Novel C-Ring-Hydroxy-Substituted Controlled Deactivation Cannabinergic Analogues

Article abstract of DOI:10.1021/acs.jmedchem.6b00717

In pursuit of safer controlled-deactivation cannabinoids with high potency and short duration of action, we report the design, synthesis, and pharmacological evaluation of novel C9- and C11-hydroxy-substituted hexahydrocannabinol (HHC) and tetrahydrocanna

Full text of DOI:10.1021/acs.jmedchem.6b00717

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Article
Novel C-Ring-Hydroxy-substituted Controlled
Deactivation Cannabinergic Analogues
Shashank Kulkarni, Spyros P. Nikas, Rishi Sharma, Shan Jiang, Carol A Paronis, Michael Z
Leonard, Bin Zhang, Chandrashekhar Honrao, Srikrishnan Mallipeddi, Jimit Girish Raghav,
Othman Benchama, Torbjörn U.C. Järbe, Jack Bergman, and Alexandros Makriyannis
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.6b00717 • Publication Date (Web): 01 Jul 2016
Downloaded from http://pubs.acs.org on July 5, 2016
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Novel C-Ring-Hydroxy-substituted Controlled Deactivation
Cannabinergic Analogues
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†
,†
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Shashank Kulkarni, Spyros P. Nikas,* Rishi Sharma, Shan Jiang, Carol A. Paronis, Michael
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†
Z. Leonard, Bin Zhang, Chandrashekhar Honrao, Srikrishnan Mallipeddi, Jimit Girish Raghav,
†
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‡
,†,#
Othman Benchama, Torbjörn U.C. Järbe, Jack Bergman, and Alexandros Makriyannis.*
†
Center for Drug Discovery and Departments of Chemistry & Chemical Biology and
Pharmaceutical Sciences, Northeastern University, Boston, Massachusetts 02115, United States
‡
McLean Hospital, Harvard Medical School, Belmont, Massachusetts 02478, United States
#
King Abdulaziz University, Jeddah, 22254, Saudi Arabia
*
To whom correspondence should be addressed.
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ABSTRACT
In pursuit of safer controlledꢀdeactivation cannabinoids with high potency and short duration of
action we report the design, synthesis, and pharmacological evaluation of novel C9ꢀ and C11ꢀ
hydroxyꢀsubstituted hexahydrocannabinol (HHC) and tetrahydrocannabinol (THC) analogues in
which a seven atom long side chain, with or without 1'ꢀsubstituents, carries a metabolically labile
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',3'ꢀester group. Importantly, in vivo studies validated our controlled deactivation approach in
rodents and nonhuman primates. The lead molecule identified here, namely butylꢀ2ꢀ
[(6aR,9R,10aR)ꢀ1ꢀhydroxyꢀ9ꢀ(hydroxymethyl)ꢀ6,6ꢀdimethylꢀ6a,7,8,9,10,10aꢀhexahydroꢀ6Hꢀ
benzo[c]chromenꢀ3ꢀyl]ꢀ2ꢀmethylpropanoate (AM7499), was found to exhibit remarkably high in
vitro and in vivo potency with shorter duration of action than the currently existing classical
cannabinoid agonists.
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Introduction
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Modulation of the two cannabinoid receptors CB1 and CB2 by the plantꢀderived (ꢀ)ꢀꢁ ꢀ
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3
tetrahydrocannabinol [(ꢀ)ꢀꢁ ꢀTHC] and its synthetic analogues is a promising therapeutic
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4ꢀ
approach to treat an array of indications including pain, inflammation, CNS disorders, and cancer.
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Unfortunately, widespread use of cannabinergic agents in therapy has been hampered due to
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target related adverse side effects as well as poor pharmacokinetic (PK) properties. While seeking
to obtain novel THCꢀbased analogues with improved druggability and safety, we reported on a
controlled deactivation/detoxification approach. To this effect, we have combined the “soft”
analogue/drug concept of enzymatic inactivation with the “depot effect” which is related to the
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2ꢀ15
compound’s lipophilicity as well as its tissue distribution and retention.
Specifically, we have
shown that incorporation of a metabolically labile carboxy ester group (soft spot) at strategic
positions within the THC structure leads to potent and efficacious CB1 agonists (2, Figure 1) that
are bioꢀconverted to inactive metabolites by plasma esterases while the rate of hydrolytic cleavage
can be accordingly modulated using stereochemical features adjacent to the ester group (enzymatic
effect). Also, it is well established that the pharmacological time of action of a lipophilic drug
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molecule, such as ꢁ ꢀ and ꢁ ꢀTHC, can greatly exceed its biochemical halfꢀlife. This is the result of
a “depot effect” due to which when the drug molecule is administered it is sequestered in the fatty
tissues before being slowly released into the circulation to start exerting its action. The depot effect
is dependent on the compound’s polar characteristics and can be regulated by varying its log P and
PSA values. Thus, higher lipophilicity of a molecule is associated with longer residency in fatty
tissue, while enhancement of the compound’s polar features is expected to moderate its lipophilic
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2ꢀ15
character and reduce the depot effect.
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A major goal of this project is to develop controlledꢀdeactivation cannabinoids with faster
onset/offset and shorter duration of action than currently existing THC analogues. Such compounds
can be useful as tools to study the pharmacological relevance of the precise, shortꢀterm, activation
of CB receptors and to help develop better designed and more predictable animal models. Towards
this goal, we have recently focused on controlledꢀdeactivation cannabinergic templates with
enhanced polar characteristics that are associated with lower lipophilicity and, thus, less of the
depot effect. In an initial study, we observed that incorporation of polar groups (e.g. cyanoꢀ and
imidazolylꢀgroups) at the carboxyester side chain pharmacophore of THCꢀlike analogues increases
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its stability toward esterases. This suggests that the depot effect and enzymatic actions are not
working synergistically for optimal short duration of action when both the soft spot and the polar
group are incorporated in the side chain of the molecule. For this reason we sought to design second
generation controlled deactivation ligands that retain the metabolically labile ester group at the 2'ꢀ
position of the side chain while the polar moiety, in the form of a hydroxyl group, is introduced at
the C9 or C11 positions of the tricyclic cannabinoid structure (3, Figure 1). The choice of the
hydroxyl group and the relative stereochemistry at the C9 is based on our earlier lead optimization
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6ꢀ21
and pharmacophore refinement work in the classical cannabinoid prototype.
An additional goal
of the present work was to explore the validity of our controlled deactivation approach by obtaining
information related to the modulation of the depot effect and its role in the in vivo pharmacokinetic
profile of the analogues in rodents and monkeys.
All synthesized compounds were characterized biochemically by determining their in vitro CB1 and
CB2 receptor binding affinities, functional activities, and assessment of their in vitro metabolic
stability toward mouse and rat plasma esterases. The most successful compounds were evaluated
for their hypothermic and analgesic effects in rodents while a key compound was further tested in
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drug discrimination experiments in squirrel monkeys. Our data show that the C1'ꢀgemꢀdimethyl
analogues exhibit very high affinities for the CB1 and CB2 receptors and that they are susceptible
to enzymatic deactivation by plasma esterases while their hydrolytic metabolites showed no or very
low cannabinergic activity. In vitro and in vivo characterization suggested that three of the
analogues identified in this study are potent CB1/CB2 agonists and exhibit CBꢀmediated
hypothermic effects. The analogue with the most promising activity and short duration of action
profile, namely AM7499 (3b), was further tested in the analgesia assay in mice and drug
discrimination experiments in squirrel monkeys. The in vivo data show that our lead compound is a
remarkably potent CB receptor agonist with significantly faster onset and shorter duration of action
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than the less polar and nonꢀhydrolyzable congener 11ꢀOHꢀꢁ ꢀTHCꢀDMH (3g, Table 1).
Importantly, drug discrimination experiments in nonꢀhuman primates validated our controlled
deactivation approach in which compound 3b exhibited approximately 3ꢀfold greater potency and
approximately 4ꢀtimes shorter duration of action when compared to non hydrolyzable congener 11ꢀ
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OHꢀꢁ ꢀTHCꢀDMH.
Chemistry
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Synthesis of the 11ꢀOHꢀ(ꢀ)ꢀꢁ ꢀTHC ester derivative 3a is depicted in Scheme 1. The chiral 4ꢀ
hydroxymyrtenyl pivalate 6 was prepared in three steps from (1R, 5S)ꢀmyrtenol (5) using the
2
3
method by Liddle et al. , while carboxylic acid 8 was obtained in two steps from nitrile 7 by
15
following procedures we reported earlier. Methyl ester formation produced 9 (72% yield) which
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was treated with boron tribromide to give the corresponding resorcinol 10 in 71% yield.
Condensation of 10 with the chiral terpenoid alcohol 6 in the presence of boron trifluoride diethyl
23,24
etherate provided the diester 11 in 40% yield.
Saponification of all ester groups in 11 gave the
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precursor carboxylic acid 4a (68% yield) which upon treatment with 1ꢀbromobutane and sodium
bicarbonate under microwave conditions afforded the respective ester 3a in 51% yield (nonꢀ
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optimized).
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Stereoselective synthesis of the 11ꢀOHꢀ(ꢀ)ꢀhexahydrocannabinol ester analogue 3b and the
respective carboxylic acid metabolite 4b is shown in Scheme 2. Deprotection of the phenolic
hydroxyl groups in 8 led to carboxylic acid 12 (98% yield) which upon microwave assisted
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esterification gave resorcinol 13 in an excellent yield (92%).
