Synthesis and biological evaluation of a conformationally free seco-analogue of the immunosuppressant FR901483

Article abstract of DOI:10.1016/S0968-0896(99)00250-3

The synthesis of an azaspirocyclic analogue of FR901483, phosphate 2Figure 1Scheme 1Reagents and conditions: (i) 4-methoxyphenethylamine, molecular sieves; then allylmagnesium bromide, Et₂O:CH₂Cl₂, 71%; (ii) I₂,

Full text of DOI:10.1016/S0968-0896(99)00250-3

Bioorganic & Medicinal Chemistry 7 (1999) 2891±2897
Synthesis and Biological Evaluation of a Conformationally Free
seco-Analogue of the Immunosuppressant FR901483
J. Bonjoch,a,* F. Diaba,a G. Puigbo,a D. Sole,a
V. Segarra,b L. Santamarõa, b J. Beleta, b H. Ryder b and J.-M. Palacios b
aLaboratory of Organic Chemistry, Faculty of Pharmacy, University of Barcelona, 08028-Barcelona, Spain
bAlmirall Prodesfarma, Research Center, Cardener 68-74, 08024-Barcelona, Spain
Received 17 June 1999; accepted 19 July 1999
AbstractÐThe synthesis of an azaspirocyclic analogue of FR901483, phosphate 2, is described based on the implementation of a
key 5-endo aminocyclization promoted by iodine for direct functionalization of the 1-azaspiro[4.5]decane ring at the C-3 atom.
Compound 2 has no inhibitory activity in the cell proliferation assays reported. # 1999 Elsevier Science Ltd. All rights reserved.
Introduction
can be seen in the superimposition showed in Figure 2.
However, it is worth pointing out the absence in 2 of the
hydroxyl group present in FR901483 and the slight
separation of the methoxyphenyl moieties.
FR901483 (1) is a novel immunosuppressant recently
isolated from the fermentation broth of Cladobotryum
sp. No. 11231 and characterized by the Fujisawa
Laboratories.1 Immunosupressive agents display a wide
variety of structures,2,3 but none are related to that of 1.
This compound has a tricyclic ring system unprece-
dented among natural products, consisting of a 2-aza-
bicyclo[3.3.1]nonane nucleus fused to a pyrrolidine
moiety, resulting in a spiro framework. Additionally, 1
has a phosphate unit, which plays an important role in
this compound's immunosuppressive activity. Recently,
the unique structure of FR901483 has been the subject
of two synthetic approaches directed to its tricyclic ring
system,4,5 the Snider approach5 reaching the demethyl-
amino derivative of 1.
In this paper, we describe the synthesis and biological
evaluation of the seco-analogue 2 (RˆBn),6 introducing
a new synthetic procedure to obtain its azabicyclyc
framework.
Results and Discussion
Synthesis
Our procedure for the synthesis of 1-azaspiro-
[4.5]decanes (for other approaches leading to this aza-
bicyclic system by N(1)±C(2) bond formation in the last
step, see refs 7±12; for other synthetic entries, starting
from carbocyclic compounds, see inter alia, refs 13±20)
consists of the treatment of a homoallylamine (i.e. 3)
with iodine (for successful examples of the scarce reac-
tions of amino cyclization promoted by iodine, see refs
21 and 22; for iodine initiated cyclization of unsaturated
nitrogen containing compounds other than amines, see
refs 23±25), which promotes the electrophilic cyclo-
functionalization of the unsaturated amine to give a
pyrrolidine ring.26 The required homoallylamine was
prepared in a one-pot procedure involving the for-
mation of an imine from the monoethylene acetal of
1,4-cyclohexanedione and 4-methoxyphenylethylamine
followed by the addition of allylmagnesium bromide
(Scheme 1).27 Treatment of 3 with iodine promoted the
Part of our ongoing research project towards the
synthesis of FR901483 (1) is concerned with simplifying
the structure and increasing the ¯exibility of the mole-
cule; this has led to the consideration of the spiro deri-
vative 2 (RˆMe, Fig. 1). The seco-analogue 2 has a
conformational mobility that 1 lacks, and constitutes an
interesting target for pharmacological evaluation. Con-
sidering the chair axial±axial conformation for com-
pound 2, a good overlap between pyrrolidine and
methylamino nitrogen, as well as for the phosphate unit,
Key words: Immunological activity; natural products; phosphonic
acid and derivatives; poliycyclic heterocyclic compounds.
* Corresponding author. Fax: +34-934021896; e-mail: bonjoch@
farmacia.far.ub.es
0968-0896/99/$ - see front matter # 1999 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(99)00250-3

2892
J. Bonjoch et al. / Bioorg. Med. Chem. 7 (1999) 2891±2897
reduction of the resulting ketone 7 to stereoselectively
a€ord the alcohol 8; (iii) protection of the secondary
amino group, in order to avoid an oxidative process in
later steps; (iv) assembly of the phosphomonoester
group, which was achieved by phosphorylation of the
hydroxyl group of 9 with O,O-bisbenzyl-N,N-diisopro-
pyl phosphoramidate in the presence of tetrazole fol-
lowed by MCPBA oxidation;30 (v) ®nally, the benzyl
phosphates of 10 were hydrogenolyzed and the resulting
monoester 11 was treated with TFA to a€ord the dia-
mino derivative 2, which was isolated as its dipotassium
salt. The relative con®guration at C-5 and C-8, which
was generated in the ketone reduction step, was inferred
taking into account the preferred conformation of
ketone 7 (the same as that of the corresponding related
acetal 5), and the fact that the NaBH4 reduction furn-
ished an equatorial alcohol, as expected. Additionally,
NOESY experiments upon 2 agreed with the con®gura-
tion and conformation depicted in Scheme 1.
