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Lipophilic 5 5-O-Dinucleoside--hydroxybenzylphosphonic Acid Esters as Potential Prodrugs of 2 3-Dideoxythymidine (ddT).

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Lipophilic 5’,5’-O-Dinucleoside-a-hydroxybenzylphosphonic Acid Esters a s Potential Prodrugs
of 2’,3’-Dideoxythymidine (ddT) **
By Chris Meier *
Nucleoside analogues are widely used in antiviral
chemotherapy. Inside the cell the nucleoside analogues must
be metabolized into their nucleoside triphosphates by three
phosphorylation steps (Scheme I),[’] since only the triphosphates act as inhibitors of the viral polymerases or initiate
elongation termination after incorporation into a growing
viral DNA chain.[’] The structural differences between the
nucleoside analogues and the natural substrate could result
in the inhibition of the phosphorylating enzymes (kinases).
For this reason the conversion of the anti-HIV-active nucleoside analogue 2’,3’-dideoxythymidine (ddT 1) into its 5’monophosphate (ddTMP) by thymidine kinase is the rate
limiting step: once the ddTMP is formed, the subsequent
conversions are not inhibited.
products under alkaline conditions: a) a-Hydroxybenzylphosphonates (2) may undergo phosphonate-phosphate
rearrangement to the corresponding phosphotriester derivatives (3) in alkaline medium. Carbanion-stabilizing substituents in the aromatic system promote this rearrangement.[51 In 3 the ArCH,-0 bond is the most labile
phosphate ester bond, and hydrolysis leads to the nucleoside
phosphodiester 4 (Scheme 2).[61 Diester 4 is enzymatically
degradable into the 5’-monophosphate 5 and 1. b) In competition with the phosphonate-phosphate rearrangement, a
direct cleavage mechanism of the a-hydroxybenzylphosphonates (2) leading to the ddT-H-phosphonate diester 6 and the
aldehydes 7 occurs. This direct cleavage should be dominant
if the aromatic ring bears a carbanion-destabilizing, electron-donating substituent. The H-phosphonate diester 6 is
easily hydrolyzable into the H-phosphonate monoester 8
and l.[’]Alternatively, 6 might be oxidized intracellularly to
give the phosphodiester 4@’(Scheme 2).
2
Ar)-y-05hdT
-1
OH 05ddT
spontaneous a-hydroxyphosphonate-phosphate
rearrangement
,&o,H
q$bdT
H
2’,3’-dideoxythymidine ddT 1
-o-~;owT
ddT monophosphate
x
2
9
0
0
0 ddT
7
spontaneous or
enzymatic
oxidation
spontaneous
hydrolysis
no membrane-
4
inhibillon of the reserve
ILUlSpofl
/
9
i4
Hence, for nucleoside analogues like 1, intracellular delivery as their 5’-monophosphates from prodrugs should bypass this metabolic limitation and consequently improve
their biological activity. Lipophilic nucleoside phosphotriesters were studied with the aim of releasing the nucleoside
monophosphate by spontaneous or enzymatic degradat i ~ n . [In
~ ]all reported cases the controlled cleavage of the
masking groups of the prodrugs is not be satisfactorily
solved. Apart from the controlled hydrolysis, the lipophilicity of the prodrugs is important for passive transport
through biomembranes and through the blood- brain barrier. Only one prodrug form has been described for ddT
recently.f41
This communication reports on the synthesis of di-5‘,5’-0(2’,3’-dideoxythymidine)-a-hydroxybenzylphosphonate esters (2) with different substituents at the aromatic ring, on
their partition coefficients in an octanol/water mixture, and
on hydrolysis studies in phosphate buffer solution (pH 7.5)
at 37 “C. Starting from 2, two completely different mechanisms should lead to phosphorylated or phosphonylated
Dr. C. Meier
Institut fur Organische Chemie der Universitit
Marie-Curie-StrdOe 11, D-60439 Frankfurt am Main (FRG)
Telefax: Int. code +(69)5800-9148
This work was supported by the Deutschen Forschungsgemeinschaft (ME
1161/1-2). the Fonds der Chemischen Industrie. and the Otto-RohmStiftung. I am grateful to Prof. Dr. Joachim Engels for his encouragement
and his support.
