Note: Descriptions are shown in the official language in which they were submitted.
W~9~/21~90 PCT/U~92/Q4795
` 20387~7 ::
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P~RINB BA3X ~ODIFI~D 2'-D~O~Y~IBONUC~EO~ID~8, ~;
~8B I~ TnIPLX~ F~R~X~ OLI~O~CL~OTID~8 ~i~
A~D YROC~8~ ~OR PR$RARI~ T~ ~AN~
FIELD OF THE INVENTION
-- .
This invention relates to the synthesis of ~ovel ~ ` -
purine base modified 2'-deoxyribonucleosides which are ! `` `
useful as constituents of oligonud eotides capable of
forming a triple helix with a double stranded DNA~
~ ~ BACKGROUND
Although the existence o~ triple helical structures ~ -
i5 well documented, triplex formation is a relatively new ~;
concept as the basis for a program of pharmaceutical
intervention. Recent work has made considerable progress
. .
towards the development of triplex forming
oligonucleotides (TFO) reagénts which bind to DNA in a
sitè selective manner. This work has demonstrated that
synthetic DNA oligonucleotides targeted to repeating A or
G segments within a DNA~gene can form stable triplexes at
acidic~ pH. The~formation of these triple helices is
;based upon hydrogen bonding of T to the AT~bases in a DNA
double helix or duplex~and a protonated cytosine (C~) to
a~G~ dupl~ex. The~requirement~or protonation of cytosine
while~conceptually important is not of physiological
25~ significance since the pH ~of the C+GC triplex is almost
3. Moreover, this triplex is not stable at physiological
pH. Attempts to solve this pro~lem have included
substituting cytosine with 5-methylcytosine and 5-
bromouridine. These substitutions increased the apparent
pH to allow triplex formation in the~pH range between 6
and 7, however, this range is still not physiologic.
Therefore, when considering the potential biological
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~V92/2~90 PCT/US92~795
2~78~ ;
-2-
utili~y of TFOs, it is rather important to ex~mi~e
nuc}eoside modifications or other nucleoside congeners
which would allow for sequence specific triplex ~ormation
at physiological pH (i.e., pH 7.4 to pH 7.8).
~uring the past several years the prospect for
oligonucleotide based therapy has been proposed: Initial
experiments were d~signed to determine the site
selectivity for triplex for~ation along a sequence of
DNA. The control region of the c-myc oncogene was used
as a model. In these early experiments it was shown
that, at physiological pH, a discrete 27 base long
oligonucleotide had the capacity to form a stable triple
helix with high sele~tiv~ty. The synth~tic
oligon~cleotide was designed to bind in the major groove
to the purine rich strand o~ the DNA c-myc oncogene
target and wa~ stabilized by T binding to AT; A binding
to AT; and G binding to GC. Subsequently it has been
shown that GGC and TAT triplets result in stabilized
triple helix formation in A or G rich sites within DNA.
Additional refinements in triple helix ~orming
oligonucleotide design has shown that significant
stability enhancement can be achieved at most DNA target
; sites by forming triple helix compounds so that they bind
antiparallel rather than parallel to the purine rich
strand of the DNA. Thus, triple helix forming ligands
~an be de~igned which bind to purine-rich gene promoter
seg~ents at physiQlogical pH, with dis~ociakion con~tants
in the 10 9 - 10 7 M range.
The goal o~ TFO design is to develop molecules which
can bind to any dupl~x DNA sequence, without regard for
purine content or other symmetry conaideration. H-
bonding of the Hoogsteen or reverse Hoogsteen type occurs
with~purine bases in the major groove of an underlying
duplex. Consequently, a polypurine/polypyrimidine duplex
~: .
'~
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,.,-- ..
wos2/2~o
2~8~787
.
_3_
target presents an orderly ar~ay of bond forMers
positioned upon one side of the major helix groove. At
sites of CG cr TA inversion, the corresponding purine
r target base is placed upon the opposing half of the major
groove and can be reached for the purposes of H-bonding
only (a) upon extension of the TF0 backbone, (b) by
distension of the duplex, or (c) both.
