Synthesis of an α-aminophosphonate nucleoside as an inhibitor of S-adenosyl-L-homocysteine hydrolase

Article abstract of DOI:10.1016/S0960-894X(01)00789-2

A phosphonic acid analogue of S-adenosyl-L-homocysteine was prepared by a novel method and the epimeric mixture separated. Preliminary studies indicate that each epimer causes time-dependent inactivation of S-adenosyl-L-homocysteine hydrolase, however each presented distinct kinetic characteristics.

Full text of DOI:10.1016/S0960-894X(01)00789-2

Bioorganic & Medicinal Chemistry Letters 12 (2002) 457–460
Synthesis of an ꢀ-Aminophosphonate Nucleoside as an Inhibitor
of S-Adenosyl-L-Homocysteine Hydrolase
Jennifer A. Steere, Peter B. Sampson and John F. Honek*
Department of Chemistry, University of Waterloo, Waterloo, Ontario, Canada N2L 3G1
Received 26 September 2001; accepted 16 November 2001
Abstract—A phosphonic acid analogue of S-adenosyl-l-homocysteine was prepared by a novel method and the epimeric mixture
separated. Preliminary studies indicate that each epimer causes time-dependent inactivation of S-adenosyl-l-homocysteine hydro-
lase, however each presented distinct kinetic characteristics. # 2002 Elsevier Science Ltd. All rights reserved.
S-Adenosyl-l-methionine (AdoMet) serves as a methyl
donor for a variety of transmethylation reactions.1a,b In
these reactions, the methyl group from AdoMet is
transferred to various acceptor molecules such as pro-
teins, nucleic acids, phospholipids, and small molecules
by specific methyltransferase enzymes.2a,b AdoMet-
dependent methyltransferase reactions are indirectly
controlled by the intracellular levels of S-adenosyl-l-
homocysteine (AdoHcy), as AdoHcy is a potent feed-
back inhibitor of AdoMet-dependent methyltransfer-
ases.3a,b In eukaryotes, the intracellular levels of
AdoHcy and the ratio of AdoHcy/AdoMet are regu-
lated by the hydrolysis of AdoHcy to adenosine (Ado)
and homocysteine (Hcy) by the enzyme S-adenosyl-l-
homocysteine hydrolase (EC 3.3.1.1) (Fig. 1).4a,b Inhi-
bition of AdoHcy hydrolase leads to a rapid accumula-
tion of intracellular AdoHcy and the consequent
inhibition of AdoMet-dependent transmethylation
reactions. Failure to regulate the intracellular levels of
AdoHcy leads to cellular toxicity.
ded methyltransferases that are involved in the forma-
tion of this methylated cap structure can be inhibited
and viral replication can be slowed. The recent deter-
mination of the crystal structure of the rat liver10 and
human11 AdoHcy hydrolases have further contributed
to our understanding of this enzyme.
There have been very few studies on inhibitors of
AdoHcy hydrolase with variations in the amino acid
portion of AdoHcy.12 We wish to investigate further
the importance of the amino acid portion of AdoHcy
in regards to its inhibitory activity as well as providing
information about the key components of AdoHcy
hydrolase interactions with substrates and inhibitors.
In this regard, we were interested in the bioisosteric
replacement of the carboxylate by a phosphonate moiety.
The phosphonic acid moiety has long been established
as a bioisostere of carboxylic acids13 and organophos-
phorus analogues have been shown to be inhibitors of
numerous metabolic processes.14a,b,c The phosphonic
and phosphinic acid analogues of AdoHcy and AdoMet
have previously been prepared by Khomutov and co-
workers.15 The compound, 50-deoxy-50-chloroadenosine
In recent years, AdoHcy hydrolase has become an
attractive target for drug design since its inhibitors have
been shown to exhibit antiviral,5a,b,c antiparasitic,6
antiarthritic,7 and immunosuppressive effects.8a,b The
antiviral activity associated with inhibiting AdoHcy
hydrolase arises due to the observation that most plant
and animal viruses require a methylated cap structure at
the 50-terminus of their mRNA for viral replication.9
Thus, by inhibiting AdoHcy hydrolase, the virus-enco-
*Corresponding author. Fax: +1-519-746-0435; e-mail: jhonek@
uwaterloo.ca
Figure 1. The reaction catalyzed by S-adenosyl-l-homocysteine
hydrolase.
