Effect of C-ring modifications in benzo[c]quinolizin-3-ones, new selective inhibitors of human 5α-reductase 1

Article abstract of DOI:10.1016/S0968-0896(01)00012-8

The synthesis and the inhibition potency of ocatahydro- and decahydrobenzo[c]quinolizin-3-one derivatives 3-7, as new non-steroidal selective inhibitors of human enzyme 5α-reductase type 1, are reported. These compounds differ from the recently reported enzo[c]quinolizin-3-one inhibitors 2 by the presence of a fully or partially saturated C-ring. Compounds 3 and 4, with a double bond in the C-ring, were prepared by sequential rearrangement-annulation of isoxazolines 19 and 20. C-ring saturated compounds 5-7 were prepared by the lewis acid-promoted. Mannich-Michael tandem reaction of Danishefsky diene with the appropriate N-t-Boc iminium ion. Inhibition experiments were carried out in 5αR-1 and 5αR-2 expressed by CHO cells. Among the prepared compounds, octahydrobenzo9c0quinolizin-3-one3, with a double bond at the position 6a-10a, was a potent and selective inhibitor of human 5αR-1 IC₅₀ = 58 nM). The introduction of a tert-butylcarboxyamide at the position 8 compounds 5-7. The extended planarity of the most potent benzo[c]quinolizin-3-ones as well as favorable interactions of the C-ring unsaturation with the enzyme active site could account for the inhibition activity of these compounds. Copyright

Full text of DOI:10.1016/S0968-0896(01)00012-8

Bioorganic & Medicinal Chemistry 9 (2001) 1385±1393
E€ect of C-ring Modi®cations in Benzo[c]quinolizin-3-ones,
New Selective Inhibitors of Human 5ꢀ-reductase 1
Antonio Guarna,a,* Ernesto G. Occhiato,a Fabrizio Machetti,a
Andrea Trabocchi,a Dina Scarpi,a Giovanna Danza,b Rosa Mancina,b
Alessandra Comercib and Mario Seriob
aDipartimento di Chimica Organica `U. Schi€' and Centro di Studio sulla Chimica e la Struttura dei Composti
Á
Eterociclici e loro Applicazioni, C.N.R., Universita di Firenze, Via G. Capponi 9, I-50121 Firenze, Italy
Á Á
Dipartimento di Fisiopatologia Clinica, Unita di Endocrinologia, Universita di Firenze, Viale Pieraccini 6, I-50134 Firenze, Italy
b
Received 13 November 2000; accepted 5 January 2001
AbstractÐThe synthesis and the inhibition potency of octahydro- and decahydrobenzo[c]quinolizin-3-one derivatives 3±7, as new
non-steroidal selective inhibitors of human enzyme 5a-reductase type 1, are reported. These compounds di€er from the recently
reported benzo[c]quinolizin-3-one inhibitors 2 by the presence of a fully or partially saturated C-ring. Compounds 3 and 4, with a
double bond in the C-ring, were prepared by sequential rearrangement-annulation of isoxazolines 19 and 20. C-ring saturated
compounds 5±7 were prepared by the Lewis acid-promoted Mannich-Michael tandem reaction of Danishefsky diene with the
appropriate N-t-Boc iminium ion. Inhibition experiments were carried out on 5aR-1 and 5aR-2 expressed by CHO cells. Among the
prepared compounds, octahydrobenzo[c]quinolizin-3-one 3, with a double bond at the position 6a±10a, was a potent and selective
inhibitor of human 5aR-1 (IC50=58 nM). The introduction of a tert-butylcarboxyamide at the position 8 (compound 4) was dele-
terious for the inhibition activity. The lack of the double bond in the C-ring reduced strongly the inhibition activity of compounds
5±7. The extended planarity of the most potent benzo[c]quinolizin-3-ones as well as favorable interactions of the C-ring unsatura-
tion with the enzyme active site could account for the inhibition activity of these compounds. # 2001 Elsevier Science Ltd. All
rights reserved.
Introduction
deprivation, could be used in a pharmacological treat-
ment of DHT-related diseases.9 Some potent inhibitors
are based on the steroidal structure of testosterone,
modi®ed with the introduction of a nitrogen atom in the
A or B ring as in 4-azasteroid and 6-azasteroids.
Recently, we have described the synthesis and the bio-
logical evaluation of a novel class of azasteroids having
as a new feature the nitrogen atomat position 10 of the
steroidal skeleton,10,11 with 19-nor-10-azasteroid 1 (as a
9:1 mixture of Á9(11) and Á8(9) isomers) (Fig. 1) being a
potent dual inhibitor of the isoenzymes 5aR-1 and 5aR-
2. Since the presence of all the four fused A±D steroidal
rings in an inhibitor seems to be not essential for the
activity toward this enzyme,12,13 we have recently pre-
pared a novel class of non-steroidal compounds having
the benzo[c]quinolizin-3-one skeleton 2 which resulted
in very potent and selective inhibitors of 5aR-1 iso-
enzyme.14
Steroid 5a-reductase (EC 1.3.99.5) is a systemof two
isoenzymes (5aR-1 and 5aR-2) involved in the
NADPH-dependent conversion of testosterone to the
more potent androgen dihydrotestosterone (DHT).1
Human 5a-reductases 1 and 2 are di€erently expressed
in various tissues: type 1 is found mainly in liver, skin
and scalp; type 2 in prostate, seminal vesicle and epidi-
dymis.1 It is now well established that the formation of
DHT in these tissues is related to several human endo-
crine diseases and skin disorders such as acne, andro-
genic alopecia, benign prostatic hyperplasia (BPH) and
prostatic carcinoma in men, and hirsutism in women.2À8
The role of DHT in the maintenance of these patholo-
gies has prompted several synthetic e€orts toward
potent and selective 5a-reductases inhibitors which,
blocking the formation of DHT without testosterone
Since there is now clinical evidence that dual inhibitors
could give great bene®t in the treatment of BPH,15 and
because only few examples of non-steroidal dual inhibi-
tors of 5aR have been reported so far,1c,9 we decided to
*Corresponding author. Tel.: +39-55-2757611; fax: +39-55-2476964;
e-mail: guarna@chimorg.uni®.it
0968-0896/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(01)00012-8

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A. Guarna et al. / Bioorg. Med. Chem. 9 (2001) 1385±1393
investigate if non-steroidal octahydro-(1H)-benzo[c]qui-
nolizin-3-ones 3 and 4 (Fig. 1) and decahydrobenzo
[c]quinolizin-3-ones 5±7, which di€er fromthe previous
series of tetrahydrobenzo[c]quinolizin-3-one 2 by the
presence of an aliphatic C-ring, could maintain the
inhibitory potency toward 5aR-1 typically associated to
the tricyclic structure 2 while displaying at the same time
some activity toward the other isozyme. In this paper we
therefore describe the synthesis and the evaluation of the
inhibitory activity of compounds 3±7 against human
5aR-1 and 5aR-2 expressed in recombinant CHO cells.
À78 ꢀC in order to have complete reduction of ester 11
to alcohol 13. However, in these conditions, reduction
of the amide to amine also occurred to some extent
(25%). Alcohol 13 was oxidized to aldehyde 14 by
Swern reaction and the crude aldehyde was ®nally con-
verted to oxime under the usual conditions providing
compound 16 in 35% overall yield after chromato-
graphic puri®cation.
