Note: Descriptions are shown in the official language in which they were submitted.
' 1339483
PRO~u~l-lON OF 2', 3' -DIDEOXY-2', 3' -DIDEHYDRONUCLEOSIDES
RACR~ROUND OF THE lNv~NllON
FIELD OF THE lNv~NLlON
This invention relates to improved processes to produce
2',3'-dideoxy-2,'3'-didehydronucleosides.
DESCRIPTION OF THE R~CK~ROUND AND RELATED REFERENCES
Acquired immunodeficiency syndrome (AIDS) is the result
of an infection by human immunodeficiency virus(es) (HIV).
This retrovirus shows a specific tropism for the
helper/inducer T cells2 leading to their depletion. The
resultant immunosuppression predisposes HIV patients to
life-threatening opportunistic infections.
Although at present there is no cure for AIDS, one
nucleoside derivative, 3'-azido-3'-deoxythymidine (AZT,
RetrovirTM), has already proved to be an efficacious agent
in the treatment of AIDS in clinical trials and has been
licensed by the appropriate regulatory agency for use in
patients with AIDS. 3 A number of other chemical and
biological agents have been reported to have biological
activity against HIV. 2',3'-Dideoxycytidine (ddC), 2',3'-
dideoxyadenosine (ddA),4 2',3'-dideoxy-2',3'-
didehydrocytidine (d4C),s suramine and its analogs,6
ribavarin, 7 foscarnet, 8 HPA-23,9 d-penicilllamine,10
castanospermine,11 fusidic aid,12 3'-azidoguanosine (AZG) ,13
and 3'-fluoro-3'-deoxythymidine (FDDT)14 are all reported to
be active against HIV.
A number of reports have appeared in the literature
which have shown that 2',3'-dideoxy-2',3'-didehydrothymidine
(d4T) possesses in vitro activity against HIV in several
cell lines.15
2',3'-dideoxy-2',3'-didehydrothymidine (d4T) has been
prepared by Horwitz et al. by two different routes. 16-17 The
first of these synthetic routes involves subjecting the
3',5'-anhydro derivative of thymidine to elimination
reaction conditions. The second of these routes involves
1339~83
subjecting the 5'-O-protected 2,3'-anhydro nucleoside
derivative of thymidine to ring-opening elimination reaction
conditions.
The use of anhydro nucleosides as intermediates for
nucleoside synthesis is well precedented in the literature
in the art to which the present invention pertains.18
With the recent discovery of the potency of 2',3'-
dideoxy-2',3'-didehydrothymidine (d4T) as an anti-HIV agent,
a process which allows 2',3'-dideoxy-2',3'-didehydro
nucleosides, including d4T, to be prepared cheaply on a
large scale becomes important.
The Horwitz route to produce d4T from the 3',5'-anhydro
compoundl6 is not feasible on a large scale because complete
removal of the large volume of DMSO used in scale-up of the
Horwitz procedure is very difficult to achieve and requires
high vacuum (O.O1 mmHg and heating for the temperature range
of about 40-50~C) for an extended period of time. These
conditions lead to cleavage of the glycosidic bond to give
thymine as an undesired side product. Also, prolonged
exposure to basic conditions, which are required when
solvents other than DMSO (e.g. THF, DMF) are used leads to
decomposition of d4T to, again, give thymine as an undesired
side product.
The alternative Horwitz procedure requires protection
of the 5'-OH position before formation of the 2,5'-anhydro-
nucleoside. This 2,5'-anhydronucleoside can be opened to
give the 5'-O-protected nucleoside.
The desired 2,3'-anhydro nucleoside can be prepared
directly by reacting thymidine with diethyl-(2-chloro~ 2
trifluoroethyl)amine.19
-- 2
1339483
SUMMARY OF THE lNV~N-llON
This invention is a process for producing
2',3'-dideoxy-2',3'-didehydronucleosides of the formula
R4
X ~ Y
O~ N
HO ~ O J
in high yields and on a relatively large scale.
DETAILED DESCRIPTION OF THE lNV~NllON
In one generic aspect, this invention is in a process
for producing a 2',3'-dideoxy-2',3'-didehydronucleoside
represented by the formula R4
X Y
O N
HO-~ O
~7
wherein the base moiety is a member selected from the group
of unsubstituted and substituted bases consisting of
pyrimidine, aza-pyrimidine, and deaza-pyrimidine; X is
selected from N and C-H; Y is selected from C-R5 and N; Z is
selected from C-H and N; R4 is selected from OH and NH2; and
Rs is selected from H, unsubstituted and halo-substituted
alkyl having the formula CnH2nA, and unsubstituted and
halo-substituted alkenyl having the formula -(CH2)m-CH=CHA
wherein m is an integer selected form 0, 1, 2 and 3, n is an
integer selected from 1, 2, and 3 and A is selected from H,
F, C1, Br, and I, comprising the steps of:
(a) converting a 2'-deoxynucleoside represented by the
formula
-- 3
1339 183
R4
X ~ Y
O N
H
HO
to a reactive 3',5~-anyhydro-2'-deoxynucleoside intermediate
represented by the formula
R~
X Y
O N
p~O ~J
\O
and
(b) converting in the presence of strong base said
reactive 3',5'-anhydro-2'-deoxynucleoside from step (a)
above to said 2',3'-dideoxy-2',3'-didehydronucleoside;
the improvement comprising:
(i) reacting said 3',5'-anhydro-2'deoxynucleoside
with a strong base selected from KOtBu, nBuLi, NaH, and LDA
in the presence of a polar solvent selected from DMSO, THF,
DMF, DME, and mixtures thereof;
(ii) triturating the resulting salt in the
presence of an organic solvent;
(iii) collecting the solid crude salt intermediate
from step (ii);
(iv) dissolving the salt from step (iii) in water;
(v) neutralizing the salt from step (iv); and
(vi) obtaining the solid nucleoside free base
product.