Coupling of 13 with a mixture of
chiral terpene diacetates 14 in the presence of pꢀtoluenesulfonic acid (pꢀTSA) gave norpinanone 15
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in moderate yield (27%). We had explored a variety of solvents, acids and conditions in order to
improve the yield to no avail. In addition to the desired product 15, this reaction produces a mixture
of byproducts where the (+)ꢀapoverbenone (14a) and the resorcinol monoacetate 13a were found to
be the major ingredients. We and others have observed that this condensation works well with
resorcinols bearing electron donating alkyl side chains with bulky groups at the benzylic position
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including the 5ꢀ(1,1ꢀdimethylheptyl)resorcinol.
This, coupled with our earlier investigations
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on the mechanism of this condensation, led us to postulate that presence of the carboxyester group
in 13 decreases the electron density of the aromatic ring and slows down the rate of the initial
FriedelꢀCrafts allylation reaction. Thus, the acid catalyzed decomposition of diacetates 14 to give
1
4a as well as the mono acetylation of 13 to give 13a, become important side reactions. Currently
studies to circumvent the problem of the moderate yield of this synthetic step are ongoing in our
laboratory. Treatment of 15 with catalytic amounts of trimethylsilyl triflate in
dichloromethane/nitromethane gave 16 with the required 6aR, 10aR stereochemistry in 67%
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yield.
The free phenolic hydroxyl in 16 was protected as the tertꢀbutyldimethylsilyl (TBS) ether
(87% yield). Ketone 17 was exposed to (methoxymethylene)triphenylphosphorane to give enol
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ether 18 in 63% yield as a 2:3 mixture of two geometric isomers (18a and 18b see experimental
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section) based on H NMR analysis. Methyl vinyl ether 18 was hydrolyzed with wet trichloroacetic
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acid (97% yield), and the diastereomeric mixture of C9 aldehydes 19 (5:2 ratio by H NMR) was
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16,18
epimerized to produce the βꢀequatorial isomer 20 in 78% yield.
Reduction with sodium
borohydride afforded the intermediate alcohol 21 in 85% yield. Subsequent treatment with tetraꢀnꢀ
butylammonium fluoride gave the final compound 3b in high yield (90%). The metabolite acid 4b
was prepared by alkaline hydrolysis of the ester 3b using lithium hydroxide in dioxane/water (85%
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yield).
The intermediate side chain carboxylated ketone 16 served as the starting point for the synthesis of
the C9 alcohols 3c and 3d (Scheme 3). Thus, reduction of the C9 keto group in 16 with sodium
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borohydride led to the βꢀequatorial hydroxyl compound 3c along with traces (~ 4% by H NMR) of
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the respective αꢀaxial isomer (3d). This was followed by flash column chromatography
purification to give pure 3c in 84% yield. Alkaline hydrolysis of the ester side chain in 16 provided
ketoꢀacid 22 (63% yield) which upon treatment with sodium borohydride afforded the acid
metabolite 4c in 81% yield after purification from traces of the αꢀaxial diastereomer 4d. Reduction
of 22 with potassium triꢀsecꢀbutylꢀborohydride (Kꢀselectride) was very selective and gave the αꢀ
axial alcohol 4d exclusively (91% yield). Alkylation of the respective carboxylate anion with 1ꢀ
bromobutane under microwave heating led to the corresponding ester 3d (42% yield). It should be
noted that the stereochemistry of the hydroxyl groups of 3c/3d and 4c/4d isomeric pairs was
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assigned on the basis of H NMR (500 MHz) spectral data as in our earlier work with close related
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systems. Thus, in compounds 3c and 4c, the peak halfꢀwidth for the C9 protons was found to be
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5ꢀ26 Hz while in compounds 3d and 4d was 9.5 Hz (see experimental section). This correlates
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well with an axial C9 proton in the former two compounds and an equatorial C9 proton in the latter
two.
Synthesis of the side chain carboxylated analogues lacking the C1'ꢀgemꢀdimethyl group are
summarized in Scheme 4. In a similar fashion, terpenylation of resorcinol 23 (23% yield) was
followed by dibenzo[b,d]pyran ring closure to give methyl ester 25 (57% yield). The sequence
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methyl ester hydrolysis, esterification, hydride reduction, worked as expected and provided the C9
equatorial alcohol 3e in good overall yield. Sodium borohydride and Kꢀselectride reductions were
employed to synthesize the requisite C9 alcohols 3f, 4e, and 4f from the C9 ketones 27 and 26.
Cannabinoid receptor affinities
The abilities of compounds 3aꢀ3f and 4aꢀ4f to displace the radiolabeled CB1/CB2 agonist CPꢀ
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5,940 from membranes prepared from rat brain (source of CB1) and HEK 293 cells expressing
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either mouse CB2 or human CB2 were determined as described earlier,
and inhibition constant
values (Ki) from the respective competition binding curves are listed in Table 1 in which the nonꢀ
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hydrolyzable prototype 11ꢀOHꢀꢁ ꢀTHCꢀDMH (3g), as well as the first generation carboxyꢀꢁ ꢀTHC
analogue 2a, are included for comparison. The use of two CB2 receptor preparations was aimed at
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addressing species differences that we observed earlier. The compounds included in this study are
C9ꢀ and C11ꢀhydroxyꢀsubstituted hexahydrocannabinol (HHC) and tetrahydrocannabinol (THC)
analogues in which a seven atom long side chain, with or without 1'ꢀsubstituents carries a 2', 3'ꢀester
group. In agreement with our rational design, the hydrolytic metabolites 4aꢀ4f have no significant
affinities for CB1 and CB2 receptors, thus minimizing the possibility of undesirable cannabinoid
receptor related side effects.
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Examination of the binding data of 11ꢀOHꢀꢁ ꢀTHCꢀDMH (3g), the first generation carboxyester
THC compound 2a, and the side chain carboxylated 11ꢀOHꢀTHC analogue 3a, suggests that
simultaneous addition of carboxyester functionality at the side chain and hydroxyl group at C11 of
the prototype maintains very high binding affinities for both the CB1 and CB2 receptors. This holds
true when the tetrahydrocannabinol Cꢀring is converted to the fully saturated hexahydrocannabinol
ring with the hydroxymethyl substituent occupying the equatorial position (analogue 3b). Likewise,
the one carbon shorter homologue 3c exhibits somewhat higher binding affinities for both CB
receptors. However, conversion of the C9 equatorial hydroxyl group (seen in 3c) to the axial
hydroxyl (3d) results in a distinct reduction (~ 20ꢀ30 fold) in the binding affinities for CB1 and
CB2. This indicates that the relative stereochemistry of the hydroxyl group at C9 can affect the
ligand’s affinity within the side chain carboxylated hexahydrocannabinol class of compounds. A
comparison of the binding data of the two diastereomers 3e and 3f with their 1',1'ꢀdimethyl
congeners 3c and 3d demonstrates the striking effects of the C1'ꢀgemꢀdimethyl substitution. Thus,
analogues 3c and 3d (0.1 nM < Ki < 7 nM) exhibit 2ꢀ3 orders of magnitude higher binding affinities
when compared to 3e and 3f that bind weakly at CB receptors (238 nM < Ki < 750 nM).
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Overall, this SAR study shows that C9ꢀ and C11ꢀhydroxyꢀsubstituted hexahydrocannabinol and
tetrahydrocannabinol analogues with a 1',1'ꢀdimethylꢀ2'ꢀcarboxyester group [ꢀC(CH ) ꢀC(O)Oꢀ] on
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the side chain pharmacophore exhibit remarkably high affinities for both the CB1 and CB2
receptors. It should be noted that we have observed similar SAR trends with the side chain
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carboxyester THC analogues.
However, it appears that the HHC scaffold is more sensitive to
the presence of the C1'ꢀgemꢀdimethyl substitution in regards of CB receptor recognition.
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In vitro plasma stability studies
Enzymatic labile moieties such as ester, thioester, carbamate, or phosphate groups that are
incorporated into the parent drug molecules can be targeted by esterase enzymes expressed through
body organs and other tissues as well as blood. Esterases are a heterogeneous group of enzymes that
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are
classified
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as
cholinesterases
(including
acetylcholinesterases
and
butyrylcholinesterases), paraoxonases and carboxyesterases. Human serum albumin also exhibits
esterase activity towards phenyl esters such as aspirin (acetylsalicylic acid). Notably, esterase
activity is higher in the blood of small rodents than in large animals and humans while
13,30
carboxyesterases are found in mice and rat but not in human plasma.
In this study, analogues
with the highest binding affinities for CB1 and CB2 were assessed for their in vitro plasma stability
1
3,15
toward mouse and rat plasma esterases as detailed in the Experimental Section.
A comparison
of the halfꢀlives (t , Table 1) of the C9ꢀ and C11ꢀhydroxyꢀsubstituted analogues (3a, 3b, 3c and
1
/2
3
d) with the first generation side chain carboxylated compound 2a show that incorporation of a
polar hydroxyl group at the Cꢀring of the prototype reduces the stabilities of the analogues toward
esterases for both the mouse and the rat plasma. In addition, a comparison of the compounds’ halfꢀ
lives in mouse and rat plasma indicates some species differences with the rat plasma being less
1
5
active. This observation parallels those we reported earlier for carboxyester THC analogues.