0960-894X/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(01)00789-2

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J. A. Steere et al. / Bioorg. Med. Chem. Lett. 12 (2002) 457–460
was alkylated with g-mercapto-a-aminopropyl phos-
phinic or phosphonic acid in the presence of liquid
ammonia to afford the desired analogue in unspecified
yield. Preliminary studies pertaining to the biological
evaluation of these compounds were reported on epi-
meric material. Both the phosphinic and phosphonic
acid analogues of AdoMet and AdoHcy were shown to
inhibit AdoMet decarboxylase. With respect to RNA
methyltransferase, the AdoMet derivatives appeared to
act as substrates, whereas the AdoHcy analogues acted
as inhibitors of the enzyme.
give fully protected AdoHcy analogue 6 in 63% yield.
As was expected, 1H NMR analyses of the phosphonate
adduct 6 revealed that the compound was generated as a
1:1 mixture of epimers. Full deprotection was achieved
with trimethylsilyl iodide (TMSI)19a,b to generate the
desired compound 7 in 75% yield. The AdoHcy ana-
logue 7 was purified on DOWEX 50W-X8 (elution:
H2O and then 1N NH 4OH) and reverse-phase HPLC
served to separate the 90-epimers.20
Kinetic Studies
We have successfully synthesized the a-aminophos-
phonate AdoHcy analogue by a novel method and have
separated the epimeric mixture into each individual
epimer (A and B). The epimeric mixture and epimers A
and B were evaluated against recombinant rat liver
AdoHcy hydrolase.
The recombinant rat liver AdoHcy hydrolase (MV
1304/pUCSAH) was purified as previously determined
by Gomi and coworkers with minor modifications.21
Enzyme activity was measured in the hydrolytic direc-
tion, through the use of 5,50-dithiobis (2-nitrobenzoic
acid) (DTNB), as described previously.22 The Km for
S -adenosyl-l-homocysteine was determined to be 9.7
mM and the Vmax was 1.1 mmol/min/mg which compares
favourably with previously reported values.22 The com-
pound, 50-deoxy-50-isobutylthioadenosine (SIBA), is a
well studied time-dependent inhibitor of AdoHcy
hydrolase.23 When tested, we found it to have a kinact of
0.0488 minꢀ1 and a Ki of 104.7 mM in our system.
Chemistry
Preparation of the phosphonate nucleoside is outlined
in Scheme 1. Diphenylmethylamine was reacted with
formalin to give 2,4,6-tris(benzhydryl)hexahydro-1,3,5-
triazine 1 in 99% yield upon recrystallization.16 Addi-
tion of diethylphosphite to 1 gave diethyl N-(di-
phenylmethyl)methylphosphonate
2 in 96% yield.
Table 1. Inhibition constants for epimers A and B with AdoHcy
hydrolasea
Compound 2 was oxidized with DDQ in benzene in the
presence of crushed 4 A molecular sieves to give the
imine 3 in 99% yield.17 Next, compound 3 was alkylated
with excess 1,2-dibromoethane to give 4 in 38% yield.
The 50-thio nucleoside analogue 5 was prepared as pre-
viously described.18 The thiolate anion of 5 was gener-
ated from methanolic potassium hydroxide and
compound 4 was added to perform the alkylation to
Compd
Ki (mM)
kinact (minꢀ1
)
A
B
345ꢂ3
0.0049
0.0145
1094ꢂ49
aThe inhibition parameters were calculated from the Kitz and Wilson
plots shown in Figure 2.