The 1,3-dipolar cycloadditions were performed in one-
pot converting oximes 15 and 16 into the corresponding
a-chloro oximes by adding NaClO to an ice-cooled
solution of the oximes and Et3N. The nitrile oxides,
formed under these conditions by HCl elimination, were
trapped in situ by an excess of methylenecyclopropane,
providing isoxazoline-5-spirocyclopropanes 17 (73%)
and 18 (58%). After deprotection, the thermal rerrange-
ment of 19 and 20 was performed in re¯uxing DMF.
According to the proposed mechanism,10,16 along with
target octahydro-(1H)-benzo[c]quinolizin-3-ones 3 and 4,
open chain compounds 21 and 22, as well as benzoqui-
nolinone derivatives 23 and 24, could be obtained in the
thermal processes, all as mixtures of two regioisomers.
Chemistry
All compounds were synthesized starting from racemic
material. The common key step in the synthesis of
octahydro-(1H)-benzo[c]quinolizin-3-ones 3±4 is the
sequential rearrangement-annulation of isoxazoline-5-
spirocyclopropanes 19 and 20 (Scheme 1)16 prepared
starting fromesters 8 and 9, respectively, the latter as a
1:1 mixture of diastereoisomers. The transformation of
ester 8 into the corresponding E/Z mixture of oxime 15
was achieved, after protection of the carbonyl group as
a ketal, by partial reduction of 10 with DIBAL in tolu-
ene at À78 ꢀC, which provided aldehyde 12 in 48%
In the rearrangement of 19 only compounds 3 (37%) and
21 (28%) were formed. The ratio between isomers 3a and
1
.
yield, followed by treatment with NH2OH HCl in pyri-
3b seen in the H NMR spectrumwas 10:1. Compound
dine. Due to the low yield obtained in the preparation
of aldehyde 12 by partial reduction of the ester group
with DIBAL, an excess of this reagent was used at
3a, with the double bond at 6a±10a `internal' position,
was the major regioisomer. In the thermal rearrange-
ment of isoxazoline 20, a mixture of products 4, 22 and
24 was obtained, with low yields in isolated compounds
4 and 22 (18 and 21%, respectively). Compound 4 was
obtained, as in the previous case, as 10:1 mixtures of
regioisomers, with isomers 4a being the major one. In
similar rearrangements producing 10-azasteroids,10
compounds having `external' C9±C11 double bond were
predominant with respect to the corresponding regioi-
somers having `internal' C8±C9 double bond (9:1 ratio).
Ab initio HF-STO3-21G* calculations17 performed on
compounds 3a,b indicate 3a as the thermodynamically
favored isomer (by 1.25 kcal/mol). Because all the
e€orts to separate the regioisomers by chromatography
failed, racemic products 3 and 4 were isolated, char-
acterized and tested as 10:1 regioisomeric mixtures.
Racemic decahydro-(4aH)-benzo[c]quinolizin-3-ones 5
and 6 (Scheme 2) were prepared by the tandem Man-
nich-Michael addition reaction of Danishefsky's diene
with the N-t-Boc iminium ion derived from its precursor
27, in analogy to the synthesis of 19-nor-10-azasteroids
recently reported by us.18 Treatment of 27 with TiCl4
and Danishefsky's diene thus gave compound 5 having
5a relative stereochemistry, whereas its 5b isomer 6 was
obtained through the use of TMSOTf to promote the
iminium ion formation. Complete hydrogenation of 5
over Pd/C in MeOH, followed by sequential Jones and
Hg(OAc)2 oxidations a€orded compound 7 in 16%
overall yield.
IC50 determination
The inhibition potency of compounds 3±7 was eval-
uated on CHO cells expressing human 5aR-1 and 5aR-2
Figure 1.

A. Guarna et al. / Bioorg. Med. Chem. 9 (2001) 1385±1393
1387
according to the reported methods,14c,19 using tritiated
testosterone as substrate of the enzyme and NADPH as
cofactor. In Table 1 the IC50 values of 3±7 are reported.
nM). This inhibitory potency was comparable to that of
the compounds belonging to the class of benzo[c]qui-
nolinones inhibitors 2 recently reported by us (for
example, the IC50 of compounds 28 depicted in Figure 2
was 298 nM).14aÀc Instead, compound 4, which was
designed to be mimic of azasteroid 1, was a very weak,
although still selective, inhibitor of 5aR-1 (IC50=19
mM). Compared to its parent azasteroidal compound 1,
inhibitor 4 was about 150-fold less potent. Removing
the double bond in the C ring caused a strong decrease
All tested compounds were inactive towards 5aR-2
whereas they displayed inhibitory activity only towards
5aR-1, modulated by the number and position of the
double bonds. Simple octahydro-(1H)-benzo[c]quinoli-
zin-3-one 3 proved to be a potent and selective inhibitor
of 5aR-1, its IC50 value being at the nanomolar level (58
.
Scheme 1. (a) (CH2OH)2, p-TsOH, toluene; (b) DIBAL-H (1.9 equiv), toluene; (c) DIBAL-H (4.5 equiv), toluene; (d) Swern; (e) NH2OH HCl,
pyridine; (f) NaClO, Et3N, methylenecyclopropane, CH2Cl2; (g) p-TsOH, acetone; (h) H2SO4, acetone; (i) DMF, re¯ux.
Scheme 2. (a) Boc2O, Et3N, DMAP, CH2Cl2, re¯ux, 6 days; (b) LiEt3BH, THF, À78 ꢀC, then 1 M HCl in EtOH; (c) 1-Methoxy-3-[(trimethylsilyl)oxy]-
1,3-butadiene, TiCl4, CH2Cl2, 0 ꢀC, 1 h, then NaHCO3 (satd) 30 min; (d) 1-Methoxy-3-(trimethylsilyl)oxy-1,3-butadiene, Et3N, TMSOTf, CH2Cl2, 0 to
25 ꢀC, then NaHCO3 (satd) 48 h; (e) H2, Pd/C (5%); (f) Jones oxidation; (g) Hg(OAc)2, EDTA tetrasodiumsalt, 5% AcOH, H 2O, 85 ꢀC, 2 h.

