26B1 protein is not detected in human
1
1
liver and the expression of this isoform appears to be restricted to extrahepatic sites. The biological
role and expression pattern of CYP26C1 is not well characterized but this enzyme appears to be
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responsible for 9ꢀcisRA metabolism and be less critical in regulating RA concentrations than CYP26A1
and CYP26B1.
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As atRA is predominantly cleared by the CYP26 enzymes, it has been proposed that inhibition
of the CYP26 enzymes will increase atRA concentrations in target tissues, and inhibition of CYP26
1
2
enzymes during atRA or 13ꢀcisRA therapy could be used to combat disease resistance. However,
inhibitors of CYP26 that have been tested in humans (Figure 1) commonly suffer from broad spectrum
CYP inhibition resulting in untoward side effects. Examples of this include the inhibition of steroid
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metabolism by liarozole (compound 1) and the drugꢀdrug interactions seen with ketoconazole
7
(
compound 2). The lack of specificity of these inhibitors is likely due to the imidazole and triazole
moieties in the compounds that are known to coordinate with the heme iron providing additional
potency towards CYP inhibition. Although more selective triazole and imidazole containing CYP26
7
inhibitors have been developed, none of these compounds has yet reached human trials and therefore
their drugꢀdrug interaction potential in vivo is unknown. In addition, none of the existing inhibitors of
CYP26s have been evaluated for specificity between the CYP26 enzymes and therefore the benefits and
shortcomings of selective or broad CYP26 inhibition have not been established.
Figure 1. Chemical structures of previously identified CYP26 inhibitors, 1 (liarozole), 2 (ketoconazole),
13
and 3 (talarozole also called R115866 ).
1
1
CYP26 enzymes appear to have cell type and tissue specific expression, and therefore it is
likely that specific CYP26A1 or CYP26B1 inhibitors would be beneficial for increasing RA
concentrations in selected target tissues without causing broad side effects. Specifically, since
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CYP26A1 appears to be the predominant CYP26 enzyme in the liver, selective inhibition of CYP26A1
is expected to decrease systemic clearance of atRA and therefore be useful for combatting therapy
resistance in individuals being treated with atRA or 13cisRA. In addition, it is likely that use of
CYP26A1 specific inhibitor will selectively increase RA concentrations in the liver. Notably, based on
conditional knockꢀout mouse studies, CYP26B1 inhibition could be a liability for male reproduction as
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testes cell type specific CYP26B1 knockꢀout mice show adverse effects on the testes. Similarly, Tꢀcell
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specific CYP26B1 knockouts have shown that CYP26B1 regulates iTreg and Th17 cell polarization,
an effect that may be a liability for a broad CYP26 inhibitor. A CYP26A1 selective inhibitor would
avoid both of these effects and likely be beneficial when systemic atRA concentrations are targeted.
The aim of this study was to identify novel nonꢀazole inhibitors of CYP26 enzymes that would
be selective for CYP26A1 and devoid of CYP26B1 inhibition. A series of novel CYP26A1 selective
inhibitors was identified with low nM potency for CYP26A1 inhibition. The lead compound from the
series had 43ꢀfold selectivity towards CYP26A1 in comparison to CYP26B1 and other P450 enzymes
and inhibited CYP26A1 with an IC of 340 nM. The lead compound and two of its structural analogs
5
0
also inhibited atRA metabolism in HepG2 cells resulting in increased potency of atRA towards RAR
activation in these cells. The identified compounds are the first CYP26A1 selective inhibitors
discovered and have potential to become novel treatments aiming to elevate endogenous RA
concentrations in circulation and in the liver. These compounds would also be useful as cotreatment
with atRA to combat therapy resistance likely caused by increased systemic clearance of atRA and
autoinduciton of CYP26A1, particularly in the liver.
RESULTS
Chemistry. Initial coupling of 2ꢀbromoꢀ1ꢀ(5,5,8,8ꢀtetramethylꢀ5,6,7,8ꢀtetrahydronaphthalenꢀ2ꢀyl)ethanꢀ
1
ꢀone with methylꢀ4ꢀhydroxybenzoate in the presence of K CO in methyl ethyl ketone under
2 3
microwave conditions, gave ester 4 in good yield (Scheme 1). It was further hydrolyzed under basic
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conditions to afford carboxylic acid 5 in good yield. Reduction of the ketone moiety of compound 4 to
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alcohol by treatment with NaBH , followed by basic hydrolysis at the ester position, gave carboxylic
4
acid 6. Reaction of 4 with hydroxylamine hydrochloride in MeOH under reflux formed oxime isomers E
(7) and Z (8), in moderate and good yield, respectively. Zꢀisomer 8 was further hydrolyzed in aqueous
K CO to provide carboxylic acid 9.
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a
Scheme 1
HO
OH
O
O
N
N
Br
i)
O
iv)
O
O
+
O
O
O
O
O
O
4
7
8
ii)
iii), ii)
v)
OH
O
OH
N
O
O
O
OH
OH
OH
O
O
O
6
9
5
a
Reagents and conditions: (i) Methylꢀ4ꢀhydroxybenzoate, K CO /MEK, µW, 100ºC, 2×20 min; (ii)
2
3
NaOH, THF. EtOH, H O, 80ºC, 12 h; (iii) NaBH4, THF, r.t., 1 h; (iv) H NOH.HCl/MeOH, pyridine,
2
2
reflux, 6 h; (v) K CO (2M)/MeOH, reflux, 80ºC.
2
3
1
6,17
FriedelꢀCrafts acylation
of 1,1,4,4ꢀtetramethylꢀ1,2,3,4ꢀtetrahydronaphthalene (TTN) with methyl 4ꢀ
(chlorocarbonyl)benzoate and methyl 6ꢀ(chlorocarbonyl)naphthaleneꢀ2ꢀcarboxylate in CH Cl in the
2
2
presence of AlCl yielded the expected ketones 10a and 10b (Scheme 2). The ester moieties of 10a and
3
1
0b were then hydrolyzed in basic conditions to respectively afford carboxylic acids 11a and 11b in
1
7
excellent yields. Further, treatment of the ketone 10a and 10b with ethylene glycol resulted in the
formation of ketals 12a and 12b which after basic hydrolysis, afforded the expected carboxylic acids
13a and 13b. Likewise thioketalization of 10a and 10b was achieved by reaction with ethanedithiol in
the presence of boron trifluoride diethyl etherate (BF •Et O), giving the thioketals 14a and 14b in
3
2
excellent yields which after basic hydrolysis, gave carboxylic acids 15a and 15b.
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Journal of Medicinal Chemistry
a
Scheme 2
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O
O
O
O
ii)
O
OH
O
O
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2a
2b
13a
13b
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iii)
O
O
O
i)
ii)
Cl
+
O
O
OH
O
O
O
1
1
0a
0b
11a
11b
iv)
=
a
or
S
S
S
S
b
ii)
O
OH
O
O
1
1
4a
4b
1
1
5a
5b
a
Reagents and conditions: (i) AlCl , CH Cl , r.t., overnight; (ii) K CO (2M)/MeOH, reflux, 80ºC, ; (iii)
(CH OH) , pTsOH, toluene, reflux, overnight; (iv) (CH SH) , BF .Et O, CH Cl , r.t., 0.5ꢀ1h.
3
2
2
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3
2
2
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3
2
2
2
1
6
FriedelꢀCrafts acylation of TTN by 4ꢀiodobenzoyl chloride according to the previously described
18,19
procedure, afforded ketone 16 in good yield (Scheme 3). Heck
coupling reaction of 16 with methyl
acrylate under palladium catalysis proceeded smoothly to give alkene 17. Compound 17 was then
hydrolyzed under basic conditions, to give carboxylic acid 18. Hydrogenation of 17 under Pd/C
catalysis not only reduced the alkene moiety, but also the carbonyl moiety resulting in the formation of
benzylbenzene 19 which upon subsequent basic hydrolysis provided the corresponding carboxylic acid
2
0. Ketalization of 17 was carried out according to the previously described procedure, to afford ketal
1 in moderate yield which after basic hydrolysis gave carboxylic acid 22. Hydrogenation of 21 in
2
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presence of Pd/C followed by hydrolysis gave carboxylic acid 24. The ketal moiety of 23 was cleaved
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with iodine in acetone under reflux according to a modified procedure of Sun et al, to afford ketone 25
which after saponification yielded to the carboxylic acid 26. Thioketalization of 17 followed by
hydrolysis provided carboxylic acid 28. The alkene moiety of 28 was found to be resistant to
hydrogenation using PdꢀC or Wilkinson’s catalyst. Therefore, ketal 29 was obtained directly from the
thioketalization of ketone 25 which after basic hydrolysis gave carboxylic acid 30.