Interestingly, our stability data show that the isomeric C9 alcohols 3c and 3d have similar halfꢀlives
in mouse plasma whereas in rat plasma the equatorial alcohol 3c appears somewhat more stable. In
summary, our data validated the soft analogue profile of our more polar, second generation,
controlled deactivation cannabinergic ligands that also exhibit shorter metabolic halfꢀlives for
esterases when compared to the first generation side chain carboxylated THCs which carry no
hydroxy groups at the C9 or C11 positions.
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Functional characterization
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We focused on those analogues possessing the highest CB1/CB2 binding affinities (3a, 3b and 3c),
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and experiments were carried out by measuring changes in forskolinꢀstimulated cAMP, as detailed
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earlier.
Our testing results (Table 2) show that all three compounds potently decreased the levels
of cAMP, indicating that within this signaling mechanism these compounds behaved as potent
agonists at both the CB1 and CB2 receptors with the 11ꢀOHꢀHHC ester analogue 3b being the most
potent.
In vivo behavioral characterization
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1
Hypothermia testing. The hypothermic effects of the side chain carboxyester analogues 3a, 3b,
and 3c with the highest CB1 receptor affinities were compared in rats. Rectal body temperature was
measured in isolated rats over a 6 h period following drug injection (detailed procedures are given
under Experimental section). In agreement with our in vitro functional characterization, compounds
3
a, 3b and 3c all decreased core body temperature in a doseꢀdependent manner, reducing body
o
temperature by 4.7 to 7.2 C at the highest doses tested (Figure 2). For comparison, the effects of
8
the nonꢀhydrolysable congener 11ꢀOHꢀ(ꢀ)ꢀꢁ ꢀTHCꢀDMH (3g) are also shown. The ED values (in
5
0
mg/kg, with 95% CI) were 0.04 (0.001, 0.15) for 3a, 0.04 (0.02, 0.1) for 3b, 0.74 (0.05, >10) for 3c
and 0.04 (0.01, 0.12) for the 3g. Thus, the 11ꢀOHꢀanalogues 3a and 3b were equipotent with 3g,
and 3c was slightly less potent than the other three compounds. All compounds had relatively fast
onsets of drug effect. Significant decreases in body temperature typically occurred within 60 min
after injection although peak effects were not obtained until 2 to 3 hours after injection (time course
data for all tested doses of each compound are included under supporting information, Figure S1).
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A more detailed examination of the time course of the lowest doses of 3a, 3b, 3c and 3g that had
significant hypothermic effects is summarized in Figure 3. Administration of 0.1 mg/kg of 3a or 3b,
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carboxyester side chain analogues, resulted in temperature decreases of > 4 C, with significant
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recovery towards baseline within the 6 h test period. By comparison, doses of 3c and 3g that
produced an equivalent reduction in rectal temperature had effects that persisted more than 6 h.
Our hypothermia data in rats indicate that compounds 3a and 3b are equipotent and they have
similar duration of action profiles. For further studies, the HHC analogue 3b was selected over the
THC analogue 3a on the basis of the in vitro cAMP data where it behaves as a slightly more potent
agonist for both the CB1 and CB2 receptors.
Analgesia testing. To confirm the observed pharmacokinetic differences between the side chain
carboxyester analogue 3b and the nonꢀhydrolysable counterpart 3g, we used the CB1 receptor
characteristic analgesia assay. Twoꢀway ANOVA suggested a significant interaction between time
and drug [F(3, 66) = 28.94; p < 0.001]. The main effect of time was also significant [F(3, 66) =
4
.33; p = 0.008]. Postꢀhoc comparisons suggested significant differences between the two drugs (3b
and 3g) at 20, 60 and 360 min post injection (Figure 4). These differences in onꢀset and offꢀset of
effect for the two compounds are also reflected by comparisons within each ligand over time. Pairꢀ
wise comparisons suggested less analgesia at the 360 min time point compared to the three earlier
time points as well as a significant difference between the recordings at 60 and 180 min post
injection for compound 3b. Slow onꢀset of effect for compound 3g is suggested by significant
differences in analgesia scores at 20 as well as 60 min post injection and the recordings at the 180
and 360 time points. In keeping with above profile of a fast onꢀset and offꢀset for compound 3b, an
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additional dose of 1 mg/kg resulted in average MPE scores of 94, 96, 97, and 58% at the four timeꢀ
points (20, 60, 180 and 360 min post injection) examined (n=6; data not shown).
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Drug discrimination in nonhuman primates. In an effort to further explore the validity of our
controlled deactivation approach in species beyond rodents we performed drug discrimination
studies in squirrel monkeys (Saimiri sciureus) trained to discriminate 0.01 mg/kg (6aR, 9R, 10aR)ꢀ
3
ꢀ(adamantanꢀ1ꢀyl)ꢀ9ꢀ(hydroxymethyl)ꢀ6,6ꢀdimethylꢀ6a,7,8,9,10,10aꢀhexahydroꢀ6Hꢀ
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benzo[c]chromenꢀ1ꢀol (28, AM4054, see supporting information Figure S2) from saline. This
compound is a structurally related cannabinoid CB1 full agonist which was characterized in our
3
3
earlier work. It should be noted that studies with nonhuman primates are key to this project
because metabolic processes and volumes of drug distribution vary between small rodent and
34
primate species.
In these experiments, the time course of the lowest doses of our lead compound 3b (0.001 mg/kg)
and the nonꢀhydrolyzable cannabinoid 3g (0.003 mg/kg) that fully substituted for the training dose
of the CB1 agonist 28 were compared directly in the same group of subjects (Figure 5; see
Experimental section). Compound 3b produced evidence of CB1ꢀrelated discriminativeꢀstimulus
effects within 15 minutes (t1/2 = 48 min) and was fully effective in three of four subjects 1 h after
injection. Compound 3g was similar in its onset of action (t1/2 = 66 min) but did not achieve a full
effect in the group of subjects until 2 h after injection. Notwithstanding the generally comparable
onset of behavioral effects, the two compounds varied considerably in offset of action. Thus, 3b
began losing its CB1 discriminativeꢀstimulus effects 4 h after injection and was completely without
effect 16 h following treatment. On the other hand, 3g fully substituted for the training drug
stimulus (28) 24 h after injection and was completely without CB1 discriminativeꢀstimulus effects
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at the 36ꢀh time point. Based on calculated values for loss of 50% of full effect, the duration of
action for 3b was approximately 4 times shorter than for 3g. Neither compound had appreciable
effects on response rates throughout the present time course studies (Figure 5, lower panel).
Overall, our in vivo experiments in rats show that compounds 3a, 3b and 3c have in vivo
hypothermia activity and produce similar maximum effects as the structurally related cannabinoid
agonist 3g. Among the hydrolysable compounds tested, analogues 3a and 3b found to be equipotent
and they have similar duration of action profiles. Our antinociception data in mice and drug
discrimination experiments in squirrel monkeys confirmed that the more polar 11ꢀOHꢀcarboxyester
analogue 3b is equipotent and exhibits significantly shorter duration of action when compared to
the nonꢀhydrolyzable standard 3g.
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Conclusions
We recently reported on a controlled deactivation/detoxification approach for obtaining
cannabinoids with improved druggability and safety. In these earlier studies we have also provided
evidence that the pharmacological halfꢀlives of our selectively detoxified analogues can be
controlled by the joint modulation of their relative stabilities for plasma esterases as well as through
variation of their polar characteristics and thus to the depot effects. A major goal of this project is to
develop controlled deactivation cannabinoid agonists with faster onset/offset and significantly
shorter duration of action than the currently existing classical cannabinoids and also, to explore the
validity of this approach in species beyond rodents including nonhuman primates. To this extent,
we report now the design, synthesis and pharmacological evaluation of second generation, more
polar, controlled deactivation cannabinoids. These novel cannabinergic compounds are C9ꢀ and
C11ꢀhydroxyꢀsubstituted hexahydrocannabinol (HHC) and tetrahydrocannabinol (THC) analogues
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in which the seven atom long side chain, with or without substituents, carries a metabolically labile
',3'ꢀester group. Our in vitro data showed that compounds with a 1',1'ꢀdimethylꢀ2'ꢀcarboxyester
group [ꢀC(CH ) ꢀC(O)Oꢀ] on the side chain pharmacophore exhibit very high affinities for both the
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CB1 and CB2 receptors, and also behave as potent and efficacious CB1/CB2 agonists in the cAMP
assay. In agreement with our design principles, the analogues are susceptible to enzymatic
hydrolysis by plasma esterases, while their hydrolytic metabolites are inactive. Importantly, for
further work towards clinical development, in vivo studies validated our controlled deactivation
approach in rodents and nonhuman primates. The lead molecule indentified in this study, namely
butylꢀ2ꢀ[(6aR,9R,10aR)ꢀ1ꢀhydroxyꢀ9ꢀ(hydroxymethyl)ꢀ6,6ꢀdimethylꢀ6a,7,8,9,10,10aꢀhexahydroꢀ
6
Hꢀbenzo[c]chromenꢀ3ꢀyl]ꢀ2ꢀmethylpropanoate (3b), was found to exhibit remarkably high in vitro
and in vivo potency with a shorter duration of action than the currently existing classical
cannabinoid agonists.