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A. Guarna et al. / Bioorg. Med. Chem. 9 (2001) 1385±1393
of potency: the IC50 of compound 7 toward 5aR-1 was
only 4.78 mM. The same occurred with C-ring saturated
compounds 5 and 6 which were very weak inhibitors and
with IC50 values toward 5aR-1 in the 2±20 mM range.
and B rings are almost coincident. The weak inhibitory
activity of 8-N-(tert-butyl)acetamido substituted com-
pound 4 toward 5aR-1 could be explained by the fact
that, generally, lipophylic groups, such as a chlorine
atom, at the position 8 of benzoquinoline and benzo[c]-
quinolizin-3-one inhibitors, enhance the potency toward
5aR-1.1c,13,14c Therefore, the 8-N-(tert-butyl)acetamido
group might be a `wrong' substituent in a tricyclic inhi-
bitor such as 4. Moreover, due to the high number of
conformers generated by the rotations of the C-8 sub-
stituent in 4, a considerable entropy loss should occur in
the binding, thus lowering the anity of the inhibitor
toward the enzyme. Concerning C-ring saturated com-
pound 7, the decrease of its inhibitory potency toward
the enzyme appears consistent to, and even more
marked than, the decrease of activity measured in 19-nor-
10-azasteroids after reduction of the C-ring double
bond.10 The RMS ®tting after superposition of the global
minimum conformer of 7 with that of 28 was 0.235 A, a
value not signi®cantly di€erent fromthat measured after
superposition of 3a and 28 (Fig. 2). Therefore, the
decrease of inhibition activity of compound 7 can be only
in part accounted for on the basis of the ¯atness of the
skeleton, and thus favorable interactions between the C-
ring unsaturation in 3a and the enzyme active site could
have an important role in increasing the inhibitory
potency. While in benzo[c]quinolizin-3-ones 2 a higher
activity toward 5aR-1 has been always reported for com-
pounds having the double bond at the 4±4a position, for
compounds 5±7 the di€erent position of the double bond
in the A ring seems instead to have no e€ect on the inhi-
bitory potency.14 The distorted planarity of compounds 5
and 6 with respect to 28 (RMS values greater than 0.32 A)
and the lack of the C-double bond could both contribute
to the low activity displayed by these two inhibitors.
In general, the selectivity observed for tricyclic inhibitors
3±7 is in accordance with the reported ®ndings on ben-
zo[c]quinolizinones of type 2 and other tricyclic inhibitors
derived from4-azasteroids which are selective inhibitors
of isoenzyme 1.13,14 It has been suggested that, for the
these classes of inhibitors, the extended planarity of the
molecular skeleton is an important feature to have a
good inhibitory potency.14c,20 Thus, the superposition
of the minimized structures of 3±7 with that of com-
pound 28 (Fig. 2), taken as a model of the benzo[c]qui-
nolizinone inhibitors,14 would provide us with a means
of evaluating the overall planarity of the molecules.
After a Monte Carlo conformational analysis and AM1
geometry optimization,21 six conformers for compound
3±7 and two conformers for 28 were found in the 0±3
kcal/mol range. After superposition of all the skeleton
atoms of the global minimum conformers of 3a and 28,
the resulting RMS ®tting was 0.226 A (Fig. 2). There-
fore the two conformers are very close structures and
the global minimum of 3a could be considered almost as
planar as 28. As expected, the greater contribution to
the RMS value arises fromthe presence of a half-twist C
ring in 3a instead of an aromatic one, whereas the A
Table 1. IC50 values for compounds 1±7 toward human 5aR-1 and
5aR-2
Compound
IC50 (nM)
5aR-1a
5aR-2b
1
127Æ12c
58Æ15
122Æ37 nM
3d
4d
5
n.i.
n.i.
n.i.
n.i.
n.i.
19,000Æ1000
20,000Æ400
2400Æ400
4780Æ458
Conclusion
6
7
In conclusion, non-steroidal octahydro- and decahy-
drobenzo[c]quinolizin-3-one inhibitors 3±7 failed to
show dual inhibition activity toward 5aR-1 and 2, while
they displayed an interesting selectivity toward human
enzyme 5a-reductase type 1. In particular, inhibition
experiments carried out toward 5aR-1 and 5aR-2
aDetermined using CHO 1827 cells.
bDetermined using CHO 1829 cells.
cDetermined on DU-145 cells.
Figure 2. Left: superposition of global minimum conformers of compounds 3a and 28. Right: superposition of global minimum conformers of
compounds 7 and 28.

A. Guarna et al. / Bioorg. Med. Chem. 9 (2001) 1385±1393
1389
expressed by CHO cells showed that octahydrobenzo[c]-
quinolizin-3-one 3, with a double bond at the position
6a±10a, was a potent and selective inhibitor of human
5aR-1 (IC50=58 nM), with a potency comparable to
that of benzo[c]quinolizin-3-ones 2. The lack of the
double bond in the C-ring instead reduced strongly the
activity whereas the introduction of a tert-butylcarbox-
yamide at the position 8, was deleterious for the inhibi-
tion activity. These results can be only in part explained
on the basis of the extended planarity of the benzo[c]-
quinolizin-3-one skeleton,14c but also favorable interac-
tions between the C-ring unsaturation (e.g., in 3a) and
the enzyme active site should be taken into account.
methyl ester 8 (20.0 g, 109 mmol), ethylene glycol (60
mL, 1.08 mol) and p-TsOH (0.8 g, 5 mmol) were dis-
solved in toluene (550 mL) and the resulting solution
was heated under re¯ux. After 4 h the reaction was
complete and the mixture was washed with 2 N
NaHCO3, water and dried over Na2SO4. After ®ltration
and evaporation of the solvent, a crude yellow oil was
obtained. This was puri®ed by distillation under
reduced pressure, a€ording pure 10 (15.9 g, 64%).
10: oil; bp 127±130 ꢀC (2 mbar); H NMR (CDCl3) d
1
3.95±3.88 (m, 4H), 3.63 (s, 3H), 2.42±2.25 (m, 2H),
1.96±1.88 (m, 1H), 1.76±1.70 (m, 2H), 1.63±1.18 (m,
8H); MS m/z (rel. intensity) 228 (M+, 0.5), 154 (16), 98
(100); IR (CDCl3) 1739 cmÀ1
.
Experimental
3-[2-(1,3-Dioxolan-2-yl)cyclohexyl]propanal (12). To a
solution of 10 (15.7 g, 69.1 mmol) in toluene (220 mL)
cooled at À78 ꢀC, DIBAL-H (1.2 M solution in toluene,
116 mL, 135 mmol) was slowly added during 3 h. After
3 h of stirring, the mixture was poured into water (110
mL) and allowed to warm to room temperature. After
®ltration on a Celite layer, the organic phase was dried
over Na2SO4. After ®ltration and evaporation of the
solvent the residual crude oil was puri®ed by chroma-
tography (petroleumether/EtOAc, 2:1, Rf 0.30), a€ord-
ing pure aldehyde 12 (6.6 g, 48%).
All the reactions requiring dry conditions were per-
formed under nitrogen and anhydrous solvents. Chro-
matographic separations were performed under pressure
on silica gel using ¯ash-column techniques; Rf values
refer to TLC carried out on 25-mm silica gel plates
(Merck F254), with the same eluant indicated for the
column chromatography. Melting points (mp) are
uncorrect. IR spectra were recorded on a Perkin±Elmer
881 spectrophotometer in CDCl3 solution. 1H NMR
(200 MHz) and 13C NMR (50 MHz) spectra were
recorded on a Varian XL 200 instrument in CDCl3
solution. Mass spectra were carried out in EI at 70 eV
on 5790A-5970A Hewlett-Packard and QMD 1000
Carlo Erba instruments. Microanalyses were carried out
with a Perkin±Elmer 240C elemental analyzer. All chiral
compounds were prepared in racemic form.
1
12: oil; H NMR (CDCl3) d 9.75 (t, J=1.8 Hz, 1H),
3.99±3.90 (m, 4H), 2.50±2.27 (m, 2H), 2.02±1.91 (m,
1H), 1.80±1.19 (m, 10H); 13C NMR (CDCl3) d 202.8 (d),
110.4 (s), 64.6 (t), 64.4 (t), 43.9 (d), 42.2 (t), 34.4 (t), 29.1
(t), 24.4 (t), 23.6 (t), 20.8 (t); MS m/z (rel. intensity) 198
(M+, 0.1), 155 (22), 99 (100); IR (CDCl3) 2940, 1723
cmÀ1. Anal. calcd for C11H18O3: C, 66.64; H, 9.15.