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a
Scheme 3
O
iii)
iii)
OH
O
O
1
9
20
iv)
I
O
O
O
i)
ii)
+
O
OH
I
1
6
17
O
18
O
O
Cl
vi)
v)
S
S
S
S
O
O
O
O
iii)
iii)
OH
O
O
OH
O
O
21
iv)
O
22
O
28
27
O
O
O
O
iv)
iii)
O
OH
23
24
O
O
vii)
O
O
S
S
S
S
iii)
vi)
iii)
O
O
OH
OH
29
25
O
26
O
O
30
O
a
Reagents and conditions: (i) AlCl , CH Cl , r.t., overnight; (ii) methyl acrylate, Pd(OAc) , NEt , DMF,
3
2
2
2
3
1
1
00ºC, 6 h; (iii) LiOH aq. 1N, THF, reflux, 3 h or r.t., overnight; (iv) H , Pd/C, EtOH/EtOAc 20 psi, 7ꢀ
2
2 h; (v) (CH OH) , pTsOH, toluene, reflux, overnight; (vi) (CH SH) , BF .Et O, CH Cl , r.t., 0.5ꢀ1 h;
2
2
2
2
3
2
2
2
(vii) I , acetone, 4Å MS, reflux, 16 h.
2
Sulfone 34 was prepared in 4 steps starting from TTN. TTN was first converted to disulfide 31 (Scheme
2
1
4
) by treatment with chlorosulfonic acid followed by heating in presence of zinc in hydrochloric acid.
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Disulfide 31 was then reduced and coupled with ethyl 3ꢀ(4ꢀbromophenyl)propanoate in presence of
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9
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2
2
PdCl (dppf) and zinc, to give diaryl sulfide 32. Oxidation of 32 with oxone produced the expected
2
sulfone 33 which after hydrolysis under basic condition afforded carboxylic acid 34 in good yield.
a
Scheme 4
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9
0
S
S
i)
ii)
O
O
3
1
32
33
iii)
2
O
O
O
O
S
S
iv)
OH
O
O
O
3
4
a
Reagents and conditions: i) 1) Chlorosulfonic acid, 0°C to r.t., 3h, 2) Zn, EtOH, conc. HCl, reflux 45
min, r.t. overnight ; ii) ethyl 3ꢀ(4ꢀbromophenyl)propanoate, PdCl (dppf), Zn, THF, reflux, 24h; iii)
2
oxone, H O, MeOH, 0°C to r.t., 12h ; iv) LiOH 1N, THF, H O, r.t. 12h.
2
2
To explore the role of the atoms of oxygen (or sulfur) from the dioxolane (dithiolane) moiety, we
investigated the activity of a novel series of compounds with a sulfide moiety or a sulfoxide moiety as a
linker in between the two aromatic rings. Synthesis started with preparation of iodo TTN derivative 35
2
3
from TTN (Scheme 5). Diaryl sulfides 36a and 36b were then prepared by nickel catalyzed coupling
with the iodo derivative 32 in the presence of polymerꢀsupported borohydride in butanol, to respectively
24
provide the 4ꢀ and 3ꢀmercaptophenylacetic acid methyl esters. Methyl esters 36a and 36b were
obtained as mixtures with their butyl ester analogues (25% butyl ester). Biaryl sulfides 36a and 36b
were then oxidized with oxone in methanol to afford the expected biaryl sulfones 37a and 37b.
Compounds 36a, 36b, 37a and 37b were then hydrolyzed under basic conditions to afford the desired
carboxylic acids 38a, 38b, 39a and 39b respectively in good to excellent yields.
a
Scheme 5
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O
O
O
O
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
i)
I
ii)
S
iii)
S
O
O
3
3
6a
6b
37a
37b
3
5
iv)
iv)
a: ortho
b: meta
O
O
O
O
S
S
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
OH
OH
3
3
8a
8b
39a
39b
a
Reagents and conditions: i) I , H SO , HIO , AcOH, H O, 70°C ii) 3ꢀ or 4ꢀmercaptophenylacetic acid
methyl ester, borohydride, polymer supported, (bpy) NiBr , dioxane, ButOH, 130ꢀ145°C, 3 h to 5 h; iii)
oxone, H O, MeOH, 0°C to r.t., 12 h; iv) LiOH 1N, THF, H O, r.t. 12h.
2
2
4
4
2
2
2
2
2
Enzyme Inhibition and structure-activity relationship
A method was previously developed using recombinant CYP26A1 microsomes and 9ꢀcisꢀRA as a
25
substrate to test for inhibition of CYP26A1. Previously, 9ꢀcisꢀRA was chosen as the screening
substrate instead of atRA due to the lack of substrate and metabolite depletion in the screening assays
with 9ꢀcisꢀRA, and the lower affinity of 9ꢀcisꢀRA to CYP26A1 when compared to atRA allowing
25
screening of inhibitors at substrate concentration at or below the K value. Similar to CYP26A1,
m
atRA is a high affinity substrate of CYP26B1 and the metabolites formed by CYP26B1 are also
1
1
substrates of CYP26B1 confounding kinetic inhibition measurements. To allow characterization of
inhibition of CYP26B1, 9ꢀcisꢀRA turnover by recombinant CYP26B1 was also characterized. 9ꢀcisꢀRA
was found to be a substrate of CYP26B1 and the formation of the metabolite that coeluted with 9ꢀcisꢀ4ꢀ
OHꢀRA was NADPH dependent. The K and V for 9ꢀcisꢀ4ꢀOHꢀRA formation by CYP26B1 were of
m
max
5
55 nM and 3.6 pmol/min/pmol P450, respectively. Based on the K value, all compounds were
m
evaluated for CYP26B1 inhibition at a substrate (9ꢀcisꢀRA) concentration of 100 nM (concentration
<
<K to increase sensitivity and decrease the dependence of IC values on inhibition mechanism).
m
50
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Journal of Medicinal Chemistry
Table 1. IC50 values and 95% confidence intervals of the IC50 for the RAMBAs and synthetic
retinoids against CYP26A1 and CYP26B1 for compounds 1, 3, 40, and the naphthyl analogues 11b, 13b
and 15b.
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
1
R
OH
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
O
Activity (µM)
Compound
R1
_
CYP26A1
.9
1.5ꢀ2.3)
CYP26B1
0.018
1
1
3
(
(0.013ꢀ0.027)
a
a
0.0051
0.00046
_
(
0.0034ꢀ0.0068)
(0.000069ꢀ0.00085)
1
3.5
5.9
(2.6ꢀ13.3)
OH
_
(
(
8.6ꢀ21.3)
O
40
O
1.63
0.52
0.4ꢀ0.7)
11b
0.95ꢀ2.9)
0
.11
1.03
(0.62ꢀ1.7)
O
S
O
S
1
1
3b
5b
(
0.05ꢀ0.24)
0.06
0.04ꢀ0.10)
1.03
(0.69ꢀ1.5)
(
a
Inhibition constant determined based on tight binding quadratic equation.
In an effort to identify lead CYP26A1 specific inhibitors, we decided to focus our screening on
synthetic analogues of RA. The compounds were selected on the basis of structural similarity with
atRA, the endogenous substrate of CYP26A1, and a small number of analogues were prepared to obtain
an initial characterization of the structureꢀactivity relationships (SAR) of CYP26A1 binding and
selectivity. This approach was based on prior work showing that some RAR agonists bind and inhibit
25
CYP26A1. The classic RAMBAs (compounds 1 and 3) and three commercially available synthetic
2
6
27
retinoids 40 (bexarotene) (Table 1), 13a (SR11237 ) (Table 2), and 15b (MM11253 ) (Table 1), and
two close analogues 13b and 15a, were tested for their CYP26A1 and CYP26B1 inhibition potency.