1339483
In another generic aspect, this invention is a process
for producing a 2',3'-dideoxy-2',3'-didehydronucleoside
represented by the formula
X ~ Y
O~ N
~0_ 0 1
wherein the base moiety is a member selected from the group
of unsubstituted and substituted bases consisting of
pyrimidine, aza-pyrimidine, and deaza-pyrimidine; X is
selected from N and C-H; Y is selected from C-R5 and N; Z is
selected from C-H and N; R4 is selected from OH and NH2; and
R5 is selected from H, unsubstituted and halo-substituted
alkyl having the formula CnH2nA, and unsubstituted and
halo-substituted alkenyl having the formula CnHnA, wherein n
is an integer selected from 1, 2, and 3 and A is selected
from H, F, C1, Br, and I, comprising the steps of:
(a) converting a 2'-deoxynucleoside represented by the
formula
R~
X Y
0~ N
HO ~ o
HO
1339483
to a reactive 2,3'-anyhydro-2'-deoxynucleoside intermediate
represented by the formula
R~
X ~ Y
O ~~ ~ Z
H
and
(b) converting in the presence of base selected from
non-nucleophilic and nucleophilic bases said reactive
2,3'-anhydro-2'-deoxynucleoside from step (a) above to said
2',3'-dideoxy-2'3'-didehydronucleoside.
In still another generic aspect, this invention is a
process for producing 2',3'-dideoxy-2',3'-didehydronucleo-
side represented by the formula
H ~ O ~
wherein B is a member selected from the group of bases
consisting of purine, aza-purine, deaza-purine, pyrimidine,
aza-pyrimidine, deaza-pyrimidine, and triazole ring bases,
comprising the steps of:
1339483
(a) reacting a starting ribonucleoside represented by
the formula
H ~ B
HO OH
with trimethylorthoformate in the presence of a polar
solvent under anhydrous conditions to obtain a reactive
intermediate represented by the formula
H ~ B
O O
~Me
(b) subjecting the intermediate from step (a) to an
elimination reaction by treatment with p-TsOH in Ac2O at an
elevated temperature of about 120-160~C for about 4-8 hours;
and
(c) deprotecting the resulting 5'-OAc group by
treatment under mild base hydrolysis conditions.
In yet another generic aspect, this invention is a
process for producing a 2',3'-dideoxy-2',3'-didehydro-
nucleoside represented by the formula
H ~ O ~
wherein B is a member selected from the group of bases
consisting of purine, aza-purine, deaza-purine, pyrimidine,
aza-pyrimidine, deaza-pyrimidine, and triazole ring bases,
comprising the steps of
- 7 -
1339483
(a) reacting a starting ribonucleoside represented by
the formula
HO~ o B
HO OH
with a hydroxy protecting group reagent effective to
selectively protect the 5'-hydroxyl (primary hydroxyl)
group;
(b) reacting with 5'-OH-protected ribonucleoside from
step (a) with a reagent selected from 1,1-thiocarbodiimida-
zole and thiophosgene under anhydrous conditions to obtain a
reactive intermediate represented by the formula
HO~ o B
O ~.
S
(c) subjecting the intermediate from step (6) to an
elimination reaction by treatment with P(OEt) 3 in a polar
solvent at an elevated temperature of about 140-175~C for
about 0.5-4 hours; and
(d) deprotecting the resulting 5'-O-protecting group
by treatment under mild acid hydrolysis conditions.
In yet another generic aspect, this invention is a
process for producing a 2',3'-dideoxy-2',3'-didehydro-
nucleoside represented by the formula
Ho~~ o 8
1339~83
wherein B is a member selected from the group of bases
consisting of purine, aza-purine, deaza-purine, pyrimidine,
aza-pyrimidine, deaza-pyrimidine, and triazole ring bases,
comprising the steps of:
(a) reacting a starting ribonucleoside represented by
the formula
HO OH
with an acyloxyisobutyryl bromide, preferably 2-acetoxyiso-
butyryl bromide, in a polar solvent under anhydrous
conditions at an elevated temperature of about 75~-100~C for
about 1-3 hours to obtain a reactive intermediate
represented by the formula
RO~B
~r)R'O B~OR~
wherein R represents the acyloxyisobutyryl group and R'
represents the acyl group of the acyloxyisobutyryl bromide;
(b) subjecting the intermediate from step (a) to an
elimination reaction by treatment of said intermediate in an
aprotic polar solvent with Zn/Cu reagent to obtain an
intermediate represented by the formula
RO-~ O B
; and
(c) deprotecting the 5'-O-protecting group in the
intermediate from step (b) by treatment of said intermediate
g
1339483
with a mild base, preferably methanolic ammonia, to give
derived 2,3'-dideoxy-2',3'-didehydro-nucleoside.
As is described above, this invention concerns two
embodiments of a process to produce 2',3'-dideoxy-2',3'-
didehydronucleosides wherein the starting material is a 2'-
deoxynucleoside, and wherein the base component B is derived
from a member selected from the group of bases consisting of
is an unsubstituted or substituted pyrimidine, or aza-
pyrimidine, or deaza-pyrimidine, preferably an unsubstituted
or substituted pyrimidine. More preferably, the base moiety
in these two embodiments is said unsubstituted or
substituted pyrimidine corresponding to the formula and
description set forth hereinbelow with respect to suitable
unsubstituted and substituted pyrimidine bases. Still more
preferably in these two embodiments, the base moiety is
selected from thymine (5-methyl-2,6-dihydroxy-pyrimidine),
cytosine (2-hydroxy-6-aminopyrimidine), uracil (2,6-
dihydroxy-pyrimidine), and 5-ethyl- and 5-vinyl- and
5-halovinyl- and 5-halomethyl- and 5-haloethyl-2,
6-dihydroxypyrimidine-3-yl. Most preferably in these two
embodiments the base moiety is thymine.
As is described above, this invention concerns three
embodiments of a process to produce 2',3'-dideoxy-2',3~-
didehydronucleosides wherein the starting material is a
ribonucleoside and wherein the base component B is derived
from a member selected from the group of bases consisting of
unsubstituted and substituted purine, aza-purine, deaza-
purine, pyrimidine, aza-pyrimidine, deaza-pyrimidine, and
triazole ring bases. Preferably the base is selected from
purine and pyrimidine bases. More preferably, the base is a
pyrimidine base including one of the group of uracil and
thymine.