Figure 1.
iodoaminocyclization process to give 4. Thus, in only
two separate steps we gained access to the suitably
functionalized azabicyclic system. The oxidation of the
iodo derivative 4 to ketone 5 was e€ected using DMSO
and silver tetra¯uoroborate.28 The overall sequence
worked well and we obtained azaspiro 5 in 30% overall
yield. Compound 5 was analyzed spectroscopically by
means of NOESY experiments in order to elucidate its
preferred conformation, which proved to be that where
the amino group was disposed equatorially from the
carbocyclic ring.
Biological evaluation
The synthesized simpli®ed analogues of 1 (compounds
2, 7 and 8) were tested in the three lymphocyte in vitro
proliferation assays indicated in Experimental (TPA
plus IL-2, Tetanus toxoid or Phytohemegglutinin). None
of these three compounds showed inhibitory activity
at the range dose of 40 ng/mL to 4 mg/mL. The refer-
ence compound cyclosporin A showed good activity at
1 mg/mL.
Having established a simple and ecient synthetic entry
to 3,8-functionalized 1-azaspiro[4.5]decanes,29 we next
turned our attention to the preparation of the seco-
analogue of FR901483 2 (RˆBn). The subsequent steps
involved several functional transformations: (i) reduc-
tive amination to introduce the amino group at C-3; (ii)
deprotection of the acetal group in 6, followed by
Conformational studies
This lack of activity in the proliferation assay studies,
particularly for compound 2 which is the closest ana-
logue of 1, may be due to either the di€erences in
functional groups between the compounds (absence of
OH, benzyl in place of methyl) or an unsuitable con-
formation of 2, as suggested by the NMR experiments.
For a better understanding of this second possibility, a
conformational analysis of the cyclohexane ring in the
spiro moiety was carried out. We elected to evaluate this
point by calculating the di€erences in the heat of for-
mation for the two chair conformations. Comparisons
of the resultant energy minima revealed that the con-
formation 2-I is indeed favored thermodynamically vis-
a-vis the conformer 2-II; the corresponding heats of
formation di€ered by 3.2 kcal/mol (Fig. 3). Thus, as
suggested by the NMR experiments, the only conforma-
tion detected for compound 2 was that corresponding to
a chair equatorial±equatorial (conformation 2-I) since
the equilibrium constant between the two conformers is
about 1000 at room temperature.31 This may suggest
that one of the reasons for the lack of activity for
compound 2 is the inaccessibility of the correct chair
conformation.
Conclusions
Although compound 2 could in principle adopt a con-
formation that matches to a great extention that of
Figure 2. Molecular ®tting of FR901483 (green) and 2 (yellow).

J. Bonjoch et al. / Bioorg. Med. Chem. 7 (1999) 2891±2897
2893
Scheme 1. Reagents and conditions: (i) 4-methoxyphenethylamine, molecular sieves; then allylmagnesium bromide, Et2O:CH2Cl2, 71%; (ii) I2,
NaHCO3, CH2Cl2:H2O, 51%; (iii) AgBF4, DMSO, 80%; (iv) benzylamine, molecular sieves; then NaBH4 (85%); (v) HCl, THF:H2O, 79%; (vi)
NaBH4, MeOH, 75%; (vii) (Boc)2O. MeOH:Et3N, 76%; (viii) (BnO)2PN(iPr)2, tetrazole; then m-CPBA, CH2Cl2, 50ꢀC, 73%; (ix), H2, Pd/C,
MeOH, 92%; (x) TFA, CH2Cl2; then K2CO3.
substituents of the cyclohexane ring must be trans-di-
axial for the additional ring linkage. As a consequence,
the di€erent distances between the various functional
groups in 2 with respect to those in 1 may explain the
lack of activity of the seco-analogue 2.
From a synthetic standpoint, we report a new synthetic
entry to the azaspiro[4,5]decane sca€old that might
allow us to access the hitherto unprepared natural pro-
duct FR901483, using an adequate amine (i.e. tryrosine
derivative) as starting material which could allow the
elaboration of the additional ring.
Experimental
Computational procedures
Structures were built with standard bond lengths and
angles using the Chem-X32,33 molecular modelling
package. All structures were initially optimised using
the steepest descents and conjugate gradient methods.
After semiempirical optimization using the MOPAC
program,34,35 charge distributions were calculated by
Figure 3. Conformational analysis of cyclohexane ring of compound 2
(HF=heat of formation in Kcal/mol, MOPAC/AM1).
means of the AM1 method. The PULAY keyword and
the eigenvector following the (EF) routine for minimum
search with GNORM=0.1 were used in the optimisa-
tion step. Conformation analysis was performed with
fully AM1 optimization. All calculations were per-
formed on a Digital Alpha Station 3000.
structure 1 (see Fig. 1), in fact 2 adopts a trans-di-
equatorial conformational relationship between the
phosphate group and the nitrogen atom, which is
non-accessible to the natural product 1 in which both

2894
J. Bonjoch et al. / Bioorg. Med. Chem. 7 (1999) 2891±2897
Biology
H-cis), 5.00 (ddt, J=10, 2.5 and 1.5 Hz, 1H, H-trans),
5.66 (ddt, J=17, 10 and 7.5 Hz, 1H, H-gem),0 6.81 (d,
J=8.5 Hz, 2H, 30-H), 7.10 (d, J=8.5 Hz, 2H, 2 -H); 13C
NMR (74.5 MHz, CDCl3) d 30.1 and 32.5 (C-2 and C-
3), 36.2 (CH2Ar), 41.4 (CH2CH), 42.8 (CH2N), 52.7 (C-
4), 55.1 (CH3O), 64.0 (CH2O), 108.9 (C-1), 113.6 (C-30),
117.5 (ˆCH2), 129.5 (C-20), 132.3 (C-10), 134.0 (ˆCH),
157.8 (C-40). Anal. calcd for C20H29NO3: C, 72.47; H,
8.82; N, 4.23. Found: C, 72.26; H, 9.05; N, 4.30.