Indirect data suggests that dùplex DNA assumes the
A form upon triplex formation. In the A form, the major
lo groove is deep and:narrow, with dimensions well suited to
the size of a bound third strand. Thereforè, at ~ites o~
CG or TA inversion wi~hin a polypurine/polypyrimidine
domain, at most 3-5~ of tran~verse distortion is required
to acco~modate H-bonding at the "other side" of the major
groove. That mu¢h lateral distortlon can be partially
accommodated by the conformational freedom available to
the deoxyribose backbone or a duplex. Preliminary
modelling suggests that, in order to form a ctandard TAT
or GGC Hoogst~en or reverse Hoogsteen triplex at such
~o sites, distortion of the duplex binding site mi~ht also
be required.
Evidence suggests that at sites of CG or TA
inversion in an antiparallel triple helix, local triplet
for~ation gives risé to su~:stantial distortion of the
duplex. The ~present invention provides a group of
modi~ied nucleos:ide analogues.which form a ~trong triple
helix at the sites of ~A or CG inversion, without the
reguire~ent ~or binding site distortion.
Becauce o~ the biostability, tissue distribution,
cellular uptake and cost to manufacture, the exploration
and synthesis of novel nucleoside surrogates is extre~ely
important for the progression and eventual
commerriaIization of these novel compounds. These novel
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WO92/216~0 PCT/US92/04795
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2~8~78~ _4_
compounds should be very useful in treatment of many
disorders.
For example, since identification of a retrovirus
HIV-l as the etiological agent of AIDS, an intense effort
has been made to identify drugs for the treatment and
prevention of this debilitating, lethal disease.
Although a variety of purine and pyrimidine nucleoside
drugs have been shown to have in ~i~ an i-HIV activity,
only one, AZ~, has beco~e widely available for treatment
of AIDS and approved by the FDA for treatment of advanced
AIDS cases. While clinically useful in many settin~s,
AZT is associated with considerable toxicity, especially
in myelosuppression, leading to serious he~otological
side e~fects.. Additionally, AZT-resistant HIV strains
15 have been isolated from AIDS pat~nts. Because of the
toxicity of:currently used drugs, new drugs must be
discovered ~or chemical intervention of HIV infection.
A radical novel therapeutic approach, commonly referred
to "antisense" is now emerging which promises safe and
effective treatments for a broad range of viral diseases,
including HIV infection. This new approach entails the
use of polymeric agents designed to specifically bind and
inactivate selected viral genetic seguences, and thereby
halt the disea~Q process. The preliminary findings using
antisense treatments,:however, have been controversial.
The present invention, however, is based upon the premise
of a new method of trèatment; triple helix formation. In
the pre~ent invention, novel reagents are designed and ~ -
synthesized with the potential to sclectively inhibit HIV
3 0 by direct binding to proviral DNA rather than messenger
RNA, thereby forming a colinear triple helix. This
approach can also be used in treatment of a variety of
other disea~es and in modulations of genetic expressio~
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~YO92/21~gO PCT~US92/04795
2~d~5~7~
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S~MMUR!Y OF THE INVEN~ N
A object of the present invention is the provision
of novel analogues of guano ine. --~
A further object o~ the present invention is the
introduction o$ 2'-deoxyormycin A, thioguanosine and
other guanosine anaIogues into TFOs.
An additional object of the present invention is a
method for treat~ent of AIDS.
A further object of the present invention is to use
these modified TFOs for treatment o~ disease. ~`
An additional object of the prei~ent invention is ~`
modified TFOs for regulation of gene expression.
Thus, in accomp1ishing the foregoing objects, there
is provided in accordance with one aspect of the present
inve~tion a ~series of guanosine analogues, compounds
numbers 4, 10a, 10b, 14, 19 and 29 (Figures 5-9). In
addition, there are~also the specific compounds which
were intermediates~in the synthesis of these analogues.
In the preferred embodiment, the 2'-deoxyformycin A
and other guanosine analogues are incorporated into TFOs
for use in ~the treatment o~ a variety of diseases and in
; ~ regulation of proteins, hor~ones, and gene expression.
Other ànd~ further ~ objectives, features and
~advantages will be~apparent and `the invention more; ~ 25~ readily un~derstood from a reading of thè following
speci~ication~ and by~reference to the accompanying
drawings forming a part thereof, wherein the examples of ~`
the presently preferred embodiments o~ the inventian are
given for the~purpose of disclosure.