Found: C, 65.69; H, 9.25.
Methyl 3-(2-Oxocyclohexyl)propanoate (8). In a ¯ask
provided with a Dean-Stark apparatus, cyclohexanone
(21.2 g, 216 mmol) and pyrrolidine (19 mL, 227 mmol)
were dissolved under stirring in benzene (65 mL). The
solution was re¯uxed for 5.5 h, the solvent and the
excess of pyrrolidine were then distilled o€ and the
residual crude oil was dissolved in dioxane (77 mL).
Fresh distilled methyl acrylate (29.2 mL, 324 mmol) was
added under stirring and the resulting solution was
re¯uxed for 3 h. Water was then added (10 mL) and the
mixture heated again under re¯ux. After 1 h, the pro-
duct was extracted with Et2O and the organic layer
washed with diluted HCl and dried over Na2SO4. After
®ltration and evaporation of the solvent, the crude oil
obtained was puri®ed by distillation under reduced
pressure, a€ording pure 8 (20.0 g, 50%).
3-[2-(1,3-Dioxolan-2-yl)cyclohexyl]propanal oxime (15).
A solution of aldehyde 12 (6.12 g, 31.0 mmol) and
.
NH2OH HCl (2.76 g, 40.0 mmol) in pyridine (120 mL)
was stirred for 2 h at room temperature. The mixture
was extracted with Et2O and the organic layer washed
with water and dried over Na2SO4. After ®ltration and
evaporation of the solvent the crude oil obtained was
puri®ed by chromatography (petroleum ether/EtOAc,
1.5:1, Rf 0.5). Recrystallization fromEt 2O-petroleum
ether gave pure oxime 15 (4.02 g, 61%) as a 1:1 mixture
of diastereoisomers.
15: mp 74±75 ꢀC; H NMR (CDCl3) d 7.50 (s, 1H, E),
1
7.39 (t, J=6.0 Hz, 1H, Z), 7.17 (s, 1H, Z), 6.69 (t,
J=5.4 Hz, 1H, E), 3.91 (m, 4H), 2.48±2.08 (m, 2H),
1.90±1.18 (m, 11H); 13C NMR (CDCl3) d 153.2 (d, Z),
152.4 (d, E), 110.6 (s), 64.6 (t), 64.5 (t), 44.2 and 43.9
(d), 34.5 (t), 28.8 and 28.7 (t), 27.5 and 25.1 (t), 24.5 and
24.4 (t), 24.2 (t), 23.6 and 22.9 (t); MS m/z (rel. inten-
sity) 213 (M+, 2), 155 (40), 99 (100); IR (CDCl3) 3587,
3321, 1602 cmÀ1. Anal. calcd for C11H19NO3: C, 61.95;
H, 8.98; N, 6.57. Found: C, 62.31; H, 9.28; N, 6.34.
8: oil; bp 115±117 ꢀC (1 mbar); 1H NMR (CDCl3) d 3.62
(s, 3H), 2.40±2.22 (m, 5H), 2.14±1.94 (m, 3H), 1.88±1.76
(m, 1H), 1.74±1.56 (m, 2H), 1.56±1.44 (m, 1H), 1.42±
1.22 (m, 1H); 13C NMR (CDCl3) d 212.2 (s), 173.8 (s),
51.2 (q), 49.5 (t), 41.9 (d), 33.9 (t), 31.4 (t), 27.8 (t), 24.8
(t), 24.6 (t); MS m/z (rel. intensity) 184 (M+, 1), 151
(70), 124 (48), 54 (100); IR (CDCl3) 1742, 1709 cmÀ1
.
6-[2-[2-(1,3-Dioxolan-2-yl)cyclohexyl]ethyl]-4-oxa-5-aza-
spiro[2.4]hept-5-ene (17). Liquid methylenecyclopropane
(5 mL) was transferred by a double-tipped needle into a
Methyl 3-[2-(1,3-Dioxolan-2-yl)cyclohexyl]propanoate
(10). In a ¯ask provided with a Dean-Stark apparatus,

1390
A. Guarna et al. / Bioorg. Med. Chem. 9 (2001) 1385±1393
1
solution of oxime 15 (4.02 g, 18.8 mmol) and Et3N (226
mg, 2.23 mmol) in CH2Cl2 (35 mL) cooled at À60 ꢀC.
The mixture was allowed to warm to 0 ꢀC and NaClO
(8% solution, 54 mL) was slowly added in 3.5 h. The
solution was stirred for 21 h, then the phases were
separated, the aqueous layer was extracted with
CH2Cl2 (3Â25 mL) and the combined organic layers
were dried over Na2SO4. After ®ltration and evapora-
tion of the solvent, crude 17 (4.89 g, 73%) was
obtained and used without puri®cation in the next
reaction.
21: oil; H NMR (CDCl3) d 11.80 (s, 1H), 6.31 (dd,
J=9.9, 17.2 Hz, 1H, 21a), 6.28 (dd, 1H, 21b), 6.10 (dd,
J=2.2, 17.2 Hz, 1H, 21a), 6.08 (dd, 1H, 21b), 5.48 (dd,
J=2.2, 9.9 Hz, 1H, 21a), 5.45 (dd, 1H, 21b), 5.14 (s, 1H,
21b), 5.07 (s, 1H, 21a), 4.99 (s, 1H, 21b), 2.51 (t, 2H),
2.05 (m, 6H), 1.63 (m, 4H); 13C NMR (CDCl3) d 186.6
(s), 158.7 (s), 137.6 (d), 128.0 (s), 122.7 (t), 113.0 (s), 94.7
(d), 28.4 (t), 28.0 (t), 26.3 (t), 24.1 (t), 22.5 (t), 22.4 (t);
MS m/z (rel. intensity) 203 (M+, 43), 202 (100), 147
(14); IR (CDCl3): 3692, 1690, 1589, 1543 cmÀ1. Anal.
calcd for C13H17NO: C, 76.81; H, 8.43; N, 6.89. Found:
C, 76.64; H, 8.21; N, 7.00.
17: 1H NMR (CDCl3) d 3.90 (m, 4H), 2.98 (m, 2H), 2.35
(m, 2H), 1.99±1.12 (m, 11H), 1.09 (m, 2H), 0.67 (m,
2H); 13C NMR (CDCl3) d 160.1 (s), 110.5 (s), 64.6 (s),
64.6 (s), 64.5 (t), 43.9 (d), 41.9 (t), 34.4 (t), 28.9 (t), 26.3
(t), 24.8 (t), 24.3 (t), 23.6 (t), 11.4 (t, 2 C); MS m/z (rel.
intensity) 265 (M+, 10), 155 (41), 99 (95), 55 (100); IR
Methyl 3-[[2-(1,3-Dioxolan-2-yl)-5-(N-t-butyl)acetamido]-
cyclohexyl]]propanoate (11). Prepared as compound 10.
