Structural and stereoelectronic requirements for the inhibition of mammalian 2,3-oxidosqualene cyclase by substituted isoquinoline derivatives

Article abstract of DOI:10.1021/jm9504621

2,3-Oxidosqualene lanosterol-cyclase (OSC; EC 5.4.99.7) is an attractive target for the design of compounds that block hepatic cholesterol biosynthesis. (4aα,5α,6β,8aβ)-Decahydro-5,8a-dimethyl-2-(1,5,9- trimethyldecyl)-6-isoquinolinol (1) and simplified a

Full text of DOI:10.1021/jm9504621

2302
J . Med. Chem. 1996, 39, 2302-2312
Str u ctu r a l a n d Ster eoelectr on ic Requ ir em en ts for th e In h ibition of Ma m m a lia n
2,3-Oxid osqu a len e Cycla se by Su bstitu ted Isoqu in olin e Der iva tives
Martine M. Barth,† J ean L. Binet,† Didier M. Thomas,† Daniel C. de Fornel,† Soth Samreth,†
Francis J . Schuber,*,‡ and Patrice P. Renaut*,†
Laboratoires Fournier SCA, 50 rue de Dijon, 21121 Daix, France, and Laboratoire de Chimie Bioorganique, URA CNRS 1386,
Faculte´ de Pharmacie, 67400 Illkirch, France
Received J une 23, 1995X
2,3-Oxidosqualene lanosterol-cyclase (OSC; EC 5.4.99.7) is an attractive target for the design
of compounds that block hepatic cholesterol biosynthesis. (4aR,5R,6â,8aâ)-Decahydro-5,8a-
dimethyl-2-(1,5,9-trimethyldecyl)-6-isoquinolinol (1) and simplified analogs have been devised
to inhibit this enzyme by mimicking the postulated pro-C-8 high-energy intermediary
carbocation occurring during the cyclization-rearrangement pathway. In order to gain an
understanding into the mechanism by which these types of molecules inhibit OSC, we have
synthesized a series of substituted isoquinoline derivatives 3 and investigated the structural
and stereoelectronic requirements, and their stringency, that make 3 potential high-energy
intermediate analogs of OSC. Determination of the IC50 values of the different compounds,
with rat liver microsomal cyclase, allowed the study of the relative importance of (i) the nature
and the stereochemistry of the nitrogen side chain, (ii) the presence of methyl groups at C-5
and C-8a (ring junction), (iii) the presence and stereochemistry of the C-6 hydroxyl group, (iv)
the nature of the ring junction, and (v) the absolute configuration of the bicyclic system. The
resulting structure-activity relationships seem to validate the mechanism of action of these
inhibitors as analogs of a pro-C-8 high-energy intermediate and delineate the minimal
requirements for the design of efficient isoquinoline-based, or simplified, OSC inhibitors.
In tr od u ction
The cyclization is believed to be triggered by a general
acid-catalyzed epoxide ring opening assisted by a neigh-
boring π-bond.12 The concertedness of the ensuing
overall annulation and backbone rearrangement is a
matter of debate.13 However, for entropic reasons and
from experimental evidence, the reaction is more likely
to proceed through a series of discrete conformationally
rigid carbocationic intermediates.14 Thus, in some
plants, a bicyclic intermediate occurring at position pro-
C-8, besides its normal transformation into triterpen-
oids, can be trapped by a water molecule.15 Moreover
J ohnson et al. have demonstrated the dramatic ef-
ficiency of intermediary carbocation-stabilizing auxiliary
groups in biomimetic polyene cyclization reactions.16
This indicates that in the cyclization of a molecule such
as 2,3-oxidosqualene both conformational and electronic
environments, provided by the active site of the cyclase,
are of importance. Interestingly, a methylidene auxil-
iary group, when introduced at position C-29 of 2,3-
oxidosqualene, was shown to lead to a mechanism-based
type inhibitor of the cyclase,17 presumably by trapping
the intermediary proto-sterol carbocation.
Carbocationic high-energy intermediates occurring
during the reaction pathway of enzymes catalyzing, for
example, sterol biosynthesis, can be mimicked by struc-
turally related molecules which bear strategically po-
sitioned positively charged groups, such as ammonium
derivatives.18 In many cases, these analogs proved to
be very powerful inhibitors of these enzymes.19 This
approach was quite fruitful in inhibiting 2,3-oxi-
dosqualene lanosterol-cyclase.19,20 The first example
along these lines was the design of 2-aza-2,3-dihy-
drosqualene and related compounds which were thought
to mimic the charge deficiency at pro-C-2 of the sub-
strate during the oxirane ring opening.21 Importantly
when an aza group was introduced at position 2 in 4,4,-
A prime target for the design of hypocholesterolemic
drugs is HMG-CoA reductase, the major regulatory and
rate-limiting enzyme in the biosynthetic pathway of
cholesterol.1 Inhibition of cholesterol synthesis at a
later stage, however, which avoids possible limitations
in mevalonate (a metabolite involved in many other
functions2) is also of interest. Among the different
possible target enzymes, 2,3-oxidosqualene lanosterol-
cyclase (OSC; EC 5.4.99.7) is particularly attractive. In
mammalian cells, inhibitors of this enzyme affect very
efficiently the biosynthesis of cholesterol by a dual
mechanism.3-6 In addition to the direct inhibition of
lanosterol formation, inhibition of the cyclase also
results in the accumulation of (3S)-2,3-oxidosqualene
and (3S,22S)-2,3:22,23-dioxidosqualene. This latter
metabolite, which is preferentially cyclized into (24S)-
24,25-epoxylanosterol,7 leads ultimately to the forma-
tion of (24S)-24,25-epoxycholesterol, a known repressor
of HMG-CoA reductase activity.8,9 Thus, cyclase inhibi-
tors can also affect cholesterol biosynthesis via a regula-
tory pathway; this mode of action might lead to an
amplification of a normal enzyme inhibition.
Rational design of inhibitors, such as transition state
analog, relies on the knowledge of the molecular mech-
anism of the target enzyme. The transformation of all-
trans-2,3-oxidosqualene into lanosterol is a fascinating
reaction for both the chemist and enzymologist. Its
elusive mechanism has attracted much attention in the
past10 and a renewed interest during recent years.
Work by the group of Corey11 has emphasized substrate
folding for the correct cyclization-rearrangement step.
† Laboratoires Fournier SCA.
‡ URA CNRS 1386.
X Abstract published in Advance ACS Abstracts, May 1, 1996.
S0022-2623(95)00462-6 CCC: $12.00 © 1996 American Chemical Society

Inhibition of OSC by Isoquinolines
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 12 2303
Sch em e 1. General Methods A-Ca
a
Reagents: (a) R1X, K2CO3, CH3CN, reflux; (b) R9COR10
,
NaBH3CN, CH3OH; (c) R8CO2H, CDI, THF. Groups R1-R7 are
defined in Tables 1 and 3.
Sch em e 2a
F igu r e 1. 2,3-Oxidosqualene lanosterol-cyclase inhibitor 1,
designed to mimic the high-energy intermediate 2 (B-ring
closure), and the series of isoquinolines 3 studied in this
paper.
10â-trimethyl-trans-decal-3â-ol (TMD), i.e., a decalin
structure which was previously found to be a cyclase
inhibitor,22 much less inhibition was observed.23 The
same was true for the first generation of isoquinolines.21
Mimicking the postulated pro-C-8 intermediary car-
bocation 2 (Figure 1) by (4aR,5R,6â,8aâ)-decahydro-5,-
8a-dimethyl-2-(1,5,9-trimethyldecyl)-6-isoquinolinol (com-
pound 1, Figure 1) led, however, to an efficient cyclase
inhibitor.24 This indicates that these charged inhibitors
seemingly need a hydrophobic tail, which mimics the
long squalene molecule, in order to adopt a correct
positioning in the enzyme active site. Many analogs of
the other intermediary carbocationic intermediates
along the cyclization-rearrangement pathway have
since been synthesized and tested.25
Since the original publications on compound 1, other
workers have evaluated novel isoquinoline and simpli-
fied analogs.26 In order to gain an understanding into
the mechanism by which these types of molecules inhibit
OSC, in the present study, we have synthesized and
tested analogs structurally related to the proposed
intermediate 2, in an attempt to mimic the relevant
structural attributes of the latter. Thus, a methyl group
in position 5â of ring A and the carbon-carbon double
bonds of the lateral side chain were reintroduced. This
was done for the deca- and octahydroisoquinoline series.
Subsequently, we evaluated the importance of different
structural elements for the inhibition of OSC by inves-
tigating stereochemical and electronic aspects. The
generalized sum of these structures is represented by
the general structure 3 (Figure 1).
a
Reagents: (a) MVK, CH3ONa, CH3OH, 50 °C; (b) (i) (R)-(+)-
PhCH(CH3)NH2, toluene, (ii) MVK, THF, 50 °C; (c) (i) (S)-(-)-
PhCH(CH3)NH2, toluene, (ii) MVK, THF, 50 °C; (d) CH3ONa,
CH3OH, 50 °C.
of the isoquinoline with CDI-activated acid (method C).
The bicyclic derivatives required for these syntheses
were prepared by methods outlined in Schemes 2-7.