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WO 92/21690 PC~JUS92/~4795
~8~787 6~
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I:~ESC~IPTION OF THE DRAW~_S
Figure 1 is a model exp~aining triplet H-borlding at
sites o~ CG and TA inversion.
Figure 2 is a model showing the binding of formycin
as a G homologue at sites of CG inversion.
Figure 3 shows Cu-Phenanthroline hypersensitivity at
sites of CG:and TA inversion in anti-paraIlel triple
helices.
Figure 4 is the band shift analysis showing the
effect of 2--deoxyformycin (dF): substitution.
Figure 5 is a sohematic of the synthesis of 7-N-
benzoylamino-3- [ 3-O- ( ~-cyanoethyl ~ -N, N-
diisopropylaminc~phosphoramidite-5-0-dimethoxytrityl:-2-
deoxy-~-D-erythro-pentofuranosyl]pyrazolo[4,3-d]-
pyriDIidine.;~
Figure 6 is ~a~ schematic of the synthesis of 2
: isobutyrylamino-9-[3-0~ cyanoethyl ) -N, N-
diisopropylaminophosphoramidite-5-0-dimethoxytrityl-2-
:; deoxy-~-D-erytAro~-pentofuranosyl]purin-6(1H)-one and 2-
20 : ~is~obutyrylamino-~- ~ 3-0- ( ~-cyanoethyl ) -N, N-
:~ diisopropylaminophosphoramidite-5-Q-dimethoxyt~rityl-2-
deo~-~-D-erythro-pentofuranosyl]purine-6~ thione.
Figure~7 ~ lS ~a~ schematic ~of the synthesis of 2
:isobutyrylamino-6-isobutyrylthio-9-[3-Q-(,B-cyanoethyl)-
25~: ~N~,N-diisopropylaminophosphor midite-5-0-dimethoxytrityl-
2-deo~-,B~ erythro-pentofuranosyl~purine.
Figure a is a schematic of the synthesis of 2-
:~ isobutyrylamino-9- [ 3 -O- ( ~-cyan.oethyl ) -N, N-
diisopropylaminophcsphoramidite-5-Q-dimethoxytrityl-2-
deoxy~ er~thro-pento~uranosyl]purine-6(1~)-thione.
Figure 9 ~is a schematic of the synthe~is of 2-
isobut}~ amiIlo-Ns- isobutyryl -7 - [ 3 -Q- ( ~ -cyalloethyl ) -N ~ N~
~ ~ : dilsopropylaminophosphoramidite-~-O-dimethoxytrityI-2- ~ :~
: .. . .
: ~ : : : .: -
~092~2~690 ~CT/US92/0~795
208~787
; ': '' '
-7~
.
deoxy-~-D-e~ythro-pentofuranosyl]pyrrolo~3,2-d]pyrimidin- ~-
4(3H)-one. .-
Figure 10 is a schematic of the synthesis of 7-N- - .
benzoylamino-3- r 2-O-~ethyl-3-O(~-cyanoethyI)-N,N-diiso- .
propylaminophosphora~idite-5-O-dimethoxytrityl-~-D- ~-
ribofuranosyl]pyrazolo-[4,3~]pyrimidine. ' i:
The drawings and figures are not necessarily to
scale and certain features mentioned may be exaggerated
in scale or shown in schematic for~ in the:interest of
clarity and conciseness. .
DETAILE~ DESCRIP~ION OF ~E IN~ENTIQN
: It will be readily apparent to one skiIled in the ~:
art that various ~ubstitutions and ~odifications ~ay be
made to the invention~dis~lo~ed herein without departing
from the scope and the spirit of the invention.
. The t~r~ "TFO" or "triplex forming oligonucleotide"
as used herein refers to the oligonucleotides o~ the
present invention which are capable o~ forming a triplex
by binding in the major groove with a duplex DNA
s~ructur~.
As u~ed herein the term ';major ~roove" refers to one
of the grooves along the outer surface of the DNA helix -
:which is fo~ed because the sugar phosphate backbone of
the duplex :DN~ extends further from the axis than the
: bases:do. The major groove is important for binding of
regulator ~olecules to cpecific DNA se~uence~.