Experimental section
Materials. All reagents and solvents were purchased from Aldrich Chemical Company, unless
otherwise specified, and used without further purification. All anhydrous reactions were performed
under a static argon atmosphere in flameꢀdried glassware using scrupulously dry solvents. Flash
column chromatography employed silica gel 60 (230ꢀ400 mesh). All compounds were
demonstrated to be homogeneous by analytical TLC on preꢀcoated silica gel TLC plates (Merck, 60
F245 on glass, layer thickness 250 ꢀm), and chromatograms were visualized by phosphomolybdic
acid staining. Melting points were determined on a microꢀmelting point apparatus and are
uncorrected. IR spectra were recorded on a Perkin Elmer Spectrum One FTꢀIR spectrometer. NMR
1
spectra were recorded in CDCl , unless otherwise stated, on a Bruker Ultra Shield 400 WB plus ( H
3
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13
at 400 MHz, C at 100 MHz) or on a Varian INOVAꢀ500 ( H at 500 MHz, C at 125 MHz)
spectrometers and chemical shifts are reported in units of
δ relative to internal TMS. Multiplicities
are indicated as br (broadened), s (singlet), d (doublet), t (triplet), q (quartet), m (multiplet) and
coupling constants (J) are reported in hertz (Hz). Low and highꢀresolution mass spectra were
performed in School of Chemical Sciences, University of Illinois at UrbanaꢀChampaign. Mass
spectral data are reported in the form of m/z (intensity relative to base = 100). Elemental analyses
were obtained in Baron Consulting Co, Milford, CT, and were within ± 0.4% of the theoretical
values (see supporting information). Purities of the tested compounds were determined by
elemental analysis or by HPLC (using Waters Alliance HPLC system, 4.6 x 250 mm, Supelco
discovery column, acetonitrile/water with 8.5% oꢀphosphoric acid) or by LC/MS analysis using a
Waters MicroMass ZQ system [electrosprayꢀionization (ESI) with Watersꢀ2525 binary gradient
module coupled to a Photodiode Array Detector (Watersꢀ2996) and ELS detector (Watersꢀ2424)
using a XTerra MS C18, 5 µm, 4.6 mm x 50 mm column and acetonitrile/water] and were > 95%.
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Butyl 2-[(6aR,10aR)-1-hydroxy-9-(hydroxymethyl)-6,6-dimethyl-6a,7,10,10a-tetrahydro-6H-
benzo[c]chromen-3-yl]-2-methyl-propanoate (3a). A stirred mixture of carboxylic acid 4a (100
mg, 0.29 mmol), bromobutane (119 mg, 0.87 mmol), and sodium bicarbonate (37 mg, 0.43 mmol)
o
in DMF (2 mL) was heated at 165 C for 12 min using microwave irradiation. The reaction mixture
was cooled to room temperature and diluted with water and ethyl acetate. The organic layer was
separated, and the aqueous phase was extracted with ethyl acetate. The combined organic layer was
washed with brine, dried (MgSO ), and concentrated under reduced pressure. Purification by flash
4
column chromatography on silica gel (5ꢀ50% ethyl acetate in hexane) gave 3a (60 mg, 51%) as a
ꢀ1
light yellow gum. IR (neat) 3292, 2961, 1727, 1704 (s, >C=O), 1619, 1578, 1417, 1328, 1263cm ;
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H NMR (500 MHz, CDCl ) δ 6.41 (d, J = 2.0 Hz, 1H, 4ꢀH), 6.25 (d, J = 2.0 Hz, 1H, 2ꢀH), 5.73 (m
3
as d, J = 4.5 Hz, 1H, 8ꢀH), 5.42 (s, 1H, ArOH), 4.10ꢀ4.02 (d, t and d overlapping, 4H, especially
4
.08, d, J = 13.0 Hz,1H, half of an AB system, ꢀCH ꢀOH, 4.06, t, J = 6.5 Hz, 2H, ꢀC(O)OCH ꢀ,
2 2
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.03, d, J = 13.0 Hz, 1H, half of an AB system, ꢀCH ꢀOH), 3.41 (dd, J = 16.0 Hz, J = 4.5 Hz, 1H,
2
10αꢀH), 2.71 (td, J = 11.0 Hz, J = 5.0 Hz, 1H, 10aꢀH), 2.27ꢀ2.20 (m, 1H, 7αꢀH), 1.93ꢀ1.78 (m, 3H,
1
0βꢀH, 7βꢀH, 6aꢀH), 1.55 (quintet, J = 7.0 Hz, 2H, ꢀCH ꢀ of the side chain), 1.50 (s, 6H, ꢀC(CH ) ꢀ),
2 3 2
1
.39 (s, 3H, 6βꢀMe), 1.26 (quintet, 2H, ꢀCH ꢀ of the side chain), 1.10 (s, 3H, 6αꢀMe), 0.86 (t, J =
2
1
3
7.5Hz, 3H, 7'ꢀH). C NMR (125 MHz CDCl ) δ 177.3 (>C=O), 155.6 (Cꢀ1 or Cꢀ5), 154.8 (Cꢀ5 or
3
Cꢀ1), 144.5, 138.3, 121.6, 111.4, 107.3, 105.5, 77.7 (Cꢀ6), 67.2 (ꢀOꢀCH ), 65.0(ꢀOCH ꢀ), 46.2, 45.1,
2
2
3
1.5, 31.4, 30.6, 27.8, 27.7, 26.4, 26.2, 19.1 18.5, 13.8. Mass spectrum (ESI) m/z (relative intensity)
+
+
403 (M +H, 100). Mass spectrum (EI) m/z (relative intensity) 402 (M , 100), 301 (53), 217 (38).
+
Exact mass (EI) calculated for C H O (M ), 402.2406; found 402.2407. HPLC (4.6mm x
2
4
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5
2
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50mm, Supelco Discovery column, acetonitrile/water) showed purity of 98.5% and retention time
1.1min for 3a. Anal. (C H O ) C, H.
2
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5
Butyl-2-[(6aR,9R,10aR)-1-hydroxy-9-(hydroxymethyl)-6,6-dimethyl-6a,7,8,9,10,10a-
hexahydro-6H-benzo[c]chromen-3-yl]-2-methylpropanoate (3b). To a solution of 21 (75 mg,
0
.15 mmol) in anhydrous THF (3.6 mL) at ꢀ40 °C, under an argon atmosphere, was added tetraꢀnꢀ
butylammonium fluoride (0.3 mL, 0.3 mmol, 1M solution in anhydrous THF). The reaction mixture
was stirred for 30 min at the same temperature, and then quenched using a saturated aqueous NH Cl
4
solution. Extractive isolation with diethyl ether, and purification by flash column chromatography
on silica gel (20% ethyl acetate in hexane) gave 3b (52 mg, 90% yield) as a white solid. mp = 59ꢀ60
o
ꢀ1 1
C. IR (neat): 3380, 2971, 2869, 1727, 1703, 1620, 1577, 1417, 1268, 1141 cm ; H NMR (500
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MHz, CDCl ) δ 6.39 (d, J = 2.0 Hz, 1H, ArꢀH), 6.22 (d, J = 2.0 Hz, 1H, ArꢀH), 5.33 (br s, 1H,
3
ArOH), 4.05 (d, J = 6.0 Hz, 2H, 4'ꢀH), 3.56ꢀ3.49 (m, 2H, ꢀCH OH), 3.23ꢀ3.18 (m as br d, J = 13.0
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ring), 1.94ꢀ1.88 (m, 1H, Cꢀring), 1.82ꢀ1.72 (m, 1H, Cꢀring), 1.58ꢀ1.44 (quintet, s, and m,
overlapping, 9H, ꢀC(CH ) ꢀ, 5'ꢀH, Cꢀring, especially 1.49, s, 6H, ꢀC(CH ) ꢀ), 1.38 (s, 3H, 6ꢀMe),
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1
3
J = 7.5 Hz, 3H, 7'ꢀH), 0.76 (m as q, J = 11.0 Hz, 1H, Cꢀring). C NMR (100 MHz CDCl ) δ 177.1
3
(ꢀC(O)Oꢀ), 155.2 (ArCꢀ1 or ArCꢀ5), 154.9 (ArCꢀ5 or ArCꢀ1), 144.2 (tertiary aromatic), 111.0
(
tertiary aromatic), 107.2 (ArCꢀ2 or ArCꢀ4), 105.3 (ArCꢀ4 or ArCꢀ2), 68.4 (ꢀCH OH), 64.7 (ꢀ
2
OCH ꢀ), 49.3, 46.0, 40.4, 35.0, 33.0, 30.5, 29.7, 27.7, 27.4, 26.2, 26.0, 19.0, 13.6. Mass spectum
2
+
+
(ESI) m/z (relative intensity) 405 (M +H, 100). Mass spectum (EI) m/z (relative intensity) 404 (M ,
+
1
00), 361 (41), 303 (65), 265 (41). Exact mass (ESI) calculated for C H 0 (M +H), 405.2641;
2
4
37 5
+
found 405.2642. Exact mass (EI) calculated for C H 0 (M ), 404.2563; found 404.2559. HPLC
24 36
5
(4.6 x 250 mm, Supelco discovery column, acetonitrile/water) showed purity 99.5% and retention
time 10.8 min for the title compound.
