Synthesis of N-diisopropyl phosphoryl benzyl-tetrahydroisoquinoline, a new class of mitochondrial complexes I and III inhibitors

Article abstract of DOI:10.1016/S0960-894X(00)00262-6

The synthesis of N-(O,O-diisopropyl phosphoryl)-benzyltetrahydroisoquinoline (3) has been achieved in a 'one pot' procedure from imine (2) and diisopropyl-phosphorochloridate (1) generated in situ (POCl₃ + iPrOH). Compound 3 is the first benzyl

Full text of DOI:10.1016/S0960-894X(00)00262-6

Bioorganic & Medicinal Chemistry Letters 10 (2000) 1491±1494
Synthesis of N-Diisopropyl Phosphoryl Benzyl-
tetrahydroisoquinoline, a New Class of Mitochondrial
Complexes I and III Inhibitors
Inmaculada Andreu, a Nuria Cabedo, a Jose R. Tormo, a Almudena Bermejo, a
Rossella Mello b and Diego Cortes a,*
a
Â
Departamento de Farmacologõa, Farmacognosia y Farmacodinamia, Universidad de Valencia, 46100 Burjassot, Valencia, Spain
b
Â
Servicio Central de Soporte a la Investigacion Experimental, Universidad de Valencia, 46100 Burjassot, Valencia, Spain
Received 23 February 2000; accepted 5 May 2000
AbstractÐThe synthesis of N-(O,O-diisopropyl phosphoryl)-benzyltetrahydroisoquinoline (3) has been achieved in a `one pot' procedure
from imine (2) and diisopropyl-phosphorochloridate (1) generated in situ (POCl3+iPrOH). Compound 3 is the ®rst benzyltetrahy-
droisoquinoline derivative found to be a potent inhibitor of mitochondrial complexes I and III, and therefore it opens a new perspective
with this series of compounds as they can be considered as new class of antitumor agents. # 2000 Elsevier Science Ltd. All rights reserved.
Introduction
agrochemical agents, as well as with previously descri-
bed cytotoxic and antitumor natural compounds.5
Having the possibility to study the relevance of a DIPP
group in the interaction of these inhibitors with the
respiratory enzymes, we have provided the synthesis of
N-(O,O-diisopropyl phosphoryl)-1-benzyl-6-benzyloxy-7-
methoxy-1,2,3,4-tetrahydroisoquinoline (3).
The diisopropyl phosphoryl (DIPP) group has been used
recently as an amino acid protecting group.1 Indeed,
under classical Atherton±Todd conditions,2 when an a-
amino acid is treated by a dialkylphosphorochloridate
(e.g., obtained by treatment of a dialkylphosphite with
sodium hypochlorite), the N-protected a-amino acid is
obtained in high yield.1 Thus diisopropyl- and various
dialkylphosphorochloridates have been used as phos-
phorylating agents.3
Chemistry
For these reasons a `one pot' sequence under acid con-
ditions in isopropanol, starting from imine 2,6 and sub-
sequent reduction with NaBH4 after 4 h re¯ux, was
proposed and conducted. The N-phosphorylation was
achieved with success in a `one-pot' procedure under
acid medium via the reaction of POCl3 with isopropanol
to give diisopropyl phosphorochloridate (1) formed in
situ. The reaction of 1 with BDHIQ (2) and consecutive
NaBH4 reduction a€orded a 1:1 mixture of compounds
3 and 4 in 95% overall yield.7 The DIPP protecting
group can be easily cleaved, together with O-debenzy-
lation,4,6 by treatment with ethanol and concd HCl, to
a€ord the corresponding amine (5) in good yield (80%)8
(Scheme 1).
We recently accomplished the syntheses of N-alkyl-ben-
zyltetrahydroisoquinolines (BTHIQ) by a new method
incorporating a `one pot' cyclization±reduction±alkylation
sequence.4 Following this procedure, herein we descri-
bed an original method for the preparation of N-pro-
tected BTHIQ, through the generation of the DIPP
protecting group from POCl3 and isopropanol, by a
rapid, ecient and unexpensive procedure.
In our studies of new antitumor inhibitors of the mito-
chondrial electron-transport chain we decided to study,
for the ®rst time, the BTHIQ class as inhibitors of cell
respiration. Potency found for these compounds was in
accordance with the one observed for other commercial
It is interesting to note that when isopropanol is used
as a solvent in this reaction sequence, N-(O,O-diiso-
propyl phosphoryl) derivative was obtained. However,
if this alcohol is substituted by EtOH or MeOH, the
*Corresponding author. Tel.: +34-96-386-49-75; fax: +34-96-386-49-
43; e-mail: dcortes@uv.es
0960-894X/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(00)00262-6