Assays were performed with freshly isolated human
peripheral blood mononuclear cells isolated from blood
by density gradient centrifugation. Lymphocyte pro-
liferation induced by di€erent stimulators (TPA plus IL-
2, Tetanus toxoid or Phytohemagglutinin) were assessed
by tritiated thymidine uptake. In all the cases 1Â105
cells were placed in ¯at bottom wells from a microtiter
plate in triplicates in a ®nal volume of 200 mL of RPMI
1640 complete medium with or without test compound.
For the TPA plus IL-2 induced proliferation assay
nylon wool puri®ed T cells were used. Cell proliferation
was assessed on day 1 for TPA plus IL-2 and Phytohe-
magglutinin stimulation, whereas for Tetanus toxoid,
induced proliferation was tested on day 6. Cyclosporin
A was used as a control.
1-[2-(4-Methoxyphenyl)ethyl]-3-iodo-1-azaspiro[4.5]decan-
8-one ethylene acetal (4). To a solution of amine 3 (2 g,
6 mmol) in CH2Cl2 (60 mL) and 5% aqueous NaHCO3
(60 mL) was added dropwise a solution of I2 (2.28 g,
9 mmol) in CH2Cl2 (60 mL). After stirring at room
temperature for 12 h, saturated aqueous sodium thio-
sulfate was added. The mixture was extracted with
CH2Cl2. The dried organic extracts were concentrated
and chromatographed (99.5:0.5 CH2Cl2:MeOH) to give
compound 4 (1.41 g, 51%): 1H NMR (300 MHz,
CDCl3) d 1.36 (m, 1H), 1.45±1.85 (m, 7H), 2.26 (dd,
J=14 and 6.6 Hz, 1H, H-4), 2.50 (dd, J=14 and 8.2 Hz,
1H, H-4), 2.69 (m, 4H, CH2CH2), 3.23 (dd, J=10.1 and
6.9 Hz, 1H, H-2), 3.39 (dd, J=10.1 and 7.4 Hz, 1H, H-
2), 3.79 (s, 3H, CH3O), 3.91 (m, 4H, CH2O), 4.27 (dddd,
J=8.2, 7.4, 6.9 and 6.6 Hz, 1H, H-3), 6.82 (d, J=8.6 Hz,
2H, 30-H), 7.12 (d, J=8.6 Hz, 2H, 20-H); 13C NMR
(74.5 MHz, CDCl3) d 15.0 (C-3), 28.9 and 29.9 (C-6 and
C-10), 31.9 and 32.4 (C-7 and C-9), 34.5 (CH2Ar), 47.0
(C-4), 49.3 (CH2N), 55.1 (CH3O), 61.9 (C-2), 64.0 and
64.2 (CH2O), 64.6 (C-5), 107.8 (C-8), 113.6 (C-30), 129.6
(C-20), 131.8 (C-10), 157.8 (C-40). Anal. calcd for
C20H28INO3: C, 52.52; H, 6.17; N, 3.06. Found: C,
52.76; H, 6.25; N, 3.02.
Chemistry
All reactions were carried out under an argon atmo-
sphere with dry, freshly distilled solvents under anhy-
drous conditions. TLC was carried out on SiO2 (silica
gel 60 F254, Merck), and the spots were located with UV
light, iodoplatinate reagent or 1% aqueous KMnO4.
Chromatography refers to ¯ash chromatography and
was carried out on SiO2 (silica gel, SDS, 230±400 mesh
ASTM). Unless otherwise noted, drying of organic
extracts during work up of reactions was performed
over anhydrous Na2SO4. Evaporation of solvents was
accomplished with a rotatory evaporator. 1H NMR and
13C NMR spectra were recorded with a Varian 300 or a
Varian VXR-500 instrument. Chemical shifts are repor-
ted in ppm down®eld from Me4Si. IR spectra were
recorded on a Nicolet 205 FT±IR spectrophotometer,
and only noteworthy absorptions are listed. Micro-
analyses were performed by the Centro de Investigacion
y Desarrollo (CSIC), Barcelona.