)The te~m "oligonucleotides" as used herein is
defined as a molecule comprising two or more .: .
deoxyribonucleotides or ribonucleotides, pre~erably more
than 10. The exact size would depend on many ~actors
including the ~peci~icity and binding affinity. . ;
: In referring to:"ba~es" herein the term includes
both deoxyribonucleic acid~ and ribonucleic acids. The ~ :.
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WO~2/21~ PCT/U~92/04795
~8787 ~
following abbreviations are used: "A" refers to adenine
as well as its deoxyribose derivativ~s, "T" refers to
thiamine as well as its deoxyribose derivatives, "G"
refers to guanine as well as its deoxyribose derivative,
S "C" refers to cytosine as well as its deoxyribose
derivative, "F" or "N" in sequences refers to guanosine
analog~es. Some examples of analogues are compounds 4,
lOa, lOb, 14, 19, 29 and 35 (Figures 5-10). These
compounds are call~d guanosine analogues because they can
. 10 he used to replae guanosine in an oligonucleotide and
will bind with C ~o form FCG Hoogsteen and reverse
Hoogsteen triplets.
One ~mbodimant of the presen invention are the
co~pounds of Figure 5 which can be represented by the -:~
structureo ~ .
Rl~ .R2 ' ~
N~ h
: ~3
, . ",-~-'.
wherein Rl is H or -COCs~5; R2 is H or -COC6H5; and ~3 is ~:
attached by a ~ linka~e and is selected from the group
con~i6ting o~ ~ o ~ and
Si~ yO ~ ,.,,:
.,, ,~.
wherein X is H or DMT and Y is H or
NC(CH2)20~ `N ~ : '
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W~92~ gO PCT/US92/~7~5
2 0 8 8 7 8 ~
_9_
These compounds of the present invention are
synthesized by the process depicted in the schematic
illustration represented in Figure 5. In the preferred
embodiment co~pound 4 is synthesized.
Another embodim nt of the present invention are the
compounds of Figure 6 which can be represented by the
structure:
1~ ,
iBuHN
, R2~ ¦
DMTO~
. .
wherein R1 is O or S; R2 is H or p ~
Nc(cH2)2o N.y , ;.
These co~pounds of the present invention are
synthesized; by the process depicted in the schematic :-
illustration in:Figur~ 6.
. . .
Another embodiment of the present invention are the
compounds of Figure 7 whic;1 can be represented by the `
structures: S-~BU S
Ng~cN~ rN~
iB,~N 1~N N H2N ~N ~N
Rll~l a~d TBDMS
\
1-- . 1-- ~ "':'.
R20 TBDMSO
:. ~
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W092/2t690 PCT/US92/~795
.
2~78~ -
--10--
.
wherein R1 is TBDMS, DMT or U and R2 is TBD~S, H or
NC(CH~)~O~ `Ny
These compounds o~ the present invention are
synthesized:with the process depicted in the schematic
illu-tration shown~in Figure 7. ~
Anothex: alternative embodiment of the present -
invention are~the compounds of Figure 8 which can be :.;~ :
represented by the structure;
S . , ' ,
HIN ~cN~
iBuHN "bN N "
,o ~
'
` ~ ~ : R10 .: ;:
. .
:
:: wherein R~ is H or I ~ and R2 is H ..
or DMT. NC(CH~20~ 'N~
These compounds of the present invention are
synthesized by~the~process: depicted in the schematic .:
illustration of Figure 8. ;,
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~2/2~90 PC~US92/0479~ ~-
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2~8g787
Another ~alternative embodIment of the present
invention are the compounds of Figure g which can be :i
represented by the structure~
HN~
iBuHN J~N
R2--¦~o~
~ \J : :
: ~ ~ : , I
OR3 ;;
: wherein R1 is H~or~(CH3)2CH-C0-; R~ is H or D~T; R3 is H or
NC(CH~)20~ `N~
5~ ~ Thess compounds of the present invention are
~ synthesized~by the~process depicted in the schematic
:~ : illu~tration of Figure 9. : i -~- Another~alter~ative embo~iment of the present
invention:are~co~pounds 23-26 (~igure 9). - ~:
~ A~ further~embodiment:~of~:~the pre~ent invention are : :~
;the compounds~ of:Figure 10 ~hich :can be represented by
~ the~strui~ture~
: . 1 11 "
N~
: ~ R~
~: : ; . ~,.: -
~ -
.