This screening showed that both the RAMBAs and synthetic retinoids inhibited CYP26A1 and
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CYP26B1 (Table 1 and 2). The azole containing compound 3 inhibited both CYP26A1 and CYP26B1
potently whereas compound 1 was a more potent inhibitor of CYP26B1 than CYP26A1. Neither one of
these compounds provided sufficient specificity and selectivity to inhibit atRA metabolism specifically
by CYP26A1.
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Compounds 40, 13a and 15a had IC values in the micromolar range and did not demonstrate
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
50
28
significant selectivity for CYP26A1, whereas the RARγ selective antagonist 15b had a 0.061 ꢁM IC
5
0
for CYP26A1 and 1.03 ꢁM IC for CYP26B1 (Table 1) demonstrating a 17ꢀfold higher IC towards
5
0
50
CYP26B1 than CYP26A1. Compound 13b, the ketal homologue of 15b displayed the same CYP26B1
inhibitory activity but exhibited a reduced CYP26A1 activity compared to the thioketal analogues.
Replacement of the original dithiolane linker of 15b by a carbonyl in compounds 11b and 18, and
synthesis of their shorter homologues 11a and 15a resulted in a reduction of CYP26A1 inhibitory
activities and a modest enhancement of CYP26B1 inhibition for compound 11b. All these data together
indicate that the geometry of the thioketal ring and the distance between the carboxylic acid and the
thioketal moiety are crucial for the CYP26A1 inhibitory activity. Our subsequent research efforts to
complete SAR of synthetic retinoids led to the synthesis of compounds 5, 6 and 9. We decided to
incorporate polar groups in the spacer between the two aromatic moieties to explore potential hydrogen
bonding interaction with CYP26A1. The distance between the carboxylic acid and the
tetramethyltetrahydronaphthalene moiety was kept in the same range as for compound 15b. The
naphthyl moiety was replaced by a methyleneoxyphenyl moiety to minimize potential steric hindrance
that would prevent hydrogen bonding. However, such a chemical modification did not produce
significant improvement in affinity towards CYP26A1. We also explored the effect of potential steric
hindrance on compound 11b by replacing the naphthyl moiety by a styryl moiety (18) This change led
to a dramatic loss of CYP26B1 inhibition and a 2 to 3ꢀfold reduction in CYP26A1 activity. A similar
trend was observed for the CYP26B1 activity of the styryl derivative 22 whereas CYP26A1 activity was
comparable to the naphthyl analogue 13b. Surprisingly compound 28, the styryl analogue of compound
1
5b was not active towards CYP26A1. While 15b is a potent inhibitor of CYP26A1 it is not an ideal
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Journal of Medicinal Chemistry
2
8
inhibitor to be developed further due to its high RAR affinity. Therefore, 15b was chosen as a lead
compound for the design and synthesis of novel inhibitors that would be free of RAR binding (agonist
or antagonist), and inhibition of other P450s except CYP26A1. We decided to explore the effect of the
replacement of the naphthyl group bearing the carboxylic acid by a phenethyl group on CYP26 and
RAR activities. Phenethyl derivative 24 displayed a lower CYP26B1 activity compared to the naphthyl
analogue 13b while CYP26A1 activity was only reduced by 2 to 3ꢀfold. CYP26A1 inhibitory activity
for the thioketal analogue 30 was in the same range of compound 24 whereas CYP26B1 activity was
increased. We also explored whether it is possible to improve CYP26A1 inhibitory activity and
selectivity by modifying the substituents on the oneꢀcarbon between the two ring systems of compound
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
4. These will result in the modification of the spatial orientation of the two aromatic moieties. Such a
chemical modification did not produce significant improvement in CYP26 inhibition for analogues 20,
6 and 34. In the course of our initial exploratory work on the SARs for this novel series of CYP26
2
inhibitors, we also explored whether it is possible to impact the spatial positioning of the carboxylic
moiety by shortening the phenethyl moiety. The thioethers (38a, 38b) and the sulfones (39a, 39b)
lacked inhibitory activity towards CYP26A1 and CYP26B1.
Table 2. IC values and 95% confidence intervals of the IC for the novel CYP26 inhibitors
50
50
against CYP26A1 and CYP26B1.
1
R
2
R
R1
R2
Activity (µM)
Cpd
CYP26A1
CYP26B1
O
O
para
para
4
.0
0.93
(0.61ꢀ1.4)
5
O
(
(
2.5ꢀ6.3)
OH
OH
OH
O
2.8
1.4
0.77ꢀ2.6)
6
O
1.7ꢀ4.8)
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OH
O
para
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
N
3.5
1.4
9
(
2.2ꢀ5.6)
(0.4ꢀ4.8)
O
OH
O
O
O
para
para
1
1a
>25
>25
OH
OH
b
13a
7.8
12.6
O
O
S
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(2.8ꢀ21.4)
O
para
1.3
1.1
S
1
5a
(0.64ꢀ2.54)
(0.1ꢀ11.7)
OH
O
O
para
para
para
1
8
4.7
>5
OH
OH
OH
O
O
1
.7
20
1.66
0.24
(
0.8ꢀ3.5)
O
O
O
O
2
2
>10
(
0.10ꢀ0.59)
O
para
0.34
14.5
2
2
2
4
6
8
(
0.06ꢀ2.0)
>5
(3.5ꢀ59.7)
OH
O
O
O
para
para
>5
OH
OH
4
.4
S
S
>10
(
1.8ꢀ10.6)
O
O
para
0.27
0.68
S
S
3
0
(
0.15ꢀ0.47)
(0.2ꢀ2.4)
6.2
OH
OH
O
O
para
para
para
para
meta
S
S
34
>5
O
O
O
O
10.6
(5ꢀ23)
3
8a
>5
>5
>5
>5
OH
S
3
8b
>5
>5
>5
OH
OH
OH
O
O
O
O
S
S
3
9a
3
9b
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Journal of Medicinal Chemistry
b
Maximum inhibition 59% at 20ꢀM
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
In summary, in contrast to the relatively robust nanomolar inhibition of CYP26A1 by the
dithiolane and dioxolane compounds in this series, and the micromolar inhibition of CYP26A1 by
majority of the remaining compounds, the inhibitory potency of the compounds (5-39) towards
CYP26B1 was variable with only compounds 11b, 30 and 5 showing submicromolar potency towards
CYP26B1. Interestingly, compounds 5, 6, 9, 11b, 34 were more potent inhibitors of CYP26B1 than
CYP26A1 suggesting that interactions between these inhibitors and the CYP26 enzymes within the
active site are different allowing development of CYP26 isoform selective inhibitors. Compounds 15b
and 13b were approximately 10ꢀ20 fold more potent inhibitors of CYP26A1 than CYP26B1. The
replacement of the naphthyl group in the dioxolane compound 13b with a saturated or an unsaturated
two carbon chain (22 and 24) led to over 10ꢀfold decrease in the CYP26B1 inhibition with a lower
effect on CYP26A1 inhibition. Therefore, these changes increased the CYP26A1 selectivity to >40ꢀfold
between CYP26B1 and CYP26A1. In contrast, with the dithiolane compound 15b, replacement of the
naphthyl with an unsaturated or a saturated two carbon chain led to either considerable decrease in
CYP26A1 inhibition (28) or loss of CYP26A1 selectivity (30).
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
RAR activation
28
The initial lead compound in the series, 15b, is a known RAR antagonist and therefore it is
possible that also the novel compounds identified will possess offꢀtarget effects via RAR binding.