1-[2-(4-Methoxyphenyl)ethyl]-1-azaspiro[4.5]decan-3,8-
dione 8-monoethylene acetal (5). To a solution of
AgBF4 (0.68 g, 3.44 mmol) in DMSO (40 mL) was
added dropwise a solution of 4 (1.64 g, 3.6 mmol) in
DMSO (30 mL). After stirring at room temperature for
12 h, the suspension was ®ltered through Celite, and the
®ltrate partitioned between Et2O and water. The
organic extracts were washed with brine, dried, and
concentrated. Chromatography (CH2Cl2:MeOH, 99:1)
a€orded ketone 5 (0.99 g, 80%): IR (®lm) 1758; 1H
NMR (500 MHz, CDCl3) d 1.37 (dm, J=13.5 Hz, 2H,
H-6eq and H-10eq), 1.50 (td, J=13.5 and 4 Hz, 2H, H-
7ax and H-9ax), 1.73 (dm, J=13.5 Hz, 2H, H-7eq and
H-9eq), 1.88 (td, J=13.5 and 4 Hz, 2H, H-6ax and H-
10ax), 2.37 (s, 2H, H-4), 2.65 (dd, J=9 and 6.5 Hz, 2H,
CH2Ar), 2.77 (dd, J=9 and 6.5 Hz, 2H, CH2N), 3.24 (s,
2H, H-2), 3.77 (s, 3H, CH3O), 3.90 (s, 4H, CH2O), 6.81
(d, J=9 Hz, 2H, 30-H), 7.09 (d, J=9 Hz, 2H, 20-H); 13C
NMR (74.5 MHz, CDCl3) d 28.3 (C-6 and C-10), 32.4
(C-7 and C-9), 35.0 (CH2Ar), 47.7 (C-4), 49.9 (CH2N),
55.2 (CH3O), 58.9 (C-2), 62.1 (C-5), 64.2 and 64.3
(CH2O), 107.9 (C-8), 113.6 (C-30), 129.6 (C-20), 132.1
(C-10), 157.9 (C-40), 214.0 (C-3). Anal. calcd for
C20H27NO4: C, 69.54; H, 7.88; N, 4.05. Found: C,
69.59; H, 7.88; N, 4.24.
4-Allyl-4-[2-(4-methoxyphenyl)ethylamino]cyclohexanone
ethylene acetal (3). To a solution of 1,4-cyclohex-
anedione monoethylene acetal (8 g, 51.2 mmol) in
CH2Cl2 (160 mL) were added 2-(4-methoxyphenyl)-
ethylamine (9.8 mL, 66.6 mmol) and 4 A molecular
sieves (16 g). After stirring at room temperature for 4 h,
the suspension was ®ltered through Celite, and the ®l-
trate was concentrated to give the corresponding imine
(13C NMR (74.5 MHz, CDCl3) d 24.3 (CH2), 33.6
(CH2), 34.5 (CH2), 35.9 (CH2), 36.0 (CH2), 52.2
(CH2N), 54.8 (CH3O), 64.0 (CH2O), 107.5 (C-1), 113.3
(CH), 129.4 (CH), 131.8 (C), 157.6 (C), 170.6 (CN)). To
a solution of this imine in CH2Cl2 (80 mL) was added
dropwise a solution of allyl magnesium bromide
(102.4 mmol) in Et2O (120 mL). The mixture was stirred
at room temperature for 4 h, poured into saturated
aqueous NH4Cl, and extracted with CH2Cl2. The
organic extracts were washed with brine, dried, and
concentrated. Chromatography (from CH2Cl2 to 9:1
1
CH2Cl2:MeOH) yielded amine 3 (12 g, 71%): H NMR
(500 MHz, CDCl3) d 0.75 (br, 1H, NH), 1.40±1.58 (m,
6H), 1.70±1.80 (m, 2H), 2.10 (d, J=7.5 Hz, 2H,
CH2Cˆ), 2.66 (m, 4H, CH2CH2), 3.77 (s, 3H, OCH3),
3.90 (m, 4H, CH2O), 4.96 (ddt, J=17, 2 and 1.5 Hz, 1H,
3-Benzylamino-1-[2-(4-methoxyphenyl)ethyl]-1-azaspiro-
[4.5]decan-8-one ethylene acetal (6). To a solution of

J. Bonjoch et al. / Bioorg. Med. Chem. 7 (1999) 2891±2897
2895
ketone 5 (0.55 g, 1.6 mmol) in CH2Cl2 (2.8 mL) were
added benzylamine (0.34 mL, 3.2 mmol) and 4 A mole-
cular sieves (0.55 g). After stirring at room temperature
for 4 h, the suspension was ®ltered through Celite, and
the ®ltrate was concentrated to give the corresponding
imine. To a cooled (0ꢀC) solution of this imine in
MeOH (14 mL) was added NaBH4 (121 mg, 3.2 mmol).
After 2 h at 0ꢀC and 2 h at room temperature, the reac-
tion mixture was poured into saturated aqueous NH4Cl
and extracted with CH2Cl2 (5Â50 mL). The organic
extracts were dried and concentrated. Chromatography
(CH2Cl2 to 9:1 CH2Cl2:MeOH) yielded amine 6 (0.59 g,
poured into saturated aqueous NH4Cl and extracted
with CH2Cl2 (4Â25 mL). The organic extracts were
dried and concentrated. Chromatography (CH2Cl2 to
88:12 CH2Cl2:MeOH) yielded alcohol 8 (0.44 g, 75%):
1H NMR (300 MHz, CDCl3) d 1.20±1.33 (m, 4H, H-6
and H-9), 1.35 (br d, J=13.5 Hz, 1H, H-10eq), 1.42 (td,
J=13.5 and 3.2 Hz, 1H, H-10ax), 1.46 (dd, J=13 and
6 Hz, 1H, H-4), 1.57 (br s, 1H, NH), 1.83 (m, 2H, H-7),
2.11 (dd, J=13 and 8.7 Hz, 1H, H-4), 2.48 (m, 1H,
CH2N), 2.54±2.65 (m, 3H, CH2N and CH2Ar), 2.77 (dd,
J=9.5 and 4.5 Hz, 1H, H-2), 2.94 (dd, J=9.5 and 7 Hz,
1H, H-2), 3.23 (m, 1H, H-3), 3.41 (m, 1H, H-8), 3.69 (s,
2H, NCH2Ar), 3.71 (s, 3H, CH3O), 6.74 (d, J=8.5 Hz,
2H, 30-H), 7.04 (d, J=8.5 Hz, 2H, 20-H), 7.18 (m, 1H),
7.24±7.27 (m, 4H); 13C NMR (74.5 MHz, CDCl3) d 28.8
(C-6) 31.6 (C-10), 32.9 (C-9), 33.1 (C-7), 35.3 (CH2Ar),
42.7 (C-4), 50.1 (NCH2), 52.4 (NCH2Ar), 54.5 (C-3),
55.2 (CH3O), 57.2 (C-2), 62.6 (C-5), 70.6 (C-8), 113.6
(C-30), 126.9 (p-C), 128.2 and 128.4 (o-C and m-C),
129.6 (C-20), 132.8 (C-10), 140.2 (ipso-C), 157.8 (C-40).