Butyl-2-[(6aR,9R,10aR)-6a,7,8,9,10,10a-hexahydro-1,9-dihydroxy-6,6-dimethyl-6H-
benzo[c]chromen-3-yl]-2-methylpropanoate (3c). To a solution of 16 (35 mg, 0.09 mmol) in
anhydrous methanol (3 mL) at ꢀ78 °C, under an argon atmosphere, was added sodium borohydride
(3.4 mg, 0.09 mmol). The reaction mixture was stirred at the same temperature for 2 h and then
quenched by the addition of aqueous 2N HCl solution. The mixture was warmed to room
temperature, diluted with water and extracted with ethyl acetate. The organic layer was washed with
brine, dried (MgSO ), and concentrated in vacuo. Purification by flash column chromatography on
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silica gel (24% acetone in hexane) gave 3c (30 mg, 84% yield) as light yellow gum free from its
epimer 3d which is also produced in traces during the reaction (ratio 3c:3d ~ 96:4). IR (neat): 3329,
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 1
2
971, 2872, 1727, 1704, 1620, 1578, 1418, 1272, 1142 cm ; H NMR (500 MHz, CDCl ) δ 6.38 (
3
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
d, J = 2.0 Hz, 1H, ArH), 6.22 (d, J = 2.0 Hz, 1H, ArH), 6.08 (br s, 1H, ArOH), 4.04 (t, J = 6.5
Hz, 2H, 4′ꢀH), 3.86 (dddd, J = 16.0 Hz, J = 15.0 Hz, J = 4.5 Hz, J = 4.0 Hz, 1H, 9axꢀH, peak halfꢀ
width = 26 Hz), 3.51 (dddd, J = 12.0 Hz, J = 4.5 Hz, J = 2.5 Hz, J = 2.0 Hz, 1H, 10eqꢀH), 2.47
(ddd, J = 12.0 Hz, J = 11.5 Hz, J = 2.5 Hz, 1H, 10aꢀH), 2.16 (m as br d, J = 10.0 Hz, 1H, 8eqꢀH),
1.88 (dddd, J = 13.5 Hz, J = 3.5 Hz, J = 2.5 Hz, J = 2.0 Hz,, 1H, 7eqꢀH), 1.63 (br s, 1H, OH),
1
1
.57ꢀ1.50 (quintet and m, overlapping 3H, 5'ꢀH and 6aꢀH), 1.49 (s, 6H, ꢀC(CH ) ꢀ), 1.42 (dddd, J =
3 2
5.5 Hz, J = 15.0 Hz, J = 13.0 Hz, J = 3.5 Hz, 1H, 8axꢀH), 1.38 (s, 3H, 6ꢀMe), 1.25 (sextet, J = 7.0
Hz, 2H, 6′ꢀH), 1.15 (dddd, J = 13.5 Hz, J = 13.0 Hz, J = 12.0 Hz, J = 3.5 Hz, 1H, 7axꢀH), 1.12
(ddd, J = 15.5 Hz, J = 12.0 Hz, J = 12.0 Hz, 1H, 10axꢀH), 1.05 (s, 3H, 6ꢀMe), 0.85 (t, J = 7.5 Hz,
1
3
3
1
3
H, 7′ꢀH). C NMR (100 MHz, CDCl ) δ 171.5 (ꢀC(O)Oꢀ), 155.0 (ArC), 154.7 (ArC), 144.5 (ArC),
3
15.4 (ArC), 110.2 (ArC), 107.5 (ArC), 77.2 (Cꢀ6), 70.7 (>CHꢀOH), 64.6 (ꢀOCH ꢀ), 48.3, 46.0,
2
9.0, 35.6, 33.4, 30.5, 27.8, 26.2, 26.1, 26.0, 19.0, 178, 13.6. Mass spectum (ESI) m/z (relative
+
+
intensity) 391 (M +H, 100). Mass spectum (EI) m/z (relative intensity) 390 (M , 87), 329 (32), 289
+
+
(
100, M ꢀ CH (CH ) OC(O)), 265 (37), 177 (61). Exact mass (EI) calculated for C H 0 (M ),
3 2 3 23 34 5
3
90.2406; found 390.2408. HPLC (4.6 x 250 mm, Supelco discovery column, acetonitrile/water)
showed purity 99.5% and retention time 9.7 min for the title compound.
Butyl-2-[(6aR,9S,10aR)-6a,7,8,9,10,10a-hexahydro-1,9-dihydroxy-6,6-dimethyl-6H-
benzo[c]chromen-3-yl]-2-methylpropanoate (3d). The synthesis was carried out as described for
3
a using 4d (34 mg, 0.10 mmol), sodium bicarbonate (15 mg, 0.16 mmol) and 1ꢀbromobutane (35
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2
2
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5
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5
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5
5
5
6
mg, 0.25 mmol) in DMF (1.5 mL) to give 3d (17 mg, 42% yield) as light yellow gum. IR (neat):
ꢀ1 1
3
357, 2927, 2873, 1727, 1706, 1620, 1578, 1418, 1262, 1140 cm ; H NMR (500 MHz, CDCl ) δ
3
6
.41 (d, J = 2.0 Hz, 1H, ArH), 6.31 ( d, J = 2.0 Hz,1H, ArH), 4.29 (m as t, J = 3.5 Hz, 1H, 9eqꢀH,
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
peak halfꢀwidth = 9.5 Hz), 4.07 (m as t, J = 6.5 Hz, 2H, 4'ꢀH), 3.24 (ddd, J = 14.5 Hz, J = 3.0 Hz, J
= 2.5 Hz, 1H, 10eqꢀH), 2.97 (ddd, J = 12.0 Hz, J = 11.0 Hz, J = 2.5 Hz, 1H, 10aꢀH), 2.02ꢀ1.97 (m
as br d, J = 13.5 Hz, 1H, 8eqꢀH), 1.71ꢀ1.47 (m and s overlapping, 12H, 8axꢀH, 7axꢀH, 7eqꢀH, 6aꢀH,
5
'ꢀH, ꢀC(CH ) ꢀ, especially 1.51, s, ꢀC(CH ) ꢀ), 1.39 (s, 3H, 6ꢀCH ), 1.38ꢀ1.32 (m, 1H, 10axꢀH),
3 2 3 2 3
1.28 (sextet, J = 7.0 Hz, 2H, 6′ꢀH), 1.07 (s, 3H, 6ꢀCH ), 0.87 (t, J = 7.5 Hz, 3H, 7′ꢀH). Mass
3
+
+
spectum (ESI) m/z (relative intensity) 391 (M +H, 100), 289 (45, M ꢀ CH (CH ) OC(O)). Mass
3
2 3
+
+
spectum (EI) m/z (relative intensity) 390 (M , 57), 329 (55), 289 (32, M ꢀ CH (CH ) OC(O)), 183
3
2 3
+
(97), 84 (100). Exact mass (EI) calculated for C H 0 (M ), 390.2406; found 390.2408. LC/MS
2
3
34 5
analysis (Waters MicroMass ZQ system) showed purity 97.7% and retention time 5.5 min for the
title compound.
Butyl
benzo[c]chromen-3-yl] acetate (3e). The synthesis was carried out as described for 3c using 27
58 mg, 0.16 mmol) and NaBH (6.1 mg, 0.16 mmol) in MeOH (5 mL) and gave 3e (35 mg, 61%
2-[(6aR,9R,10aR)-1,9-dihydroxy-6,6-dimethyl-6a,7,8,9,10,10a-hexahydro-6H-
(
4
yield) as light yellow gum. Purification by flash column chromatography on silica gel (20ꢀ30%
acetone in hexane) gave the title compound free from its epimer 3f which is also produced in traces
ꢀ1
1
(
4ꢀ6%) during the reaction. IR (neat): 3428, 2975, 2872, 1729, 1704, 1619, 1578, 1417 cm ; H
NMR (500 MHz, CDCl ) δ 6.41 (br s, 1H, ArOH), 6.32 ( d, J = 2.0 Hz, 1H, ArH), 6.22 (d, J = 2.0
3
Hz, 1H, ArH), 4.08 (t, J = 6.5 Hz, 2H, 4′ꢀH), 3.80 (dddd, J = 16.0 Hz, J = 15.0 Hz, J = 4.5 Hz, J =
4
.0 Hz, 1H, 9axꢀH, peak halfꢀwidth = 26 Hz), 3.68 (d, J = 16.0 Hz, 1H, half of an AX system, ꢀ
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CH ꢀC(O)Oꢀ), 3.55 (d, J = 16.0 Hz, 1H, half of an AX system, ꢀCH ꢀC(O)Oꢀ), 3.51 (dddd, J = 12.0
2
2
4
5
6
7
8
9
1
1
1
1
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1
1
1
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2
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Hz, J = 4.5 Hz, J = 2.5 Hz, J = 2.0 Hz, 1H, 10eqꢀH), 2.39 (ddd, J = 12.0 Hz, J = 11.5 Hz, J = 2.5
Hz, 1H, 10aꢀH), 2.16 (m as br d, J = 10.0 Hz, 1H, 8eqꢀH), 1.87 (dddd, J = 13.5 Hz, J = 3.5 Hz, J =
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
.5 Hz, J = 2.0 Hz,, 1H, 7eqꢀH), 1.59 (quintet, J = 7.5 Hz, 2H, 5'ꢀH), 1.51 (m as td, J = 12.5 Hz, J =
2.5 Hz, 1H, 6aꢀH), 1.42ꢀ1.28 (m, s and sextet overlapping, 6H, 8axꢀH, 6′ꢀH, 6ꢀMe), 1.19ꢀ1.06 (m,
1
3
2
H, 7axꢀH, 10axꢀH), 1.05 (s, 3H, 6ꢀMe), 0.89 (t, J = 7.5 Hz, 3H, 7′ꢀH). C NMR (100 MHz
CDCl ) δ 171.8 (ꢀC(O)Oꢀ),155.4 (ArC), 154.8 (ArC), 134.2 (ArC), 116.3 (ArC), 110.8 (ArC), 103.6
3
(ArC), 77.2(Cꢀ6), 70.5(>CHꢀOH), 65.0(ꢀOCH ꢀ), 49.8, 42.0, 39.7, 35.6, 34.7, 30.6, 27.6, 26.21,
2
+
+
1
9.0, 18.4, 13.6. Mass spectum (ESI) m/z (relative intensity) 363 (M +H, 23), 345 (100, M ꢀ OH),
+
2
71 (53). Exact mass (ESI) calculated for C H O (M +H), 363.2171; found 363.2167. LC/MS
21 31
5
analysis (Waters MicroMass ZQ system) showed purity 97.3% and retention time 7.1 min for the
title compound.