Starting from 9 (32.14 g, 108 mmol), crude ketal 11
(22.2 g, 60%) was obtained as an oil. A portion (100
mg) of this crude oil was puri®ed by chromatography
(CH2Cl2/MeOH, 30:1, 1% Et3N, Rf 0.31), a€ording 11
as a 5:1 mixture of cis and trans isomers. Spectroscopic
data refer to the major isomer.
(CDCl3) 1617 cmÀ1
.
6-[2-(2-Oxocyclohexyl)ethyl]-4-oxa-5-azaspiro[2.4]hept-
5-ene (19). Isoxazoline 17 (3.64 g, 13.7 mmol) and p-
TsOH (392 mg, 2.23 mmol) were dissolved in acetone
(90 mL) and water (30 mL) and the resulting solution
was stirred at roomtemperature for 7 days. The product
was extracted with CH2Cl2, the organic phase washed
with 2 N NaHCO3 and dried over Na2SO4. After ®ltra-
tion and evaporation of the solvent, a yellow crude oil
(2.36 g) was obtained. This was puri®ed ®rst by chro-
matography (CH2Cl2/EtOAc, 12.5:1, Rf 0.35) and then
1
11: oil; H NMR (CDCl3) d 5.27 (br s, 1H), 3.88 (m,
4H), 3.60 (s, 3H), 2.40±2.20 (m, 2H), 2.00±1.81 (m, 5H),
1.76±1.43 (m, 6H), 1.28 (s, 9H), 1.16±0.91 (m, 1H); 13C
NMR (CDCl3) d 174.4 (s), 171.3 (s), 110.2 (s), 64.7 (t),
64.6 (t), 51.4 (s), 51.1 (q), 44.7 (d), 43.6 (t), 35.6 (t), 34.5
(t), 34.1 (d), 32.2 (t), 29.7 (t), 28.8 (q, 3 C), 23.7 (t); MS
m/z (rel. intensity) 341 (M+, 6), 310 (5), 185 (57), 99
(100); IR (CDCl3) 3439, 1725, 1660, 1502 cmÀ1. Anal.
calcd for C18H31NO5: C, 63.32; H, 9.15; N, 4.10. Found:
C, 63.01; H, 9.42; N, 3.99.
by recrystallization fromEt O-petroleumether, a€ord-
2
ing pure isoxazoline 19 (1.43 g, 47%).
ꢀ
1
19: m p 109 C; H NMR (CDCl3) d 2.98 (s, 2H), 2.35
(m, 3H), 2.20±1.30 (m, 10H), 1.09 (m, 2H), 0.68 (m,
2H); 13C NMR (CDCl3) d 213.2 (s), 159.9 (s), 64.8 (s),
49.7 (d), 42.1 (t), 42.0 (t), 34.0 (t), 27.9 (t), 25.9 (t), 25.8
(t), 24.9 (t), 11.4 (t, 2 C); MS m/z (rel. intensity) 221
(M+, 30), 55 (100); IR (CDCl3) 1704, 1617 cmÀ1. Anal.
calcd for C13H19NO2: C, 70.56; H, 8.65; N, 6.33. Found:
C, 70.59; H, 8.90; N, 6.36.
3-[[2-(1,3-Dioxolan-2-yl)-5-(N-t-butyl)acetamido]cyclo-
hexyl]]propanal Oxime (16). A solution of 11 (22.1 g,
64.7 mmol) in toluene (500 mL) was cooled at À78 ꢀC;
DIBAL-H (solution 1 M in toluene, 288 mL) was then
slowly added in 4 h and the resulting solution was stir-
red for 3 h. After addition of water (260 mL), the mix-
ture was allowed to warmto roomtemperature,
extracted with CH2Cl2 (4Â200 mL) and the organic
layer dried over Na2SO4. After ®ltration and evapora-
tion of the solvent a crude oil (17.2 g) was obtained (13)
which was used without puri®cation in the next reac-
2,3,5,6,7,8,9,10- and 2,3,5,6,6a,7,8,9-Octahydro-(1H)-
benzo[c]quinolizin-3-one (3a and 3b). Isoxazoline 19 (476
mg, 2.15 mmol) dissolved in dry DMF (50 mL) was
heated under re¯ux for 3 h. After distillation of the sol-
vent, a yellow crude oil (470 mg) was obtained, con-
taining a mixture of products 3 and 21. This oil was
puri®ed by chromatography (CHCl3/MeOH, 30:1)
a€ording pure 3 (163 mg, 37%, Rf 0.27) as an orange
solid and 21 (122 mg, 28%, Rf 0.48), both as mixtures of
two isomers (3a/3b, 10:1; 21a/21b, 10:1).
1
tion. H NMR (CDCl3) d 5.20 (br s, 1H), 3.94 (m, 4H),
3.62 (m, 2H), 2.10±1.10 (m, 14H), 1.32 (s, 9H).
Then, under stirring, to a solution of distilled oxalyl
chloride (10.9 mL, 125 mmol) in CH2Cl2 (270 mL),
cooled at À60 ꢀC, DMSO (15 mL, 211 mmol) was
added, followed by slow addition (25 min) of a solution
of the above crude oil in CH2Cl2 (260 mL). After 15
min, Et3N (56 mL) was slowly added in 15 min. After 5
min stirring, the mixture was warmed to room tem-
perature and washed with water (535 mL); after
separation of the phases, the aqueous one was extracted
with CH2Cl2 (3Â250 mL) and the combined organic
layers were dried over Na2SO4. After ®ltration and
evaporation of the solvent, crude aldehyde 14 (14.6 g)
was obtained, which was used without puri®cation in
the next reaction. 1H NMR (CDCl3) d 9.74 (s, 1H), 5.20
(br s, 1H), 3.92 (m, 4H), 2.55 (m, 2H), 2.10±1.10 (m,
12H), 1.32 (s, 9H).
3: mp 85±86 ꢀC; 1H NMR (CDCl3) d 5.12 (br s, 1H, 3b),
4.95 (s, 1H, 3a), 4.92 (s, 1H, 3b), 3.65 (t, J=7.5 Hz, 2H),
2.45 (m, 4H), 2.14 (m, 2H), 2.00 (m, 4H), 1.71 (m, 2H),
1.57 (m, 2H); 13C NMR (CDCl3) d 191.5 (s), 159.5 (s),
130.4 (s), 115.8 (s), 99.6 (d), 43.5 (t), 35.8 (t), 29.5 (t),
29.1 (t), 25.5 (t), 24.3 (t), 23.0 (t), 22.0 (t); MS m/z (rel.
intensity) 203 (M+, 100), 174 (56), 147 (59); IR (CDCl3)
1619, 1551, 1470 cmÀ1. Anal. calcd for C13H17NO: C,
76.81; H, 8.43; N, 6.89. Found: C, 76.72; H, 8.65; N,
6.78.