1704 0 VCH Verlagsgesellschafr mhH, 0-69451
Weinheim.1993
ddT
J
+
1
1
enzymatic
metabolization
ddMTMP enzymatic
metabolization
5
+ ddT
* I
enzymatic
degradation
[**I
spontaneous
hydrolysis
H-q-05ddT
0-
Scheme 1. I n vivo activation of 7.3’-dideoxythymidine (ddT. 1). Ar = aryl.
[*I
spontaneous
cleavage
-
I
bioactive metabolite
Scheme 2. Hydrolytic degradation pathways of 1-hydroxyphosphonatediesters
(2). Ar = aryl.
Consequently, the two mechanisms would lead in three
and two steps to phosphorylated or phosphonylated nucleoside analogues, respectively, and both precursors (the
a-hydroxy-benzylphosphonates 2 as well as the benzylphosphotriesters 3) could act as lipophilic prodrugs. Is it possible
to control the two different reaction pathways under physiological conditions by changing the substituent at the aromatic ring?
The 5’,5’-0-(2’,3’-dideoxythymidine)-H-phosphonate
diester 6, used as starting material for the synthesis of 2,[’] was
obtained in 60-80% yield in a one-pot reaction with
iPr,NPCl, (9) and two equivalents of ddT (1) via the phosphoramidite 10 as intermediate, which was directly hydrolyzed by tetrazole activation (Scheme 3).[’] Alternatively,
6 was synthesized in the same yield by esterification of 5‘0-(2’,3’-dideoxythymidine)-H-phosphonatemonoester (8)
with a second equivalent of ddT 1 through the pivaloylchloride activation procedure described previously.[g1Precursor
8 was obtained by reacting phosphorus trichloride with imidazole to yield the phosphotriimidazolide, followed by reaction with 1 and subsequent hydrolysis (Scheme 3).I’01
0570-0833/93/1212-1704$10.00+ ,2510
Angew. Chem. Int. Ed. Engl. 1993, 32, No. I2
ddT 1
a)
I
PCI3
iPr,NPCI,
9
0
II
H-T-O’ddT
0’ddT
e,
I
Ar)--{-05ddT
OH O’ddT
Scheme 3. Synthesis of a-hydroxybenzylphosphonates 2a-j. a) iPr,NP(CI), 9.
iPr,NEt. CH,CN. 0°C 15mm; b) tetrazole, water. CH,CN, 25°C. 30 min:
c ) l . imidazole. NEt,. CH,CN. 0°C. 4 h ; 2.water. CH,CN, 20’C, 1 h;
d ) pivaloyl chloride, 1. T H E 0°C. 5 min; e) 7, NEt,. 85‘C. 30 min or 7, T H E
NEt, (cat.). 20°C. 3 h.
The title compounds 2 were synthesized by two procedures
(Scheme 3): a) reaction of 6 with the benzaldehyde derivatives 7 in anhydrous triethyIamine (NEt,) at 90 “C (yield:
40-60%),[1’1 or b) reaction of 6 and 7 in anhydrous T H F in
presence of catalytic amounts of NEt, at 20 “C (yield: 90%)
(Table l).[”] Method b was the more convenient procedure.
Table 1. Synthesized 1-hydroxyphosphonates 2, their partition coefficients ( 5 )
in octano1:water. the half-lives of 2 in 10 mmol phosphate buffer (pH 7.5). and
the dependence of the hydrolysis products on the substituents.
2
x
Substituents
Y
Z
1,
2
[hl
Products
r
1
+ 8 [“A]
3 [“A]
-
a
b
H
C
H
H
H
CI
H
H
H
H
d
e
f
g
h
i
j
1
H
0.61
0.45
1.32
0.69
2.06
1.69
0.26
0.63
0.58
0.86
0.23
23
31
47
57
43
in
40
25
12
0.5
100
100
100
10
100
100
60
60
15
2
40
40
85
98
The first method allows the synthesis of the donor substituted compounds 2a-f but failed to yield acceptor-substituted benzaldehydes 7g-k. In all reactions the title compounds
2 were isolated as 1 :1 diastereomeric mixtures with respect to
the configuration at the C, atom after purification by silica
gel chromatography on a Chromatotron (Harrison Research). The diastereomers were not separable by chromatography but could be distinguished by ‘H, I3C, and 31P
NMR spectroscopy. The use of the chiral base quinidine
instead of triethylamine had no effect on the diastereomeric
ratio formed in the reaction. In no reaction was the formation of the corresponding phosphotriester observed. Consequently, these reaction conditions avoid rearrangement as a
side reaction during the synthesis.