Hence, the two most promising selective inhibitors, compounds 22 and 24, as well as compound 30
which was a potent nonꢀselective CYP26A1/B1 inhibitor, were assessed for RAR activation. We first
used a luciferaseꢀbased reporter assay to screen compounds 22, 24 and 30 for RARα and RARγ
activation at a concentration of 1 µM. Screen was accomplished using human RARα and RARγ reporter
assay kits provided by Indigo Biosciences as reported previously. No measurable RARα and RARγ
activation was detected for the three compounds (Data not shown). We then used the HepG2 cells,
2
9
which have robust atRA metabolism activity and RAR mediated effects on gene transcription to test
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the effects of compounds 22, 24 and 30 on RAR activation in a cell system. The induction of the mRNA
of the classic RAR target genes CYP26A1 and RARβ was evaluated after treatment with each of the
novel compounds in comparison to vehicle and atRA (Figure 2). As shown, none of the three
compounds resulted in activation of the pool of RARs in the HepG2 cells at a test concentration of 1
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
ꢁ
M, a concentration at least 5 times greater than the IC value of these compounds towards CYP26A1.
50
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Inhibition of RA metabolism and Enhancement of RA potency
To further determine the biological activity of the lead compounds, their efficacy to increase the potency
of atRA was tested in the HepG2 cells. It has been previously shown that atRA is efficiently
metabolized in HepG2 cells and that the metabolism of atRA in HepG2 cells is predominantly mediated
29
by CYP26A1. Therefore, RA metabolism in HepG2 cells and the activation of RARs (CYP26A1 and
RARβ induction) by atRA in these cells provide a reliable test system for cellular activity of the
compounds of interest. When HepG2 cells were treated with 100 nM atRA in combination with 1 ꢁM of
compounds 22, 24 and 30, the magnitude of induction of RARβ and CYP26A1 was significantly
increased when compared to atRA treatment alone (Figure 2), demonstrating that all three compounds
potentiate the effect of atRA in cells presumably via inhibiting atRA metabolism and increasing the
cellular concentrations of atRA. Importantly the induction of RARβ and CYP26A1 following treatment
with 100 nM atRA together with the inhibitors was significantly greater than the induction observed
following treatment with 1 ꢁM atRA alone demonstrating a considerable increase in the potency of
atRA by the inhibitors. Furthermore these data demonstrate that these compounds do not exhibit an
RAR antagonistic activity.
To test whether the increased induction of RAR target genes in the presence of the inhibitors and
atRA was due to inhibition of atRA depletion in the cells, the concentrations of atRA in the media at the
end of the 24ꢀhour treatment were measured. Table 3 summarizes the effects of the lead compounds on
atRA depletion in HepG2 cells. All three CYP26 inhibitors significantly increased atRA concentrations
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Journal of Medicinal Chemistry
in the cell media with compound 30 having the greatest effect, approximately 9ꢀfold increase in atRA
concentrations in the media. Compound 22 increased atRA concentration 3.5ꢀfold and compound 24
increased atRA concentration in the media by 2ꢀfold. This increase in atRA concentrations correlated
well with the observed magnitude of effect on potentiating atRA mediated RAR activation in cells with
compound 30 also having the greatest effect on increasing atRA mediated induction of RARβ and
CYP26A1.
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 2: Induction of RARβ and CYP26A1 mRNA in HepG2 cells as measured by rtꢀPCR. HepG2
cells were treated with 100 nM or 1 ꢁM atRA or with 1 ꢁM compound 22, 24 or 30 alone or in
combination with 100 nM atRA.
Table 3. atRA concentration in cell media after 24 hour treatment
Treatments
Vehicle
atRA in cell media (nM)
0.49 ± 0.33
1
1
1
00nM atRA
ꢁM atRA
ꢁM 22
13.2 ± 0.14
214 ± 13.6
1.09 ± 0.52
1ꢁM 24
0.27 ± 0.09
100nM atRA + 1ꢁM 22
45.5 ± 2.8*
1
1
00nM atRA + 1ꢁM 24
00nM atRA + 1ꢁM 30
25.6 ± 3.4*
113 ± 9*
*
significantly different from the 100 nM RA treatment
Selectivity for CYP26A1
To determine the selectivity of the lead compounds towards other P450 enzymes the inhibition of
CYP2D6, CYP3A4, CYP2C19 and CYP2B6 by compounds 22, 24 and 30 was evaluated. The
inhibition data is summarized in Table 4. When screened at 10 ꢁM inhibitor concentration, compounds
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2
2 and 24 were free of any CYP3A4 inhibition while compound 30 inhibited CYP3A4 by 25%. The
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5
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5
5
6
IC values for CYP3A4 inhibition for compounds 22 and 30 were 30 ꢁM and 7.5 ꢁM respectively. For
5
0
compound 24 no concentration dependent inhibition of CYP3A4 was observed up to 100 ꢁM with
maximum 40% inhibition observed, demonstrating that compound 24 is unlikely to result in CYP3A4
inhibition at concentrations required to inhibit CYP26A1. Similarly, compound 24 was free of any
significant inhibition of CYP2D6, CYP2C19 and CYP2B6 suggesting that this compound may become
a novel CYP26A1 selective inhibitor. In contrast compound 22 inhibited CYP2D6 by 50% at 10 ꢁM
concentration and compound 30 inhibited CYP2B6 by 70% at 10 ꢁM.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
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7
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9
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7
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0
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0
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4
5
6
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8
9
0
Table 4. Percent inhibition for each of the tested CYP enzymes when screened with 10 ꢁM of CYP26
inhibitors. NI indicates no inhibition observed. * shows that activity in the presence of the inhibitor was
significantly different (p<0.01) from that in control.
Compound
CYP3A4
NI
NI
75 ± 8*
CYP2D6
49 ± 7*
NI
CYP2C19
CYP2B6
NI
NI
30 ± 1*
2
2
3
2
4
0
NI
NI
NI
NI
* activity in the presence of the test inhibitor (22 or 30) was significantly different from control
DISCUSSION
Inhibition of atRA metabolism has been a potential drug target for some decades now and several
promising inhibitors of overall atRA metabolism have emerged from variety of drug development
7
programs in industry and academia. Yet, in the development of the inhibitors of atRA metabolism or
CYP26, the role of inhibition of the different CYP26 isoforms has never been addressed. In fact, the
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Journal of Medicinal Chemistry
screening data in this study shows that talarozole is a potent inhibitor of both CYP26A1 and CYP26B1
while liarozole is a preferential inhibitor of CYP26B1. This difference in their inhibition profile likely
contributes to some differences in their in vivo pharmacology and side effect profile. As CYP26A1 and
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11
CYP26B1 are expressed in a tissue specific manner, selective inhibition of either CYP26 isoform
holds great promise in organ specific targeting of the retinoid system. This study is the first to test and
identify CYP26 isoform specific inhibitors with the goal of identifying a CYP26A1 selective inhibitor
that can be used to inhibit atRA metabolism in the liver. Compound 24 has 43ꢀfold specificity towards
CYP26A1 compared to CYP26B1 and it emerged as the lead inhibitor in this novel structural series.
The selectivity of compound 24 together with compounds 15b, 13b and 22 is striking as up to now only
minor differences in the biochemical structure function between CYP26A1 and CYP26B1 have been
observed. Both enzymes use atRA as a substrate, bind atRA with high affinity and form very similar
0
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11
metabolites from atRA although at slightly different product ratios. This is despite the fact that the two
CYP26 enzymes only share about 40% sequence similarity and hence would be expected to be
structurally and functionally different. As the CYP26 enzymes have proven exceedingly difficult to
express and purify in recombinant systems, no Xꢀray crystal structure or lowꢀresolution structural
information is available for these enzymes at present. Therefore the discovery of these novel selective
inhibitors of CYP26A1 provide critical insight to the differences in the active site features and structureꢀ
activity relationships of these two enzymes and provide a lead for future development of selective
inhibitors of the two enzymes.