85%): 1H NMR (500 MHz, CDCl3)
d 1.20 (d,
J=9.5 Hz, 1H, H-6eq), 1.30 (d, J=12.5 Hz, 1H, H-
10eq), 1.46 (dd, J=13 and 6 Hz, 1H, H-4), 1.49±1.66 (m,
5H, H-6ax, H-7 and H-9), 1.69 (td, J=12.5 and 4 Hz,
1H, H-10ax), 2.12 (dd, J=13 and 8.5 Hz, 1H, H-4), 2.51
(m, 1H, NCH2), 2.56±2.68 (m, 3H, NCH2 and CH2Ar),
2.77 (dd, J=9.5 and 4.5 Hz, 1H, H-2), 2.93 (dd, J=9.5
and 7 Hz, 1H, H-2), 3.24 (m, 1H, H-3), 3.68 (s, 2H,
NCH2Ar), 3.70 (s, 3H, CH3O), 3.82 (m, 4H, OCH2-
CH2O), 6.72 (d, J=8.5 Hz, 2H, 30-H), 7.03 (d,
J=8.5 Hz, 2H, 20-H), 7.17 (m, 1H), 7.21±7.28 (m, 4H);
13C NMR (74.5 MHz, CDCl3) d 28.0 (C-6), 30.8 (C-10),
32.4 (C-7), 32.6 (C-9), 35.3 (CH2Ar), 42.0 (C-4), 50.0
(NCH2), 52.4 (NCH2Ar), 54.5 (C-3), 55.0 (CH3O), 57.2
(C-2), 62.5 (C-5), 64.1 and 64.2 (OCH2CH2O), 108.5 (C-
8), 113.6 (C-30), 126.9 (p-C), 128.2 and 128.4 (o-C and
m-C), 129.6 (C-20), 132.8 (C-10), 140.2 (ipso-C), 157.8
(C-40). Anal. calcd for C27H36N2O3.1/2H2O: C, 72.77;
H, 8.37; N, 6.29. Found: C, 73.08; H, 8.20; N, 6.69.
trans-3-[N-Benzyl-N-(tert-butoxycarbonyl)amino]-1-[2-
(4-methoxyphenyl)-ethyl]-1-azaspiro[4.5]decan-8-ol (9).
To a solution of alcohol 8 (430 mg, 1.1 mmol) in 9:1
MeOH:Et3N (7 mL) was added ditert-butyl dicarbonate
(476 mg, 2.2 mmol), and the mixture was heated at 50ꢀC
for 5 h. The solvent was removed in vacuo, and the
residue was puri®ed by chromatography (CH2Cl2 to
88:12 CH2Cl2:MeOH) to give carbamate 9 (0.41 g,
76%): IR (CHCl3) 1681; 1H NMR (300 MHz, CDCl3) d
1.15±1.75 (m, 17H), 1.80±1.96 (m, 2H), 2.22±2.44 (m,
2H), 2.45±2.64 (m, 2H), 2.72 (m, 1H), 2.82 (m, 1H), 2.94
(m, 1H), 3.46 (m, 1H, H-8), 3.78 (s, 3H, CH3O), 4.43 (br
s, 2H, NCH2Ar), 4.85 (br s, 1H, OH), 6.78 (d,
J=8.7 Hz, 2H, 30-H), 7.04 (d, J=8.7 Hz, 2H, 20-H),
7.13±7.34 (m, 5H); 13C NMR (74.5 MHz, CDCl3) d 26.1
(CH2), 28.2 (CH3), 32.6 (CH2), 32.9 (CH2), 34.8
(CH2Ar), 39.5 (C-4), 46.6 (NCH2Ar), 49.6 (NCH2), 52.3
(C-3), 54.1 (C-2), 55.1 (CH3O), 62.8 (C-5), 70.3 (C-8),
79.7 (C), 113.5 (C-30), 126.3 (p-C), 125.9 and 128.1 (o-C
and m-C), 129.4 (C-20), 132.4 (C-10), 140.2 (ipso-C),
155.9 (CO), 157.7 (C-40). Anal. calcd for C30H42N2
O4.H2O: C, 70.28; H, 8.65; N, 5.46. Found: C, 70.37; H,
8.33; N, 5.46.