Butyl
2-[(6aR,9S,10aR)-1,9-dihydroxy-6,6-dimethyl-6a,7,8,9,10,10a-hexahydro-6H-
benzo[c]chromen-3-yl] acetate (3f). The synthesis was carried out as described for 4d using 27
(40 mg, 0.11 mmol) and KꢀSelectride (0.44 mL, 1 M solution in THF) in THF (3 mL). Purification
by flash column chromatography on silica gel (20ꢀ30% acetone in hexane) gave the title compound
(21 mg, 52% yield) as light yellow gum free from its epimer 3e which is also produced in traces (3ꢀ
ꢀ1 1
5%) during the reaction. IR (neat): 3430, 2973, 1729, 1704, 1620, 1578, 1417 cm ; H NMR (500
MHz, CDCl ) δ 6.30 (d, J = 2.0 Hz, 1H, ArH), 6.23 ( d, J = 2.0 Hz,1H, ArH), 5.22 (br s, 1H ,
3
ArOH), 4.23 (m as t, J = 2.5 Hz, 1H, 9eqꢀH, peak halfꢀwidth = 9.0 Hz), 4.11 (m as td, J = 7.0 Hz, J
=
2.5 Hz, 2H, 4'ꢀH), 3.74 (d, J = 16.0 Hz, 1H, half of an AX system, ꢀCH ꢀC(O)Oꢀ), 3.58 (d, J =
2
1
6.0 Hz, 1H, half of an AX system, ꢀCH ꢀC(O)Oꢀ), 2.90 (ddd, J = 12.0 Hz, J = 11.0 Hz, J = 2.0 Hz,
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1
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H, 10aꢀH), 2.51 (ddd, J = 14.5 Hz, J = 3.0 Hz, J = 2.2 Hz, 1H, 10eqꢀH), 1.98ꢀ1.90 (m as br d, J =
3.5 Hz, 1H, 8eqꢀH), 1.70ꢀ1.47 ( m, 6H, 8axꢀH, 7axꢀH, 7eqꢀH, 6aꢀH, 5'ꢀH), 1.42ꢀ1.28 (s, m and
sextet overlapping, 6H, 10axꢀH, 6ꢀCH , 6′ꢀH, especially 1.36, s, 6ꢀCH ), 1.04 (s, 3H, 6ꢀCH ), 0.89
3
3
3
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
+
(t, J = 7.5 Hz, 3H, 7′ꢀH). Mass spectum (ESI) m/z (relative intensity) 363 (M +H, 100), 345 (153,
+
+
M ꢀ OH). Exact mass (ESI) calculated for C H 0 (M +H), 363.2171; found 363.2173. LC/MS
2
1
31 5
analysis (Waters MicroMass ZQ system) showed purity 97.8% and retention time 7.2 min for the
title compound.
2
-[(6aR,10aR)-1-Hydroxy-9-(hydroxymethyl)-6,6-dimethyl-6a,7,10,10a-tetrahydro-6H-
benzo[c]chromen-3-yl]-2-methylpropanoic acid (4a). A stirred mixture of 11 (200 mg, 0.45
mmol) and NaOH (72 mg, 1.8 mmol) in THF/water (1:1 ratio, 8 mL) was refluxed for 16 h under
argon. Volatiles were removed under reduced pressure, and the residue was acidified with aqueous
1
N HCl and diluted with ethyl acetate. The organic layer was separated, and the aqueous layer was
extracted with ethyl acetate. The combined organic phase was washed with water and brine, dried
MgSO ) and concentrated in vacuo. Purification by flash column chromatography on silica gel
(
4
(50% acetone in hexane) afforded the title compound in 68% yield (106 mg) as a light yellow gum.
ꢀ1
1
IR (neat): 3317, 2976, 2932, 1702, 1620, 1577, 1417, 1265, 1185 cm ; H NMR (500 MHz,
CDCl ) δ 7.31ꢀ6.40 (br s, 1H, ꢀOH), 6.41 (d, J = 1.5 Hz, 1H, 4ꢀH), 6.30 (d, J = 1.5 Hz, 1H, 2ꢀH),
3
5.68 (m as d, J = 3.5 Hz, 1H, 8ꢀH), 4.03 (d, J = 12.5 Hz, 1H, half of an AB system, ꢀCH OH), 3.93
2
(d, J = 12.5 Hz, 1H, half of an AB system, ꢀCH OH), 3.47 (br d, J = 14.5Hz, 1H, 10αꢀH), 2.64 (td, J
2
=
11.5 Hz, J = 4.5 Hz, 1H, 10aꢀH), 2.16 (m, 1H, 7αꢀH), 1.83ꢀ1.70 (m, 3H, 10βꢀH, 7βꢀH, 6aꢀH), 1.50
1
3
(s, 3H, 1'ꢀCH ), 1.47(s, 3H, 1'ꢀCH ) 1.36 (s, 3H, 6βꢀMe), 1.06 (s, 3H, 6αꢀMe); C NMR(125 MHz
3 3
CDCl ) δ 180 (>C=O), 155.7 (Cꢀ1 or Cꢀ5), 154.5 (Cꢀ5 or Cꢀ1), 144.0, 137.9, 121.4, 111.5, 106.6,
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05.4, 76.6 (Cꢀ6), 66.7 (ꢀCH OH), 45.8, 44.9, 31.3, 31.1, 29.6, 27.5, 27.4, 26.1, 18.3; Mass
2
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
+
+
spectrum (ESI) m/z (relative intensity) 347 (M +H, 100), 329 (M ꢀOH, 33); Mass spectrum (EI)
+
+
m/z (relative intensity) 346 (M , 19), 329 (M ꢀOH, 8), 285 (10), 247 (15), 84 (100); exact mass (EI)
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
+
calculated for C H O (M ), 346.1780; found 346.1780. HPLC (4.6mm x 250mm, Supelco
2
0
26
5
Discovery column, acetonitrile/water) showed purity of 99.8% and retention time of 6.2min for 4a.
2
-[(6aR,9R,10aR)-1-Hydroxy-9-(hydroxymethyl)-6,6-dimethyl-6a,7,8,9,10,10a-hexahydro-6H-
benzo[c]chromen-3-yl]-2-methylpropanoic acid (4b). The synthesis was carried out as described
for 22 using 3b (20 mg, 0.05 mmol) and lithium hydroxide (12 mg, 0.5 mmol) in dioxane/water (2.8
mL, 1:1 ratio). Work up and purification by flash column chromatography on silica gel (60% ethyl
acetate in hexane) gave 4b (14.5 mg, 85% yield) as a yellow gum. IR (neat): 3321, 2975, 2932,
ꢀ1
1
1
702, 1621, 1577, 1417, 1264, 1185 cm ; H NMR (500 MHz, CDCl ) δ 6.42 (d, J = 2.0 Hz, 1H,
3
ArꢀH), 6.28 (d, J = 2.0 Hz, 1H, ArꢀH), 3.53 (dd, J = 10.5 Hz, J = 5.5 Hz, half of an AB system, 1H,
CH OH), 3.45 (dd, J = 10.5 Hz, J = 8.0 Hz, half of an AB system, 1H, ꢀCH OH), 3.29ꢀ3.22 (m as
ꢀ
2
2
br d, J = 13.0 Hz, 1H, Cꢀring), 2.44ꢀ2.37 (m as td, J = 11.0 Hz, J = 2.5 Hz, 1H, Cꢀring), 1.90ꢀ1.82
(m as br d, J = 9.5 Hz, 2H, Cꢀring), 1.78ꢀ1.68 (m, 1H, Cꢀring), 1.49ꢀ1.40 (m and s, overlapping, 7H,
ꢀ
C(CH ) ꢀ, Cꢀring, especially 1.48, s, 6H, ꢀC(CH ) ꢀ), 1.35 (s, 3H, 6ꢀMe), 1.12ꢀ1.10 (m, 2H, Cꢀ
3 2 3 2
1
3
ring), 0.98 (s, 3H, 6ꢀMe), 0.74 (m as q, J = 11.0 Hz, 1H, Cꢀring). C NMR (100 MHz CDCl ) δ
3
175.6 (ꢀC(O)Oꢀ), 154.9 (ArCꢀ1 or ArCꢀ5), 155.0 (ArCꢀ1 and ArCꢀ5), 143.7 (tertiary aromatic),
1
4
11.4 (tertiary aromatic), 107.4 (ArCꢀ2 or ArCꢀ4), 105.3 (ArCꢀ4 or ArCꢀ2), 68.4 (ꢀCH OH), 49.2,
2
5.9, 40.4, 34.8, 33.0, 29.5, 27.6, 27.4, 26.1, 19.0. Mass spectrum (ESI) m/z (relative intensity) 349
+
(
M +H, 100). LC/MS analysis (Waters MicroMass ZQ system) showed purity 97.5% and retention
time 4.2 min for the title compound.