Compound 5 (Scheme 2) was prepared in a one-step
procedure using Robinson annelation27 starting from
1-(phenylmethyl)-3-methyl-4-piperidone (4) and methyl
vinyl ketone (MVK). An alternative method, via a two-
step process, according to Stork28 was used for the
synthesis of 21 (Scheme 6) starting from 1-(phenyl-
methyl)-4-piperidone (20) and ethyl vinyl ketone (EVK).
Since these methods were not suitable for the enanti-
oselective preparations of the optical isomers of 5, we
developed an original synthesis to prepare (-)-5 and
(+)-5 via a three-step procedure by reference to
D’Angelo’s work29 (Scheme 2). The first step of this
reaction between 4-piperidone derivative 4 and (+)-R-
methylbenzylamine on one hand and (-)-R-methylben-
zylamine on the other hand provided a chiral imine-
enamine equilibrium ensuring a good diastereofacial
differentiation for MVK in the subsequent Michael
reaction leading to (-)-6 and (+)-6. Bicycles (+)-5 and
(-)-5 were respectively obtained by cyclization of (-)-6
and (+)-6 with sodium methoxide in methanol. Optical
purities of these compounds were determined by NMR
analysis in the presence of the chiral Eu(hfc)3 shift
reagent and were better than 95%. The attribution of
absolute stereochemistry was done using empirical
D’Angelo’s rules.29
Ch em istr y
The compounds listed in Tables 1-3 were synthesized
by the routes shown in Scheme 1. The preparation
involved the alkylation of isoquinoline derivatives with
either halides or sulfonates in acetonitrile in the pres-
ence of K2CO3 (method A) or with carbonyl derivatives
using a reductive amination procedure with NaBH3CN
(method B). Amide derivative was obtained by acylation

2304 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 12
Sch em e 3. General Methods D-Fa
Barth et al.
Sch em e 5a
a
Reagents: (a) H2, PtO2, CH3CO2H; (b) CH3Li, THF, 0 °C.
Sch em e 6a
a
a
Reagents: (a) ROCOCl, K2CO3, reflux; (b) tert-BuOH, tert-
Reagents: (a) (i) pyrrolidine, toluene, (ii) EVK, toluene, reflux,
BuOK, CH3I; (c) Me3SiCl, NaI, CH3CN; (d) NaBH4, CH3OH, -30
°C; (e) (i-PrO)3Al, i-PrOH, reflux; (f) H2, 10% Pd/C, CH3CO2H.
(iii) CH3ONa, toluene; (b) Li, NH3, CH3I, -60 °C; (c) H2, Pd/C,
ethyl acetate.
Sch em e 4a
Sch em e 7a
a
Reagents: (a) H2SO4, isopropenyl acetate; (b) EtOH, NaBH4;
(c) EtOH, concentrated HCl; (d) CH3Li-LiBr, THF.
Reaction of racemic 5 or its enantiomers (+)-5 and
(-)-5 (Scheme 3) with ethyl or benzyl chloroformate
provided respectively 7a ,b, (+)-7a , and (-)-7a (method
D). Alkylation of these compounds according to Atwa-
ter30 with iodomethane and potassium tert-butoxide in
tert-butyl alcohol gave respectively 8a ,b, (-)-8a , and
(+)-8a (method E). Cleavage of the carbamate group
was performed using Me3SiCl/NaI methodology31 from
8, (+)-8a , and (-)-8a leading to 9, (-)-9, and (+)-9.
Reduction of 9, (+)-9, and (-)-9 with sodium borohy-
dride (method F) at -30 °C gave almost exclusively 6â-
hydroxy derivatives 10, (-)-10, and (+)-10. Reduction
of 8b to afford 6R-hydroxy derivatives was attempted
according to various literature procedures.32-35 The best
result was obtained using aluminum isopropoxide and
gave a 50:50 mixture of 6R-derivative 11a and 6â-
derivative 11b which were separated by flash chroma-
tography. Assignment of the stereochemistry of 11a ,b
was made by 1H NMR analysis using chemical shift,
coupling constant, and half-height width36 of the proton
signal borne by the C-6. Selective hydrogenolysis of 11a
in the presence of palladium on carbon afforded 12.
We prepared demethylated compound 28k via inter-
mediate 14 (Scheme 4). Deconjugation of the R,â-
unsaturated ketone 7a with isopropenyl acetate in acidic
medium afforded an unstable enol acetate37 which was
a
Reagents: (a) TsNHNH2, CH3CO2H; (b) NaH, toluene, reflux;
(c) H2, Pd/C, CH3CO2H.
reduced with NaBH4 and then treated with acid to give
13 after purification by chromatography.38 Deprotection
of 13 using MeLi and LiBr gave 14.
Decahydroisoquinolin-6-ols 18 and 19 were prepared
as indicated in Scheme 5. Reduction of 8a using method
F gave 15. Hydrogenation of 15 with PtO2 as catalyst
led to a 50:50 mixture of trans and cis isomers 16 and
17 which were separated by chromatography. The
1
nature of the ring junction was supported by H NMR
analysis by analogy with studies in steroid series.39
Compounds 18 and 19 were obtained after deprotection
of 16 and 17 using MeLi in THF.40
The preparation of 24 starting from 20 is illustrated
in Scheme 6. Robinson annelation of 20 led to the
known compound 21, which afforded 22, the pure trans-
fused ring, using the reductive alkylation procedure of
Heathcock41 or Stork.42 After stereoselective reduction
of the carbonyl function and hydrogenolysis of the
benzyl protective group, 22 gave 24.

Inhibition of OSC by Isoquinolines
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 12 2305
Ta ble 1. 1,2,3,5,6,7,8,8a-Octahydroisoquinoline Derivatives
yield, %
mp, °C (method)
OSC inh
IC50, µM
compd
R1
R2
R3
R4 R5
formulaa
f
10
H
CH3 CH3
H
OH 157-162
50
C12H21NO‚C4H4O4
>100
0.6
28a
28b
28c
28d
28e
28f
28g
28h
28i
(E)-CH(CH3)(CH2)2CHdC(CH3)(CH2)2CHdC(CH3)2 CH3 CH3
(Z)-CH(CH3)(CH2)2CHdC(CH3)(CH2)2CHdC(CH3)2 CH3 CH3
(E)-(CH2)3CHdC(CH3)(CH2)2CHdC(CH3)2
CH(CH3)(CH2)2CHdC(CH3)2
n-C12H25
n-C14H29
n-C18H37
CH2Ph
(CH2)3Ph
H
H
H
H
H
H
H
H
H
OH oilb
66 (B)
60 (B)
70 (A)
30 (A)
(A)
40 (A)
50 (A)
(A)
C25H43NO‚CH4O3Sg
C25H43NO
OH oilc
6.8
1.0
17.0
1.1
2.9
CH3 CH3
CH3 CH3
CH3 CH3
CH3 CH3
CH3 CH3
CH3 CH3
CH3 CH3
OH 118
C24H41NO‚C2H2O4
C20H35NO
C24H45NO‚C2H2O4
C26H49NO
C30H57NO
OH oild
h
OH 106-108
OH 53
OH 59-62
OH 88-105
OH 90
>100
h
C19H27NO‚C2H2O4
25
h
(A)
C21H31NO‚C2H2O4
>25
20
16.4
0.68
>25
h
28j
(E)-(CH2)3CHdC(CH3)(CH2)2CHdC(CH3)2
n-C12H25
(E)-CO(CH2)2CHdC(CH3)(CH2)2CHdC(CH3)2
CH3 CH3 OH
H
108
60 (A)
48 (A)
80 (C)
C24H41NO‚C2H2O4
h
28k
29
H
H
H
H
OH 100
C22H41NO‚C2H2O4
CH3 CH3
OH oile
C25H39NO2
TMDi
a
b
d
Analytical results are within (0.4% of theoretical values unless otherwise noted. n20D ) 1.5180. c n20 ) 1.5120. n20 ) 1.6560.
D
D
D
e n20 ) 1.5240. f Fumarate. Mesylate. TMD ) 4,4,10â-trimethyl-trans-decal-3â-ol.
g
i
Ta ble 2. Optical Isomers of Compound 28c
compd
config
mp, °C
RD (deg)
yield, % (method)
formulaa
OSC inh IC50, µM
28l
28m
6S,8aS
6R,8aR
65-73
67-75
-38.4 (c 0.63, MeOH)
+38.4 (c 0.64, MeOH)
84 (A)
86 (A)
C24H41NO‚C2H2O4‚0.5H2Ob
0.6
1.60
C
24H41NO‚C2H2O4‚0.5H2Ob
a
b
Analytical results are within (0.4% of theoretical values unless otherwise noted. Oxalate.
The preparation of 27 is illustrated in Scheme 7.