3-Benzylamino-1-[2-(4-methoxyphenyl)ethyl]-1-azaspiro-
[4.5]decan-8-one (7). To a solution of acetal 6 (1.65 g,
3.8 mmol) in THF (30 mL) was added hydrochloric acid
(10%, 83 mL). After 12 h at room temperature, the
reaction mixture was poured into 5% aqueous NaHCO3
and extracted with CH2Cl2 (5Â50 mL). The organic
extracts were dried and concentrated. Chromatography
(CH2Cl2 to 94:6 CH2Cl2:MeOH) a€orded ketone 7
(1.18 g, 79%): IR (®lm) 1714; 1H NMR (300 MHz,
CDCl3) d 1.60 (br, 1H, H-6eq), 1.75 (m, 3H, H-4, H-6ax
and H-10eq), 1.86 (td, J=13 and 5.5 Hz, 1H, H-10ax),
2.30±2.42 (m, 5H, H-4, H-7 and H-9), 2.56 (m, 1H,
CH2N), 2.62±2.72 (m, 3H, CH2N and CH2Ar), 2.89 (dd,
J=9.5 and 4.5 Hz, 1H, H-2), 3.06 (dd, J=9.5 and
7.5 Hz, 1H, H-2), 3.39 (m, 1H, H-3), 3.75 (s, 3H,
CH3O), 3.78 (s, 2H, NCH2Ar), 6.78 (d, J=8.5 Hz, 2H,
30-H), 7.07 (d, J=8.5 Hz, 2H, 20-H), 7.25 (m, 1H), 7.30±
7.34 (m, 4H); 13C NMR (74.5 MHz, CDCl3) d 30.5 (C-
6), 32.9 (C-10), 35.1 (CH2Ar), 38.6 (C-7), 38.9 (C-9),
42.1 (C-4), 49.9 (NCH2), 52.4 (NCH2Ar), 54.4 (C-3),
55.2 (CH3O), 57.2 (C-2), 61.9 (C-5), 113.6 (C-30), 127.0
(p-C), 128.2 and 128.4 (o-C and m-C), 129.5 (C-20),
132.4 (C-10), 139.9 (ipso-C), 157.8 (C-40), 211.2 (C-8).
Anal. calcd for C25H32N2O3.H2O: C, 73.14; H, 8.34; N,
6.82. Found: C, 73.41; H, 8.08; N, 6.94.
trans-3-[N-Benzyl-N-(tert-butoxycarbonyl)amino]-1-[2-
(4-methoxyphenyl)ethyl]-1-azaspiro[4.5]dec-8-yl dibenzyl
phosphate (10). To a solution of alcohol 9 (370 mg,
0.75 mmol) in CH2Cl2 (37 mL) were added 1-H-tetrazole
(158 mg, 2.25 mmol) and N,N-diisopropyl dibenzyl
phosphoramidite (0.38 mL, 1.12 mmol). The mixture
was stirred at room temperature for 5 h and then
cooled to 50ꢀC. MCPBA (98%, 197 mg, 1.12 mmol) in
CH2Cl2 (10 mL) was added. The resulting solution was
stirred at 0ꢀC for 2 h, diluted with CH2Cl2 and washed
with saturated aqueous NaHCO3 (4Â50 mL). The
organic extracts were dried and concentrated. Chroma-
tography (CH2Cl2 to 95:5 CH2Cl2:MeOH) yielded
phosphate 10 (0.41 g, 73%): IR (CHCl3) 1681; 1H NMR
(300 MHz, CDCl3) d 1.10±1.54 (m, 16H), 1.93 (m, 2H),
2.25 (m, 1H), 2.33 (m, 1H), 2.54 (m, 2H), 2.65 (m, 1H),
2.79 (m, 1H), 2.92 (m, 1H), 3.78 (s, 3H, CH3O), 4.10 (br
s, 1H), 4.40 (br s, 2H, NCH2Ar), 4.70 (br s, 1H), 4.98 (s,
trans-3-Benzylamino-1-[2-(4-methoxyphenyl)ethyl]-1-aza-
spiro[4.5]decan-8-ol (8). To a cooled (0ꢀC) solution of
ketone 7 (0.58 g, 1.5 mmol) in MeOH (20 mL) was
added NaBH4 (112 mg, 3 mmol). After 30 min at 0ꢀC
and 1.5 h at room temperature, the reaction mixture was

2896
J. Bonjoch et al. / Bioorg. Med. Chem. 7 (1999) 2891±2897
2H, OCH2Ar), 5.01 (s, 2H, OCH2Ar), 6.78 (d,
J=8.7 Hz, 2H, 30-H), 7.01 (d, J=8.7 Hz, 2H, 20-H),
7.10±7.40 (m, 15H); 13C NMR (74.5 MHz, CDCl3) d
25.9 (CH2), 28.2 (CH3), 30.5 (CH2), 31.0 (CH2), 32.8
(CH2), 34.9 (CH2Ar), 39.7 (C-4), 46.6 (NCH2Ar), 49.4
(NCH2), 52.3 (C-3), 54.1 (C-2), 55.1 (CH3O), 62.0 (C-5),
68.9 and 69.0 (OCH2Ar), 77.5 (C-8), 79.7 (C), 113.5 (C-
30), 126.3 (p-C), 125.9 and 128.1 (o-C and m-C), 127.8
(CH), 128.3 and 128.4 (CH), 129.4 (C-20), 132.5 (C-10),
135.8 and 135.9 (C), 140.3 (ipso-C), 155.9 (CO), 157.7
(C-40); 31P NMR (121.5 MHz, CDCl3)d 5.44. Anal.