The novel compounds in this series were developed using known RAR ligands as lead
compounds and by exploring synthetically feasible modifications that could reduce RAR binding
activity without compromising CYP26A1 inhibition. Replacement of the naphthyl group of the
dioxolane derivative 13b by a phenethyl moiety in compound 24 appears to have a minor impact on
CYP26A1 inhibitory activity while reducing CYP26B1 activity. The results in HepG2 cells show that
indeed the lead compounds 22, 24 and 30 were devoid of RAR modulation while maintaining the
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Page 20 of 51
CYP26 inhibition potency. In addition, the studies in HepG2 cells showed that compound 24 and the
two other potent inhibitors in this series, compounds 22 and 30, were effective at inhibiting atRA
metabolism in cell culture systems and they potentiated the activity of atRA in the cell system. To
1
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mimic the in vivo endogenous atRA concentrations in cells, the HepG2 cells were treated with 100 nM
atRA and under these conditions, the lead inhibitors increased the activity of atRA by 5ꢀ100 fold and
decreased atRA metabolism significantly. The HepG2 cells have been previously shown to be devoid of
0
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30
CYP26B1 expression although CYP26B1 expression is somewhat inducible in these cells. Similarly
HepG2 cells do not express significant levels of the common drug metabolizing enzymes such as
CYP3A4 and CYP2C enzymes. Therefore it is likely that the effect of the inhibitors on atRA activity in
these cells is solely due to CYP26A1 inhibition.
In this investigation, we present a broad range of experimental data on a novel series of retinoidꢀ
based CYP26A1 inhibitors. Within this series, a SAR could be established that showed that lipophilic
bulk induced by a dioxolane or a dithiolane on the spacer appears to be required for enhanced CYP26A1
inhibitory activity. Dioxolane analogues 22 and 24 were identified as selective CYP26A1 inhibitors.
Classic CYP26 inhibitors have all included a triazole or imidazole functional group to increase their
31,32
affinity to CYP26s via coordination to the heme iron.
However, atRA itself binds to CYP26A1 with
1
0 nM affinity suggesting that high affinity inhibitors devoid of the imidazole or triazole group prone to
broad spectrum CYP inhibition can be developed. Indeed, this study shows that nM inhibitors of both
CYP26A1 and CYP26B1 free of imidazole or triazole functionality can be synthesized. These inhibitors
were also free of inhibition of offꢀtarget CYPs such as CYP3A4, CYP2C19 and CYP2D6 at
concentrations relevant for CYP26A1 inhibition. However, these compounds do include a ketal that
may be metabolically labile and compound 22 is an unsaturated acid. These functional groups are
potential in vivo liabilities of these compounds and require further study. Nevertheless, these
compounds provide the first structural leads for selective CYP26A1 inhibition, and have the potential to
become the first selective CYP26A1 inhibitors for testing in preclinical models. Eventually these
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Journal of Medicinal Chemistry
compounds may become useful for treatment of diseases that would require increasing systemic or liver
atRA concentrations or in inhibiting atRA metabolism when atRA is administered clinically to combat
therapy resistance that develops due to CYP26A1 induction by atRA.
1
2
3
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9
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1
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0
EXPERIMENTAL SECTION
General Experimental methods. Moisture sensitive reactions were performed in an inert, dry
atmosphere of argon in flame dried glassware. Air sensitive liquids were transferred via syringe or
cannula through rubber septa. Reagent grade solvents were used for extraction and flash
chromatography. THF was distilled from Na/benzophenone under argon; dichloromethane (CH Cl ) and
2
2
chloroform (CHCl ) were distilled from CaH under argon. All other reagents including 2ꢀbromoꢀ1ꢀ
3
2
(5,5,8,8ꢀtetramethylꢀ5,6,7,8ꢀtetrahydronaphthalenꢀ2ꢀyl)ethanꢀ1ꢀone and 1,1,4,4ꢀtetramethylꢀ1,2,3,4ꢀ
tetrahydronaphthalene and solvents which were purchased from commercial sources, were used directly
without further purification. The progress of reactions was checked by analytical thinꢀlayer
chromatography (Sorbent Technologies, Silica G TLC plates w/UV 254). Flash column chromatography
was performed using prepacked Biotage SNAP/ZIP cartridges on a Biotage Isolera One instrument.
Microwave reactions were performed using a Biotage Initiator instrument. The solvent compositions
1
reported for all chromatographic separations are on a volume/volume (v/v) basis. HNMR spectra were
recorded at 400 or 500 MHz and are reported in parts per million (ppm) on the δ scale relative to
13
tetramethylsilane as an internal standard. CNMR spectra were recorded at 100 or 125 MHz and are
reported in parts per million (ppm) on the δ scale relative to CDCl . Melting points were determined on
3
a Stuart melting point apparatus from Bibby Scientific Limited and are uncorrected. LCꢀUV and high
resolution mass spectrometry (HRMS) analyses were conducted on a Waters ACQUITY UPLCꢀseries
TM
liquid chromatography system equipped with a diode array detector and LCT PREMIER XE time of
flight (TOF) mass spectrometer operated on the electrospray ionization mode. Compounds were
separated using a Phenomenex (Torrance, CA) NXꢀC18 column (50 x 4.60 mm) with 3 ꢁm particle size
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Page 22 of 51
and gradient elution at mobile phase flow rate of 0.4 mL/min with solvents A: water with 0.1% formic
acid and B: acetonitrile. The gradient was from 90% A 10% B to 2% A 98% B over 6.6 min then held
isocratic till 13 min followed by return to initial conditions by 15 min. Compound purity was assigned
on the basis of UV detection data at 254ꢀnM detection by comparing relative peak areas of the signals.
All final compounds were more than 95% pure.
1
2
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9
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0
General procedure for ester hydrolysis using K CO (method A)
2
3
To a solution of ester (0.25 mmol) in MeOH (4 mL) was added aqueous K CO (0.4 mL, 2.0 M)
2
3
dropwise. The reaction mixture was stirred at 80°C for 1 hour. Completion of reaction was assessed by
TLC. The reaction was then cooled (0 °C), acidified to pH 2 with 1.0 N HCl, and extracted with EtOAc
(
3 x 10 mL). The combined organic phases were dried (MgSO ) and concentrated under reduced
4
pressure.
General procedure for ester hydrolysis using LiOH (method B)
To a solution of the ester (0.11 mmol) in anhydrous THF (0.5 mL) was added aqueous LiOH (0.25 mL,
1
.0 N) dropwise. The reaction mixture was refluxed for 2 hours or stirred overnight at room
temperature. The reaction was then cooled (0 °C), acidified to pH 2 with 1.0 N HCl, and extracted with
EtOAc (3 x 10 mL). The combined organic phases were dried (MgSO ) and concentrated.
4
General procedure for Friedel-Crafts acylation (method C)
Acyl chloride (11.7 mmol) and 1,1,4,4ꢀtetramethylꢀ1,2,3,4ꢀtetrahydronaphthalene material (2.0 g, 10.6
mmol) were weighed into a roundꢀbottomed flask equipped with a stir bar and 24 mL dichloromethane
added. Stirring commenced and AlCl (3.10 g, 23.3 mmol) was added slowly. After completion of the
3
addition, stirring was continued at room temperature overnight. The reaction was then poured into an ice
solution (40 mL), acidified with a 20% HCl solution (10 mL) and the organic layer removed. Then
extracted further with 2x40 mL EtOAc. The organic layers were combined and washed with water (40
mL) and brine respectively, dried over MgSO , filtered and concentrated under reduced pressure.
4
General procedure for formation of dioxolane (method D)
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Journal of Medicinal Chemistry
Diarylketone (2.0 mmol) was treated with ethylene glycol (2 mL) and a catalytic amount of pTsOH (20
mg) in toluene (10 mL) at 145°C overnight using a Dean Stark trap. After cooling to room temperature,
the mixture was washed with saturated NaHCO (aq.), brine, dried over MgSO and concentrated under
1
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9
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1
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6
3
4
reduced pressure.
General procedure for formation of dithiolane (method E)
To a solution of diarylketone (1.14 mmol) in CH Cl (2 mL) at 0°C under argon, was added a solution
0
1
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0
2
2
of (CH SH) (144 ꢀL, 1.71 mmol) in CH Cl (0.5 mL) followed by BF •Et 0 (217 ꢀL, 1.71 mmol). The
2
2
2
2
3
2
resulting mixture was stirred at 0°C for 1 h and then warmed to room temperature overnight. The
reaction was quenched by pouring the mixture into saturated NaHCO , and the mixture was extracted
3
with 20 mL CH C1 . The combined organic layers were dried (MgSO ) and concentrated.