A. Guarna et al. / Bioorg. Med. Chem. 9 (2001) 1385±1393
1391
A solution of this crude oil (14.6 g) in pyridine (210 mL)
.
was added to a solution of NH2OH HCl (13.7 g, 196.9
(t), 11.4 (t, 2 C); MS m/z (rel. intensity) 334 (M+, 22),
277 (15), 55 (100); IR (CDCl3) 3440, 1707, 1665, 1505
cmÀ1. Anal. calcd for C19H30N2O3: C, 68.23; H, 9.04;
N, 8.38. Found: C, 68.21; H, 9.00; N, 8.60.
mmol) in pyridine (107 mL) and the resulting mixture
was stirred at room temperature for 20 h. The mixture
was poured into CH2Cl2 (800 mL) and washed with
water; after separation of the phases, the aqueous one
was extracted with CH2Cl2 (3Â200 mL) and the com-
bined organic layers were dried over Na2SO4. After ®l-
tration and evaporation of the solvent, crude 16 (11.3 g)
was obtained. This was puri®ed by chromatography
eluting with CHCl3/MeOH, 50:1, 1% Et3N, and then
with CHCl3/MeOH, 3:1, 1% Et3N (Rf 0.32), a€ording
oxime 16 (7.41 g, 35%) as a 1:1 mixture of E/Z diaster-
eoisomers. Spectral data refer to the major E and Z
diastereoisomers.
2,3,5,6,7,8,9,10- and 2,3,5,6,6a,7,8,9-Octahydro-(1H)-8-
(N-t-Butyl)acetamido-benzo[c]quinolizin-3-one (4a and
4b). A solution of isoxazoline 20 (947 mg, 2.83 mmol)
in DMF (109 mL) was heated under re¯ux for 3 h. After
distillation under reduced pressure of the solvent, a
crude oil containing a mixture of 4, 22 and 24 was
obtained. Chromatographic separation (CH2Cl2/MeOH,
25:1, 1% NH3) a€orded pure 4 (161 mg, 18%, Rf 0.32)
and 22 (188 mg, 21%, Rf 0.43) both as mixtures of two
isomers (4a/4b, 10:1, and 22a/22b).
1
1
16: oil; H NMR (CDCl3) d 8.80 (br s, 1H, E), 8.40 (br
4: oil; H NMR (CDCl3) d 5.29 (br s, 1H), 4.94 (s, 1H,
s, 1H, Z), 7.36 (t, J=5.8 Hz, 1H, Z), 6.64 (m, 1H, E),
5.29 (br s, 1H), 3.89 (m, 4H), 2.34±2.08 (m, 2H), 2.01±
1.61 (m, 8H), 1.50±1.12 (m, 3H), 1.30 (s, 9H), 1.03±0.84
(m, 1H); 13C NMR (CDCl3) d 171.6 (s), 152.7 (d) and
152.0 (d), 110.3 (s), 64.8 (t), 64.7 (t), 51.2 (s), 44.8 and
43.6 (d), 35.4 and 35.6 (t), 34.2 (d), 32.7 (t), 29.7 (t), 28.8
(q, 3 C), 27.4 (t), 25.1 (t), 22.7 (t); MS m/z (rel. intensity)
326 (M+, 2), 309 (13), 268 (4), 99 (100); IR (CDCl3)
3588, 3550±3000 (br), 3443, 1657, 1508 cmÀ1. Anal.
calcd for C17H30N2O4: C, 62.55; H, 9.26; N, 8.58.
Found: C, 62.27; H, 9.20; N, 8.30.
4a), 4.91 (s, 1H, 4b), 3.67 (m, 2H), 2.59±2.38 (m, 4H),
2.30±1.70 (m, 11H), 1.33 (s, 9H); 13C NMR (CDCl3) d
191.7 (s), 171.1 (s), 159.6 (s), 130.2 (s), 114.7 (s), 99.9
(d), 51.3 (s), 43.7 (t), 35.8 (t), 35.4 (t), 30.8 (d), 29.6 (t),
28.9 (t), 28.8 (t), 28.8 (q, 3 C), 25.4 (t), 24.3 (t); MS m/z
(rel. intensity) 316 (M+, 92), 315 (41), 259 (12), 244 (17),
202 (98), 201 (100); IR (CDCl3): 3441, 1662, 1621, 1551,
1509 cmÀ1. Anal. calcd for C19H28N2O2: C, 72.12; H,
8.92; N, 8.85. Found: C, 72.01; H, 8.70; N, 8.90.
1
22: oil; H NMR (CDCl3) d 11.78 (br s, 1H), 6.26 (dd,
J=15.4, 9.9 Hz, 1H), 6.08 (dd, J=15.4, 2.2 Hz, 1H),
5.45 (dd, J=9.9, 2.2 Hz, 1H), 5.36 (br s, 1H), 5.07 (s,
1H), 2.80±1.62 (m, 13H), 1.30 (s, 9H); 13C NMR
(CDCl3) d 181.8 (s), 171.2 (s), 158.8 (s), 137.7 (s), 126.6
(d), 123.0 (t), 111.9 (s), 94.9 (d), 51.3 (s), 34.5 (d), 34.2
(t), 32.4 (t), 31.3 (t), 28.7 (q, 3 C), 28.7 (t), 25.9 (t), 24.0
(t); MS m/z (rel. intensity) 316 (M+, 41), 315 (68), 259
(17), 202 (81), 201 (100); IR (CDCl3) 3437, 1705, 1660,
1588, 1504 cmÀ1. Anal. calcd for C19H28N2O2: C, 72.12;
H, 8.92; N, 8.85. Found: C, 72.07; H, 8.90; N, 9.00.
6-[2-[2-(1,3-Dioxolan-2-yl)-5-(N-t-butyl)acetamido]cyclo-
hexyl]ethyl]-4-oxa-5-azaspiro[2.4]hept-5-ene (18). Pre-
pared as compound 17. Starting fromoxime 16 (7.40 g,
22.6 mmol), isoxazoline 18 (4.96 g, 58%) was obtained
as a crude oil used without puri®cation in the next
reaction.
1
18: H NMR (CDCl3) d 5.31 (br s, 1H), 3.86 (m, 4H),
2.92 (br s, 2H), 2.40±2.17 (m, 2H), 1.97±1.47 (m, 8H),
1.40±0.92 (m, 6H), 1.27 (s, 9H), 0.64 (m, 2H); 13C NMR
(CDCl3) d 171.3 (s), 160.1 (s), 110.2 (s), 64.7 (s), 64.1 (t,
2 C), 51.1 (s), 44.6 (d), 43.7 (t), 42.0 (t), 35.6 (t), 34.4 (t),
34.1 (d), 29.6 (t), 28.7 (q, 3 C), 26.3 (t), 24.8 (t), 11.5 (t, 2
C).
24: 1H NMR (CDCl3) d 7.30 (br s, 1H); 5.30 (br s, 1H);
3.60±3.40 (m, 2H); 2.60±1.50 (m, 15H); 1.31 (s, 9H).
N-(tert-Butoxycarbonyl)-octahydroquinolin-2-one (26).
To a solution of lactam 25 (4.0 g, 26 mmol) in CH2Cl2
(90 mL) were added Et3N (3.6 mL, 26 mmol), Boc2O
(34.6, g 156 mmol) and DMAP (3.17, 52 mmol) and the
resulting solution was heated at re¯ux for 6 days. Water
(90 mL) was added, and after separation of the phases,
the aqueous layer was extracted with CH2Cl2 (3Â30
mL). The combined organic phases were washed with 1
M KHSO4 (30 mL), NaHCO3 (satd) (30 mL), and brine
(30 mL) and dried over Na2SO4. After ®ltration and
evaporation of the solvent the crude residue was puri®ed
by ¯ash chromatography (CH2Cl2 then CH2Cl2/MeOH,
50:1) to give N-Boc-octahydroquinolin-2-one 26 (5.55g,
92%) as a clear oil.