WO~2/2~gO PCT/US92/0~79~ :
2 ~ ~ ~ 7 ~
wherein R1 is attached by a p linkage and is selected
fro= the group consisting of~
>~s~X ~ ZO~
; wherein X is H or CH~; Y is H, o~ NC(CH2)20
and Z~is ~ or D~T. ,~
Thesa ;compounds of tha present invention are :
synthesized by ~hé process depicted in th schematic
illustration represented in Figure lO. In the preferred :
embodiment, compound:35 is synthesized.
In the preferred embodiment, there is a TFO ` .
~: ~ lO comprising an oligonucleotide capable of orming a triple~. `
:~ helix with a double-stranded DNA wherein at least one . ; :
base of said oligonucleotide is replaced with at least : :
one of the Compounds 4, lOa, lOb, 14, l9, 29 and 35 `~:
tFigures 5-lO).:~
15 ~ ~ Addltional embodlments o~ ~he present invention are :.-:`
he ~ ~ use ~ ~ of TFOs: containing compounds of :the present
: invention:for:the~inhibition of HIV-l, and for altering
: the~ :expressio~n~ ~of~ genes selected from the group
: consisting of epidermal growth factor receptor, mouse
insulin receptor; among others. : :
A further embodim2nt o~ the present invention i5 a :
TFO containing a guanosine analogue. Specific - ;
embodiments of these TFOs are selected from the group `;:
consisting:of:~SEQ. I.D. No~.:l, SEQ. I.D. No. 2, SEQ. I.D. ~.
: 25 No. 3, SEQ. and I.D. No. 4.~ :
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~2/21~0 PCT/VS92/04795
^.`,.;~. 2D~787
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Another embodiment of the present invention is a ~Fo
for treating AIDS co~prising SEQ. I.D. No. 4.
Another e~bodiment of the present invention is a
triplex forming oligonucleotide for for~ing a colinear
triplex with a target sequence in a duplex DNA. The TFo
is comprisad of a nu~i}eotide sequence o~ at least about
20 nucleotides. The sequence includes G, T and guanosine
analogues. In the TF0, ~ is used when the complementary
location in the duplex DNA target possesses a G in the
lo orienting (~ore purine rich) strand. Guanosine analogues
are used instead of G when the duplex DNA target
possesses a C in the orienting strand. T is used when
: the co~plementary location in ~ha duplex DNA target is an
AT base base. In general the TF0 can be designed so as
~o ~or~ the~ above ba~ed triplets wi~h either a net
parallel or antiparallel TF0 strand orientation with
reæpect to the orienting strand of the duplex DNA target.
However, the antiparallel TPO isomer is often favored for
targets with a:GC to AT base pair ratio substantially
greater tha~ one while the parallel isomer is often
favored for targets with a GC to AT base pair ratio
substantially less th:an one. Both TF0 iso~ers can be
equally favored~for targets with a GC to AT base pair
ratio:near to one.
In a preferr~:embodi~ent of the present invention
~her~ i~ a ~arget~sequ~nce in a duplex ~NA co~prising a
nuclaotide sequence o~ at least about 20 nucleotides;
said nucleotide sequence including G, T, and guanosine
analogue, wh~rein G is u4ed when the complementary
location in the duplex DNA i9 a GC base pair, ~ i~ used
when the complementary location in the duplex DNA is an
AT base pair and guanosine analogue is used when there is
a CG inversion within the duplex DNA target site; said
sequence binding anti-parallel to the purine strand in
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WO 92/216gO Pcr/uss2Jo4?ss
2088787 ~
-14-
the duplex DN~ target ~it~ and said binding forming TAT,
GGC and FCG Hoogsteen or reverse Hoogsteen triplets.
The following examples are offered by way of
illustration and are not intended to li~it the inYention
in any manner. In examples, all percentages are by
weight, if ~or solids and by volumes, if for liquids and
- all temperatures are in degrees Celsius unless ~therwise
notedO
Example 1
~ ~ ;" ~,
phosphor~midi~e derivative.
A synthetic procedure to convert formycin to the
corresponding 2'-deoxyribonucleoside (dF), ~ollowed by
activation to the ~corresponding ~locked DMT-
phosphoramidite was developed.