-- 10
133~83
Suitable unsubstituted and substituted purine bases
include those purine bases represented by the structural
formula
R
~O~
R~
wherein Rl and R2 may be the same or different and are
selected from hydrogen, hydroxy, halo (F, C1, Br), amino,
monoalkylamino, dialkylamino, alkoxy and cyano groups
wherein the alkyl moiety is selected from Cl-C3 alkyl groups.
Suitable unsubstituted and substituted pyrimidine bases
include those pyrimidine bases represented by the structural
formula
R5
R6
R N
H ~
wherein, R3 is selected from hydroxy, amino and
sulfhydryl groups; R4 is selected from hydroxy, amino and
sulfhydryl groups; R5 is selected from hydrogen, Cl-C3 alkyl,
C2-C3 alkenyl, C2-C3 haloalkenyl having from 1 to 5 halo
groups as defined herein, C2-C3 alkynyl, alkoxy wherein the
alkyl moiety has 1-3 carbon atoms, cyano and halo (F, Cl, Br
and I).
When derived from purine bases, representative of B are
the following:
6-aminopurin-9-yl
2-aminopurin-9-yl
2,6-diaminopurin-9-yl
- 11 -
13~9483
2-amino-6-hydroxypurin-9-yl (guanin-9-yl)
6-hydroxypurin-9-yl
ln addition to the above, the B component may be
2-halopurin-9-yl, 6-halopurin-9-yl, or 2,6-dihalopurin-9-yl,
in which event the base component need not be activated, for
example, completely silylated, in order to undergo the
condensation or coupling reaction in step (e).
When derived from pyrimidine bases, representative of B
are the following:
2, 4-dihydroxyprimidin-1-yl
5-methyl-2, 4-dihydroxypyrimidin-1-yl
5-ethyl-2, 4-aminopyrimidin-1-yl
2-hydroxy-4-aminopyrimidin-1-yl
5-vinyl-2, 4-dihydroxypyrimidin-1-yl
5-halovinyl-2, 4-dihydroxypyrimidin-1-yl
5-haloethyl-2, 4-dihydroxypyrimidin-1-yl
5-haloethyl-2, 4-dihydroxypyrimidin-1-yl
The above-mentioned 5-methyl and 5-ethyl substituents
are representative of 5-alkyl substituents and the 5-vinyl
substituent is representative of 5-alkenyl substituents.
Examples of halo-groups on the 5-halovinyl (or
5-haloalkenyl) group include 1 to 4 F, C1, and Br groups.
In the first-mentioned embodiment of the process
according to this invention, the first step involves the
preparation of a reactive 3',5'-anhydro-2'-deoxynucleoside,
intermediate from a starting 2'-deoxynucleoside. This known
intermediate is obtained by reacting a corresponding
2'-deoxynucleoside with sufficient, conventional activating
hydroxyl protecting group reagent, e.g. MsCl or TsCl under
conventional conditions to obtain a 3',5'-0-protecting
group-2'-deoxynucleoside first intermediate and reacting
said first intermediate with strong base selected from KOH
and NaOH in a solvent selected from water and ethanol.
Although any well known activating hydroxyl protecting group
reagent useful in the art to which this invention pertains
may be used, mesyl chloride is most advantageously used
according to the procedure of Horwitz et al.
- 12 -
1339483
The second step of this embodiment involves an H-atom
elimination reaction in the presence of strong base as is
known and in the presence of polar solvent whereby the
3',5'-anhydro ring is opened and the 2'-ene double bond is
formed to obtain the desired 2',3'-dideoxy-2',3'-didehydro-
nucleoside.
The invention in this first-mentioned embodiment of the
process according to this invention is in the improvements
in the selection, and handling and processing of the
reactants and intermediates and products. Said improvement
comprlses:
(i) reacting said 3',5'-anhydro-2'-deoxynucleoside
with a strong base selected from KOtBu, nBuLi, NaH, and LDA
in the presence of a polar solvent selected from DMSO, THF,
DMF, DME, and mixtures thereof and, preferably, at a
temperature in the range of about 18~-80~C, more preferably
about 18~-22~C;
(ii) triturating the resulting salt in the presence of
an organic solvent;
(iii) collecting the solid crude salt intermediate from
step (ii);
(iv) dissolving the salt from step (iii) in water;
(v) neutralizing the salt from step (iv); and
(vi) obtaining the solid nucleoside free base product.
In addition to the above-mentioned improvements in step
(b), we have in step (a) found that by reducing the volume
of solvent in the KOH addition and then by concentrating the
slurry following neutralization to about 20~ of its original
volume, the desired intermediate precipitates and can be
collected by filtration whereas the by-product KCL salt
remains dissolved in the solvent. This obviates the need for
hot acetone treatment of the completely evaporated step (a)
reaction mixture to recover the reactive intermediate.
An alternative approach to making the desired 2',3'-
dideoxy-2',3'-didehydronucleosides from the corresponding
2'-deoxynucleoside would be to start from the corresponding
ribonucleoside. Accordingly, we have discovered that we can
- 13 -
1339483
treat uridine, with trimethyl orthoformate to obtain the
corresponding ortho ester reactive intermediate.
In step (ii) of the above first-mentioned embodiment,
the solvent used may be any organic solvent compatible with
the reactants and intermediate salt resulting from step (i).
Preferably, the solvent is selected from toluene, acetone
and ethyl acetate, most preferably toluene. The temperature
of the trituration procedure in step (ii) preferably is
about 0~-10~C, most preferably about 0~-4~C.
In the second-mentioned embodiment of the process
according to this invention, the first step involves the
preparation of the known reactive 2,3'-anhydro-2'-deoxy-
nucleoside intermediate by reacting a starting corresponding
2-deoxynucleoside with a strong base effective to form a
2,3'-anhydro-2'-deoxynucleoside. A suitable reagent to
accomplish this ring formation is the known reagent,
diethyl(2-chloro-1,1,2-triFluoroethyl)amine. This known
intermediate has been reacted with nucleophiles to obtain
substituted nucleosides such as, for example, 3'-azido-2',
3'-dideoxythymidine (AZT). 20
The second step of this embodiment involves not
nucleophilic addition but, rather, the ring opening reaction
of the anhydro ring of the above reactive intermediate.