Using the Bamford-Stevens43,44 reaction, 26 was ob-
tained from 22, and a subsequent catalytic hydrogena-
tion of 26 led to 27.
detrimental for the inhibition as shown by compounds
28f,g. Aryl-substituted alkyl chains are also not suit-
able for a good inhibition. Similar findings have already
been described with other cyclase inhibitors.25b,26a That
suggests that the active site of OSC contains a pocket
which can only tolerate moieties closely related to the
linear part of intermediate 2. Interestingly, the amide
29 is as potent as the corresponding amine 28c despite
the absence of the fully charged nitrogen atom. A simi-
lar observation was made by Wannamaker et al. with a
related series of compounds.26a It seems therefore pos-
sible that to mimic 2, a full charge is not an absolute
requisite and a dipole, provided by an amide or a sulf-
oxide,25f when suitably positioned could also interact
with the putative group from the OSC active site that
stabilizes the pro-C-8 carbocationic intermediate. In-
terestingly, the amide analog of 2-aza-2,3-dihydro-
squalene, which was designed to mimic the high-energy
intermediate generated in the A-ring formation, was a
far less potent inhibitor than the parent compound.21
Then we focused our attention on the methyl groups
of the isoquinoline ring. Comparison of 28e,k clearly
shows that substitution of the carbon C-5 is important
for the inhibition, and results published for compound
145 seem to indicate that the equatorial methyl plays
probably the major role. On the other hand, removal
of the C-8 axial methyl group in the trans-decahydroiso-
quinoline series led to a 9-fold increased inhibition (28n
vs 28o, Table 3).
Resu lts a n d Discu ssion
2,3-Oxidosqualene lanosterol-cyclase inhibition was
measured using rat liver microsomes and synthetic
(R,S)-2,3-oxidosqualene. The inhibition potencies of our
compounds, expressed as IC50 values, are presented in
Tables 1-3. We have first examined the influence of
the nature of the substituent linked to the nitrogen. The
secondary amine 10 is totally devoid of inhibitory
activity at a concentration as high as 100 µM. By sharp
contrast, 28a which bears a side chain which has the
closest resemblance to that of the intermediate 2 is a
good inhibitor with an IC50 of 0.6 µM. Interestingly 28b,
the Z-isomer of 28a , is about 10-fold less potent.
Moreover, removal of the terminal isoprenyl unit of 28a
afforded a 28-fold less active compound 28d . Due to the
presence of the methyl group carried by the side chain
R to the nitrogen, 28a ,b,d are mixtures of diastereoi-
somers. In order to simplify the interpretation of results
obtained with such compounds, we synthesized the
corresponding normethylated analog of 28a , 28c, which
is a single racemate. Its IC50 is 1.0 µM. A simple C12
alkyl straight chain with the same carbon condensation
as the substituent of 28c led to compound 28e which
has the same potency. Lengthening of the chain is

2306 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 12
Ta ble 3. Decahydroisoquinoline Derivatives
Barth et al.
yield, %
(method)
OSC inh
IC50, µM
compd
R1
R5
R6
R7
mp, °C
formulaa
28n
28o
28p
28q
(E)-(CH2)3CHdC(CH3)(CH2)2CHdC(CH3)2 OH HR
H
97-98
72 (A)
60 (A)
61 (A)
66 (A)
C23H41NO‚C2H2O4‚0.5H2Ob
C24H43NO‚C2H2O4‚1.5H2Ob
C24H43NO‚C2H2O4‚H2Ob
C23H45N‚C7H8O3Sc
0.10
0.9
0.7
(E)-(CH2)3CHdC(CH3)(CH2)2CHdC(CH3)2 OH HR CH3 136
(E)-(CH2)3CHdC(CH3)(CH2)2CHdC(CH3)2 OH Hâ CH3 124
N-C12H25
H
HR
H
121-124
>25
a
b
Analytical results are within (0.4% of theoretical values unless otherwise noted. Oxalate. c Tosylate.
F igu r e 2. Lineweaver-Burk plots of the inhibition of rat microsomal oxidosqualene cyclase by different concentrations of compound
28a . The insets show the plot of intercepts of the lines obtained in the Lineweaver-Burk plots versus the concentrations of
compound 28a . The concentrations of 28a were (1) 1.2, ([) 0.6, (2) 0.2, and (b) 0 µM. A: curves are plotted according to a
noncompetitive inhibition; Km ) 33 ( 3.5 µM, Vmax ) 0.57 ( 0.04 nmol min-1 mg-1 of proteins, and Ki ) 0.40 ( 0.02 µM. B:
curves are plotted according to a competitive inhibition; Km ) 29 ( 6 µM, Vmax ) 0.49 ( 0.06 nmol min-1 mg-1 of proteins, and
Ki ) 0.30 ( 0.04 µM.
The nature of the ring junction has been also inves-
tigated. Comparison of compounds 28c,o,p shows that
they are equally potent.
We then considered the orientation of the hydroxyl
group at position 6 with compounds 28j,c. As expected,
the R-isomer 28j is 20-fold less active than the â-isomer
28c. Finally, removal of the hydroxyl group led to 28q
(Table 3) which is totally inactive at 25 µM.
It is worth noting that all the above-mentioned data
were obtained with racemic compounds. Since the
mimicked intermediate 2 has the depicted absolute
configuration, it was necessary to verify the importance
of the absolute configuration on the inhibitory potency
of our compounds. We chose to synthesize the enanti-
omers of 28c as one of the best representative of
intermediate 2. The IC50 of 28l (Table 2) which has the
“natural” stereochemistry is 0.6 µM, whereas that of
28m which has the “unnatural” stereochemistry is 1.6
µM. This small difference in activity between the two
enantiomers was quite unexpected. Thus, the different
results obtained here raise the question: Do these
molecules really mimic a high-energy intermediate
binding to the active site of the cyclase? The answer
has to take into account several kinds of arguments that
are discussed below.
Kinetic studies were undertaken to find out whether
our compounds were competitive inhibitors. Using rat
liver microsomal OSC, we found using Lineweaver-
Burk plots (Figure 2) that the leading compound 28a ,
contrary to our expectation, seems to be a noncompeti-
tive inhibitor (Ki: 0.4 µM) (Figure 2, plot A) rather than
a competitive one (Figure 2, plot B) when using the
Akaike criterion test.46 It was nevertheless difficult to
distinguish between competitive and noncompetitive
inhibition. However, when using the method of joint
saturation (Figure 3) with increasing concentrations of
substrate and inhibitor, at a fixed ratio S/I, it appears
that V(S+I) is finite at high concentrations of substrate,
albeit smaller than VS. This result is consistent with a
competitive inhibition, on account of the counterproduc-
tive competition between substrate and inhibitor,
whereas with a noncompetitive inhibition, joint satura-
tion by substrate and inhibitor will entrap all the
enzyme into the unproductive ESI complex and V(S+I)
will fall to zero.47
These contradictory results underline the difficulty
in obtaining meaningful kinetic data, which rely on
steady state kinetic equations, when dealing with
membrane-associated enzymes (or integrated in mi-
celles) and hydrophobic substrate and inhibitors. The

Inhibition of OSC by Isoquinolines
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 12 2307
nitrogen of the ring and its side chain, and the equato-
rial C-5 methyl group, it appears from simple molecular
model examination of the enantiomers 28l,m that these
four elements can be easily superimposed. On the other
hand, the C-8a methyl groups are opposite in their
orientation as are the axial C-5 methyl groups. It is
interesting to note that the C-8a methyl group appears
to be detrimental for activity and that the C-5 axial
methyl group is not essential. Although at that point,
it is difficult to conclude definitely, we do think that our
results are compatible with the initially postulated
hypothesis. It should also be kept in mind that, al-
though formation of rings A and B can be rate-deter-
mining in biomimetic cyclization of 2,3-oxidosqualene
analogs,14,25f,50 we still ignore the relative heights of the
energy barriers occurring along the OSC-catalyzed
reaction pathway, and consequently it is hard to predict
the interaction energy that could be expected by mim-
icking a transient intermediate such as 2.
F igu r e 3. V versus [2,3-oxidosqualene] plots (using rat liver
microsomal enzyme) in the absence of compound 28a (b) and
in the presence of increasing concentrations of a mixture of
2,3-oxidosqualene and compound 28a at a fixed ratio of 60 (O).
Con clu sion
presence of the lipid environment could have a profound
effect on the apparent ability of the cyclase to bind
hydrophobic compounds, whose respective amounts in
the aqueous and membrane phases are determined by
their partition coefficient.48 The partitioning or micel-
lization may affect the apparent association constants
by making an unknown portion of substrate or inhibitor
unavailable for binding. It is worth mentioning that it
is the case with either an aqueous- or a lipid-faced
enzyme active site. In such anisotropic systems, non-
competitive inhibition has been found with OSC inhibi-
tors,21 whereas with purified enzyme, (4R,5R,6â,8aâ)-
decahydro-5,8a-dimethyl-2-(1-oxododecyl)-6-isoquino-
linol, which is closely related to our series, has been
found competitive with an apparent Ki of 28 nM.26a
Furthermore, inhibitors such as 28a designed to mimic
high-energy intermediates (HEI), could also be slow-
binding inhibitors with slow kon and koff rate constants,
compared to diffusion rates, during the formation of the
enzyme-inhibitor complex.49 The observed noncom-
petitive behavior may therefore reflect a reduced amount
of free enzyme. When the concentration of inhibitor
increases, a fraction of OSC accumulates as an initial
collision E-I complex, or as a more tight E-I* complex,
which does not noticeably dissociate. To give support
to this hypothesis, measurement of rate constants for
inhibitor-enzyme formation and dissociation with puri-
fied enzyme would be necessary.