Using commercially available formycin A(1), 2'-
deoxyformycin A can be prepared by a variety of known
procedures. One method of synthesis of 2'-deoxyformycin
~ A is ~escribed by T. C. Jain, et al., J. org. Chem.
38:3179 (1973~.~ This procedure involves reaction of 1
with acetoxyisobutyryl bromide, followed by ammonolysis
and reductive d-halogenation. This approach produces a
mixture of 2'-deoxyformycin A and the 3'-deoxy isomer,
; with the latter ~predominating ~y a ratio of more than
2:1. An alternative procedure is described by Rosowsky,
et ;al., J. ~N~d. Cham. 28:1096 (1985). This is a
deoxygenation procedure and employs
phenoxythiocarbonylation o~ the 2'-hydroxyl group of the
corresponding 3',5'-protected ~. 3',5'-Protection of
was accomplished by using 1,3-dichloro-1,1,3,3-
tetraisopropyldisilQxane, which in the presence of a
proton acceptor gave the cyclic disilox~ derïvative.
Acylation of the 2'-hydroxyl of the disiloxy derivative
.
. :, .
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~Y~ 92~2~690 PCI/llS92/04795
,~, 2~787.
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--15-- ;
with phenoxythiocarbonyl chloride, followed by reductive
cleavage of the phenoxythiocarbonyl group with tri-n- ;
butyltin hydride in the presence o~
azobisisobutyronitrile gave the viable intermediate 7-
a~ino-3 t 3, 5-O- ( 1, 1, 3, 3-tetraisopropyl-1, 3-
disiloxanediyl-2-d~oxy-B-Dori~ofuranosyl)]pyrazolo ~4,3-
d]pyrimidine (2).
.
Example 2
r ~1~,~5~u,sopropyl-1,3-~lsil~2~ne~iyl-2-deoxy-~-D-
(3)
To a cooled~ ~0-5C) solution o~E 1.5 g of colapo~Lnd
(2) (2.92 ~moles) in 25 ~l anhydrous pyridine was added
1.36 ml benzoyl chloride (11.7 mmol~s~ with stirring.
After stirring the reaction mixture at room temperature
for 2.5 h, it was partitioned between water and
dichloromethane. The orqanic layer ~as separated, dri~d
over anhydrous Na2SO6,and then evaporated to dryness under
reduced pressure. The residue was azeotroped with ~`
toluene ~4 x 50 ml) to r~DIove last traces of pyridin2,
and purified~by chromatoqraphy over a silica gel column ~ :
using a gradisnt ~of ~I2Cl2-- MeOH ~0~1%) as the eluent.
The homogeneous fractions were collected and evaporated
under reduced pr~ssure to yield 1.45 g (70.7%) of the
~itle compound; mp 74-75C. IR (~Br): t)max 1720 (C50) ~ , '
3240 (NH) c:m l; W (MeOH): Amax 230 nm (~24,000), 318 nm
(~14,500); 1H NMR (DMSO-d6): ~ 0.98 {br s, 28 H,
2[(CHs)2CH]2Si}, 2.40-2.90 (m, 2 H, Cc,~ and C2,,~), 3.7-4.0
(m, 3 H, C4,~ and C5,C~2), 4.90 (q, 1 H, C~,~), 5.50 tdd,
1 H, Cl,~), 7.30-8.15 (m, 10 H, 2 COC6~s), 9.07 (s, 1 H,
Cs~) and 12.0 tbr s, 1 H, N~). ~L- Calcd. for
- C3~s~47N5o6si2: C, 6I.59; H, 6~75; N, 9.97. ~ound: C,
61.68; H, 6.81: N, 9.86. ~ `
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~0~2/216gO PCT/~S92/04795
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53g787 ~ . ' :.
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Example 3
Synthesis of 7-N-Benzoylamino-3-(2 deoxy-B-D-
erythro-~entofuranosyl)~y~a~olor4,3.-dlpyri~idine ~6).
To a solution Of (3) (1.35 g, 1.96 mmoles) in
anhydrous tetrahydrofuran (30 ml) was added tetra-n-
butylammoniu~ ~luoride (8 ml, 1~ solution in THF) at O~C.