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I. Andreu et al. / Bioorg. Med. Chem. Lett. 10 (2000) 1491±1494
corresponding N-ethyl and N-methyl derivatives were
obtained due to in situ formation of PO(OEt)3 or PO
(OMe)3, respectively.4,9
The molecular weight for compound 3 was determined by
low resolution LSIMS (Cs+ at 30 kV) mass spectrometry
(m/z 524 for [MH]+ corresponding to C30H38NO5P).
Under EI conditions the molecular ion fragments were
quickly detected. High resolution mass spectrometry
(HREIMS) allowed us to con®rm the proposed struc-
ture for compound 3 by exactly measuring the masses of
the signi®cant daughter ions formed.10 Losses of benzyl
groups, propene molecules and phosphorous acid are
involved in the observed fragmentation pattern. Com-
pound 3 was characterized on the basis of its spectroscopic
Scheme 2.
with regard to the N-diisopropyl phosphoryl moiety.
Two important NOE's were observed between H-8 and
both H-1 and one of the CH-isopropyl protons, as well
as negative NOE's which were observed between the H-
1/OMe-7 and the CH-isopropyl/OMe-7. All these data
revealed an out-of-plane orientation of the 1-benzylic
moiety, and a spatial proximity of one of the isopropyl
groups to the isoquinoline skeleton (Schemes 3 and 4).
1
data (1D and 2D NMR experiments: H, 13C, DEPT,
NOEDIFF, NOESY, COSY 45, COSY LR and HM
QC),11 which indicated that the N-phosphoryl group
should be disposed as a phosphorimonium form
(Scheme 2).
Bioactivities
As phosphate esters and N-(O,O-diisopropyl phosphoryl)
derivatives can be considered as good transport systems
in several lipid membranes,12 and due to their ability of
dislocating electron charges, we decided to assay 3 and
their related compounds (2 and 4) as well as the corre-
sponding N-methyl analogue (6),13 as inhibitors of the
electron transport in the mitochondrial respiratory chain.14
The 31P NMR (CDCl3, 100 MHz) spectrum of 3 showed
an absorption at dP=7.04, characteristic of a phosphoryl
moiety. In the 13C NMR (CDCl3, 100 MHz) spectrum of
3, three methyne carbons appear as doublets (2JCP=6,
2
2JCP=5.5 and JCP=5 Hz), at dC=70.7, dC=70.1 (2
CH, isopropyl) and dC=55.7 (CH-1), respectively, just
as four isopropyl±methyl groups appear as four doub-
lets (3JCP=6 Hz), between dC=23.8 and dC=23.5.
Synthesized 2±4, and 6 were initially found to be inhibi-
tors of the integrated respiratory chain NADH oxidase
enzymatic activity that includes respiratory chain com-
plexes I, III and IV (see IC50 in Fig. 1).15
In addition, the NOEDIFF's observed for 3 were in
accordance with an anti conformation of 1-benzyl group
Once we observed that BTHIQ's inhibited the respiratory
transport-chain at a micromolar range, we decided to
Scheme 1.
Scheme 3.