Suitable reagents to effect this ring opening are either
non-nucleophilic bases such as tetrabutyl ammonium fluoride
or nucleophilic bases selected from KOtBu, NaOH, KOH and the
like.
In the third-mentioned embodiment of the process
according to this invention, the first step involves
reacting a starting ribonucleoside with trimethylortho-
formate in the presence of a polar solvent under anhydrous
133g~83
conditions2ln to obtain an ortho ester reactive intermediate
represented by the formula
H ~ ~
O O
~Mc
The next step involves subjecting the reactive
intermediate to an elimination23 reaction by treatment with
an organic acid, e.g. p-TsOH, in Ac2O at an elevated
temperature of about 120~-160~C for about 4-8 hours.
Finally, the last step involved deprotecting the resulting
5'-OAc group of the ortho ester reactive intermediate by
treatment under mild base hydrolysis conditions. ISb
In the fourth-mentioned embodiment of the process
according to this invention, the first step involves
reacting a starting ribonucleoside with one of any
conventional hydroxy protecting groups effective to
selectively protect the 5'-hydroxyl group (i.e., the primary
hydroxyl group as distinguished from the sugar-ring-bound
secondary hydroxy groups). The second step involves reacting
the 5'-0 protected ribonucleoside with one of
1,1-thiocarbonyldiimdazole and thiophosgene under anhydrous
conditions to obtain a reactive thiocarbonate intermediate
represented by the formula
HO~B
O ~ O
S
The next step involves subjecting the reactive
intermediate to an elimination reaction by treatment with
P(OEt) 3 in a polar solvent at an elevated temperature of
- 15 -
1339483
about 140~-175~C for about 0.5-4 hours. Finally, the fourth
and last step involves deprotecting the resulting 5'-O-
protecting group of the reactive thiocarbonate intermediate
by treatment under mild acid hydrolysis conditions.
In the fifth and last-mentioned embodiment of the
process according to this invention, the first step involves
reacting a starting ribonucleoside with 2-acetoxyisobutyryl
bromide24 to obtain the reactive intermediate represented by
the formula
R ~ O ~
(Br)R'O B~OR')
Next, the reactive intermediate mixture is reacted with
Zn/Cu reagent in an aprotic polar solvent to effect
elimination to obtain the desired 2',3'-dideoxy-2',3'-
didehydronucleoside product. 25
SCHEME I illustrates schematically typical,
representative processes according to our present invention.
Route A illustrates the first-mentioned embodiment
proceeding through the 3',5'-anhydro, or "oxetane", reactive
intermediate. Route B illustrates the second-mentioned
embodiment proceeding through the 2,3'-anhydro reactive
intermediate. Both of Routes A and B start from a
2'-deoxynucleoside. Route C illustrates the third- and
fourth-mentioned embodiments proceeding from a starting
ribonucleoside through an ortho ester or thiocarbonate
reactive intermediate, respectively. Route D illustrates the
last-mentioned embodiment proceeding through a
3'-O-acetyl-2'-bromo-2'-deoxynucleoside and/or
3~-bromo-2'-0-acetyl intermediate.
1339483
SCHEME I
ROUIE A:
~CH O~;~ O~;~
HO~ ~ ~ i > ~ ~ HO--~y
HO MeO WT
THYMIDINE
ROUTE B:
NN ~ ~ al o~CK~
HO~ HO'~ ~ H
OH
ROUTE C:
NO~
~-MErHYl,UR,tDtNE o o
R
R _ OE~--S
ROUrE D:
1~l ~01 1~l
o"N~~3 o~ N o~3
HO~ > RO--~ > 3. HO--
OH OH (B~)R' O Dr(OR')
-- 17 --
1339483
The problems presented when attempting to practice the
so-called "oxetane" route according to Horwitz et al. which
proceeds through the 3~,5~-anhydro intermediate are
associated mainly with scale-up of the final elimination
reaction. On larger scales removal of the solvent leads to
thymine elimination on account of prolonged exposure to
heat. The use of larger amounts of base also gives thymine
as an undesired product.
The specific improvements made to this "oxetane" route
which constitute one embodiment of our invention involve (1)
utilizing a much smaller amount of water in converting the
mesylate to the oxetane, thereby carrying-out the reaction
under more concentrated conditions, and (2) modifying the
work-up of the final step wherein we precipitate by means of
trituration the potassium salt from the final step involving
KOtBu/DMSO treatment of the reactive intermediate. In
addition, the workup of step (a) has been simplified to
neutralizing the reaction, reducing the amount of water and
collecting the resulting product. This is much easier than
completely removing the water, suspending the resulting
salts in hot acetone, filtering, then stripping the
filtrate. This allows the product to be collected,
neutralized and re-collected. The advantage of our
improvements over the literature procedures is that removal
of large volumes of DMSO under vacuum is not required and,
also, that lengthy heating which destroys the product but
which otherwise is required to remove DMSO under vacuum is
avoided. As a result of these improvements, relatively pure
product is obtained on bulk scale.
The problems in the literature methods for producing
the desired 2',3'-dideoxy-2',3'-didehydronucleosides by way
of the 2,3'-anhydro reactive intermediate involve the use of
diethyl(2-chloro-1,1,2-trifluoroethyl)amine, a fluoramine
reagent that is difficult to make and which requires
specialized equipment. Alternatively, the literature reports
a lengthy 4-step procedure involving 5'-0-tritylation,
3'-0-mesylation, detritylation, and anhydro formation. The
- 18 -
1~39 183
literature reports formation of various products and
side-products other than the desired
2',3'-dideoxy-2',3'-didehydronucleoside products
The specific improvements made in the published route
proceeding through the 2,3'-anhydro reactive intermediate
involve the discovery that the use of the reagent,
tetrabutyl ammonium fluoride (TBAF), under non-nucleophilic
conditions affords the desired product cleanly and in high
yields. Alternatively, the use of KOtBu/DMSO and NaOH/DMF,
but not NaCN/DMF or DBU/DMF or NaOH/MeOH or KOtBu/BuOH, in
place of TBAF in THF or DMF affords the desired product
although yields are lower and some undesired side-product
(3'-epi-thymidine) was obtained with the use of NaOH/DMF.