In the present study we have synthesized a series of
compounds in order to investigate the structural re-
quirements for the isoquinolinol-based inhibitors of the
2,3-oxidosqualene lanosterol-cyclase. The resulting struc-
ture-activity relationships seem to validate the mech-
anism of action of these inhibitors as analogs of a high-
energy intermediate. In the course of this work, we
discovered that besides crucial elements that were
present in the initially designed compounds others are
not essential, and are even detrimental, for a good
inhibition. These findings could be helpful for the
correct understanding of the mechanism of action of the
cyclase and for the design of structurally simplified
inhibitors; some examples will be described elsewhere.
Exp er im en ta l Section
Gen er a l Meth od s. Melting points were determined on a
Bu¨chi melting point apparatus and are uncorrected. IR
spectra were measured on a Perkin Elmer 782 spectropho-
tometer. 1H and 13C NMR spectra were obtained on a Bruker
AC 300 spectrometer using tetramethylsilane as internal
reference. Structural assignments for all new compounds are
consistent with their spectra. Specific rotation determinations
were obtained using a Perkin Elmer 241 polarimeter. El-
emental analyses were performed on a Perkin Elmer 240c
apparatus. Molecular formulas followed by the symbols C,H,N
indicate that elemental analyses were found to be within
(0.4% of the theoretical values for C, H, and N.
1,3,4,7,8,8a -H exa h yd r o-8a -m et h yl-2-(p h en ylm et h yl)-
6(2H)-isoqu in olin on e (5). To a solution of 1 L of 0.55 M
sodium methoxide in methanol was added 101.5 g (0.5 mol) of
4. After the mixture was stirred at room temperature for 45
min, it was cooled at 5 °C and 62.3 mL (0.75 mol) of 3-buten-
2-one was added over a period of 2 h; then the mixture was
stirred overnight at room temperature. Concentrated hydro-
chloric acid (55 mL) was added, and the mixture was evapo-
rated. The residue was partitioned between water and ether,
and the organic layer was washed with water, dried (MgSO4),
and concentrated. The crude material was purified by chro-
matography on silica gel and eluted with CH2Cl2-ether (9:1)
Most of the elements of the structure-activity rela-
tionships observed with the present series of compounds
are in agreement with the hypothesis of mimicking 2.
Indeed, good inhibitors are obtained when the side chain
on the nitrogen has a length and a stereochemistry
similar to that of 2. C-5 methyl groups appear impor-
tant for activity, as is the case for the presence of a C-6
hydroxyl group with â-configuration. The negative
influence of the C-8a methyl group is more difficult to
explain, although it can be noticed that the correspond-
ing methyl group of 2 is not fully axial so it does not
occupy the same region of space as does the C-8a methyl
of 28a .
to afford a solid (37.8 g, 37%): mp 106 °C; IR (KBr, 1%) ν (cm-1
)
1670, 1600, 1500; 1H NMR (300 MHz, CDCl3) δ 7.33 (m, 5H),
5.73 (d, J ) 1.5 Hz, H5), 3.60 (d, J ) 13.4 Hz, H10), 3.43 (d, J
) 13.4 Hz, H10), 3.02 (m, H3), 2.72 (m, H4), 2.63 (dd, J ) 2,
11.2 Hz, H1), 2.45 (m, H7), 2.37 (m, H7), 2.15 (m, H4), 2.05 (m,
H3), 1.82 (d, J ) 11.2 Hz, H1), 1.78 (m, H8), 1.63 (m, H8), 1.38
(s, 3H); 13C NMR (CDCl3) δ 200 (C6), 168 (C4a), 124 (C5), 67
(C1), 63 (C10), 55 (C3), 38 (C8a), 35 (C8), 34 (C7), 33 (C4), 23 (C9).
Anal. (C17H21NO) C,H,N.
Results dealing with the absolute configuration of the
ring system are more puzzling and could be explained
as follows. If we assume that the key features for the
recognition of the inhibitors by the active site of the
cyclase are the oxygen of the hydroxyl group, the

2308 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 12
Barth et al.
(3R)-3-Met h yl-3-(3-oxob u t yl)-1-(p h en ylm et h yl)-4-p i-
p er id on e [(-)-6)]. To a solution of 30 g (0.15 mol) of 4 in
300 mL of anhydrous toluene was added 23 mL (0.18 mol) of
(R)-(+)-R-methylbenzylamine. The mixture was then stirred
under reflux for 20 h with elimination of water. Toluene and
excess of amine were evaporated to give 46 g of imine as a
yellow oil. The crude oil was dissolved in 300 mL of anhydrous
THF; then 15 mL (0.18 mol) of methyl vinyl ketone and 300
mg of hydroquinone were added. The mixture was stirred at
50 °C, sheltered from light for 2 days, and then diluted with
1 N HCl, made basic by adding 1 N NaOH, and extracted with
ethyl acetate. The organic layer was washed with brine, dried
(MgSO4), and concentrated. The crude material was chro-
matographed with toluene-AcOEt (9:1) to give 27.6 g (68%)
tert-butoxide. The stirred mixture was heated at 50 °C for 1
h; then a solution of 44 mL (0.7 mol) of methyl iodide in 50
mL of tert-butyl alcohol was added. After addition, the mixture
was heated at 50 °C for a further 1 h and then allowed to cool
at room temperature overnight. The mixture was poured onto
water and extracted with ethyl acetate. The extracts were
washed with water, dried (MgSO4), and concentrated. The
residue was purified by flash chromatography eluting with
31
toluene-AcOEt (9:1) to give 8b as an oil (23.6 g, 62%): nD
1.5375; IR (neat) ν (cm-1) 1710; 1H NMR (300 MHz, CDCl3) δ
7.36 (m, 5H), 5.56 (s) and 5.48 (total 1H), 5.18 (s, 2H), 4.34
(m, 1H), 3.90 (dd, J ) 12.5, 34.9 Hz, 1H), 3.71 (t, J ) 15.5 Hz,
1H), 2.73 (dd, J ) 11.9, 18.8 Hz, 1H), 2.56-2.43 (m, 2H), 1.77
(m, 2H), 1.25 (s, 6H), 1.10 (s) and 1.07 (s) (total 3H).
3,5,6,7,8,8a -H exa h yd r o-6-oxo-5,5,8a -t r im et h yl-2(1H )-
isoqu in olin eca r boxylic Acid Eth yl Ester (8a ). This com-
pound was obtained from 7a as described above (65%): mp
65-67 °C; IR (KBr, 1%) ν (cm-1) 1710; 1H NMR (300 MHz,
CDCl3) δ 5.53 (d, J ) 17.5 Hz, H4), 4.31 (m, 1H), 4.19 (m, 2H),
3.87 (d, J ) 12.4, 4.0 Hz, 1H), 3.68 (dd, J ) 7.8, 18.3 Hz, 1H),
2.62 (m, 1H), 2.57 (m, 1H), 2.45 (m, 1H), 1.77 (m, 2H), 1.30 (t,
J ) 7.2 Hz, 3H), 1.26 (s, 6H), 1.09 (s, 3H). Anal. (C15H23NO3)
C,H,N.
of (-)-6 as a yellow oil: [R]20 ) -52.7° (c ) 1.4, CHCl3); IR
D
1
(neat) ν (cm-1) 1720, 1600, 1500; H NMR (300 MHz, CDCl3)
δ 7.35 (m, 5H), 3.59 (d, J ) 13.2 Hz, 1H), 3.50 (d, J ) 13.2 Hz,
1H), 2.89 (m, 1H), 2.58 (m, 3H), 2.37 (m, 1H), 2.24 (m, 4H),
2.11 (s, 3H), 1.72 (m, 1H), 0.99 (s, 3H).
(3S)-3-Meth yl-3-(3-oxobu tyl)-1-(p h en ylm eth yl)-4-p ip er -
id on e [(+)-6]. Following the procedure described above, 4 was
reacted with (S)-(-)-R-methylbenzylamine and then with
methyl vinyl ketone to give (+)-6 as an oil (75%): [R]20
+47.7° (c ) 0.77, CHCl3).
)
D
(8a S)-3,4,6,7,8,8a -H exa h yd r o-6-oxo-5,5,8a -t r im et h yl-
2(1H)-isoqu in olin eca r boxylic Acid Eth yl Ester [(-)-8a ].
Following method E (-)-8a was obtained from (+)-7a as a
white solid (68%): mp 61 °C; [R]19D ) -42.6° (c ) 0.93, CHCl3).
(8a R)-3,4,6,7,8,8a -H exa h yd r o-6-oxo-5,5,8a -t r im et h yl-
2(1H)-isoqu in olin eca r boxylic Acid Eth yl Ester [(+)-8a ].
(+)-8a was prepared from (-)-7a according to method E: mp
(8aS)-1,3,4,7,8,8a-Hexah ydr o-8a-m eth yl-2-(ph en ylm eth -
yl)-6(2H)-isoqu in olin on e [(+)-5]. To a solution of 26 g (0.09
mol) of (-)-6 in 100 mL of anhydrous methanol was added
200 mL of MeONa (8% in methanol). The mixture was stirred
at 50 °C overnight and then diluted with water and extracted
with ethyl acetate. The organic layer was washed with brine,
dried (MgSO4), and concentrated. The crude material was
purified by chromatography with toluene-AcOEt (8:2) to
afford 19.7 g (81%) of (+)-5 as a light yellow powder: mp 87
61 °C; [R]19 ) +42.5°(c ) 0.85, CHCl3).