The partition coefficients of the new compounds (2) in
octanol/water mixture[’31 allows an estimation of their
lipophilic properties (Table 1). All compounds showed partition coefficients a factor 1.13 to 9 higher than that of 1
A n g m Chiwi. I n r . Ed. EngI. 1993. 32. N o I 2
(0.23).[‘41Consequently, the passive transport across a membrane should be easier for these compounds than for the free
nucleoside.
In hydrolysis studies in which the disappearance of the
a-hydroxybenzylphosphonates (2) in 10 mmolar phosphate
buffer (pH 7.5) was monitored at 37 “C, a striking effect of
the different substituents was observed (Table
All ahydroxyphosphonates disappeared following pseudo-firstorder kinetics. Derivatives containing strong electron-withdrawing substituents in the aromatic residue rearranged in
aqueous solution into the corresponding 0-benzyl-0,O-dinucleosidephosphates 3 (Table 1, Scheme 2). To my knowledge, this is the first time that compounds of type 2 rearranged into 3 under aqueous, physiological conditions
without enzyme mediation. Benzylphosphotriesters 3
showed further selective degradation into the 5’,5’-dinucleosidephosphodiester 4. In this case the rate-determining
step is the hydrolysis of the formed phosphotriester 3 to yield
4.Phosphodiesters of type 4 are degraded in vivo enzymatically to yield the monophosphate 5 and ddT (1). Thus, z-hydroxyphosphonates (2) can, in principle, act as lipophilic
prodrugs of 5’-nucleoside monophosphates.
Furthermore, it was shown that compounds 2a-2f bearing weak electron-withdrawing groups or electron-donating
substituents were selectively cleaved to form the 5‘,5’-dinucleoside-H-phosphonate diesters 6 and the aldehydes 7 under the same conditions (Table 1, Scheme 2). The rate of this
direct cleavage was controlled by the substituent : the
stronger the electron-donating properties of the substituent,
the faster the direct cleavage. Under the studied reaction
conditions 6 was rapidly hydrolyzed into the 5I-ddT-phosphonate monoester 8 and ddT.[’] In this degradation pathway the direct cleavage is the rate-determining step. This
pathway, which competes with the rearrangement, opens the
possibility of the design of lipophilic prodrugs of 5’-nucleoside-H-phosphonate monoesters of type 8. This result is
important because recently it was shown that compounds of
type 8 exhibit selectivity indices comparable with and even
better than their parent nucleoside analogues in anti-HIV
tests.“ 61
In conclusion, these results show that it should be possible
to deliver products with biological activity by spontaneous
hydrolysis from 2a-j. Furthermore, it is possible to control
the reaction pathway (rearrangement versus direct cleavage)
and the rate of the reaction with the substituents at the aromatic ring. Consequently, the new 5‘,5’-dinucleoside-a-hydroxybenzylphosphonate esters 2 are potential prodrugs of
phosphorylated or phosphonylated derivatives of antiviral
nucleoside analogues like 1. We are currently investigating
the hydrolysis of 2 in 10% fetal calf serum (RPMI-culture
medium) and their antiviral activity against HIV-1 and
HIV-2.
Experimental Procedure
6 : Dried 2’,3-dideoxythymidine (1,4.0 mmol) was dissolved in anhydrous acetonitrile (60 mL). After addition of diisopropyl(ethy1)amine (6.0 mmol), the
solution was cooled in an ice bath. iPr,NPCI, (3.0 mmol) was added to the
stirred solution within Smin. After this mixture had been stirred at room
temperature for 10 min. tetrazole (4.0 mmol) and water (80 pL) were added.
After 15 min the solvent was evaporated under high vacuum. The reaction
mixture was purified on a Chromatotron on silica gel plates (solvent gradient
of ethyl acetate and methanol; 0 % to 30% methanol). The 2’,3’-ddT-H-phosphonate diester 6 was isolated as a colorless solid in 80% yield.
2: 6 (1.6 mmol) and 7 (4.8 mmol) were dissolved in anhydrous THF (40 mL).