Thus, the processes according to this invention are
useful for the preparation of a variety of
2'3'-dideoxy-2',3'-didehydronucleosides, especially
pyrimidine and purine nucleosides, having antiviral,
antimetabolic, and antineoplastic activity as well as
activity against human immunodeficiency viruses.
The following examples illustrate but a few
representative embodiments of the processes according to
this invention and are set forth to teach those skilled in
the pertinent art how to practice this invention and are not
to be construed as limiting in scope. All parts and
percentages are by weight and temperatures are in degrees
Celsius unless otherwise specified.
Biological data, including anti-HIV data, of d4T
produced by a process according to this invention are set
forth in TABLE I. These data are consistent with data
published.
References
1. (a) Barre-Sinoussi, F; Chermann, J.C.; Rey, R.;
Nugeyre, M.T.; Ch~m~ret, S.; Gruest, C.; Dauguet, C.;
Axler-Blin, C.; Rouzioux, C.; Rozenbaum, W.;
Montagnier, L. Science (Washington, D. C. ) 1983, 220,
868-871. (b) Broder, S.; Gallo, R.C. N. Engl . J. Med.
- 19 -
133948~
1984, 311, 1292-1297. (c) Broder, si Gallo, R.C. Annu.
Rev. Immunol. 1985, 3, 321-336.
2. Popovic, M.; Sarngadharan, M.G.; Read E.; Gallo, R.C.
Science (Washington D.C.J 1984, 224, 497-500. (b)
Gallo, R.C.; Sarngadharan, M.G.; Popovic, M.; Shaw,
G.M.; Hahn, B.; Wong-Stahl, F.; Robert-Guroff, M.;
Salahaddian, Z., Markham, P.D. Prog. Allergy 1986, 37,
1-45.
3. Fischl, M.A.; Richman, D.D.; Grieco, M.H.; Gottlieb,
M.S.; Volberding, P.A.; Laskin, O.L.; Leedom, J.M.;
Groopman, J.E.; Mildvan, D.; Schooley, R.T.; Jackson,
G.G.; Durack, D.T.; King, D. New Engl. J. Med., 1987,
317, 185.
4. Mitsuya, H.; Broder, S. Proc. Natl. Acad. Sci. U.S.A.
1986, 83, 1911-1915.
5. (a) Lin, T.S.; Shinazi, R.; Chen, M.S.; Kinney-Thomas,
E.; Prusoff, W.H. Biochem. Pharmacol. 1987, 36, 311.
(b) Balzarini, J.; Pauwels, R.; Herdewijn, P.; De
Clercq, E.; Cooney, D.A.; Kang, G-J.; Dalal, M.; Johns,
D.G.; Broder, S. Biochem. Biophys. Res. Comm. 1986,
140, 735.
6. Cheson, B.D.; Levine, A.D.; Mildvan, D,; Kaplan, L.D.;
Wolfe, P.; Rios, A.; Groopman, J.; Gill, P.;
Volberding, P.A.; Poiesz, B.J.; Gottlieb, M.S.; Holden,
H.; Volsky, D.J.; Silver, S.S.; Hawkins, M.J. ~. Amer.
Med. Assoc. 1987, 258, 1347.
7. (a) Balzarini, J.; Mitsuya, H.; De Clercq, E.; Broder
S. Int. J. Med, 1986, 37 451. (b) McCormick, J.B.;
Getchell, J.B.; Mitchell, S.W.; Hicks, D.R. Lancet
1984, ii, 1367.
8. (a) Sarin, P.S.; Taguchi, Y.; Sun, D.; Thornton, A.;
Gallo, R.C.; Oberg, B. Biochem. Pharmacol. 1985, 34,
4075. (b) Sandstrom, E.G.; Kaplan, J.C.; Byington,
R.E.; Hirsch, M.S. Lancet 1985, i, 480.
9. Lane, H.C.; Fauci, A.S. Ann. Intern. Med. 1985, 103,
714.
- 20 -
1~3g483
10. Chandra, P.; Sarin, P.S. Arznrim-Forsch/Drug Res. 1986,
36, 184.
11. Tyms, A.S.; Berrie, E.M.; Ryder, T.A.; Nash, R.J.;
Hegarty, M.P.; Taylor, D.L.; Mobberley, M.A.; Davis,
J.M.; Bell, E.A.; Jeffries, D.A.; Taylor-Robinson, D.;
Fellows, L.E. Lancet 1987, ii, 1025.
12. Faber, V.; Newell, A.; Dalgleish, A.G.; Malkovsky, M.
Lancet 1987, ii, 827.
13. (a) Hartmann, H.; Hunsmann, G.; Eckstein, F. Lancet
1987, i, 40. (b) Baba, M.; Pauwels, R.; Balzarini, J.;
Herdewiijn, P.; De Clercq, E. Biochem. Biophys. Res.
Comm. 1987, 145, 1080.
14. (a) Herdewijn, P.; Balzarini, J.; De Clercq, E.;
Pauwels, R.; Baba, M.; Broder, S.; Vanderhaeghe, H. J.
Med. Chem. 1987, 30, 1270. (b) Mattes, E.; Lehm~nn, C.;
Scholz, D.; von Janta-Lipinski M.; Gaertner, K.;
Rosenthal, H.A.; Langen, P. Biochem. Biophys. Res.
Comm. 1987, 148, 78. (c) Polski, B.; Gold, J.M.W.;
Hardy, W.D.; Baron, P.A.; Zuckermann, E.E.; Chou, T-C.;
Levine, S.M.; Flomenberg, N.; Wang, L.; Watanabe, K.A.;
Fox, J.J.; Armstrong, D. 27th ICAAC 1987, Abstract 368,
pl61.