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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
2
-[(6aR,9R,10aR)-6a,7,8,9,10,10a-Hexahydro-1,9-dihydroxy-6,6-dimethyl-6H-
benzo[c]chromen-3-yl]-2-methylpropanoic acid (4c). The synthesis was carried out as described
for 3c using 22 (55 mg, 0.16 mmol) and sodium borohydride (25 mg, 0.66 mmol) in methanol (6
mL) at room temperature and gave the title compound as a light yellow gum in 81% yield (43 mg).
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 1
IR (neat): 3338, 2975, 1695, 1622, 1418 cm ; H NMR (500 MHz, CD OD) δ 6.35 ( d, J = 2.0 Hz,
3
1
H, ArH), 6.27 (d, J = 2.0 Hz, 1H, ArH), 3.75 (dddd, J = 15.5 Hz, J = 15.0 Hz, J = 4.5 Hz, J = 4.0
Hz, 1H, 9axꢀH, peak halfꢀwidth = 25 Hz), 3.53 (dddd, J = 12.0 Hz, J = 4.5 Hz, J = 2.5 Hz, J = 2.0
Hz, 1H, 10eqꢀH), 2.45 (ddd, J = 12.0 Hz, J = 11.5 Hz, J = 2.5 Hz, 1H, 10aꢀH), 2.12 (m as br d, J =
1
1.0 Hz, 1H, 8eqꢀH), 1.90 (dddd, J = 13.5 Hz, J = 3.5 Hz, J = 2.5 Hz, J = 2.0 Hz,, 1H, 7eqꢀH),
1.50ꢀ1.34 (s, s and m, overlapping, 11H, ꢀC(CH ) ꢀ, 6aꢀH, 10axꢀH, 6ꢀMe, especially 1.47, s, ꢀ
3
2
C(CH ) ꢀ and 1.34, s, 6ꢀMe), 1.21 (dddd, J = 15.5 Hz, J = 15.0 Hz, J = 13.0 Hz, J = 3.5 Hz, 1H,
3
2
8
axꢀH), 1.05 (s, 3H, 6ꢀMe), 0.97 (dddd, J = 13.5 Hz, J = 13.0 Hz, J = 12.0 Hz, J = 3.5 Hz, 1H, 7axꢀ
+
+
+
H). Mass spectum (ESI) m/z (relative intensity) 335 (M +H, 100), 317 (54, M ꢀOH), 289 (84, M ꢀ
+
COOH). Mass spectum (EI) m/z (relative intensity) 334 (M , 97), 316 (79), 273 (97), 233 (100),
+
149 (87). Exact mass (EI) calculated for C H 0 (M ), 334.1780; found 334.1777. HPLC (4.6 x
1
9
26 5
2
50 mm, Supelco discovery column, acetonitrile/water) showed purity 98.3% and retention time 5.4
min for the title compound. LC/MS analysis (Waters MicroMass ZQ system) showed purity 98.4%
and retention time 4.2 min for the title compound.
2
-[(6aR,9S,10aR)-6a,7,8,9,10,10a-Hexahydro-1,9-dihydroxy-6,6-dimethyl-6H-benzo[c]
chromen-3-yl]-2-methylpropanoic acid (4d). KꢀSelectride (0.96 mL, 1 M solution in THF) was
added under an argon atmosphere to a solution of 22 (80 mg, 0.24 mmol) in dry THF (12 mL) at
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room temperature. The reaction mixture was stirred at the same temperature for 2 h and then it was
quenched with aqueous 1N HCl solution and diluted with ethyl acetate. The organic layer was
separated and the aqueous layer was extracted with ethyl acetate. The combined organic phase was
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
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5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
washed with water and brine, dried (MgSO ) and concentrated under reduced pressure. Purification
4
by flash column chromatography on silica gel (38% acetone in hexane) gave compound 4d (73 mg,
ꢀ1 1
9
1% yield) as light yellow gum. IR (neat): 3327, 2978, 1697, 1622, 1420 cm ; H NMR (500 MHz,
CD OD) δ 6.34 (d, J = 2.0 Hz, 1H, ArH), 6.27 ( d, J = 2.0 Hz,1H, ArH), 4.13 (m as t, J = 3.5 Hz,
3
1H, 9eqꢀH, peak halfꢀwidth = 9.5 Hz), 3.40 (ddd, J = 14.5 Hz, J = 3.0 Hz, J = 2.5 Hz, 1H, 10eqꢀH),
2
.94 (ddd, J = 12.0 Hz, J = 11.0 Hz, J = 2.5 Hz, 1H, 10aꢀH), 1.96ꢀ1.91 (m, 1H, 8eqꢀH), 1.71ꢀ1.46 (
m and s overlapping, 10H, 8axꢀH, 7axꢀH, 7eqꢀH, 6aꢀH, ꢀC(CH ) ꢀ, especially 1.47, s, ꢀC(CH ) ꢀ),
3 2
3 2
1.36 (s, 3H, 6ꢀCH ), 1.24ꢀ1.16 (m, 1H, 10axꢀH), 1.09 (s, 3H, 6ꢀCH ). Mass spectum (ESI) m/z
3
3
+
+
+
(
relative intensity) 335 (M +H, 100), 317 (27, M ꢀOH), 289 (29, M ꢀCOOH). Mass spectum (EI)
+
m/z (relative intensity) 334 (M , 37), 316 (29), 273 (42), 233 (19), 149 (100). Exact mass (EI)
+
calculated for C H 0 (M ), 334.1780; found 334.1784. LC/MS analysis (Waters MicroMass ZQ
1
9
26 5
system) showed purity 96.7% and retention time 4.3 min for the title compound.
2
-[(6aR,9R,10aR)-1,9-Dihydroxy-6,6-dimethyl-6a,7,8,9,10,10a-hexahydro-6H-
benzo[c]chromen-3-yl] acetic acid (4e). The synthesis was carried out as described for 3c using 26
(25 mg, 0.082 mmol) and NaBH (12.4 mg, 0.32 mmol) in MeOH (5 mL) at room temperature and
4
gave 4e (17 mg, 65% yield) as light yellow gum. Purification by flash column chromatography on
silica gel (40% acetone in hexane) gave the title compound free from its epimer 4f which is also
ꢀ1 1
produced in traces (4ꢀ6%) during the reaction. IR (neat): 3350, 2973, 1694, 1622, 1419 cm ; H
NMR (500 MHz, CDCl ) δ 6.32 ( d, J = 2.5 Hz, 1H, ArH), 6.21 (d, J = 2.5 Hz, 1H, ArH), 3.78
3
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1
1
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2
2
2
2
2
2
2
2
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3
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4
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5
5
5
6
(
dddd, J = 16.0 Hz, J = 15.0 Hz, J = 4.5 Hz, J = 4.0 Hz, 1H, 9axꢀH, peak halfꢀwidth = 26 Hz), 3.62
d, J = 16.0 Hz, 1H, half of an AB system, ꢀCH ꢀC(O)Oꢀ), 3.54 (d, J = 16.0 Hz, 1H, half of an AB
(
2
system, ꢀCH ꢀC(O)Oꢀ), 3.49 (dddd, J = 12.0 Hz, J = 4.5 Hz, J = 2.5 Hz, J = 2.0 Hz, 1H, 10eqꢀH),
2
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
.39 (ddd, J = 12.0 Hz, J = 11.5 Hz, J = 2.5 Hz, 1H, 10aꢀH), 2.14 (m as br d, J = 10.0 Hz, 1H,
8eqꢀH), 1.87 (dddd, J = 13.5 Hz, J = 3.5 Hz, J = 2.5 Hz, J = 2.0 Hz,, 1H, 7eqꢀH), 1.51 (m as td, J
12.5 Hz, J = 2.5 Hz, 1H, 6aꢀH), 1.39ꢀ1.28 (m and s overlapping, 4H, 8axꢀH, 6ꢀMe), 1.18ꢀ1.06
m, 2H, 7axꢀH, 10axꢀH), 1.00 (s, 3H, 6ꢀMe). Mass spectum (ESI) m/z (relative intensity) 307
=
(
+
+
+
(M +H, 98), 289 (100, M ꢀOH). Exact mass (ESI) calculated for C H 0 (M +H), 307.1545;
1
7
23 5
found 307.1549. LC/MS analysis (Waters MicroMass ZQ system) showed purity 96.7% and
retention time 3.6 min for the title compound.