6-[2-[2-Oxo-5-[(N-t-butyl)acetamido]cyclohexyl]ethyl]-4-
oxa-5-azaspiro[2.4]hept-5-ene (20). Crude isoxazoline 18
(4.92 g, 13.1 mmol) was dissolved in acetone (150 mL)
and H2SO4 (1.7 M solution in acetone, 9.8 mL) was
slowly added, under vigorous stirring, at roomtem-
perature. When the reaction was complete, Na2CO3 was
added up to pH 7; after ®ltration and evaporation of the
solvent, crude 20 was obtained. This was puri®ed by
chromatography, eluting with CH2Cl2/MeOH, 60:1 and
then 20:1 (Rf 0.28), a€ording pure 20 (1.45 g, 33%) as a
mixture of cis and trans isomers. Spectroscopic data
refer to the major isomer.
1
1
20: oil; H NMR (CDCl3) d 5.25 (br s, 1H), 2.98 (br s,
26: H NMR (CDCl3) d 3.24 (m, 1H), 2.46±2.40 (m,
2H), 2.45±2.34 (m, 5H), 2.18±1.93 (m, 4H), 1.80±1.66
(m, 2H), 1.45±1.01 (m, 3H), 1.33 (s, 9H), 1.09 (m, 2H),
0.68 (m, 2H); 13C NMR (CDCl3) d 212.0 (s), 170.7 (s),
159.5 (s), 64.6 (s), 51.1 (s), 48.4 (d), 43.4 (t), 42.0 (t), 41.2
(t), 39.6 (t), 34.0 (d), 33.3 (t), 28.6 (q, 3 C), 25.8 (t), 25.6
2H), 1.96±1.89 (m, 1H), 1.76±1.63 (m, 5H), 1.41 (s, 9H),
1.27±1.24 (m, 5H); 13C NMR (CDCl3) d 170.7 (s), 153.9
(s), 83.4 (s), 61.9 (d), 40.0 (d), 33.5 (t), 31.6(t), 31.4 (t),
27.7 (t), 27.6 (q, 3 C), 25.4 (t), 24.4 (t); MS m/z (rel.
intensity) 197 (42), 154 (100), 153 (84), 138 (72), 110

1392
A. Guarna et al. / Bioorg. Med. Chem. 9 (2001) 1385±1393
(96); IR (CDCl3) 2937, 1735, 1660. Anal. calcd for
C14H23NO3: C, 66.37; H, 9.15; N, 5.53. Found: C,
66.07; H, 8.90; N, 5.36.
and the combined organic layers were dried over
Na2SO4. After ®ltration the solution was concentrated
under reduced pressure and the resulting residue was
puri®ed by chromatography (AcOEt, then AcOEt/
MeOH 10:1, then AcOEt/MeOH 1:1) to give 42 mg
(11% from 27, Rf=0.28) of 6 as a yellowish oil.
N -(tert -Butoxycarbonyl)-2-ethoxy -octahydroquinoline
(27). To a solution of N-Boc lactam 26 (2.32 g, 9.16
mmol) in anhydrous THF (25 mL) cooled at À78 ꢀC
was added dropwise a 1 M solution of LiEt3BH in THF
(18 mL) over 5 min. The reaction mixture was stirred at
À78 ꢀC for 15 min and then 1 M HCl in EtOH was
added dropwise until pH 3±4 was reached, immediately
followed by the addition of 4 mL of EtOH. The mixture
was allowed to warmto 0 ꢀC, and after 30 min stirring,
was diluted with CH2Cl2 (120 mL), the organic layer
was washed with water (120 mL), NaHCO3 (satd) (120
mL), brine (120 mL) and dried over Na2SO4. After ®l-
tration and evaporation of the solvent, 27 (2.5 g) was
obtained as an oil that was suciently pure to be used
in the next step.
1
6: H NMR (CDCl3) d 7.15 (d, J=7.4 Hz, 1H) 4.89 (d,
J=7.4 Hz, 1H), 3.75 (m, 1H), 3.18±3.13 (m, 1H), 2.86±
2.80 (m, 1H), 2.41±2.38 (m, 2H), 1.63±1.50 (m, 6H),
1.63±1.50 (m, 6H); 13C NMR (CDCl3) d 191.0 (s), 149.9
(d), 96.7 (d), 59.8 (d), 53.6 (d), 42.1 (d), 37.2 (t), 30.7 (t),
25.5 (t), 25.3 (t), 25.0 (t); IR (CDCl3) 3050, 2937, 1709,
1571 cmÀ1; MS m/z (rel. intensity) 205 (M+, 1), 162
(27), 110 (17), 86 (64), 84 (100). Anal. calcd for
C13H19NO: C, 76.06; H, 9.33; N, 6.82. Found: C, 76.16;
H, 9.04; N, 6.90.
2,3,5,6,6a,7,8,9,10, 10a-Decahydro-(1H)-benzo[c]quino-
lizin-3-one (7). Compound 5 (130 mg, 0.63 mmol) was
dissolved in EtOH (20 mL), Pd/C (5%) (170 mg) was
added, the apparatus ¯ushed three times with H2 and
the mixture stirred at 25 ꢀC for 21 h under H2 atmo-
sphere. The mixture was ®ltered through a Celite layer
and then concentrated under reduced pressure obtaining
the corresponding satd alcohol which was suciently
pure to be used in the next step. Thus, to a solution of
the crude alcohol (130 mg) in acetone cooled at 0 ꢀC was
added dropwise a solution prepared dissolving CrO3
(150 mg, 1.5 mmol) and 96% H2SO4 (0.28 mL) in water
(2 mL) until the colour of the reaction mixture turned
deep orange. After stirring the reaction mixture for 5
min, water (20 mL) was added and the mixture extrac-
ted with CH2Cl2. The organic phase was washed with
5% NaHCO3 aqueous solution, brine and then dried
over Na2SO4. After evaporation of the solvent the cor-
responding 3-oxo compound was obtained (103 mg) in
suciently pure formto be used in the next step. 1H
NMR (CDCl3) d 3.60±3.42 (m,1H), 3.28 (dm, J=6.4
Hz, 1H), 2.40±2.01 (m, 4H), 1.90±1.04 (m, 15H); MS
m/z (rel. intensity) 207 (M+, 8), 164 (100) IR (CDCl3)
2935, 1712 cmÀ1. The above compound (103 mg) was
dissolved in 5% AcOH (12 mL) aqueous solution.
EDTA (832 mg, 2.88 mmol) and Hg(OAc)2 (637mg,
2.00 mmol) were added and the resulting solution was
heated 2 h at 85 ꢀC under vigorous stirring. After cool-
ing to roomtemperature, the solution was ®ltered on a
Celite layer and Na2CO3 (satd) was added up to pH 8.
The product was extracted with CH2Cl2 (5Â25 mL) and
the organic layer washed with brine and dried over
Na2SO4. After ®ltration and evaporation of the solvent,
the crude material was puri®ed by chromatography,
eluting with acetone, a€ording pure 7 (17 mg, 16%) as a
pale yellow solid.