I. Andreu et al. / Bioorg. Med. Chem. Lett. 10 (2000) 1491±1494
1493
Scheme 4. Fully energy-minimized structure perspective of 3 by MM2
calculations.
Figure 2.
Acknowledgements
This research has been supported by the Spanish DGI-
CYT under grant SAF 97-0013. We wish to thank Dr.
Francisco Esteban and Dr. Adrien Cave for critical
analysis of the NMR data. We also wish thank the
Spanish Mininisterio de Educacion y Cultura for the
scholarship grant to J.R.T.
Figure 1. IC50 of NADH oxidase activity for compounds 2±4 [2 (2.91
Æ 0.23) mM, 3 (1.69 Æ 0.27) mM, 4 (3.88 Æ 0.54) mM, 6 (2.70 Æ 0.28)
mM ].
perform a series of time-course assays to ®nd out the
target enzyme inhibited within the respiratory com-
plexes. For these purposes we selected the most potent
inhibitor of the series, the N-diisopropyl phosphoryl-
BTHIQ (3) at a ®xed high-concentration of 75 mM.
References and Notes
1. Brands, K. M. J.; Wiedbrauk, K.; Williams, J. M.; Dolling,
U. H.; Reider, P. J. Tetrahedron Lett. 1998, 39, 9583.
2. Atherton, F. R.; Todd, A. R. J. Chem. Soc. 1947, 674.
3. Hemenway, M. S.; Olivo, H. F. J. Org. Chem. 1999, 64, 6312.
4. Andreu, I.; Cortes, D.; Protais, P.; Cassels, B. K.;
Chagraoui, A.; Cabedo, N.Bioorg. Med. Chem. 2000, 8, 859.
5. (a) Zafra-Polo, M. C; Gonzalez M. C.; Tormo, J. R;
Estornell, E; Cortes, D; J. Nat. Prod. 1996, 59, 913. (b)
Bermejo, A.; Tormo, J. R.; Cabedo, N.; Estornell, E.; Figa-
dere, B.; Cortes, D. J. Med. Chem. 1998, 41, 5158. (c) Gonza-
lez, M. C.; Lavaud, C.; Gallardo, T.; Zafra-Polo, M. C.;
Cortes, D. Tetrahedron 1998, 54, 6079.
6. 1-Benzyl-6-benzyloxy-7-methoxy-3,4-dihydroisoquinoline (2)
was prepared as previously described by Cabedo, N.; Protais,
P.; Cassels, B. K.; Cortes, D. J. Nat. Prod. 1998, 61, 709.
7. Preparation of compound 3: A solution of 1-benzyl-6-benzy-
loxy-7-methoxy-3,4-dihydroisoquinoline (2, 50 mg, 0.14 mmol) in
(CH3)2CHOH (10 mL) was treated with POCl3 (0.3 mL, 3.21
mmol) and the mixture re¯uxed for 4 h. NaBH4 (7 mg, 0.18 mmol)
was added in small portions to the reaction mixture over 1h at
rt, which was then stirred for 15 h. The acid isopropanolic
solution, after removing the solvent under reduced pressure,
was dissolved in H2O, basi®ed with NH3 aq (pHꢀ9) and
extracted with CH2Cl2. The organic layer was washed with
H2O and brine, dried over anhydrous Na2SO4 and con-
centrated. The residue was puri®ed through 60 H silicagel
column (toluene/EtOAc/DEA 96:2:2) to a€ord compounds 3
(29.3 mg, 40%) and 4 (28.6 mg, 55%). Compound 4 was pre-
viously described in ref 4.
The cytochrome c oxidase activity (complex IV)5b was
not a€ected by 3, and this activity was completely
blocked by cyanide (2 mM), a speci®c inhibitor of this
enzyme. This result seems to give evidence that 3 could
inhibit the respiratory chain at complex I and/or III
levels.
Figures 2A and 2B show the replotted traces of the e€ect
found for 3 on the time-course oxidation of NADH and
decylubiquinol, both represent the selective enzymatic
activity inhibition of complexes I and III, respectively.5c,16
Indeed the target enzymes within the respiratory chain
of 3 resulted to be both NADH:decylubiquinone
oxidoreductase (complex I) and decylubiquinol:cyto-
chrome c oxidoreductase (complex III). Our results
seem to show that the presence of a diisopropylphos-
phoryl moiety increases the potency inside this new kind
of respiratory chain inhibitors. This discovery opens a
new ®eld to study this large class of natural and syn-
thetic compounds as inhibitors of cell respiration focus-
ing in their possible development as new antitumor
agents.