Scheme 1. (i) Benzene reflux, Dean-Stark, 99%, (ii) diethylphosphite, 96%; (iii) DDQ, 4 A sieves, benzene, 99%, (iv) n-BuLi, THF, ꢀ78 ꢁC,
BrCH2CH2Br (7 equiv) 38%; (v) MeOH, KOH, 63%; (vi) TMSI, H2O, 75%.

J. A. Steere et al. / Bioorg. Med. Chem. Lett. 12 (2002) 457–460
459
the inhibitor during the experiment or could occur if a
generated product was a good inhibitor of the enzyme.26
In both cases, the earlier time points are more depend-
able. Further studies need to be performed in order to
determine the cause of this biphasic process. However, it
was determined that both epimers were not substrates
for AdoHcy hydrolase as there was no turnover of
either epimer at a concentration of 2.5 mM when incu-
bated for 2 h with the enzyme. The Ki observed for
epimer B was much larger than that for epimer A, which
may be explained if epimer B contained the unnatural
d-configuration on the amino acid side chain. Attempts
to obtain the X-ray structure of one of the epimers is
ongoing in our laboratory.
Both epimer A and B appear to inactive the enzyme
irreversibly since the lost enzymatic activity could not
be restored after dialysis (24 h) against 10 mM potas-
sium phosphate buffer, pH 7.2.
This is the first evaluation of a phosphonate nucleoside
as a probe for the enzyme AdoHcy hydrolase. It is
expected that the phosphonate group will have sub-
stantial dianionic character throughout these experi-
ments, as the pH of the assay buffer is 7.2. Hence, the
effect of pH on the extent and type of inhibition by
these analogues as well as the structure determination of
one of the epimers are the focus of ongoing research.
Acknowledgements
We gratefully acknowledge Drs. R. Aksamit and G.
Cantoni for the Escherichia coli MV1304/pUCSAH
used in these studies and the Natural Sciences and
Engineering Research Council of Canada for financial
assistance. Graduate student funding in the form of an
OGSST scholarship to J.A.S. and OGS to P.B.S. is also
gratefully appreciated. We would also like to thank Tim
Jones and Jan Venne for conducting MS and NMR
studies, respectively.
Figure 2. Time-dependent inactivation of AdoHcy hydrolase with (a)
epimer A and (b) epimer B. The enzyme was preincubated for the
indicated times at 37 ꢁC in the presence of 250 mM (*), 400 mM (Á),
500 mM (^), 750 mM (&) or 1000 mM (!) of the appropriate epimer.
At the indicated time points, residual enzyme activity was deteꢀrm1 ined
in the hydrolytic direction as stated in the text. Inset: plot of kobs ver-
sus [inhibitor]ꢀ1 from which the Ki and kinact values were calculated.
The values for epimer B were determined for time points ꢃ15 min.
Data were the average of triplicate measurements. This was repro-
ducible throughout a number of repeated trials.
References and Notes
1. (a) Chiang, P. K.; Gordon, R. K.; Tal, J.; Zeng, G. C.;
Doctor, B. P.; Pardhasaradhi, K.; McCann, P. P. FASEB J.
1996, 10, 471. (b) Yuan, C. S.; Liu, S.; Wnuk, S. F.; Robins,
M. J.; Borchardt, R. T. Adv. Antiviral Drug Des. 1996, 2, 41 .
2. (a) Takusagawa, F.; Fujioka, M.; Spies, A.; Schowen, R. L.
In Comprehensive Biological Catalysis: A Mechanistic Refer-
ence; Sinnott, M., Ed.; Academic: San Diego, 1998; p 1. (b)
Drahovsky, D.; Boehm, T. L. J. Int. J. Biochem. 1980, 12, 523.
3. (a) Wolfe, M. S.; Borchardt, R. T. J. Med. Chem. 1991, 34,
1521. (b) Wolfe, M. S.; Cantoni, G. L.; Chiang, P. K. In Nat-
ural Sulfur Compounds. Novel Biochemical and Structural
Aspects; Cavallini, D., Gaulle, G. E., Zappia, V., Eds.; Plenum:
New York, 1980; p 67.