D
1,3,5,7,8,8a -Hexa h yd r o-5,5,8a -tr im eth yl-6(2H)-isoqu in -
olin on e (9). To a solution of 2.7 g (10.3 mmol) of 8a in 25
mL of CH3CN were added 4.6 g (30.7 mmol) of sodium iodide
and 2.6 mL (20.5 mmol) of trimethylsilyl chloride. The mixture
was stirred under reflux for 3 days and concentrated. The
residue was diluted with 1 N HCl and extracted with ethyl
acetate. The aqueous layer was made basic by adding 10 N
NaOH and extracted with ethyl acetate. The organic layer
was washed with brine, dried (MgSO4), and concentrated. The
crude material was purified by chromatography with CH2Cl2-
MeOH-NH4OH (8:2:0.5) to give 1.62 g (83%) of 9 as a white
°C; [R]20 ) +209.2° (c ) 0.81, CHCl3).
D
(8aR)-1,3,4,7,8,8a-Hexah ydr o-8a-m eth yl-2-(ph en ylm eth -
yl)-6(2H)-isoqu in olin on e [(-)-5]. Following the procedure
described above, (-)-5 was obtained (83%): mp 87 °C; [R]20
) -206.7° (c ) 0.8, CHCl3).
D
3,4,6,7,8,8a -Hexa h yd r o-8a -m eth yl-6-oxo-2(1H)-isoqu in -
olin eca r boxylic Acid P h en ylm eth yl Ester (7b). Meth od
D. A stirred mixture of 35 g (0.14 mol) of 5, 99 mL (0.7 mol)
of phenylmethyl chloroformate, and 28.6 g (0.2 mol) of potas-
sium carbonate in 300 mL of chloroform was refluxed over-
night; it was cooled and filtered. The filtrate was washed with
water and dried (MgSO4). Evaporation of solvent under
reduced pressure afforded 7b as a solid (36 g, 87%): mp 128
°C; IR (KBr, 1%) ν (cm-1) 1710, 1675; 1H NMR (CDCl3, 300
MHz) δ 7.37 (m, 5H), 5.77 (s, H5), 5.19 (d, J ) 12.3 Hz, 1H),
5.14 (d, J ) 12.3 Hz, 1H), 4.38 (m, 1H), 4.05 (m, 1H), 2.83 (m,
1H), 5.65-2.39 (m, 4H), 2.23 (m, 1H), 1.78 (m, 2H), 1.24 (m,
3H).
1
crystals: IR (neat) ν (cm-1) 3300, 1715; H NMR (300 MHz,
CDCl3) δ 5,57 (t, J ) 3.1 Hz, H4), 3.44 (d, J ) 1 Hz, H3, H3),
3.17 (m large, NH), 2.80 (d, J ) 12.4 Hz, H1), 2.61 (d, J ) 12.4
Hz, H1), 2.59 (ddd, J ) 17.9, 7.1, 10.3 Hz, H7), 2.52 (ddd, J )
17.9, 7.7, 3.7 Hz, H7), 1.76 (ddd, J ) 13.4, 10.3, 7.7 Hz, H8),
1.65 (ddd, J ) 13.4, 7.1, 3.7 Hz, H8), 1.25 (s, 6H), 1.14 (s, 3H).
(8a R)-1,3,5,7,8,8a -H exa h yd r o-5,5,8a -t r im et h yl-6(2H )-
isoqu in olin on e [(+)-9]. Following the same procedure as
above, (+)-9 was obtained from (+)-8a as a colorless oil
3,4,6,7,8,8a -Hexa h yd r o-8a -m eth yl-6-oxo-2(1H)-isoqu in -
olin eca r boxylic Acid Eth yl Ester (7a ). Following the
procedure described above, 5 was reacted with ethyl chloro-
formate to give 7a as a solid (90%): mp 98 °C; IR (KBr, 1%) ν
(82%): [R]19 ) +0.38° (c ) 1.03, CHCl3).
D
(8a S)-1,3,5,7,8,8a -H exa h yd r o-5,5,8a -t r im et h yl-6(2H )-
isoqu in olin on e [(-)-9]. Following the same procedure as
above (-)-9 was obtained from (-)-8a as a colorless oil (80%):
1
(cm-1) 1690, 1660, 1620, 1480; H NMR (300 MHz, CDCl3) δ
[R]19 ) -0.36° (c ) 1.15, CHCl3).
D
5.80 (s, H5), 4.35 (m, H3), 4.18 (m, H11, H11), 4.05 (m, H1), 2.84
(m, H3), 2.62 (m, H4, H7), 2.53 (m, H1), 2.49 (m, H4), 2.24 (m,
H7), 1.83 (m, H8, H8), 1.29 (m, 3H), 1.27 (s, 3H); 13C NMR
(CDCl3) δ 198.6 (C6), 165.3 (C10), 155.5 (C4a), 125.2 (C5), 61.6
(C11), 55.5 (C1), 44.1 (C3), 37.2 (C8a), 33.6 (C4, C8), 31.6 (C7),
21.2 (C12), 14.7 (C9). Anal. (C13H19NO3) C,H,N.
(6â,8a â)-1,2,3,5,6,7,8,8a -Oct a h yd r o-5,5,8a -t r im et h yl-6-
isoqu in olin ol (10). Meth od F . To a solution of 1.43 g (7.4
mmol) of 9 in 100 mL of anhydrous methanol at -20 °C was
added 370 mg (9.8 mmol) of NaBH4. The mixture was stirred
at -20 °C for 1 h and concentrated. The residue was diluted
with water and extracted with ethyl acetate. The organic layer
was washed with brine, dried (MgSO4), and concentrated. The
product was purified by recrystallization of the fumarate salt
in methanol-diisopropyl ether to afford 700 mg (50%) of the
(8a S)-3,4,6,7,8,8a -H exa h yd r o-8a -m et h yl-6-oxo-2(1H )-
isoqu in olin eca r boxylic Acid Eth yl Ester [(+)-7a ]. Fol-
lowing method D, (+)-5 was reacted with ethyl chloroformate
to give (+)-7a as a white solid (73%): mp 106 °C; [R]19
+200.0° (c ) 0.79, CHCl3).
)
amine 10 as a white powder: mp 146 °C; IR (KBr, 1%) ν (cm-1
)
D
1
3350, 1470; H NMR (300 MHz, CDCl3) δ 5.53 (dd, J ) 2.6,
3.4 Hz, H4), 3.47 (dd, J ) 2.6, 24.5 Hz, H3), 3.39 (dd, J ) 3.4,
24.5 Hz, H3), 3.26 (dd, J ) 4.8, 11.2 Hz, H6), 2.64 (d, J ) 12.2
Hz, H1), 2.44 (d, J ) 12.2 Hz, H1), 1.84-1.70 (m, H7, H7, OH,
NH), 1.44 (dt, J ) 13.2, 3.4 Hz, H8), 1.24 (s, 3H), 1.21 (m, H8),
1.14 (s, 3H), 1.05 (s, 3H). Anal. (C12H21NO) C,H,N.
(8a R)-3,4,6,7,8,8a -H exa h yd r o-8a -m et h yl-6-oxo-2(1H )-
isoqu in olin eca r boxylic Acid Eth yl Ester [(-)-7a ]. Fol-
lowing method D, (-)-5 was reacted with ethyl chloroformate
to give (-)-7a as a white solid (90%): mp 106 °C; [R]19
-202.0° (c ) 0.87, CHCl3).
)
D
3,5,6,7,8,8a -H exa h yd r o-6-oxo-5,5,8a -t r im et h yl-2(1H )-
isoqu in olin eca r boxylic Acid P h en ylm eth yl Ester (8b).
Meth od E. To a solution of 35 g (0.12 mol) of 7b in 300 mL
of tert-butyl alcohol was added 39.4 g (0.35 mol) of potassium
(6rR,8a rR)-1,2,3,5,6,7,8,8a -Octa h yd r o-5,5,8a -tr im eth yl-
6-isoqu in olin ol [(+)-10]. Following method F (+)-10 was
obtained from (+)-9 as a white powder (46%): mp 144 °C;
[R]19 ) +102.6° (c ) 0.71, CHCl3).
D

Inhibition of OSC by Isoquinolines
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 12 2309
(6âS,8a âS)-1,2,3,5,6,7,8,8a -Octa h yd r o-5,5,8a -tr im eth yl-
6-isoqu in olin ol [(-)-10]. Following method F (-)-10 was
and then poured onto 50 mL of saturated ammonium chloride
aqueous solution. The aqueous solution was extracted with
ethyl acetate, and the organic layer was evaporated to give
14 as a brown oil (700 mg, 56%): IR (NaCl) ν (cm-1) 3425,
prepared from (-)-9 (47%, white powder): mp 144 °C; [R]19
D
) -102.8° (c ) 0.73, CHCl3).