After stirring the reaction mixture at room temperature
for 2 h, the solvent was evaporated under reduced
pressure and the residue was purified by chromatography
over a silica gel column using a gradient of CX2C12- MeOH
(0~4%) as the eluent. The homogeneous frac ions were
collected and evaporated under reduced pressure to yield
0.46 g t68~j of:analytically pure (); mp 250-251C. IR
(KBr): ~max l6g0 (C=O), 3100-3500 (OH, NH) cm1; W(MeOHj:
lmax 248 nm ~El4,500), 320 nm (~13,700); 9H NMR (D~SO-d6):
: ~ 2.00-2.20; (m, l ~, C2,~), 2.60-Z.~0 (m, l H, C2,~),
3.40~3.70 (m, 2 H,: C5,C~2), 3.90 (m, l H, C4,~), 4.40 (d,
l H, ~,~), 5.15 (br s, 2 H, 3' and 5' 0~), 5.50 (dd, l
H,:C1,_), 7.50-~.15 (m, 5 H, COC~), 8.75 (s, 1 H, C~
ll.80 (br s,~ 1 H, N1~) and 13.00 (br s, 1 H, N7~). Anal.
:. Calcd. for C1rH1~N504 ~ 1j4 H2O : C, 56.74; H, 4.90; N,
19.46. Found: ~C, 56.93; H, 4.96; N, 19.35.
~xample 4
nthesis of 7-N-~enzoyla~ino-3-(5-0-
25 : dim~thoxyt~i~yl ~ ~ nosyl)-
: ~ DvrazQlor~ d]p~ s)-
To a solution~of ~6) (0.4 g, 1.12 mmoles) in
anhydrous pyridine (10 ml) was added 4,4'-dimethoxytrityl
chloride ~0.46 g, 1.35 mmoles) at room temperature with
30 stirring. After stirring for 3 h, ~he reaction mixture
was partitioned between C~2Cl2 and wa~er (50 ~l of each).
The organic layer was separated, dried over anhydrous
Na2SO4and evaporated under reduced pres ure. The residue
was azaotroped with toluene ~4 x 50 ml) to remove last
.
~;,.,,;.
.:
-~
' ' ' '; ' ' ' ' ' ' ' " ' " ' ' : ' ~ , ! . .
W092/~1690 ~T~US92/047g5
208~ 78 7 ~
~17 ::
traces of pyridine, and puri~ied by chromatography over
a silica gel column using a gradient of CH2Cl2-MeOH (0-2%)
as the eluent. The homogeneous fractions were collected
and evaporated under reduced pressure to yield 0.26 g
(35.3%) of pure titl~ co~pound; mp 107-10~C... IR (KBr)
: umax 1690 (C=03, 3200-3400 (OH, NH~ cm~; W (MeOH): :
Amax 238 nm (~31,700), 320 nm (el8,000); lH NMR.tDMSO-d6~: .
2.05-2.30 and 2.:75-2.95 (2m, 2 H, C2,~ and C2,~), 3.70
(s, 3 H, OCM3), 3.71 (S, 3 H, OCH3), 3.98 (m, 2 H, C5,CH2), : ~:~
4.40 (m, 1 H, Cb,H), 5.20 (m, 1 H, C3,~), 5.S5 (dd, 1 H,
Cl,~), 6.70-8.30 (m, 18 H, COC~ and DMT), 8.65 (5, 1 H, .
C~, 11.85~br s, 1 H, Nl~) and 13.10 (br s, 1 H, N7H). :
. . '. '
Example 5 .:.
SYnthesi$ o~ 7-N-Be~oy~mi ~ .r ."~ "
N,N-diis~opropylami~oDhos~horami~e-5-0-dimetho~ytrityl-
2-deoxy~ eryt~*o-~çnt~ nosyll~y~aZolo~4 t 3-
d]pyrimidine (4).
To a solution o~ (5) (0.16 g, 0.2 m~oles) and ~.
N,N-diisopropylethylamine (0.14 ml, 0.83 m~oles) in
anhydrous di~hloromethane (3 ~1) w~s added with stirring
2-cyanoethyl-N,N-diisopropylchlorophosphora~idite (56
0.25 mmoles) undar an argon atmosphere. After 30 ~in an
additional 45~1 of the phosphorylating agent was added.