2
2
4
General procedure for hydrogenation or hydrogenolysis (method F)
Alkene (515 mg, 1.36 mmol) was placed in a hydrogenation apparatus equipped with a magnetic stir bar
and EtOH/EtOAc (30 mL) added followed by Pd/C (240 mg). H gas was introduced at 20 psi pressure
2
and reacted at room temperature for 7 hrs. The black solution was filtered using a celite pad and
concentrated under reduced pressure.
General procedure for coupling thioaryl to aryl halides (method G)
To a mixture of thioaryl (0.71 mmol,1 eq.) and aryl halides (0.71 mmol, 1 eq.) in dioxane (5 mL) and
butanol (5 mL), were added the borohydride, polymer supported (650 mg, 2.5 – 5 mmol/g, 3 eq.) and
(
bpy) NiBr (70 mg, 0.2 eq.). The mixture was heated to 145°C under an atmosphere of nitrogen. The
2 2
polymer beads were removed by filtration, and the mixture was concentrated to dryness before
purification by flash chromatography using the following gradient system,
cyclohexane/dichloromethane): (20/80) to (0/100).
(
General procedure for oxidation of sulfide to sulfone (method H)
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Page 24 of 51
To a solution sulfide (0.26 mmol) in THF (7 mL), was added a solution of oxone (475 mg, 0.775 mmol,
eq.) in water (4 mL). The reaction was stirred overnight at room temperature. Water (10 mL) was
1
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9
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6
3
added and the mixture was extracted with dichloromethane (3×50 mL).
Methyl-4-(2-oxo-2-(5,5,8,8-tetramethyl-5,6,7,8-tetrahydronaphthalen-2-yl)ethoxy)benzoate (4). A
mixture 500 mg (1.62 mmol) of the commercially available 2ꢀbromoꢀ1ꢀ(5,5,8,8ꢀtetramethylꢀ5,6,7,8ꢀ
tetrahydronaphthalenꢀ2ꢀyl)ethanꢀ1ꢀone, 271 mg of the commercially available methyl 4ꢀ
0
1
2
3
4
5
6
7
8
9
0
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2
3
4
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7
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9
0
hydroxybenzoate (1.78 mmol) and K CO (246 mg, 1.78 mmol) in 10 ml of methyl ethyl ketone was
2
3
heated 2 times at 100°C for 20 minutes in the microwave and then filtered and concentrated on a rotary
evaporator under vacuum. The product was purified by crystallization with a (heptane/ethyl acetate:
1
70/30) solution to give the expected compound (450 mg; 73%) as a white solid. mp = 112ꢀ114°C. H
NMR (500 MHz, CDCl ) δ 7.98 (d, J = 8.9 Hz, 2H), 7.97 (d, J = 1.9 Hz, 1H), 7.72 (dd, J = 8.3, 1.9 Hz,
3
1
H), 7.43 (d, J = 8.3 Hz, 1H), 6.95 (d, J = 8.9 Hz, 2H), 5.32 (s, 2H), 3.88 (s, 3H), 1.71 (s, 4H), 1.32 (s,
1
3
6H), 1.31 (s, 6H). C NMR (126 MHz, CDCl ) δ 193.34, 166.63, 161.72, 152.00, 145.85, 131.74,
3
1
31.58, 127.14, 126.68, 125.02, 123.32, 114.32, 70.48, 51.87, 34.80, 34.70, 34.58, 34.42, 31.73, 31.53.
+
4
+
MS (TOF ESI+): m/z calcd for C H O (M+H) calcd. 381.21, found 381.20.
24 29
4
-(2-Oxo-2-(5,5,8,8-tetramethyl-5,6,7,8-tetrahydronaphthalen-2-yl)ethoxy)benzoic acid (5).
Compound 4 (200 mg, 0.53 mmol) was stirred at 80°C with sodium hydroxide (200 mg) in a mixture of
EtOH, THF and water (10 mL, 10 mL and 1.5 mL) for 12 hours. The reaction was then cooled at r.t.,
acidified to pH 2 with 1.0 N HCl, and extracted with EtOAc (3 x 20 mL). The combined organic phases
were dried (MgSO ) and concentrated under reduced pressure. The residue was purified by
4
crystallization in a mixture of heptane and EtOAc (70/30) to provide 149 mg (77%) of a white solid.
1
mp = 162ꢀ163°C. H NMR (400 MHz, CDCl ) δ 8.06 (d, J = 8.9 Hz, 2H), 7.98 (d, J = 1.7 Hz, 1H), 7.72
3
(dd, J = 8.3, 1.8 Hz, 1H), 7.43 (d, J = 8.3 Hz, 1H), 6.97 (d, J = 8.9 Hz, 2H), 5.34 (s, 2H), 1.72 (s, 4H),
13
1
.32 (s, 6H), 1.31 (s, 6H). C NMR (101 MHz, CDCl ) δ 193.30, 170.88, 162.50, 152.11, 145.95,
3
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Journal of Medicinal Chemistry
1
32.40, 131.80, 127.21, 126.77, 125.07, 122.38, 114.51, 70.56, 34.86, 34.77, 34.66, 34.47, 31.77, 31.57.
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
+
4
+
HPLC (t= 8.12 min, 96%). MS (TOF ESI+) for C H O (M+H) calcd. 367.20, found 367.19.
2
3
27
4
-(2-Hydroxy-2-(5,5,8,8-tetramethyl-5,6,7,8-tetrahydronaphthalen-2-yl)ethoxy)benzoic acid (6). To
a solution of compound 4 95 mg (0,25 mmole) dissolved in 3 mL of THF under nitrogen, was carefully
added 10 mg of sodium borohydride(0,25 mmole).The mixture was stirred 3 h at room temperature. The
residue was taken up in 10 mL water, and the aqueous layer was extracted with 3x10 mL EtOAc. The
combined organic extract was washed with 30 mL water and 30 mL brine respectively. The organic
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
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6
7
8
9
0
1
2
3
4
5
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8
9
0
solution was dried (MgSO ), filtered, and concentrated to give 75 mg (79%) of a colorless oil. The
4
resulting ester (70 mg, 0.18 mmol) was stirred at 80°C with sodium hydroxide (70 mg) in a mixture of
EtOH, THF and water (3mL, 3mL and 0.5 mL) for 12 hours. The reaction was then cooled at r.t.,
acidified to pH 2 with 1.0 N HCl, and extracted with EtOAc (3 x 10 mL). The combined organic phases
were dried (MgSO ) and concentrated under reduced pressure. The residue was purified by
4
crystallization in a mixture of heptane and EtOAc (70/30) to provide 40 mg (60%) of a white solid. mp
1
=
156ꢀ158°C. H NMR (400 MHz, CDCl ) δ 8.06 (d, J = 8.8 Hz, 2H), 7.38 (d, J = 1.5 Hz, 1H), 7.34 (d,
3
J = 8.1 Hz, 1H), 7.21 (dd, J = 8.1, 1.6 Hz, 1H), 6.97 (d, J = 8.9 Hz, 2H), 5.11 (dd, J = 8.3, 3.4 Hz, 1H),
13
4
1
.23 – 4.04 (m, 2H), 1.69 (s, 4H), 1.35 – 1.22 (m, 12H). C NMR (101 MHz, CDCl ) δ 171.39, 162.97,
3
45.28, 145.17, 136.35, 132.39, 126.92, 124.46, 123.41, 122.09, 114.37, 73.45, 72.63, 35.07, 34.99,
+
+
34.35, 34.19, 31.87, 31.85, 31.83. HPLC (t= 8.34 min, 97%). MS (TOF ESI+) for C H O (M+H)
2
3
29
4
calcd. 369.21, found 369.21.