1
1625, 1460; H NMR (300 MHz, CDCl3) δ 5.41 (m, H4), 3.54
(6R,8a â)-3,5,6,7,8,8a -H exa h yd r o-6-h yd r oxy-5,5,8a -t r i-
m eth yl-2(1H)-isoqu in olin eca r boxylic Acid P h en ylm eth -
yl Ester (11a ) a n d (6â,8a â)-3,5,6,7,8,8a -Hexa h yd r o-6-h y-
dr oxy-5,5,8a-tr im eth yl-2(1H)-isoqu in olin ecar boxylic Acid
P h en ylm eth yl Ester (11b). A solution of 7.76 g (23.7 mmol)
of 8b in 100 mL of 2-propanol with 9.8 g (48 mmol) of
aluminum isopropoxide was stirred and heated for 7 h. The
mixture was brought to dryness, and the resulting residue was
stirred with 150 mL of 1 N HCl for 0.5 h and then extracted
with ethyl acetate. The organic layer was washed with brine,
dried (MgSO4), and concentrated under vacuum. Flash chro-
matography with cyclohexane-AcOEt (7.5:2.5) of the residue
gave the 6â-hydroxy derivative 11b as an oil (3 g, 38%).
Further elution provided the 6R-hydroxy derivative 11a as an
oil (2.4 g, 30%).
(m, H6), 3.23 (m, H3, H3), 2.76 (d, J ) 12.6 Hz, H1), 2.47 (d, J
) 12.6 Hz, H1), 2.32 (m, H5), 2.16 (m, H5), 1.88 (m, H7, OH,
NH), 1.60 (m, H7), 1.47 (m, H8), 1.22 (m, H8), 1.13 (s, 3H).
(6â,8a â)-3,5,6,7,8,8a -H exa h yd r o-6-h yd r oxy-5,5,8a -t r i-
m et h yl-2(1H )isoq u in olin eca r b oxylic Acid E t h yl E st er
(15). Following method F 15 was obtained from 8a as a
colorless oil (80%): IR (neat) ν (cm-1) 3480, 1700; 1H NMR
(300 MHz, CDCl3) δ 5.40 (d, J ) 13.3 Hz, 1H), 4.17 (m, 1H),
4.09 (m, 2H), 3.70 (m, 1H), 3.60 (m, 1H), 3.18 (dd, J ) 4.5,
11.2 Hz, 1H), 2.38 (m, 1H), 2.30 (s, OH), 1.79-1.64 (m, 2H),
1.44 (m, 1H), 1.20 (m, 3H), 1.16 (m, 1H), 1.09 (s, 6H), 0.95 (s, 3H).
(4a r,6â,8a â)-Octa h yd r o-6-h yd r oxy-5,5,8a -tr im eth yl-2-
(1H)-isoqu in olin eca r boxylic Acid Eth yl Ester (16) a n d
(4aâ,6â,8aâ)-Octah ydr o-6-h ydr oxy-5,5,8a-tr im eth yl-2(1H)-
isoqu in olin eca r boxylic Acid Eth yl Ester (17). To a solu-
tion of 3.07 g (11.5 mmol) of 15 in 15 mL of ethyl acetate were
added 40 mL of acetic acid and 620 mg of platinum oxide. The
mixture was stirred under 1 atm of H2 at room temperature
for 8 h. The catalyst was filtered and the filtrate evaporated
to give a mixture of 16 and 17. They were separated by
chromatography with toluene-AcOEt (8:2) to afford 1.3 g of
16 (42%) and 1.6 g of 17 (52%) as colorless oils.
11a : IR (neat) ν (cm-1) 3485, 1700; 1H NMR (300 MHz,
CDCl3) δ 7.33 (m, 5H), 5.47 (m) and 5.40 (m) (total 1H), 5.16
(s, 2H), 4.30 (m, 1H), 3.88-3.70 (m, 2H), 3.57 (m, 1H), 2.67
(dd, J ) 12.9, 16.9 Hz, 1H), 2.14 (m, 1H), 1.67 (m, 2H), 1.25-
0.83 (m, 11H).
11b: IR (neat) ν (cm-1) 3480, 1700; 1H NMR (300 MHz,
CDCl3) δ 7.33 (m, 5H), 5.53 (m) and 5.46 (m) (total 1H), 5.16
(s, 2H), 4.30 (m, 1H), 3.88-3.65 (m, 2H), 3.28 (m, 1H), 2.54
(dd, J ) 13.0, 18.1 Hz, 1H), 1.80 (m, 2H), 1.53 (m, 1H), 1.27
(d, J ) 10.4 Hz, 1H), 1.19-1.15 (m, 7H), 1.03 (m, 3H).
(6r,8a â)-1,2,3,5,6,7,8,8a -Octa h yd r o-5,5,8a -tr im eth yl-6-
isoqu in olin ol (12). A mixture of 2.1 g (6.37 mmol) of 11a in
3 mL of acetic acid with a catalytic amount of 10% palladium
on carbon was stirred under 1 atm of H2 pressure for 8 h. The
catalyst was filtered off and the filtrate evaporated. The
resulting residue was partitioned between 15% aqueous
sodium hydroxide solution and ethyl acetate. The extracts
were washed with brine, dried (MgSO4), and evaporated in
vacuo to afford 12 as a solid (0.89 g, 72%): mp 136 °C; IR (KBr)
ν (cm-1) 3185; 1H NMR (300 MHz, CDCl3) δ 5.48 (s, 1H), 3.55-
3.23 (m, 3H), 2.60 (m, 2H), 2.19-2.02 (m, 3H), 1.68-1.53 (m,
2H), 1.35-0.84 (m, 10H).
16: IR (neat) ν (cm-1) 3500, 1750, 1700, 1450; 1H NMR (300
MHz, CDCl3) δ 4.35 (m, 1H), 4.13 (m, 2H), 3.75 (m, 1H), 3.27
(dt, J ) 10.7, 5.5 Hz, 1H), 2.65 (m, 1H), 2.33 (m, 1H), 1.72 (m,
2H), 1.55 (m, 2H), 1.45 (m, 1H), 1.36 (d, J ) 5.5 Hz, OH), 1.27
(m, 3H), 1.17 (m, 1H), 0.99 (s, 3H), 0.95 (s, 3H), 0.78 (s, 3H).
Anal. (C15H27NO3) C,H,N.
17: IR (neat) ν (cm-1) 3500, 1750, 1700, 1440; 1H NMR (300
MHz, CDCl3) δ 4.12 (m, 3H), 3.56 (m, 2H), 2.63 (m, 2H), 1.75
(m, 3H), 1.65 (m, 1H), 1.62 (s, 1H, OH), 1.53 (m, 1H), 1.31 (m,
2H), 1.27 (m, 3H), 1.11 (s, 3H), 1.07 (s, 3H), 1.01 (s, 3H). Anal.
(C15H27NO3) C,H,N.
(4a r,6â,8a â)-Deca h yd r o-5,5,8a -tr im eth yl-6-isoqu in oli-
n ol (18). To a solution of 740 mg (2.75 mmol) of 16 in 10 mL
of anhydrous THF was added, at 0 °C, 9.6 mL (13.4 mmol) of
1.4 M methyllithium in ether. The mixture was stirred at 0
°C for 1 h and concentrated. The residue was diluted with 5
mL of water and extracted with ethyl acetate. The organic
layer was dried (MgSO4) and concentrated to afford 18 (540
mg, 99%) as a light yellow oil: 1H NMR (300 MHz, CDCl3) δ
3.26 (m, 1H), 3.20 (m, 1H), 2.58 (m, 1H), 2.52 (d, J ) 11.9 Hz,
1H), 2.25 (d, J ) 11.9 Hz, 1H), 1.80-1.55 (m, 2H), 1.47 (m,
2H), 1.36 (m, 1H), 1.26 (m, 1H), 1.23 (d, J ) 6.2 Hz, 1H, OH),
1.14 (m, 1H), 1.07 (s, 3H), 0.96 (s, 3H), 0.93 (s, 1H), 0.79 (s,
3H).
(6â,8a â)-3,5,6,7,8,8a -Hexa h yd r o-6-h yd r oxy-8a -m eth yl-
2(1H)-isoqu in olin eca r boxylic Acid Eth yl Ester (13).