calcd for C44H55N2O7P.1/8H2O: C, 69.80; H, 7.36; N,
3.70. Found: C, 69.40; H, 7.31; N, 3.73.
(m, 5H); 13C NMR (74.5 MHz, DMSO-d6) d 29.3
(CH2), 29.7 (CH2), 30.3 (CH2), 30.7 (CH2), 35.8 (CH2),
48.8 (CH2), 49.3 (CH2), 52.1 (CH), 55.3 (CH3), 60.5
(CH2), 70.9 (C), 72.3 (CH), 114.2 (CH), 117.1 (q, C)
129.0 (CH), 129.1 (CH), 129.3 (CH), 130.1 (CH), 132.1
(C), 158.4 (C), 159.1 (q, C). ISMS: 475 (M+H). This
tri¯uoroacetate (50 mg) was dissolved in MeOH (12 mL)
containing K2CO3 (30 mg). The solvent was removed to
give the dipotassium salt of 2: 1H NMR (300 MHz,
CD3OD) d 1.26±1.66 (m, 8H), 2.13 (br, 2H), 2.23 (dd,
J=13 and 8.6 Hz, 1H), 2.56 (m, 1H), 2.60±2.72 (m, 3H),
2.78 (dd, J=9.5 and 5.7 Hz, 1H), 3.01 (dd, J=9.5 and
7.4 Hz, 1H), 3.25 (m, 1H), 3.70±3.75 (m, 5H), 3.90 (br,
1H), 6.79 (d, J=8.7 Hz, 2H), 7.08 (d, J=8.7 Hz, 2H),
7.20±7.40 (m, 5H); 13C NMR (74.5 MHz, CD3OD) d
30.5 (CH2), 32.6 (CH2), 32.9 (CH2), 32.9 (CH2), 36.0
(CH2Ar), 43.1 (C-4), 51.4 (NCH2), 53.1 (NCH2Ar), 55.3
(OMe), 55.7 (C-3), 57.8 (C-2), 64.3 (C-5), 73.7 (d,
JC,P=4.5 Hz, C-8), 114.7 (C-30), 128.2 (p-C), 129.5 and
129.6 (o-C and m-C), 130.6 (C-20), 133.8 (C-10), 140.2
(ipso-C), 157.8 (C-40).
In some runs, the corresponding N-oxide was formed as
a minor byproduct:
trans-3-[N-Benzyl-N-(tert-butoxycarbonyl)amino]-8-(di-
benzylphosphoroxy)-1-[2-(4-methoxyphenyl)ethyl]-1-aza-
1
spiro[4.5]decane 1-oxide. H NMR (300 MHz, CDCl3) d
1.20±1.65 (m, 15H), 1.90±2.10 (m, 2H), 2.28 (m, 1H),
2.32±2.48 (m, 2H), 2.87 (m, 1H), 3.13 (m, 2H), 3.44±3.62
(m, 2H), 3.78 (s, 3H, CH3O), 3.88 (m, 1H), 4.05 (m,
1H), 4.80±5.20 (m, 6H), 6.83 (d, J=8.6 Hz, 2H, 30-H),
7.13 (d, J=8.6 Hz, 2H, 20-H), 7.18±7.37 (m, 15H); 13C
NMR (74.5 MHz, CDCl3) d 28.1 (CH3), 29.3 (CH2),
30.2 (CH2), 30.3 (CH2), 33.8 (CH2), 46.5 (NCH2Ar),
50.4 (C-3), 55.2 (CH3O), 61.7 (CH2N), 68.9 and 69.0 (C-5),
69.2 and 69.3 (OCH2Ar), 69.8 (C-2), 76.0 and 76.1 (C-8),
80.4 and 81.3 (C), 114.1 (C-30), 126.3 (p-C), 125.9 and
128.2 (o-C and m-C), 127.9 (CH), 128.5 and 128.6 (CH),
129.8 (C-20), 135.6 and 135.7 (C), 140.6 (ipso-C), 156.5
(CO), 158.3 (C-40).
Acknowledgments
Support for this research was provided by DGES
(Spain) through Grant PB97-0877. Thanks are also due
to the `Comissionat per a Universitats i Recerca' (Gen-
eralitat de Catalunya) for Grant SGR97-00166 and to
the MEC (Spain) for a fellowship to G.P.
References and Notes
trans-3-[N-Benzyl-N-(tert-butoxycarbonyl)amino]-1-[2-
(4-methoxyphenyl)ethyl]-1-azaspiro[4.5]dec-8-yl dihydro-
genphosphate (11). A mixture of phosphate 10 (280 mg,
0.37 mmol) and 10% Pd/C (140 mg) in MeOH (40 mL)
was stirred under 1 atm H2 for 12 h. The suspension was
®ltered through Celite, and the ®ltrate concentrated to
give phosphate 11 (195 mg, 92%): IR (®lm) 3420, 1690,
1661; 1H NMR (200 MHz, CD3OD) d 1.10±2.20 (m,
21H), 2.42 (br s, 1H), 2.80 (br s, 2H), 3.32 (m, 1H), 3.67
(s, 3H), 3.85 (br s, 1H), 4.32 (br s, 1H), 4.38 (s, 2H), 6.76
(d, J=8.4 Hz, 2H), 6.98±7.35 (m, 7H); 13C NMR
(74.5 MHz, CD3CN) d 25.6 (CH2), 28.5 (CH3), 30.0
(CH2), 31.2 (CH2), 36.5 (CH2), 38.2 (CH2), 50.1 (CH2),
51.0 (CH2), 53.7 (CH), 55.9 (CH3), 73.1 (CH), 74.1 (C),
81.4 and 82.0 (C), 115.0 (CH), 127.8 (CH), 129.4 (CH),
131.1 (CH), 139.9 (C), 156.9 (C), 159.6 (C). ISMS: 576
(M+H).
1. Sakamoto, K.; Tsujii, E.; Abe, F.; Nakanihi, T.; Yamashita,
M.; Shigematsu, N.; Izumi, S.; Okuhara, M. J. Antibiot. 1996,
49, 37.
2. Romo, D.; Rzasa, R. M.; Shea, H. A.; Park, K.; Langenhan,
J. P.; Sun, L.; Akhiezer, A.; Liu, J. O. J. Am. Chem. Soc. 1998,
120, 12237 and references therein.
3. Hamilton, G. S.; Steiner, J. P. J. Med. Chem. 1998, 41, 5119.
4. Yamazaki, N.; Suzuki, H.; Kibayashi, C. J. Org. Chem.
1997, 62, 8280.