15. (a) Lin, T.S.; Chen, M.S.; Gao, Y-S.; Ghazzouli, I.;
Prusoff, W.H. ~. Med. Chem. 1987, 30, 440. (b) Lin,
T.S.; Shinazi, R.F.; Prusoff, W.H. Biochem. Pharmacol.
1987, 17, 2713. (c) Baba, M.; Pauwels, R.; De Clercq,
E.; Desmyter, J.; Vandeputte, M. Biochem. Biophys. Res.
Comm. 1987, 142, 128. (d) Balzarini, J.; Kang, G-J.;
Dalal, M.; Herdewjin, P.; De Clercq, E.; Broder, S.;
Johns, D.G. Mol. Pharmacol. 1987, 32, 162. (e)
~m~moto, Y.; Nakashima, H.; Matsui, T.; Matsuda, A.;
Ueda, T.; Yamamoto, N. Amtimicrob. Agents Chemother.
1987, 31, 907.
16. Horwitz, J.; Chua, J. in "Synthetic Procedures in
Nucleic Acid Chemistry" (Vol. 1); Zorbach, W.W.; Tipson
R.S. (eds); Interscience, New York, p. 344.
1339483
17. Horwitz, J.; Chua, J.; Da Rooge, M.A.; Noel, M.;
Klundt, I. L. J. Org. Chem. 1966, 31, 205.
18. Fox, J.J.; Miller, N.C. J. Org. Chem., 1963, 28, 936.
19. Kowollik, G.; Gaertner, K.; Langen, P. Tetrahedron
Lett., 1969, No. 44, 3863.
20. Glinski, R.P.; Khan, M.S.; Kalamas, R.L.; Sporn, M.B.
J. Org. Chem., 1973, 38, 4299.
21. Davisson, V.J.; Davis, D.R.; Dixit, V.M.; Poulter,
C.D., J. Org. Chem., 1987, 52, 1794.
22. Griffin, B.E.; Jarman, J.; Reese, C.B.; Sulston, J.
Tetrahedron, 1967, 23, 230.
23. Ando, M.; Ohhara, H.; Takase, K. Chem. Lett., 1986,
879.
24. Jain, T.C.; Jenkins, I.D.; Russell, A.F.; Verheyden,
J.P.H.; Moffatt, J.H., J. Org. Chem., 1974, 39, 80.
Experimental
Melting points were determined on an Electrothermal
capillary apparatus and are uncorrected. TLC was performed
on silica gel 60 F-254 plates purchased from E. Merck and
Co., and column chromatography was performed on flash silica
gel (40 uM particle size, Baker), Elemental analysis were
performed by the analytical department, Bristol Myers,
Wallingford. lH and l3C NMR spectra were recorded on a AM360
BrukerNMR spectrometer using tetramethylsilane as the
internal standard; chemical shifts are recorded in parts per
million. Analytical HPLC was performed on a Waters C18
reverse phase column.
3',5'-Di-O-(methanesulfonyl)thymidine
A 3 L, 3 necked round-bottomed flask was equipped with
an overhead stirrer and paddle, a 500 mL dropping funnel and
a Claisen adapter containing a drying tube and a
thermometer. Thymidine (200g, 0.82M) and pyridine (750 mL)
were added to the flask. The mixture was stirred and warmed
with a water bath (20 mins) to give a clear solution. The
- 22 -
1339~83
solution was then cooled in an ice bath to 0-3~C and the
dropping funnel was charged with methanesulfonyl chloride
(206.5 g, 1.08 M). The methanesulfonyl chloride was then
added dropwise over 40 minutes with no noticeable exotherm.
The solution was stirred at 0~C for 1 hr and then stored at
5~C for 18 hours. The light brown mixture was then poured
onto rapidly stirred water (3L) containing ice (approx. 500
g). The desire product crystallised immediately. After
stirring for 0.5 hours, the product was collected by
filtration and washed several times with water (3 x 100mL).
The white solid was then dried under vacuum overnight (crude
weight, 322 g, 98~ yield). The product was recrystallised
from hot acetone to give 267 g of a white solid (81~ yield),
mp 169-171~C (lit. 170-171~C). lH NMR (360 MHz, d6-DMSO)
11.40 (s, lH, NH), 7.50 (s, lH, H-6), 6.21 (t, lH, H-1'),
5.29 (m, lH, H-3'), 4.45 (m, 2H, H-5'), 4.35 (m, lH, H-4'),
3.31 (s, 6H, SO2C_3), 2.50 (m, 2H, H-2'), 1.78 (s, 3H, CH3).
Analysis (C12H18N2OgS2) C, H, N.
1-(3,5-Anhydro-2-deoxy-~-D-threo-pentofuranosyl)thymine
3l,5l-Di-O-(methanesulfonyl)thymidine (248 g, 0.62 M)
was added in portions to a stirred solution of sodium
hydroxide (74.7 g, 1.87 M) in water (1.6 L). On addition the
reaction mixture became a yellow-orange solution. This
stirred solution was then heated to reflux for 2 hours. Once
the reaction mixture had cooled to room temperature, 6N
hydrochloric acid (lOO mL) was added. The reaction mixture
was concentrated in-vacuo by removing 1.3 L of water. The
resulting slurry was cooled in an ice bath for 2 hours. The
solid was then filtered and washed sparingly with ice water,
and then vacuum dried to constant weight (103.7 g, 74~). The
product 3, mp 188~-190~C (lit. 190-193~C) was used without
further purification. lH NMR (360 MHz, d6-DMSO) 11.35 (s, lH,
~Trademark
133~48~
N_), 8.01 (s, lH, H-6), 6.49 (q, lH, H-l'), 5.47 (m, lH,
H-3'), 4.88 and 4.67 (m, 2H, H-5'), 4.22 (d, lH, H-4'), 2.47
(m, 2H, H-2') 1.77 (S, 3H, CH3). I3C NMR (75 MHz, d6-DMSO)
163.64 (C2), 151.10 (C4), 136.57 (C6), 109.62 (C5), 88.29
(C4'), 86.85 (Cl'), 79.83 (C3'), 75.14 (C5'), 37.17 (C2'),
12.33 (CH3). Analysis (CloHl2N2O4) C, H~ N-
1-(2,3-Dideoxy-~-D-glycero-pent-2-enofuranosyl)thymine
To a 3-necked, 1 L round-bottomed flask equipped with a
mechanical stirrer, thermometer and nitrogen inlet was added
dry DMSO (400 mL) and oxetane (90.0 g, 0.402 M). To this
solution was added 97~ KOtBu (74 g, 0.643 M) in 1.5 g
portions over 25 minutes. The temperature was maintained
between 18~C and 22~C by means of an external ice bath.