A
solution of 12 g (0.05 mol) of 7a in 125 mL of isopropenyl
acetate and 1 mL of concentrated H2SO4 was heated at 110
°C while acetone, which was produced, was slowly distillated
for 2 h. After cooling, the solution was evaporated to give a
black oil which was used without purification. This oil was
dissolved in 170 mL of ethanol, and the stirred solution was
cooled to -10 °C; then 2.83 g (75 mmol) of sodium borohydride
was added portionwise. After completion of the addition, the
ice bath was removed and the reaction mixture was stirred at
room temperature for 24 h. Solvent was evaporated, and the
residue was partitioned between water and ethyl acetate. The
organic layer was washed with water, dried (MgSO4), and
evaporated to give an oil. Purification by silica gel column
chromatography with toluene-AcOEt (8:2) gave 4.95 g (47%)
of a mixture (6:4) of 3,5,6,7,8,8a-hexahydro-6â-hydroxy-8a-
methyl-2(1H)-isoquinolinecarboxylic acid ethyl ester and
3,4,6,7,8,8a-hexahydro-6â-hydroxy-8a-methyl-2(1H) isoquino-
linecarboxylic acid ethyl ester. This mixture was dissolved in
100 mL of ethanol with 1 mL of concentrated HCl, and the
solution was refluxed for 2 h. After cooling, solvent was
evaporated and the resulting residue was purified by silica
gel column chromatography with toluene-AcOEt (7:3) to give
13 as a white powder (2 g, 40%): mp 78-80 °C; IR (KBr) ν
(cm-1) 3400, 1700, 1440; 1H NMR (300 MHz, CDCl3) δ 5.32
(d, J ) 10.3 Hz, 1H), 4.17 (m, 3H), 3.96 (d, J ) 12.8 Hz) and
3.82 (d, J ) 12.8 Hz) (total 1H), 3.56 (m, 2H), 2.60 (m, 1H),
2.39 (m, 1H), 2.16 (m, 1H), 1.90 (m, 1H), 1.58 (m, 3H), 1.26
(m, 4H), 1.10 (s, 3H).
(4a â,6â,8a â)-Deca h yd r o-5,5,8a -tr im eth yl-6-isoqu in oli-
n ol (19). Following the procedure described above, 19 was
obtained from 17 (100%) as a light yellow oil: 1H NMR (300
MHz, CDCl3) δ 3.08 (m, 1H), 2.46 (s, 2H), 2.44 (m, 1H), 2.03
(m, 1H), 1.78-1.54 (m, 5H), 1.40 (m, 1H), 1.27 (m, 2H), 1.10
(s, 3H), 1.02 (s, 3H), 0.99 (s, 3H).
1,3,4,7,8,8a -H exa h yd r o-5-m et h yl-2-(p h en ylm et h yl)-6-
(2H)-isoqu in olin on e (21). To a solution of 15 g (79 mmol)
of 20 in 70 mL of anhydrous toluene was added 10 mL (120
mmol) of pyrrolidine. The mixture was stirred under reflux
for 20 h with elimination of water. Toluene and excess of
amine were removed under reduced pressure. The residue was
diluted in 70 mL of anhydrous toluene, and then 7.9 mL (79
mmol) of ethyl vinyl ketone and 100 mg (0.9 mmol) of
hydroquinone were added. The mixture was stirred under
reflux, sheltered from light, for 20 h. Then 200 mg (3.7 mmol)
of sodium methoxide was added, and the mixture was stirred
under reflux for 2 h. The solution was diluted with a saturated
solution of ammonium chloride, made basic by adding NaOH,
and extracted with ethyl acetate. The organic layer was
washed with brine, dried (MgSO4), and concentrated. The
crude material was chromatographed with toluene-AcOEt (7:
3) to afford 15.3 g (76%) of 21 as a yellow oil which crystallized
in pentane: mp 70 °C (lit.19 mp 67 °C); IR (neat) ν (cm-1) 1720,
(6â,8aâ)-1,2,3,5,6,7,8,8a-Octah ydr o-8a-m eth yl-6-isoqu in -
olin ol (14). A solution of 1.8 g (7.7 mmol) of 13 in 20 mL of
THF was cooled to 0 °C, and 17.5 mL (38 mmol) of 2.2 M
methyllithium-lithium bromide complex in ether were care-
fully added. The reaction mixture was stirred at 0 °C for 2 h
1
1680, 1660, 1600, 1570, 1560, 750, 700; H NMR (300 MHz,

2310 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 12
Barth et al.
CDCl3) δ 7.31 (m, 5H, Ph), 5.57 (d, J ) 13.1 Hz, H10), 3.51 (d,
J ) 13.1 Hz, H10), 3.03 (m, H1, H3), 2.77 (dt, J ) 10, 1, 2.4 Hz,
H4), 2.62 (m, H8a), 2.47 (dt, J ) 16.1, 4.0 Hz, H7), 2.30 (m, H4,
H7), 2.05 (ddd, J ) 2.9, 11.0, 13.8 Hz, H3), 1.91 (m, H8), 1.79
(s, H9, H9′, H9′′), 1.74 (d, J ) 11.0 Hz, H1), 1.52 (m, H8); 13C
NMR (75 MHz, CDCl3) δ 199.2 (C6), 156.1 (C5), 138.2 (C4), 129.4
(C11), 129.0 (C12, C16), 128.2 (C13, C15), 127.2 (C14), 62.6 (C10),
60.4 (C1), 53.2 (C3), 37.8 (C8a), 36.7 (C7), 30.5 (C4), 26.0 (C8),
10.5 (C9). Anal. (C17H21NO) C,H,N.
portionwise. After completion of the addition, the mixture was
refluxed for 4 h. The mixture was cooled, poured onto water,
and then extracted with ethyl acetate. The organic phases
were washed with water, dried (MgSO4), and evaporated. The
oil was purified by chromatography with toluene-acetone (9.5:
0.5) to give the compound 26 as an oil (3.2 g, 72.7%): IR (NaCl)
ν (cm-1) 1500, 730, 700; 1H NMR (300 MHz, CDCl3) δ 7.32 (m,
5H), 5.48 (m, 1H), 5.36 (m, 1H), 3.48 (s, 2H), 3.11 (m, 2H),
1.92 (m, 2H), 1.78-1.39 (m, 5H), 1.02 (m, 1H), 0.96 (s, 3H),
0.88 (s, 3H).
(4a r,8a â)-Oct a h yd r o-5,5-d im et h yl-2-(p h en ylm et h yl)-
6(2H)-isoqu in olin on e (22). To 250 mL of freshly distilled
ammonia was added 0.5 g (71.4 mmol) of lithium at -60 °C.
A solution of 5 g (19.6 mmol) of 21 in 80 mL of THF was added
dropwise. The mixture was stirred for 0.5 h, and 20 mL (321
mmol) of methyl iodide was added. Ammonia was evaporated,
and the mixture was stirred at room temperature for 2 days.
The mixture was diluted with water and extracted with ethyl
acetate. The organic layer was washed with brine, dried
(MgSO4), and concentrated. The crude material was purified
by chromatography with toluene-AcOEt (7:3) to give 2.1 g
(4ar,8aâ)-Decah ydr o-5,5-dim eth ylisoqu in olin e (27). To
35 mL of ethanol in a Parr hydrogenation bottle were added
2.85 g (11.1 mmol) of 26, 250 mg of 10% palladium on carbon,
and 0.7 mL of acetic acid. The mixture was hydrogenated
under 45 psi of hydrogen pressure at 50 °C for 6 h. The
mixture was filtered and the filtrate evaporated. The residue
was partitioned between 1 N NaOH and ethyl acetate; the
organic layer was washed with H2O, dried (MgSO4), and
concentrated to give 27 as an oil (1.4 g, 75%): IR (NaCl) ν
1
(cm-1) 3270, 1460; H NMR (300 MHz, CDCl3) δ 3.15 (dm, J
(50%) of 22 as a yellow solid: mp 84 °C; IR (KBr, 1%) ν (cm-1
)
) 12 Hz, 1H), 3.04 (s, NH), 2.96 (dd, J ) 2.9, 11.9 Hz, 1H),
2.59 (dt, J ) 2.7, 12.2 Hz, 1H), 2.21 (t, J ) 10.9 Hz, 1H), 1.64
(dm, J ) 10.9 Hz, 1H), 1.55-1.30 (m, 5H), 1.26-1.12 (m, 3H),
0.86 (m, 4H), 0.82 (s, 3H).
1710, 1500, 1460, 750, 700; 1H NMR (300 MHz, CDCl3) δ 7.30
(m, 5H, Ph), 3.54 (d, J ) 13.1 Hz, H11), 3.47 (d, J ) 13.1 Hz,
H11), 2.99 (dm, J ) 10.5 Hz, H3), 2.92 (dm, J ) 10.9 Hz, H1),
2.63 (dt, J ) 6.3, 14.0 Hz, H7), 2.27 (ddd, J ) 2.3, 4.5, 14.0 Hz,
H7), 2.00-1.79 (m, H3, H8a, H8), 1.63-1.55 (m, H1, H4, H4), 1.30
(m, H8), 1.09 (m, H4a), 1.07 (s, 3H), 1.05 (s, 3H); 13C NMR (75
MHz, CDCl3) δ 215.7 (C6), 138.3 (C12), 129.1 (C13, C17), 128.2
(C14, C16), 127.0 (C15), 63.2 (C11), 60.0 (C1), 53.8 (C3), 50.4 (C4a),
47.5 (C5), 37.4 (C7), 35.3 (C8a), 30.9 (C8), 25.7 (C4), 21.6-20.36
(C9, C10). Anal. (C18H25NO) C,H,N.
(6â,8a â)-2-Dod ecyl-1,2,3,5,6,7,8,8a -octa h yd r o-5,5,8a -tr i-
m eth yl-6-isoqu in olin ol (28e). Meth od A. A solution of 0.5
g (2.5 mmol) of 9 (as a base), 0.7 g (2.7 mmol) of dodecyl
bromide, 0.7 g of potassium carbonate, and a catalytic amount
of sodium iodide in 20 mL of 4-methyl-2-pentanone was
refluxed for 12 h. After cooling, the mixture was filtrated, the
filtrate concentrated under reduced pressure, and the residue
purified by chromatography with isopropyl ether to give an
oil which was converted into oxalate and recrystallized from
ethanol-ether to give 28e (0.5 g, 50%): mp 106-108 °C. Anal.