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I. Andreu et al. / Bioorg. Med. Chem. Lett. 10 (2000) 1491±1494
8. Cleavage of the benzyloxy and DIPP protective groups: N-
Diisopropyl phosphoryl BTHIQ 3 (15 mg, 0.028 mmol) was
re¯uxed for 3 h with a mixture of equal volumes of ethanol
and concentrated HCl (7 mL). The reaction solution was made
basic (pHꢀ9) and extracted with CH2Cl2. The organic layer
was washed with H2O, dried, concentrated and puri®ed over a
J0=7Hz, H-1), 5.08 (2H, s, OCH2Ph-6), 6.04 (1H, s, H-8), 6.59
(1H, s, H-5), 7.17±7.43 (10H, m, 2Ph); 13C NMRÃ (CDCl3, 100
MHz) d 147.0 and 146.9 (C-6 and C-7), 138.8 and 137.2 (C-10
and C-100), 130.0±125.4 (2Ph), 129.8 and 125.5 (C-4a and C-
8a), 114.3 (C-5), 110.9 (C-8), 71.0 (OCH2Ph-6), 70.7 and 70.1
(2CH, CH(CH3)2), 55.6 (C-1), 55.5 (OCH3-7), 43.3 (CH2-a),
37.2 (C-3), 27.5 (C-4), 23.8, 23.7, 23.5, 23.0 (4 CH3,
CH(CH3)2); LSIMS m/z 524 [MH]+; EIMS m/z (%) 432 (97),
390 (28), 348 (100), 257 (30), 176 (12), 91 (82). ÃThe assigments
were made by COSY 45, COSY LR, NOEDIFF, NOESY,
DEPT and HMQC; see also Scheme 3.
60
H
silicagel column (CH2Cl2/EtOAc/MeOH/NH4OH
40:50:10:0.1) to a€ord 1-benzyl-6-hydroxy-7-methoxy-1,2,3,4-
tetrahydroisoquinoline (5, 6 mg, 80%). The spectroscopic data
of this compound have been reported in ref 4.
9. Holy, A.; Gunter, J.; Dvorakova, H.; Masojõdkova, M.;
Andrei, G.; Snoeck, R.; Balzarini, J.; De Clercq, E. J. Med.
Chem. 1999, 42, 2064.
12. Sprecher, M.; Breslow, R.; Philosof-Oppenheimer, R.;
Chavet, E. Tetrahedron 1999, 55, 5465.
10. HREIMS (20 eV) of 3: m/z 432.19428 (432.19399 calcd for
[C23H31NO5P]+); m/z 390.14580 (390.14704 calcd for [C20H25
NO5P]+); m/z 348.10158 (348.10009 calcd for [C17H19NO5
P]+); m/z 257.04465 (257.04531 calcd for [C10H12NO5P]+); m/z
176.07072 (176.07115 calcd for [C10H10NO2]+); m/z 91.05838
(91.05477 calcd for [C7H7]+).
13. N-Methyl-1-benzyl-7-methoxy-1,2,3,4-tetrahydroisoquino-
line (6) was prepared as previously described by Andreu, I. et al.4
14. Enzymatic respiratory titrations were performed in beef
heart submitochondrial particles (SMP) by addition of
increasing concentrations of each inhibitor. IC50 values were
the ®nal compound concentrations in the assay cuvette that
yielded an average of 50% inhibition of the NADH oxidase
activity.5
15. In all the assayed compounds, full inhibition of the
NADH oxidase activity was reached at 10 mM. The residual
activity beyond this concentration was the same as that of the
nonsensitive NADH oxidation by other inhibitors.5c
16. Estornell, E.; Tormo, J. R.; Barber, T. Biochem. Biophys.
Res. Commun. 1997, 240, 234.
11. Compound 3: C30H38NO5P; IR (®lm) nmax 2917, 2849,
1517, 1453, 1384, 1246 (PˆO), 1115, 1090, 1011 (P-O-C), 980
1
cm
;
31P NMR (CDCl3, 100 MHz) d 7.04 (phosphoryl
1
group); H NMR* (CDCl3, 400 MHz) d 1.02, 1.16, 1.20, and
1.27 (12H, 4d, J=6Hz, CH(CH3)2), 2.49 and 2.88 (2H, 2m,
CH2-4), 2.98 and 3.17 (2H, 2 dd, J=14Hz, J0=7 Hz, CH2-a),
3.23 and 3.50 (2H, 2m, CH2-3), 3.54 (3H, s, OCH3-7), 4.07 and
4.43 (2H, 2 sept, J=6Hz, CH(CH3)2), 4.81 (1H, dd, J=14Hz,