5. Snider, B. B.; Lin, H.; Foxman, B. M. J. Org. Chem. 1998,
63, 6442.
6. Since initial attempts to prepare the methylamino derivative
were troublesome, we decided to work with the benzylamino
derivative.
7. Hill, R. K. J. Org. Chem. 1957, 22, 830.
8. Mo€ett, R. B. J. Am. Chem. Soc. 1957, 79, 3186.
9. Bryce, M. R.; Gardiner, J. M. Tetrahedron 1988, 44, 599.
10. Gardiner, J. M.; Bryce, M. R.; Bates, P. A.; Hursthouse,
M. B. J. Org. Chem. 1990, 55, 1261.
trans-3-(Benzylamino)-1-[2-(4-methoxyphenyl)ethyl]-1-
azaspiro[4.5]dec-8-yl dihydrogenphosphate (2). To a
solution of carbamate 11 (165 mg, 0.29 mmol) in
CH2Cl2 (13 mL) was added at 0ꢀC TFA (3.3 mL,
43 mmol). After 12 h at room temperature, the solvent
and the excess of acid were removed in vacuo to give the
tri¯uoroacetic acid salt of 2 (170 mg, quantitative): IR
11. Miller, R. D.; Goelitz, P. J. Org. Chem. 1981, 46, 1616.
12. Sawamura, M.; Nakayama, Y.; Tang, W.-M.; Ito, Y. J.
Org. Chem. 1996, 61, 9090.
13. Overman, L. E.; Kakimoto, M.; Okazaki, M. E.; Meier, G.
P. J. Am. Chem. Soc. 1983, 105, 6622.
14. Watanabe, Y.; Ueno, Y.; Tanaka, C.; Okawara, M.; Endo,
T. Tetrahedron Lett. 1987, 28, 3953.
15. Fujimoto, R. A.; Boxer, J.; Jackson, R. H.; Simke, J. P.;
Neale, R. F.; Snowhill, E. W.; Barbaz, B. J.; Williams, M.;
Sills, M. A. J. Med. Chem. 1989, 32, 1259.
1
(®lm) 2800 (br), 1668; H NMR (200 MHz, CD3OD) d
1.40±2.30 (m, 10H), 2.80±3.20 (m, 3H), 3.40 (m, 1H),
3.67 (s, 3H), 3.60±4.00 (m, 2H), 4.00±4.30 (br, 4H), 6.79
(d, J=8.8 Hz, 2H), 7.13 (d, J=8.8 Hz, 2H), 7.30±7.50
16. Kawase, M.; Kitamura, T.; Kigugawa, Y. J. Org. Chem.
1989, 54, 3394.

J. Bonjoch et al. / Bioorg. Med. Chem. 7 (1999) 2891±2897
2897
17. Pearson, W. H.; Szura, D. P.; Postich, M. J. J. Am. Chem.
Soc. 1992, 114, 1329.
18. Trost, B. M.; Marrs, C. M. J. Am. Chem. Soc. 1993, 115,
6636.
27. The addition of Grignard reagents to ketimines is not well
precedented because imines have a propensity to enolize. For
notable exceptions, see: Spero, D. M.; Kapadia, S. R. J. Org.
Chem. 1997, 62, 5537 and ref 26.
19. Coldham, I.; Warren, S. J. Chem. Soc., Perkin Trans 1
1993, 1637.
20. Shepard, M. S.; Carreira, E. M. Tetrahedron 1997, 53,
16253.
21. Wilson, S. R.; Sawicki, R. A.; Hu€man, J. C. J. Org.
Chem. 1981, 46, 3887.
22. Tanner, D.; Sellen, M.; Backvall, J.-E. J. Org. Chem. 1989,
54, 3374.
28. Lemal, D. M.; Fry, A. J. J. Org. Chem. 1964, 29, 1673.
29. During the development of our work a procedure has
appeared in the literature, involving seven steps, to obtain 3,8-
difunctionalized 1-azaspiro[4.5]decanes in the context of the
synthesis of the natural compound TAN1251A, a novel anti-
muscarinic agent: Nagumo, S.; Nishida, A.; Yamazaki, C.;
Murashige, K.; Kawahara, N. Tetrahedron Lett. 1998, 39,
4493.
23. Fujita, M.; Kitagawa, O.; Suzuki, T.; Taguchi, T. J. Org.
Chem. 1997, 62, 7330.
24. Knight, D. W.; Redfern, A. L.; Gilmore, J. Synlett 1998,
731.
25. Lee, W. S.; Jang, K. C.; Kim, J. H.; Park, K. H. Chem.
Comm. 1999, 251.
26. The reaction of allylation upon imines followed by ami-
nocyclization promoted by PhSeX gives azetidines or pyrroli-
dines depending on the number of reagent equivalents used:
Berthe, B.; Outurquin, F.; Paulmier, C. Tetrahedron Lett.
1997, 38, 1393.
30. Yu, K.-L.; Fraser-Reid, B. Tetrahedron Lett. 1988, 29, 979.
31. We acknowledge a reviewer's comments concerning this
question.
32. Chem-X, version Jan97, Chemical Design Limited,
Oxfordshire OX7 5SR, UK.
33. Murrall, N. W.; Davies, E. K. J. Chem. Inf. Comut. Sci.
1990, 30, 312.
34. MOPAC/AM1 (Program no. 506, version 6.0, Quantum
Chemistry Program Exchange, QCPE, Bloomington, IN,
USA).
35. Stewart, J. P. J. J. Comput-Aided Mol. Des. 1990, 4, 1.