(E)-Methyl-4-(2-(hydroxyimino)-2-(5,5,8,8-tetramethyl-5,6,7,8-tetrahydronaphthalen-2-yl)ethoxy-
)benzoate (7). The Eꢀisomer was recrystallized from EtOAc/heptane (3/7) to give 69 mg (24%) of a
1
white solid. mp = 146ꢀ149°C. H NMR (500 MHz, CDCl ) δ 8.21 (s, 1H), 7.98 (d, J = 9.0 Hz, 2H),
3
7.59 (d, J = 1.8 Hz, 1H), 7.40 (dd, J = 8.3, 1.9 Hz, 1H), 7.35 (d, J = 8.3 Hz, 1H), 6.98 (d, J = 9.0 Hz,
1
3
2
1
H), 4.95 (s, 2H), 3.88 (s, 3H), 1.69 (s, 4H), 1.28 (s, 6H), 1.25 (s, 6H). C NMR (126 MHz, CDCl ) δ
3
66.79, 161.93, 153.08, 146.73, 144.85, 131.56, 127.49, 126.93, 126.48, 125.51, 123.11, 114.55, 69.32,
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Journal of Medicinal Chemistry
Page 26 of 51
+
+
5
1.91, 34.88, 34.83, 34.34, 34.29, 31.78, 31.65. MS (TOF ESI+) for C H NO (M+H) calcd. 396.22,
24 30 4
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
found 396.21.
(Z)-Methyl-4-(2-(hydroxyimino)-2-(5,5,8,8-tetramethyl-5,6,7,8-tetrahydronaphthalen-2-
yl)ethoxy-)benzoate (8). A solution of compound 5 (275 mg, 0.72 mmol) in MeOH (7 mL) was treated
with hydroxylamine hydrochloride (100 mg, 1.45 mmol) and pyridine (235 ꢀL, 2.9 mmol), and the
mixture was heated at reflux for 6 h. The mixture was cooled to room temperature, and the MeOH was
removed in vacuo. The residue was taken up in 20 mL water, and the aqueous layer was extracted with
3x30 mL EtOAc. The combined organic extract was washed with 20 mL water and 20 mL brine
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
respectively. The organic solution was dried (MgSO ), filtered, and concentrated. The residue was
4
purified by flash column chromatography (heptaneꢀethyl acetate 100:0 v/v increasing to 70:30 v/v)to
yield the 2 isomers E and Z. The Zꢀisomer was recrystallized from EtOAc/heptane to give 165 mg
1
(71%) of a white solid. mp = 123ꢀ125°C. H NMR (500 MHz, CDCl ) δ 8.79 (s, 1H), 7.97 (d, J = 8.8
3
Hz, 2H), 7.60 (d, J = 1.9 Hz, 1H), 7.39 (dd, J = 8.3, 1.9 Hz, 1H), 7.29 (d, J = 8.3 Hz, 1H), 6.98 (d, J =
13
8
.8 Hz, 2H), 5.32 (s, 2H), 3.88 (s, 3H), 1.67 (s, 4H), 1.27 (s, 6H), 1.26 (s, 6H). C NMR (126 MHz,
CDCl ) δ 166.81, 161.81, 155.20, 146.73, 144.91, 131.59, 130.12, 126.69, 125.35, 124.01, 123.09,
3
+
+
1
14.25, 59.86, 51.91, 34.93, 34.85, 34.30, 34.29, 31.78, 31.65. MS (TOF ESI+) for C H NO (M+H)
24 30 4
calcd. 396.22, found 396.21.
(Z)-4-(2-(Hydroxyimino)-2-(5,5,8,8-tetramethyl-5,6,7,8-tetrahydronaphthalen-2-
yl)ethoxy)benzoic acid (9). Compound 8 (100 mg, 0.25 mmol) was hydrolysed according to method A.
The residue was purified by flash column chromatography (DCMꢀMeOH 100:0 v/v increasing to 95:5
1
v/v) to provide a white solid (25 mg, 27%). mp = 208ꢀ211°C. H NMR (500 MHz, CDCl ) δ 7.96 (d, J
3
=
8.9 Hz, 2H), 7.58 (d, J = 1.9 Hz, 1H), 7.48 (s, 1H), 7.39 (dd, J = 8.3, 1.9 Hz, 1H), 7.28 (d, J = 8.3 Hz,
1H), 6.99 (d, J = 8.9 Hz, 2H), 5.33 (s, 2H), 1.68 (s, 4H), 1.26 (s, 6H), 1.25 (s, 6H). HPLC (t= 8.14 min,
+
+
9
5%). HRMS (TOF ESI+) for C H NO (M+H) calcd. 382.2018, found 382.2012.
23 28 4
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Journal of Medicinal Chemistry
Methyl-4-(5,5,8,8-tetramethyl-5,6,7,8-tetrahydronaphthalene-2-carbonyl)benzoate
(10a).
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Compound 10a was prepared according to method C starting from 2.0 g of the commercially available
1,1,4,4ꢀtetramethylꢀ1,2,3,4ꢀtetrahydronaphthalene (10.6 mmol). The crude was crystallized from hot
EtOAc by addition of 20 mL MeOH, resulting in white crystals after cooling. Yield 2.15 g (58%). mp =
1
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
140ꢀ142°C. H NMR (400 MHz, CDCl ) δ 8.15 (d, J = 8.4 Hz, 2H), 7.83 (d, J = 8.4 Hz, 2H), 7.79 (d, J
3
=
1.8 Hz, 1H), 7.54 (dd, J = 8.2, 1.9 Hz, 1H), 7.41 (d, J = 8.2 Hz, 1H), 3.97 (s, 3H), 1.72 (s, 4H), 1.32
1
3
(
s, 6H), 1.29 (s, 6H). C NMR (101 MHz, CDCl ) δ 195.88, 166.40, 150.78, 145.30, 141.88, 134.15,
3
132.91, 129.67, 129.39, 128.89, 127.35, 126.70, 52.40, 34.81, 34.74, 34.41, 31.74, 31.60. MS (TOF
+
+
ESI+) for C H O (M+H) calcd. 351.20, found 351.20.
2
3
27
3
Methyl-6-(5,5,8,8-tetramethyl-5,6,7,8-tetrahydronaphthalene-2-carbonyl)-2-naphthoate
(10b).
Compound 10b was synthesized according to method C starting from 875 mg of 1,1,4,4ꢀtetramethylꢀ
1
,2,3,4ꢀtetrahydronaphthalene (4.65 mmol). The compound 10b crystallized from hot EtOAc by
1
addition of 20 mL MeOH, resulting in white crystals after cooling (1.94 g, 69%). mp = 134ꢀ136°C. H
NMR (400 MHz, CDCl ) δ 8.67 (s, 1H), 8.30 (s, 1H), 8.13 (dd, J = 8.6, 1.6 Hz, 1H), 8.06 (d, J = 8.5 Hz,
3
1
H), 7.98 (dd, J = 8.5, 1.6 Hz, 2H), 7.97 (d, J = 8.6 Hz, 1H), 7.87 (d, J = 1.8 Hz, 1H), 7.60 (dd, J = 8.2,
1
3
1
.8 Hz, 1H), 7.44 (d, J = 8.2 Hz, 1H), 4.01 (s, 3H), 1.74 (s, 4H), 1.34 (s, 6H), 1.31 (s, 6H). C NMR
(
101 MHz, CDCl ) δ 196.21, 166.86, 150.47, 145.26, 137.33, 134.59, 134.45, 134.13, 130.91, 130.68,
3
129.49, 129.33, 128.94, 127.44, 126.69, 126.66, 126.08, 52.40, 34.85, 34.78, 34.73, 34.43, 31.76, 31.63.
+
+
MS (TOF ESI+) for C H O (M+H) calcd. 401.21, found 401.21.
2
7
29
3
4
-(5,5,8,8-Tetramethyl-5,6,7,8-tetrahydronaphthalene-2-carbonyl)benzoic acid (11a). Compound
10a (60 mg, 0.17 mmol) was hydrolyzed according to method A. The resultant oil was triturated with
1
heptanes to yield compound 11 as a white solid (52 mg, 91%). mp = 194ꢀ195°C. H NMR (400 MHz,
CDCl ) δ 8.24 (d, J = 8.3 Hz, 2H), 7.87 (d, J = 8.3 Hz, 2H), 7.81 (d, J = 1.8 Hz, 1H), 7.55 (dd, J = 8.2,
3
1
3
1
.8 Hz, 1H), 7.42 (d, J = 8.2 Hz, 1H), 1.73 (s, 4H), 1.33 (s, 6H), 1.30 (s, 6H). C NMR (101 MHz,
CDCl ) δ 195.84, 170.91, 150.97, 145.41, 142.74, 134.03, 131.89, 130.05, 129.75, 128.91, 127.42,
3
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1
26.76, 34.81, 34.77, 34.74, 34.43, 31.76, 31.61, 26.91. HPLC (t= 8.82 min, 100%). HRMS (TOF
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
+
+
ESI+) for C H O (M+H) calcd. 337.1804, found 337.1811.