2
-[(6aR,9S,10aR)-1,9-Dihydroxy-6,6-dimethyl-6a,7,8,9,10,10a-hexahydro-6H-
benzo[c]chromen-3-yl] acetic acid (4f). The synthesis was carried out as described for 4d using 26
20 mg, 0.065 mmol) and KꢀSelectride (0.65 mL, 1 M solution in THF) in THF (5 mL). Purification
(
by flash column chromatography on silica gel (40% acetone in hexane) gave the title compound (18
mg, 90% yield) as light yellow gum free from its epimer 4e which is also produced in traces (3ꢀ5%)
ꢀ1 1
during the reaction. IR (neat): 3337, 2972, 1695, 1622, 1419 cm ; H NMR (500 MHz, CDCl +
3
CD OD) δ 6.31 (d, J = 2.0 Hz, 1H, ArH), 6.22 ( d, J = 2.0 Hz,1H, ArH), 4.21 (m as br S, 1H, 9eqꢀ
3
H, peak halfꢀwidth = 9.0 Hz), 3.70 (d, J = 16.0 Hz, 1H, half of an AB system, ꢀCH ꢀC(O)Oꢀ), 3.57
2
(d, J = 16.0 Hz, 1H, half of an AB system, ꢀCH ꢀC(O)Oꢀ), 2.90 (ddd as td, J = 12.0 Hz, J = 2.0 Hz,
2
1
1
1
H, 10aꢀH), 2.49 (br d, J = 14.0 Hz, 1H, 10eqꢀH), 1.98ꢀ1.90 (m as br d, J = 13.5 Hz, 1H, 8eqꢀH),
.70ꢀ1.23 (m and s overlapping, 8H, 8axꢀH, 7axꢀH, 7eqꢀH, 6aꢀH, 10axꢀH and 6ꢀCH , especially
3
+
.36, s, 6ꢀCH ), 1.04 (s, 3H, 6ꢀCH ). Mass spectum (ESI) m/z (relative intensity) 307 (M +H, 65),
3
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+
+
2
89 (100, M ꢀOH). Exact mass (ESI) calculated for C H 0 (M +H), 307.1545; found 307.1540.
17 23 5
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
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4
4
4
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5
5
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5
5
5
5
5
6
LC/MS analysis (Waters MicroMass ZQ system) showed purity 96.5% and retention time 3.7 min
for the title compound.
0
1
2
3
4
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9
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Methyl 2-(3,5-dimethoxyphenyl)-2-methylpropanoate (9). A suspension of carboxylic acid 8
(11.4 g, 50.9 mmol), MeI (36.2 g, 225 mmol) and K CO (10.6 g, 76 mmol) in anhydrous acetone
2 3
(200 mL) was stirred overnight at room temperature under argon. Insoluble materials were filtered
off and the filtrate was evaporated under reduced pressure. The residue was diluted with diethyl
ether and water and the ethereal layer was separated. The aqueous layer was extracted with diethyl
ether and the combined organic phase was washed with water and brine, dried (MgSO ) and
4
concentrated in vacuo. Purification by flash column chromatography on silica gel (5ꢀ20% AcOEt in
hexane) gave the title compound as colorless oil in 72% yield (8.75 g). IR (neat) 3412, 2962, 2932,
ꢀ1 1
1
728 (s, >C=O), 1702 cm ; H NMR (500MHz, CDCl ) δ 6.47 (d, J = 2.0 Hz, 2H, ArH), 6.33 (t, J
3
1
3
=
2.0 Hz, 1H, ArH), 3.79 (s, 6H, OMe), 3.66 (s, 3H, COOMe), 1.33 (s, 6H, ꢀC(CH ) ꢀ). C NMR
3 2
(
100 MHz CDCl ) δ 181.8 (>C=O), 160.7, 146.2, 104.5, 98.4, 55.3 (ꢀOMe), 46.3, 26.2. Mass
3
+
spectrum (ESI) m/z (relative intensity) 239 (M +H, 100).
Methyl 2-(3,5-dihydroxyphenyl)-2-methylpropanoate (10). To a stirred solution of methyl ester
o
9 (5.0 g, 21.0 mmol) in dry CH Cl (210 mL) at ꢀ78 C, under an argon atmosphere, was added
2
2
boron tribromide (65.1 mL of 1M solution in CH Cl ). Following the addition, the reaction
2
2
o
temperature was gradually raised over a period of 1 hour to 0 C, and the stirring was continued at
that temperature until the reaction was completed (2.5 h). Unreacted boron tribromide was
o
destroyed by the addition of MeOH at ꢀ78 C. The resulting mixture was warmed to room
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2
2
2
2
2
2
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3
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3
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6
temperature, and volatiles were removed in vacuo. The residue was dissolved in diethyl ether and
washed with water and brine and dried (MgSO ). Solvent evaporation and purification by flash
4
column chromatography on silica gel (10ꢀ40% AcOEt in hexane) gave 10 as a pale yellow
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
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8
9
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9
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6
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9
0
ꢀ1
1
semisolid material in 71% yield (3.1 g). IR (neat) 3180, 1725 (s, >C=O), 1701cm ; H NMR
(500MHz, CDCl ) δ 6.41 (d, J = 2.0 Hz, 2H, ArH), 6.24 (t, J = 2.0 Hz, 1H, ArH), 5.67 (br s, 2H,
3
1
3
OH), 3.66 (s, 3H, COOMe), 1.52 (s, 6H, ꢀC(CH ) ꢀ). C NMR (100 MHz CDCl ) δ 177.6 (>C=O),
3
2
3
1
56.9, 147.5, 105.5, 101.4, 52.5 (ꢀOMe), 46.4, 26.2. Mass spectrum (ESI) m/z (relative intensity)
+
211 (M +H, 100).
[(6aR, 10aR)-1-Hydroxy-3-(1-methoxy-2-methyl-1-oxopropan-2-yl)-6,6-dimethyl-6a,7,10,10a-
tetrahydro-6H-benzo[c]chromen-9-yl] methyl pivalate (11). To a stirred solution of resorcinol
1
0 (1.0 g, 4.76 mmol) and pivalate ester 6 (1.32 g, 5.24 mmol) in anhydrous CH Cl (24 mL) at ꢀ20
2 2
o
C, under an argon atmosphere, was added boron trifluoride diethyl etherate (3.53 mL, 28.6 mmol)
o
dropwise. Stirring was continued at ꢀ20 C for 1 h and then at room temperature for 1 h. The
reaction mixture was quenched with saturated aqueous NaHCO solution and diluted with CH Cl .
3
2
2
The organic layer was separated and the aqueous layer was extracted with CH Cl . The combined
2
2
organic phase was washed with water and brine, dried (MgSO ) and concentrated in vacuo. The
4
residue was purified by flash column chromatography on silica gel (15% ethyl acetate in hexane) to
give 11 (846 mg, 40% yield) as a light yellow gum. R = 0.3 (20% acetone in hexane). IR (neat)
f
ꢀ1
1
3
418, 2954, 1725 (s, >C=O), 1705cm ; H NMR (500MHz, CDCl ) δ 6.43 (d, J = 2.0 Hz, 1H, 4ꢀ
3
H), 6.31 (d, J = 2.0 Hz, 1H, 2ꢀH), 5.77 (m as d, J = 4.0 Hz, 1H, 8ꢀH), 4.53 (d, J = 13.0 Hz, 1H, half
of an AB system, ꢀCH Oꢀ), 4.50 (d, J = 13.0 Hz, 1H, half of an AB system, ꢀCH Oꢀ), 3.67 (s, 3H, ꢀ
2
2
OMe), 3.42 (dd, J = 16.0 Hz, J = 4.0 Hz, 1H, 10αꢀH), 2.73 (td, J = 11.0 Hz, J = 5.0 Hz, 1H, 10aꢀH),
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.26 (m as d, J = 16.5 Hz, 1H, 7αꢀH), 1.96ꢀ1.82 (m, 3H, 10βꢀH, 7βꢀH, 6aꢀH), 1.52 (brs, 6H, ꢀ
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
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5
5
5
5
5
5
5
6
C(CH ) ꢀ),1.41(s, 3H, 6βꢀMe), 1.23(brs, 9H, ꢀC(CH ) ) 1.13 (s, 3H, 6αꢀMe); Mass spectrum (ESI)
3 2
3 3
+
+
+
m/z (relative intensity) 445 (M +H, 90), 385 (M ꢀCOOMe, 45), 342 (M ꢀ(CH ) COOH, 100), 283
3
3
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
+
(
M ꢀ(CH ) COOH, ꢀCOOCH , 51).
3 3 3
2
-(3, 5-Dihydroxyphenyl)-2-methylpropanoic acid (12). To a stirred solution of 8 (2.54 g, 11.33
mmol) in dry CH Cl (75 mL) at ꢀ78 °C, under an argon atmosphere, was added boron tribromide
2
2
(39.6 mL, 39.6 mmol, 1M solution in CH Cl ). Following the addition, the reaction mixture was
2
2
gradually warmed to room temperature and stirred for 3 h. Unreacted boron tribromide was
quenched by the addition of methanol and ice at 0 °C. The resulting mixture was warmed to room
temperature, and volatiles were removed in vacuo. The residue was diluted with diethyl ether and
washed with saturated aqueous NaHCO solution, water and brine. The organic layer was dried
3
(
MgSO ), filtered, and concentrated under reduced pressure. Purification by flash column
4
chromatography on silica gel (40% diethyl ether in hexane) gave 12 (2.17 g, 98% yield) as white
15
solid with physical, spectral and analytical data identical to those reported earlier.
Butyl-2-(3,5-dihydroxyphenyl)-2-methylpropanoate (13). A stirred mixture of 12 (2.0 g, 10.20
mmol), bromobutane (2.1 g, 15.30 mmol) and sodium bicarbonate (1.05 g, 12.24 mmol) in
o
anhydrous DMF (3 mL) was heated at 165 C for 12 min using microwave irradiation. The reaction
mixture was cooled to room temperature and diluted with water and ethyl acetate. The organic layer
was washed with brine, dried (MgSO ), and concentrated in vacuo. The residue was
4
chromatographed on silica gel (50% ethyl acetate in hexane) to give 13 (2.5 g, 92% yield) as a light
ꢀ1
1
brown viscous oil. IR (neat): 3370, 2963, 1698, 1600, 1465, 1271, 1142 cm ; H NMR (500 MHz,
ACS Paragon Plus Environment