After the addition was complete the reaction was stirred for
a further 1 hour and no further rise in temperature was
observed and TLC indicated that the reaction was
approximately 90~ complete. The reaction was stirred at 21~C
for 16 hours, after which time TLC indicated that the
reaction was complete. The viscous solution was poured onto
cold (4~C) toluene (3L), resulting in a beige colored
precipitate. The temperature of the mixture rose to 7~C upon
addition of the DMSO solution. The mixture was occasionally
swirled over 20 minutes, then filtered on a 18.5 cm Buchner
funnel. The collected yellowish solid was washed twice with
cold toluene and allowed to dry under suction for 1 hour.
The solid was dissolved in 300 mL of water, whereupon two
layers formed. The mixture was placed in a separatory funnel
and the upper layer (containing residual toluene) was
discarded. The aqueous layer was placed in a 1 L beaker
equipped with a pH probe, magnetic stirring bar and
thermometer. The temperature was cooled to 10~C by the use
of an external ice bath. Concentrated HCl was added dropwise
to the stirred solution at a rate in which the temperature
was kept below 15~C. After the addition of HCl (50.5 mL,
0.61 M) the pH = 7 + 0.1 and a precipitate began to form. To
- 24 -
1339483
this thick mixture was added potassium chloride (70 g) and
stirring was continued at 5~C for 1 hour. The precipitate
was collected and sucked dry for 2 hours, then air dried for
16 hours. The solid was crushed up and slurried in hot
acetone (500 mL) and filtered. The residue in the filter
paper was rinsed with hot acetone (2 x 200 mL), then
slurried again with hot acetone (300 mL), filtered, and
washed once more with hot acetone (2 x 100 mL). The combined
filtrate was concentrated to dryness to give 51.3 g (57~) of
d4T as an off-white solid, mp 165~-166~C.
[~]20D -46.1 (c0.7, water).
lH NMR (360 MHz, d6-DMSO) 11.29 (s, lH, NH), 7.63 (s,
lH, H-6), 6.80 (d, lH, J = 1.2 Hz, H-l'), 6.38 (d, lH, J =
5.9 Hz, H-3'), 5.90 (dd, lH, J = 1.1, 4.7 Hz, H-3'), 5.01
(m, lH, OH), 4.76 (s, lH, H-4'), 3.60 (dd, 2H, J = 4.8, 3.6
Hz, H-5'), 1.71 (d, 3H J = 1.2 Hz, CH3). 13C NMR (75 MHz,
d6-DMSO) 164.42 (C4), 151.30 (C2), 137.23 (C2'), 135.36
(C3'), 126.35 (C6), 109.33 (C5), 89.15 (Cl'), 87.56 (C4'),
62.41 (C5'), 12.15 (C5CH3). MS m/e (methane DCI) (relative
intensity) 225 (M+H, 20), 207 (15), 193 (8), 155 (13), 127
(100), 99 (20). IR (cm~l) 3463, 3159, 3033, 1691, 1469,
1116, 1093.
Anal. (CloHl2N2O4) C,H,N.
1-(2,3-Dideoxy-~-D-glycero-pent-2-enofuranosyl)thymine
Tetrabutyl ammonium fluoride (0.22 mL, 0.22 mM, 1.0 M)
was added to a suspension of the anhydronucleoside (25 mg,
O.ll mM) in dry THF (3 mL). After stirring at 22~C for 3
hours, the TLC showed only starting material. The mixture
was heated to reflux for 18 hours, at which time the
reaction appeared to be complete. After cooling, the
solvents were removed in-vacuo and the residue was dissolved
in CH2Cl2/MeOH/NH4OH (90:10:1). Purification was performed on
a 20 mm flash chromatography column, eluting with
CH2Cl2/MeOH/NH4OH (90:10:1). Concentration of the fractions
containing the product afforded 18 mg (72~) of d4T.
- 25 -
' 13~9 183
1-(5-O-Trityl-2,3-thiocarbonylribofuranosyl)uracil
5'-O-Trityluridine (10.6 gm., 22 mM) was added to a dry
250 mL round-bottomed flask under an argon atmosphere. Dry
tetrahydrofuran (110 mL) was added and the reaction mixture
stirred until it became homogeneous. When 1,1-thiocarbonyl-
diimidazole (4.3 gm., 27 mM) was added to the solution the
reaction became yellow and was then allowed to stir at room
temperature for 72 hours. The solvent was removed in-vacuo
and the resulting syrup flash chromatographed on silica with
ethyl acetate/hexane (75:25) as eluent. The produce was
isolated and then recrystallized from absolute ethanol to
give an off white powder (0.8 gm., 77~).
lH NMR (360 Mhz, CDC13) 8.9 (br s, lH, NH), 7.3 (m, 16H,
3xC6H5,H6), 5.7 (d, lH, H5), 5.6 (m, 2H, H2', H3'), 5.4 (m,
lH, H1'), 3.4 (q, 2H, H5').