4. (a) de la Haba, G.; Cantoni, G. L. J. Biol. Chem. 1959, 234,
603. (b) Chiang, P. K. In Methods in Pharmacology; Paton, D.
M., Ed.; Plenum: New York, 1985; Vol. 6, p 127.
5. (a) Bray, M.; Driscoll, J.; Huggins, J. W. Antiviral Res. 2000,
45, 135. (b) Liu, S.; Wolfe, M. S.; Borchardt, R. T. Antiviral
Res. 1992, 19, 247. (c) De Clercq, E.; Bergstron, D. E.; Holy,
A.; Montgomery, J. A. Antiviral Res. 1984, 4, 119.
The inactivation of AdoHcy hydrolase by the epimeric
mixture of the phosphonate nucleoside appeared to
occur by a complex process that was investigated fur-
ther by studying each individual epimer (A and B). Each
epimer was tested in a time-dependent manner. Kinetic
constants for epimers A and B were determined
according to the method of Kitz and Wilson24 and are
listed in Table 1.
Epimer A proved to inactivate the rat liver AdoHcy
hydrolase in a time-dependent and pseudo-first-order
process, as shown in Figure 2. The Ki and kinact values
are listed in Table 1. Epimer B also proved to be time-
dependent, but biphasic in nature, showing pseudo-first-
order kinetics only in the first period of inactivation
(Fig. 2). This biphasic process has been observed quite
frequently by other AdoHcy hydrolase inhibitors25a,b
and could be due to a decrease in the concentration of

460
J. A. Steere et al. / Bioorg. Med. Chem. Lett. 12 (2002) 457–460
6. Bitonti, A. J.; Baumann, J.; Varvi, T.; McCarthy, J. R.;
McCann, P. P. Biochem. Pharmacol. 1990, 40, 601.
7. Wolos, J. A.; Frondorf, K. A.; Esser, R. E. J. Immunol.
1993, 151, 526.
8. (a) Saso, Y.; Conner, E. M.; Teegarden, B. R.; Yuan, C. S.
Pharmacology 2001, 296, 106. (b) Wolos, J. A.; Frondorf,
K. A.; Davies, G. F.; Jarvi, E. T.; McCarthy, F. R.; Bowlin,
T. L. J. Immunol. 1993, 150, 3264.
(d, 1H, J=5.6 Hz, H-10), 4.70–472 (m, 1H, H-20), 4.18–4.21
(m, 1H, H-30), 4.07–4.11 (m, 1H, H-40), 2.94–3.10 (m, 2H, H2-
50), 2.65–2.85 (m, 3H, H-90, H2-70), 2.05–2.10 (m, 1H, H-80),
1.82–1.92 (m, 1H, H-80); 13C NMR (125 MHz, DMSO) d 155.6
(C-6), 152.7 (C-2), 149.3 (C-4), 139.8 (C-8), 118.7 (C-5), 87.0
(C-10), 83.5 (C-20), 72.7 (C-30), 72.1(C-4 0), 34.5 (C-50), 29.7 (C-
70), 28.6 (C-80), (C-90 obscured by DMSO); 31P NMR
(80.0 MHz, DMSO)
d 14.1; high-resolution FAB m/z
9. Banerjee, A. K. Microbiol. Rev. 1980, 44, 175.
10. Hu, Y.; Komoto, J.; Huang, Y.; Gomi, T.; Ogawa, H.;
Takata, Y.; Fujioka, M.; Takusagawa, F. Biochemistry 1999,
38, 8323.
11. Turner, M. A.; Yuan, C. S.; Borchardt, R. T.; Hershfield,
M. S.; Smith, G. D.; Howell, P. L. Nat. Struct. Biol. 1998, 5,
369.
12. Chiang, P. K.; Richards, H. H.; Cantoni, G. L. Mol.
Pharmacol. 1977, 13, 939.