While stirring freshly distilled triethylamine (20 pL) was added. After 4 h at
room temperature no starting material 6 could be detected by TLC. The reaction mixture was neutralized by addition of acetic acid (20 pL), and the solvent
was evaporated. The crude product was purified on a Chromatotron on silica
gel plates (gradient of dichloromethane and methanol; 0% to 15% methanol).
fi> VCH Verlug.rResells~liufl
mhH, 0.69451 Weinheim, 1993
0570-0833~93~12/2-17O5
$ lO.OO+ .25:O
1705
The his-(5'.5'-0-2'.3'-dideoxythymidine) a-hydroxybenzylphosphonate esters 2
were obtained after lyophilization. as colorless solids in 95% yield.
Received. July 8th. 1993 [Z61971El
German version: Angels. Cl7en?.1993. 105. 1854
[I] P. A. Furman. J. A. Fyfe. M. H. St.Clair. K. Weinhold, J. L. Rideout,
G. A. Furman, S. N. Lehrman. D. P. Bolognesi, S. Broder, H. Mitsuya.
D. W. Barry, Proc. Nut/. Acud. Sci. USA 1986.83, 8333-8337.
[2] a) H. Mitsuya. K. J. Weinhold, P. A. Furman, M. H. St.Clair, S. UsinoffLehrman, R. C. Gallo, D. Bolognesi. D. W. Barry. S. Broder, Proc. Nut/.
Acud. Sci. U S A 1985.82.7096;b) 0 .Heidenreich. F. Eckstein. Nuclposid~*.s
Nucleotide.s 1991, 10. 535.
131 a) D.Farquhar, Bioorg. Chpm. 1984, f2.118- 123: h) C . McGuigan, R. N .
Pathirdna. J. Balzarini, E. DeClercq. J. Med. Chem. 1993.36, 1048-1052:
C) E.F. Hahn, M. Buddo, A. M. Mian, L. Resnik in Nucleoside Anulogues
us Anrir,iru/ Agrnrs iACS S v p . Ser. 401. Ed.: J. C . Martin), Am. Chem.
Soc., Washington DC. 1989. pp. 156-169; d) F. Puech, A. Pompon, I.
Lefehvre, G . Gosselin. J. L. Imhach. Bfoorg. Mrd. Chem. Lrrt. 1992, 2.
603-606: e) A. Namane. C . Gouyette, M. P. Fillion, G. Fillion, T. HuynhDinh. J Med. Cliem. 1991, 34, 1830-1837; f ) C. Meier. T. Huynh-Dinh.
Bioorg. Mrcl. Chrm. Lefl. 1991, 1, 527-530: g) C.Meier, J. M. Neumann,
F. Andre. Y Henin. T. Huynh-Dinh. J. Org. Chem. 1992, 57, 7300-7307.
[4] S. I. Shimizu. J. Balzarini. E. DeClercq. R. T. Walker. Nucleosides N u ~ l e o rides 1992, 11. 583-594.
[5] a) F. Hammerschmidt, E. Schneyder. E. Zhiral, CAem. Ber. 1980, 113.
3891-3897: h ) A . F. Janzen, O.C. Vaidya. ibid. 1973, 51, 1136-1138:
c) I. J. Borowitz, J. Am. Chem. Soc. 1972, 94. 1623-1628; d) W. F. Barthel,
P. A. Giang, S. A. Hall, ;bid. 1955, 77, 2424-2427.
J. W. Engels, E.-J. Schliiger, J. Med. Chem. 1982. 20. 907-911.
C.Meier. Bioorg. Med. Chrm. Lerr., submitted.
H-phosphonate dresters are easily converted chemically into the phosphodiesters (see H-phosphonate methodology for oligonucleotide synthesis).
Cytochrome P450 oxidase might he responsible for an intracellular oxidation, hut no detailed studies about this possibility have been published yet.
P. J. Garegg. I. Lindh. T. Regherg, J. Stawinski, R. Stromberg. Telruhedron
Leu. 1986, 27. 4051 -4054.
a) P. J. Garegg, T. Regherg, J. Stawinski, R. Stromherg, CArm. Scr. 1985.
25, 280-282; h) ihid. 1986, 26, 59-62.
J. Jacques, M. Leclercq, M.-J. Brrenne, Terruhedron 1981,37, 1727-1733.
K. Kondo, N. Ohnishr. K. Takemoto, H. Yoshida, K. Yoshida, J Org.
Chem. 1992.57, 1622-1625.