2
2
25
3
6-(5,5,8,8-Tetramethyl-5,6,7,8-tetrahydronaphthalene-2-carbonyl)-2-naphthoic
acid
(11b).
Compound 10b (220 mg, 0.55 mmol) was hydrolyzed according to method A. The crude was
crystalized from cyclohexane to provide compound 11b as a white solid (90 mg, 95%). mp = 224ꢀ
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
25°C. H NMR (400 MHz, MeOD) δ 8.70 (s, 1H), 8.30 (s, 1H), 8.15 (d, J = 8.5 Hz, 1H), 8.08 (d, J =
8
.5 Hz, 1H), 7.98 (t, J = 8.0 Hz, 2H), 7.86 (s, 1H), 7.61 (d, J = 8.2 Hz, 1H), 7.46 (d, J = 8.2 Hz, 1H),
1
3
1.75 (s, 4H), 1.35 (s, 6H), 1.32 (s, 6H). C NMR (101 MHz, MeOD) δ 196.84, 168.40, 150.59, 145.17,
1
1
36.98, 134.34, 134.32, 134.12, 130.94, 130.80, 129.70, 129.45, 129.30, 128.88, 127.30, 126.59,
26.33, 126.27, 34.65, 34.58, 34.26, 31.53, 31.40. HPLC (t= 9.40 min, 95%). HRMS (TOF ESI+) for
+
3
+
C H O (M+H) calcd. 387.1960, found 387.1998.
26
27
Methyl-4-(2-(5,5,8,8-tetramethyl-5,6,7,8-tetrahydronaphthalen-2-yl)-1,3-dioxolan-2-yl)benzoate
(12a). Compound 10a (705 mg, 2.01 mmol) was treated treated according to method D. After workꢀ
up,the residue was purified by flash column chromatography (cyclohexaneꢀEtOAc 100:0 v/v increasing
1
to 60:40 v/v) to yield a white solid, 641 mg (81%). mp= 148ꢀ148°C. H NMR (400 MHz, CDCl ) δ 8.00
3
(d, J = 8.3 Hz, 2H), 7.61 (d, J = 8.3 Hz, 2H), 7.42 (d, J = 1.8 Hz, 1H), 7.23 (d, J = 8.3 Hz, 1H), 7.17 (dd,
J = 8.2, 1.8 Hz, 1H), 4.14 – 4.07 (m, 2H), 4.07 – 3.97 (m, 2H), 3.90 (s, 3H), 1.65 (s, 4H), 1.23 (s, 12H).
1
3
C NMR (101 MHz, CDCl ) δ 166.88, 147.43, 144.86, 144.65, 138.21, 129.61, 129.47, 126.45, 126.17,
3
1
23.82, 123.25, 109.22, 64.90, 52.07, 35.04, 34.99, 34.31, 34.09, 31.83, 31.76. MS (TOF ESI+) for
+
4
+
C H O (M+H) calcd. 395.22, found 395.23.
25
31
Methyl-6-(2-(5,5,8,8-tetramethyl-5,6,7,8-tetrahydronaphthalen-2-yl)-1,3-dioxolan-2-yl)-2-
naphthoate (12b). Compound 11b (570 mg, 1.42 mmol) was treated according to method D. The
residue was purified by flash column chromatography (cyclohexaneꢀEtOAc 100:0 v/v increasing to
1
6
0:40 v/v) to yield a white solid (557 mg, 88%). mp= 182ꢀ184°C. H NMR (400 MHz, CDCl ) δ 8.57
3
(s, 1H), 8.07 (s, 1H), 8.05 (dd, J = 8.6, 1.5 Hz, 1H), 7.89 (dd, J = 8.5, 5.1 Hz, 2H), 7.67 (dd, J = 8.6, 1.6
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Journal of Medicinal Chemistry
Hz, 1H), 7.52 (s, 1H), 7.24 (d, J = 9.1 Hz, 2H), 4.19 – 4.11 (m, 2H), 4.11 – 4.02 (m, 2H), 3.97 (s, 3H),
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
1
3
1
.65 (s, 4H), 1.25 (s, 6H), 1.23 (s, 6H). C NMR (101 MHz, CDCl ) δ 167.22, 144.81, 144.63, 142.46,
3
138.37, 135.12, 132.07, 130.67, 129.38, 128.63, 127.59, 126.45, 125.43, 125.35, 124.55, 123.85,
+
1
23.47, 109.51, 64.98, 52.20, 35.06, 35.00, 34.32, 34.10, 31.85, 31.77. MS (TOF ESI+) for C H O
29 33 4
(MH+) calcd. 445.24, found 445.23.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
4
-(2-(5,5,8,8-Tetramethyl-5,6,7,8-tetrahydronaphthalen-2-yl)-1,3-dioxolan-2-yl)benzoic acid (13a).
Compound 12a (236 mg, 0.59 mmol) was hydrolyzed according to method A. The crude was crystalized
from cyclohexane and washed to provide compound 13a as a white solid (161 mg, 72%). mp = 242ꢀ
1
2
7
4
1
43°C. H NMR (400 MHz, CDCl ) δ 12.05 (bs, 1H), 8.07 (d, J = 8.5 Hz, 2H), 7.65 (d, J = 8.5 Hz, 2H),
3
.44 (d, J = 1.8 Hz, 1H), 7.24 (d, J = 8.3 Hz, 1H), 7.18 (dd, J = 8.3, 1.9 Hz, 1H), 4.15 – 4.08 (m, 2H),
13
.08 – 3.99 (m, 2H), 1.65 (s, 4H), 1.24 (s, 6H), 1.23 (s, 6H). C NMR (101 MHz, CDCl ) δ 171.55,
3
48.46, 144.93, 144.72, 138.13, 130.14, 128.67, 126.50, 126.30, 123.79, 123.24, 109.19, 64.94, 35.05,
+
35.00, 34.33, 34.11, 31.84, 31.77. HPLC (t= 8.85 min, 98%). HRMS (TOF MS ES+) for C H O
4
2
4
29
(MH+) calcd. 381.2066, found 381.2038.
6
-(2-(5,5,8,8-Tetramethyl-5,6,7,8-tetrahydronaphthalen-2-yl)-1,3-dioxolan-2-yl)-2-naphthoic acid
(13b). Compound 12b was hydrolyzed according to method B. The crude oil was purified by flash
column chromatography (cyclohexaneꢀEtOAcꢀHOAc 100:0:0.1 v/v increasing to 80:20:0.1 v/v) to
1
provide a white solid (30 mg, 63%). mp = 222ꢀ223°C. H NMR (400 MHz, CDCl ) δ 8.68 (s, 1H), 8.12
3
(d, J = 8.6 Hz, 2H), 7.94 (dd, J = 8.5, 4.6 Hz, 2H), 7.70 (dd, J = 8.6, 1.3 Hz, 1H), 7.52 (s, 1H), 7.24 (d, J
1
3
=
6.1 Hz, 2H), 4.17 (m,, 2H), 4.13 – 4.03 (m, 2H), 1.65 (s, 4H), 1.25 (s, 6H), 1.23 (s, 6H). C NMR
(101 MHz, CDCl ) δ 172.09, 144.86, 144.67, 142.95, 138.32, 135.60, 132.02, 131.77, 129.58, 128.82,
3
1
26.68, 126.48, 125.60, 125.52, 124.60, 123.87, 123.46, 109.50, 65.01, 35.06, 35.00, 34.33, 34.11,
+
+
3
1.85, 31.77. HPLC (t= 7.74 min, 95%). HRMS (TOF ESI+) for C H O (M+H) calcd. 431.2222,
28 31 4
found 431.2222.
Methyl-4-(2-(5,5,8,8-tetramethyl-5,6,7,8-tetrahydronaphthalen-2-yl)-1,3-dithiolan-2-yl)benzoate
(14a). Compound 10a (400 mg, 1.14 mmol) was treated according to method E. The crude residue was
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