5'0-Trityl-2',3'-dideoxy-2',3'-didehydrouridine
1-(5'-0-Trityl-2',3'-
thiocarbonylribofuranosyl)uracil(6.0 gm., 11.5mM.) was added
to triethyl phosphite (30 mL). The triethyl phosphite had
been preheated to 160~C. The reaction mixture was heated at
160~C for 1 hour. The solvent was then removed in-vacuo and
then the resultant glassy solid was flash chromatographed on
silica with ethyl acetate/hexane (75:25) as eluent. The
desired product was isolated from the column and then
recrystallized from ethyl acetate/hexane and then collected
as a white solid (2.0 gm. 40~). M.p. 188~-191~C.
lH NMR (360 Mhz, CDC13) 8.95 (br2, lH, NH), 8.00 (d, lH,
H6), 7.5 (m, 15H, 3xC6Hs), 7.2 (m, lH, H1') 6.7 (m, lH,
H2'), 6.05 (m, lH, H3'), 5.2 (dd, lH, H5), 5.10 (br s, lH,
H4'), 3.6 (m, 2H, H5').
1~3~83
2',3'-Dideoxy-2',3'-didehydrouridine (d4U)
5'0-trityl-2',3'-dideoxy-2',3'-didehydrouridine (0.5
gm., 1.1 mM) was dissolved in a chloroform (lO mL) and
methanol (2 mL) mixture containing 2~ p-toluenesulphonic
acid. The reaction mixture was stirred at room temperature
for 0.75 hours, and then neutralized with 2N NaOH (0.5 mL).
The solvent was removed in-vacuo and the residue
chromatographed on silica using chloroform/acetone (2:1) as
eluent. The desired product was isolated as a white
crystalline solid with the same physical and spectroscopic
characteristics as d4U produced by an alternative method.
M.p. 155~C.
1H NMR (360 Mhz, D2O/DMSO) 7.8 (d, lH, H6), 6.7 (m, lH,
H1'), 6.37 (m, lH, H2'), 5.8 (m, lH, H3'), 5.56 (d, lH, H5),
4.7 (m, lH, H4'), 3.6 (m, 2H, H5'). 13C NMR (70 Mhz,
D2O/DMSO) 163 (C4), 151 (C2), 141 (C2'), 135 (C3'), 126
(C6), 101 (C5), 89 (C1'), 87 (C4'), 62 (C5').
2',3'-Methoxymethylideneuridine
Uridine (50 gm., 0.205M) was added to a 1 L
round-bottomed flask under an nitrogen atmosphere. Dry
freshly distilled tetrahydrofuran (500 mL), pyridinium
p-toluene sulphonate (5 gm., 20 mM) were added to the
reaction mixture. Trimethyl orthoformate (109 gm., 1.03 M)
was then added slowly via an addition funnel. The reaction
mixture was left to stir for 18 hours. at ambient
temperature during which time the reaction became
homogeneous. Water (18 gm., lM) was added and the reaction
stirred for a further 0.5 hours. after which time pyridine
(20 mL) was added. The reaction was stirred at ambient
temperature for another 18 hours and the solvents then
removed in-vacuo. The resultant white solid was flash
chromatographed on silica to give the desired product as a
white solid (40 gm. 68 ~).
M.p. 188~-190~C. (lit. 189~-190~C).
- 27 -
5'-O-Acetyl-2',3-dideoxy-2',3'-didehydrouridl~n8e
The methoxymethylidene compound (11.8 gm., 41 mM) was
dissolved in acetic anhydride (110 mL) and p-toluene
sulphonate (20 mgs) added and the reaction heated to 140~C
for 6 hours. The reaction was allowed to cool and
triethylamine (1 mL) added. The solvents were removed in -
vacuo and the product was chromatographed on silica using
chloroform/acetone (4:1) as eluent to give the desired
product as a clear oil.
lH NMR (360 Mhz, DMSO) 11.3 (br s, 1 H, NH), 7.4 (d,
lH, H6), 6.8 (m, lH, Hl'), 6.4 (m, lH, H2'), 5.9 (m, lH,
H3'), 5.6 (d, lH, H5), 5.0 (m, lH, H4') 4.2 (m, 2H, H5'),
2.0 (s, 3H, CH3).
5'-O-Acetoxyisobutyrl-3'-O-acetyl-2'-bromo-2'-deoxyuridine
Uridine (5.0 g, 0.021M) was suspended in acetonitrile
(9O mL) and 2-acetoxyisobutyrylbromide (12.85 g, 0.063M)
added over 15 minutes and the reaction heated at 80~C for 3
hours. The homogeneous solution was cooled to room
temperature and the solvent removed in-vacuo. The resulting
syrup was dissolved in EtOAc (200 mL) and washed with NaHCO3
(3 x 100 mL). The organic layer was dried over MgSO4 and the
solvent removed in-vacuo. Chromatography on sio2
(75~EtOAc/25~Hex) gave 6.7 g of a white foam.
(67~) m.p. 68~-70~C (m.s. m + 477)
2',3'-dihydro-2',3'-dideoxyuridine (D4U)
The bromouridine (2 g, 4.2 mM) was dissolved in 3 ml
DMF and was added dropwise to a slurry of Zn/Cu (0.70 g,
10.5 mM.) in dry DMF (25 mL). The reaction was stirred for
2.5 hours at room temperature when no starting material was
observed by TLC. The reaction was filtered through celite
and the filtrate concentrated in-vacuo in a high-vacuum
system at 20~C. The resulting white solid (1.1 g, 85~) was
- 28 -
' 1339483
dissolved in MeOH and cooled to 0~C with an ice-water bath.
Anhydrous ammonia was bubbled in for 20 minutes and the
solution warmed to 60~C over 18 hours. TLC revealed a spot
corresponding to D4U. The solvents were removed and the
resulting a 0.5 gr. (55~) of the desired product.
m.p. 155~C (Lit:154~-155~C).
TABLE 1
Comparative in vitro anti-HIV efficacya and
cellular toxicityb of AZT and d4T
Compound ID50C(~M) TCID50d(~M)
AZT0 0.45 54.0
d4T 0.33 39.0
(Example p. 28)
a The antiviral test was performed on HIV (LAV
strain)-infected CEM cells.
b The cellular toxicity was measured in CEM cells.
c The 50~ inhibitory dose.
d The 50~ tissue culture inhibitory dose.
- 29 -