1
27: H NMR (CDCl3) d 5.38 (d, J=6.2 Hz, 1H), 3.64±
3.27 (m, 2H), 3.16 (m, 1H), 2.26±2.04 (m, 1H), 2.00±1.50
(m, 12H), 1.41 (s, 9H), 1.15 (t, J=7.0 Hz, 3H); MS m/z
(rel. intensity) 238 (M+- 45, 11), 181 (100), 136 (51),
57(91).
3,4, 5,6,6a,7,8,9,10,10a-Decahydro-(4aꢀH)-benzo[c]quino-
lizin-3-one (5). To a stirred solution of 1-methoxy-3-
[(trimethylsilyl)oxy]-1,3-butadiene (276 mL, 1.42 mmol)
and 27 (200 mg, 0.71 mmol) in CH2Cl2 (5 mL) cooled at
0 ꢀC, TiCl4 (155 mL, 1.42 mmol) was added drop wise
under nitrogen atmosphere. After 60 min at 0 ꢀC,
NaHCO3 (satd) (4 mL) was added and the resulting
mixture stirred 30 min at room temperature. After
separation of the phases, the product was extracted with
CH2Cl2 (3Â15 mL) and the combined organic layers
dried over Na2SO4. After ®ltration and evaporation of
the solvent, crude 5 was obtained. This was puri®ed by
chromatography, eluting with acetone (Rf=0.43),
a€ording pure 5 (16 mg, 11% from 26).
5: 1H NMR (CDCl3) d 7.16 (d, J=7.8 Hz, 1H), 4.96 (d,
J=7.8 Hz, 1H), 3.43±3.37 (m, 1H), 2.72±2.60 (m, 2H),
2.33±2.21 (m, 2H), 2.14±1.41 (m, 6H), 1.37±1.09 (m,
6H); 13C NMR (CDCl3) d 191.7 (s), 148.5 (d), 98.2 (d),
65.5 (d), 59.2 (d), 42.9 (d), 42.3 (t), 32.7 (t), 31.8 (t), 30.9
(t), 30.3 (t), 29.2 (t), 25.4 (t); IR (CDCl3) 2935, 1709,
1626, 1579 cmÀ1; MS m/z (rel. intensity) 205 (M+, 1),
162 (27), 110 (17), 86(64), 84 (100). Anal. calcd for
C13H19NO: C, 76.06; H, 9.33; N, 6.82. Found: C, 75.99;
H, 9.64; N, 6.44.
3,4,5,6,6a,7,8,9,10,10a-Decahydro-(4aꢁH)-benzo[c]qui-
nolizin-3-one (6). To a solution of 27 (500 mg, 1.76
mmol), 1-methoxy-3-(trimethylsilyl)oxy-1,3-butadiene
(0.69 mL, 3.53 mmol), and TEA (0.110 mL, 0.79 mmol)
in CH2Cl2 (3 mL) cooled at 0 ꢀC was added dropwise
TMSOTf (0.5 mL, 3.53 mmol). The mixture was
allowed to warmto roomtemperature and kept under
stirring for 45 min. Then NaHCO3 (satd) was added (4
mL) and the resulting reaction mixture was stirred for
48 h at roomtemperature. After separation of the pha-
ses, the product was extracted with CH2Cl2 (3Â30 mL),
1
7: H NMR (CDCl3) d 4.89 (s, 1H), 3.58 (m, 1H), 3.37
(dt, J=11.3, 4.0 Hz), 3.14 (m, 1H), 2.10±0.90 (m, 15H);
13C NMR (CDCl3) d 191.6 (s), 128.2 (d), 100.0 (s), 64.0
(d), 45.2 (t), 41.6 (t), 35.9 (t), 32.6 (d), 31.6 (t), 28.5 (t),
27.1(t), 25.5 (t), 25.4 (t); MS m/z (rel. intensity) 205
(M+, 42), 162 (100), 134 (38). IR (CDCl3) 3050,
2920,1719,1560. Anal. calcd for C13H19NO: C, 76.06; H,
9.33; N, 6.82. Found: C, 76.26; H, 9.16; N, 6.64.

A. Guarna et al. / Bioorg. Med. Chem. 9 (2001) 1385±1393
1393
Biological assays
2, 1±29. (c) Abell, A. D.; Henderson, B. R. Curr. Med. Chem.
1995, 2, 583. (d) Li, X.; Chen, C.; Singh, S.; Labrie, F. Steroids
1995, 60, 430. (e) Frye, S. V. Curr. Pharm. Des 1996, 2, 59.
10. Guarna, A.; Belle, C.; Machetti, F.; Occhiato, E. G.; Payne,
A. H.; Cassiani, C.; Comerci, A.; Danza, G.; De Bellis, A.; Dini,
S.; Marrucci, A.; Serio, M. J. Med. Chem. 1997, 40, 1112.
11. Guarna, A.; Occhiato, E. G.; Machetti, F.; Marrucci, A.;
Danza, G.; Serio, M.; Paoli, P. J. Med. Chem. 1997, 40, 3466.
12. Hartmann, R. W.; Gohring, S. Eur. J. Med. Chem. 1994,
29, 807.
The IC50 determinations toward 5aR-1 expressed in
CHO 1827 and 1829 cells were carried out as repor-
ted.14c In the inhibition experiments, the inhibitors were
added in the range 10À9±10À5 M for 3 and 5±7 and
10À7À10À3 M for 4. Each determination was performed
in duplicate.
13. (a) Jones, C. D.; Audia, J. E.; Lawhorn, D. E.; McQuaid,
L. A.; Neubauer, B. L.; Pike, A. J.; Pennington, P. A.; Stamm,
N. B.; Toomey, R. E.; Hirsch, K. S. J. Med. Chem. 1993, 36,
421. (b) Mook, R. A., Jr.; Lackey, K.; Bennet, C. Tetrahedron
Lett. 1995, 36, 3969. (c) Abell, A. D.; Brandt, M.; Levy, M. A.;
Holt, D. A. Bioorg. Med. Chem. Lett. 1994, 4, 2327. (d)
Brandt, M.; Levy, M. A.; Holt, D. A. Bioorg. Med. Chem.
Lett. 1996, 6, 481.
14. (a) Guarna, A.; Occhiato, E. G.; Scarpi, D.; Tsai, R.;
Danza, G.; Comerci, A.; Mancina, R.; Serio, M. Bioorg. Med.
Chem. Lett. 1998, 8, 2871. (b) Guarna, A.; Occhiato, E. G.;
Scarpi, D.; Zorn, C.; Danza, G.; Comerci, A.; Mancina, R.;
Serio, M. Bioorg. Med. Chem. Lett. 2000, 10, 353. (c) Guarna,
A.; Machetti, F.; Occhiato, E. G.; Scarpi, D.; Comerci, A.;
Danza, G.; Mancina, R.; Serio, M.; Hardy, K. J. Med. Chem.
2000, 43, 3718.
Acknowledgements
The authors thank Ministero dell'Universita e della
Ricerca Scienti®ca e Tecnologica (MURST - COFIN
1998-2000), CNR (Target Project on Biotechnology,
grants 99.00381.PF49 and 97.00482.PF49), and the
Ares-Serono for ®nancial support. Mr. Sandro Papaleo,
Mrs. Brunella Innocenti and Mrs. Cristina Faggi are
acknowledged for their technical assistance.
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