13. Burger, A. Prog. Drug Res. 1991, 37, 287.
14. (a) Lowther, W. T.; Zhang, Y.; Sampson, P. B.; Honek,
J. F.; Matthews, B. W. Biochemistry 1999, 38, 14810. (b)
Lejczak, B.; Kafarski, P.; Zygmunt, J. Biochemistry 1989, 28,
3549. (c) Atherton, F. R.; Hassall, C. H.; Lambert, R. W. J.
Med. Chem. 1986, 29, 29.
[421.09080 (M+1); (calcd for C13H22SPN6O6) 421.12586].
Epimer B: HPLC (C18: 25 mm ꢄ 10 cm); retention time 22
min at flow rate 3 mL/min, (30% CH3CN/H2O); 1H NMR
(500 MHz, DMSO) d 8.38 (s, 1H, H-2), 8.08 (s, 1H, H-8), 5.86
(d, 1H, J=5.5 Hz, H-10), 4.63–4.68 (m, 1H, H-20), 4.11–4.18
(m, 1H, H-30), 3.99–4.03 (m, 1H, H-40), 2.86–2.92 (m, 2H, H2-
50), 2.65–2.85 (m, 3H, H-90, H2-70), 1.92–2.04 (m, 1H, H-80),
1.76–1.82 (m, 1H, H-80); 13C NMR (125 MHz, DMSO) d 153.4
(C-6), 153.2 (C-2), 152.6 (C-4), 140.3 (C-8), 116.3 (C-5), 87.8
(C-10), 84.9 (C-20), 73.7 (C-30), 73.2 (C-40), 34.2 (C-50), 31.2 (C-
70), 29.7 (C-80), (C-90 obscured by DMSO); 31P NMR
(80.0 MHz, DMSO)
d 13.2; high-resolution FAB m/z
[421.08658 (M+1); (calcd for C13H22SPN6O6) 421.12586].
21. Gomi, T.; Date, T.; Ogawa, H.; Fujioka, M.; Akasmit,
R. R.; Backlund, P. S.; Cantoni, G. L. J. Biol. Chem. 1989,
264, 1618.
22. Mehdi, S.; Jarvi, E. T.; Koehl, J. R.; McCarthy, J. R.;
Bey, P. J. Enzym. Inhib. 1990, 4, 1 .
15. Syrku, V. I.; Zavalova, L. L.; Khomutov, R. M. Bioorg.
Khim. 1986, 12, 839.
16. Smolin, E. M.; Rapoport, L. In The Chemistry of Hetero-
cyclic Compounds; s-Triazenes and Derivatives; Weissberger,
A., Ed.; Interscience: New York, 1959; p 473.
23. Chiang, P. K.; Richards, H. H.; Cantoni, G. L. Mol.
Pharmacol. 1977, 13, 939.
17. Sampson, P. B.; Honek, J. F. Org. Lett. 1999, 1, 1395.
18. Baddiley, J.; Jamieson, G. A. J. Chem. Soc. 1955, 1085.
19. (a) Rudisill, D. E.; Whitten, J. P. Synthesis 1994, 851. (b)
TMSI (6 equiv) from a new ampoule must be utilized. Old
TMSI that contained red colouration was ineffective.
20. Epimer A: HPLC (C18: 25 mm ꢄ 10 cm) retention time 18
min at flow rate 3 mL/min, (30% CH3CN/H2O); 1H NMR
(500 MHz, DMSO) d 8.38 (s, 1H, H-2), 8.18 (s, 1H, H-8), 5.90
24. Kitz, R.; Wilson, I. B. J. Biol. Chem. 1962, 237, 3245.
25. (a) Guillerm, G.; Guillerm, D.; Vandenplas-Witkowki, C.;
Rogniaux, H.; Carte, N.; Leize, E.; Van Dorsselaer, A.; De
Clercq, E.; Lambert, C. J. Med. Chem. 2001, 44, 2743. (b)
Yuan, C. S.; Wnuk, S. F.; Liu, S.; Robins, M. J.; Borchardt,
R. T. Biochemistry 1994, 33, 12305.
26. Silverman, R. B. In Methods in Enzymology; Purich, D.
L., Ed.; Academic: New York, 1995; Vol. 249, p 240.