(C24H45NO‚C2H2O4) C,H,N.
(4ar,6â,8aâ)-Decah ydr o-5,5-dim eth yl-2-(ph en ylm eth yl)-
6-isoqu in olin ol (23). Following method F, 23 was obtained
starting from 22 as a solid (95%): mp <40 °C; IR (neat) ν
1
(cm-1) 3400, 1600, 1470, 1460; H NMR (300 MHz, CDCl3) δ
7.29 (m, 5H, Ph), 3.50 (d, J ) 13.2 Hz, H11), 3.45 (d, J ) 13.2
Hz, H11), 3.23 (dd, J ) 4.2, 11.5 Hz, H6), 2.97 (dm, J ) 11.0
Hz, H3), 2.81 (dm, J ) 8.8 Hz, H1), 1.87 (dt, J ) 2.3, 11.0 Hz,
H3), 1.75-1.32 (m, H7, H4, H8a, H1, H7′, H4′, H8, OH), 1.01 (m,
(6â,8a â)-2-(1,5,9-Tr im eth yl-(E)-4,8-d eca d ien yl)-1,2,3,5,-
6,7,8,8a -octa h yd r o-5,5,8a -tr im eth yl-6-isoqu in olin ol (28a ).
Meth od B. Acetic acid was added to a solution of 9.8 g (50
mmol) of 9 in 100 mL of methanol in order to adjust the pH of
the mixture to 7.6; then 3.5 g (55 mmol) of sodium cyanoboro-
hydride was added. After the mixture stirred at room tem-
perature for 3 days, the reaction was quenched with HCl. Then
the mixture was evaporated under reduced pressure. The
residue was partitioned between 10 N NaOH and ether. The
organic layer was washed with water, dried (MgSO4), and
evaporated to give an oil which was purified by flash chroma-
tography with CH2Cl2-ether (8:2). The resulting oil was
treated with a stoichiometric amount of methanesulfonic acid
in ether, and the mixture was evaporated and then lyophilized
to give 28a (15.5 g, 66%) as an oil. Anal. (C25H43NO‚CH4O3S)
C,H,N.
H8), 0.96 (s, 3H), 0.77 (s, 3H), 0.68 (m, H4a). Anal. (C18H27
-
NO‚0.25H2O) C,H,N.
(4a r,6â,8a â)-Deca h yd r o-5,5-d im et h yl-6-isoq u in olin ol
(24). To a solution of 1.5 g (5.5 mmol) of 23 in 40 mL of
methanol were added 400 mg of 5% Pd/C and 35 µL (6 mmol)
of acetic acid. The mixture was stirred under 50 psi of H2
pressure at 35 °C for 6 h. The catalyst was filtered and the
filtrate evaporated. The residue was diluted with NaOH and
extracted with ethyl acetate. The organic layer was washed
with brine, dried (MgSO4), and evaporated to give 0.81 g (81%)
of 24 as a light yellow solid: mp 108 °C; IR (KBr, 1%) ν (cm-1
)
3400, 3250, 3100, 1450; 1H NMR (300 MHz, CDCl3) δ 3.23 (dd,
J ) 4.5, 11.6 Hz, H6), 3.14 (dm, J ) 12.0 Hz, H3), 2.98 (ddd, J
) 1.1, 3.9, 11.9 Hz, H1), 2.53 (dt, J ) 3.3, 12.0 Hz, H3), 2.16
(dd, J ) 10.6, 11.9 Hz, H1), 1.75-1.64 (m, H7, H4, OH), 1.64-
1.53 (m, H7′), 1.46 (m, H8), 1.32 (m, H8a), 1.22 (m, H4), 1.02 (m,
H8′), 0.96 (s, 3H), 0.82 (m, H4a), 0.77 (s, 3H).
(6â,8a â)-2-(5,9-Dim eth yl-(E)-4,8-d eca d ien oyl)-1,2,3,5,6,-
7,8,8a -oct a h yd r o-5,5,8a -t r im et h yl-6-isoq u in olin ol (29).
Meth od C. To a solution of 1 g (5.1 mmol) of 5,9-dimethyl-
(E)-4,8-decadienoic acid in 20 mL of THF was added 1 g (6.7
mmol) of carbonyldiimidazole. The resulting mixture was
cooled to 0 °C, and a solution of 1 g (5 mmol) of 9 in 5 mL of
THF was added. The mixture was stirred at room tempera-
ture overnight. Solvent was evaporated, and the residue was
taken up with water and ether. The organic layer was washed
with water, dried (MgSO4), and evaporated. The crude residue
was purified by flash chromatography with CH2Cl2 as eluent
(4a r,8a â)-5,5-Dim et h yl-1,3,4,4a ,5,7,8,8a -oct a h yd r o-2-
(p h en ylm eth yl)-6(2H)-isoqu in olin on e N-[(4-Meth ylp h en -
yl)su lfon yl]h yd r a zon e (25). To a solution of 15.4 g (56.7
mmol) of 22 in 100 mL of acetic acid was added 11.6 g (62.3
mmol) of N-[(4-methylphenyl)sulfonyl]hydrazine portionwise.
The mixture was heated at 50 °C for 1 h and then allowed to
be stirred for 24 h at room temperature. Acetic acid was
evaporated, and the resulting residue was partitioned between
ethyl acetate and 1 N NaOH. The extracts were concentrated,
and the residue was purified by chromatography with toluene-
acetone (7.5:2.5) to give 25 as a solid (7.85 g, 31%): mp 173
to afford 29 as an oil (1.4 g, 80%): nD20 1.5240. Anal. (C25H35
-
NO2) C,H,N.
P r ep a r a tion of Ra t Liver Micr osom a l 2,3-Oxid osqu a -
len e Cycla se. Male Wistar rats weighing 250 g were used.
Food was withdrawn 4 h before animals were killed. The
livers were quickly removed, minced, and homogenized in ice
cold 0.25 M sucrose in a Potter-Elvehjem type Teflon homog-
enizer at 2000 rpm. Homogenates were centrifuged for 15 min
at 16000 gmax, and the supernatants collected were centrifuged
1
°C; IR (KBr) ν (cm-1) 3210, 1600, 1500, 1325, 1160; H NMR
(300 MHz, CDCl3) δ 7.85 (d, J ) 8.3 Hz, 2H), 7.29 (m, 7H),
7.14 (s, NH), 3.46 (dd, J ) 13.1, 19.8 Hz, 2H), 2.97 (d, J )
10.6 Hz, 1H), 2.82 (d, J ) 9.9 Hz, 1H), 2.55 (m, 1H), 2.44 (s,
3H), 1.99-1.41 (m, 7H), 1.07 (s, 3H), 1.03-0.81 (m, 5H).
(4a r,8a â)-5,5-Dim et h yl-1,2,3,4,4a ,5,8,8a -oct a h yd r o-2-
(p h en ylm eth yl)isoqu in olin e (26). A solution of 7.63 g (17.3
mmol) of 25 in 150 mL of toluene was cooled at 0 °C, and 2.4
g (80 mmol) of NaH (80% dispersion in mineral oil) was added
again at 16000 gmax
. Then the two-thirds upper part of the
supernatant was collected and centrifuged at 100000 gmax for
1 h. Microsomal pellets were resuspended in one-half the
initial volume and then centrifuged for 1 h at 100000 gmax
.
Pellets of washed microsomes were collected and fractionated

Inhibition of OSC by Isoquinolines
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 12 2311
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in small aliquots stored at -80 °C without loss of activity for
at least 2 months.
2,3-Oxid osqu a len e La n oster ol-Cycla se (OSC) Assa y.
We used the original following procedure: A standard assay
mixture consisted of a total volume of 500 µL containing 150
µg of microsomal protein and 100 µM (R,S)-2,3-oxidosqualene.51
Incubation was for 1 h at 37 °C; then the reaction was stopped
by adding 1 mL of 7% methanolic KOH and 20 ng of
stigmasterol as internal standard. After 0.5 h at 80 °C for
saponification, nonsaponifiable lipids were extracted twice
with n-hexane and then evaporated and derivatized with 75
µL of BSTFA-1% TMCS and 25 µL of pyridine. TMCS ethers
were chromatographed on a 30 m OV1 column at 260 °C.
Retention times of TMCS-lanosterol and -stigmasterol ethers
were respectively 17 and 15.5 min. The rat liver microsomal
OSC has an apparent Km for 2,3(S)-oxidosqualene of 30 µM.
The inhibitory potencies of compounds were expressed as the
concentration at which 50% inhibition of OSC activity was
observed. Enzyme was also assayed in the presence of several
different substrate and inhibitor concentrations or at different
substrate/inhibitor ratios. The resulting V vs [S] curves were
evaluated with EZ-Fit Enzyme Kinetic Model Fitting Software
kindly provided by F. Perrella.46
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Ack n ow led gm en t . The authors wish to thank
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