S. Rim, K. L. Audus. R. T. Borchardt, lnr. J. Phurm. 1986.32, 79-85
J. Balzarini. J. M. Cools. E. DeClercq, Biochrm. Biophys. Res. Cotnniun.
1989, 158.413-422.
For the hydrolysis studies 2 ( I mg) was dissolved in phosphate buffer,
pH 7.5 (4 mL) and incubated at 27 "C. The composition of hydrolysis
aliquots (1 50 pL samples) was determined after acidification by reversedphase-HPLC (UV detection at 269 nm).
A . A. Krayevsky. N. €3. Tarussova, Q.-Y Zhu. P. Vidal. T.C. Chou. P.
Baron, B. Polsky. X.-J. Jiang. J. Matulic-Adamic, I. Rosenberg, K. A .
Watanahe, Nucleosides Nucleatides 1992. 11, 177-196.
lowed by a palladium-mediated coupling with a silylprotected acetylene and exhaustive terminal deprotection
(Scheme 1).
f
H
R = (Alkyl)$i
Scheme 1. Possible synthesis of tetraethynylallene 3.
As a model for the first step, we investigated the reaction
of the trialkynyl alcohols 4a, b, which were readily prepared
by treatment of the corresponding dialkynylketones with
monoprotected lithium acetylides in hexane at 20 0C.[31Reaction of triisopropylsilyl (TIPS)-protected 4a with SOCI, in
CH,Cl, at - 78 "C for 20 min afforded dialkynylchloroallene 5 in 86% yield. However, in a similar reaction with
trimethylsilyl (TMS)-protected 4b, the corresponding
chloroallene 7a could not be isolated because it rapidly
dimerized to give 6 a in 76% yield. Similarly, reaction of 4b
with HBr in hexane at 20°C ( 5 h) in the presence of one
equivalent of CuBrL4]gave the brominated dimer 6 b in 70%
yield. The different reactivity of 4a and 4b can be explained
by the bulkiness of the TIPS groups, which prevent the
dimerization of chloroallene 5.
OH
k
4a, R = TiPS
5
4b, R = T M S
TMS
\
TM S,
Tricyclo[6.2.0.03 6]deca-1,3,6,8-tetraene:
A Remarkably Stable para-Quinodimethane from
a Novel Rearrangement Reaction**
3
By Jan-Dirk van Loon, PauE Seiler, and Frangois Diederich*
In the course of our studies towards the development of
new carbon networks,['' we recently became interested in the
synthesis of peralkynylcumulenes.[21A possible route towards tetraethynylallene 3, a molecule that could serve as a
building block for a three-dimensional carbon lattice,["
comprises the conversion of alcohol 1 into haloallene 2, fol[*] Prof. F. Diederich, Dr. J:D. van Loon, P. Seller
Lahoratorium fur Organische Chemre
ETH-Zentrum
Universitiitstrasse 16, CH-8092 Zurich (Switzerland)
Telefax: Int. code (1)261-3524
[**I This work was supported by the Schweizerischen Nationalfonds zur Forderung der Wissenschaftlichen Forschung. J.-D. v. L. thanks the Dutch
National Science Foundation (NWO) for a postdoctoral fellowship.
+
1706
VCH Vrr/ugsg~~'s~~ll.schufr
m b H , 0-69451 Weiuheini.1993
TM S'
TMS/
6a,X = CI
7a,X = CI
6b,X = Br
7b.X = Br
In order to determine the structure of the dimer, a lowtemperature X-ray analysis was performed on crystals of 6 b
(Fig. l).['] All bond lengths and angles are within the expected range, except the very long C(l)-C(2) bond (1.626(5) A)
in the cyclobutane ring.[61In the puckered four-membered
ring the torsional angle between the two exocyclic C = C
bonds is 44.3'. As a result the dienyne system C(18)=C(3)C(4)=C(22)-C(23)-C(24) is arranged in a helix, the handedness of which is determined by the configuration at the
057o-083319311212-1706S 10.00+ .25/0
Angels. Chcm.
hi.
Ed. Engl. 1993. 32, No. 12
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acid, potential, lipophilic, esters, prodrugs, hydroxybenzylphosphonic, dideoxythymidine, dinucleoside, ddt
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