~fter stirring~fo~ an additiQnal 15 min, the reaction
mixture wa~ dilute~ with 10% NEt3 in EtOAc ~olution (50
: ml) and wa~hed with aqueous saturated NaHCO3 solution (10 ; i
ml). The aqueous layer was separated and reextracted
with 10% NEt3-EtOAc. The combined organic layers were
dried over anhydrous Na2SO4 and evaporated to dryness
under reduced pressure. A solution of the residue in 10%
NEt3-EtOAc was passed through a silica gel column packed : :
in c~2cl2:EtaAc:NE~ (45:45:10) eluting with the same .
solvent syseem. The ppropriate, ho~ogeneous fractions
. ~:
:
~092/~l6~ PCT/US~2tO4795 ~ :.
. ' ~ ~ ~ .
2 ~ 8 87 g7 ~
-18-
were collected and evaporated under reduced pressure.
The resid~e was dissolved in a small amount of CH2Cl2 (~
ml) and the product was precipitated by the addition to
dry hexanes to yield 0.18 g 186.2~). lH NMR (CDC13):
1.20 ~m, ~2 H, N(C~2)z], 2.40-2.70 (m, 2 ~, C2,H and
C2"R), 2.80-4.0 (~, 6 H, OCH2C~2CN, Cs,CH2), 3.75 (s, 6 H,
2 OC~3), 4.35 (m, 1 H, C4, ), 4.75 (m, 1 H, C3,~), s.75 (q,
1 H, C1,~), 6.50-8.20 (m, 18 H, COCO~ and D~T), 8.64 a~d
8.65 (2 s, 1 H, CsH of the iso~rs), 12.20 (br s, 1 H,
N7H); P N~R (CDCl3 ): ~ 148.36. A~l. Calcd. for
C47H53N~7P. 1/2H20: C, 65.11; H, 6.28; N, 11.31; P, 3.57.
Found: C, 65.16, H, 6.32; N, IO.87; P, 3.70.
,
Example 6
A New Sy~thesis_o~ 2-Ami~-9-~2-deoxy-~-D-erythro-
Pe-ntouranosyl~purin-6(lHL-o~e (8).
To a suspension of 2-amino-9-(2-deoxy-~-D-erythro-
pentofuranosyl)purine-6(1H)-thione (7) ER. H. Iwamoto, E.
M. Acton, and L~. Good~an, J. Ned. ~hQm., 6, 684 (1963)]
(2.0 g, 7.06 mmoles) in water (60 ml) were added NH40H
(30%, 20 ml), followed by H202 (30%, 2.8 ~1). After
sti.rring at room temperature ~or 45 min, the reaction
mixture was concentrated to half the original volume and
~ allowed to ~tand at 25C for an hour. The product that
: 25 cry~tallized ou~:from the aqueous solu~ion was collected
by ~iltration and :the filtrate was neutralized with
Dow~x-50 (H) resin. The resin was removed by
filtration, washed thoroughly with water ~5 x 25 ml) and
the combined filtrates were evaporated under reduced
pressure to give a colorless ~olid. The solid was
combined with the one obtained as above and crystallized
~ro~ water to yield 1.62 g (86%) of the title compound,
mp > 295C [Lit. mp:> 300C, M. J. Robins and R. K.
Robins, J. org. Chem., 34, 2160 (1969)]. IR (X~r): ~max
.....
' ':; ' '
` ~
~ ': , ' , :'
- W~2/21~90 P~T/US92~79~
~ 2~88787
--19-- . . .
1740 (C=o), 3150, 3310, 3440 (OH, NH, NH2) cm1; W: Amax
(pH1) 254.5 nm (~10,800); Amax (pH 7) 254 nm (613,000);
lmax (pH 11) 263 nm (~10,000); H NMR (DMSO-d6) : ~ 2.20
(m, 1 H, C2,H) 2070 (m, 1 H, C2~), 3.45 (t, 2 H, Cs,CH2),
4.05 (m, 1 H, C4~, 4.30 (m, 1 H, C3,H), 4.80 (t, 1 H,
CslO~, 5.50 (d, 1 H, ~,OH), 6.10 (dd, 1 H, J = 3.0 ~x,
Cl,H), 6.50 (s, 2 H, N~2), 8.0 (s, 1 H, C~), 10.64 (s, 1
~, Nl~). Anal. Calcd. for C1~H13NsO4.1~3 H20 : C, 43.95; H,
5.04; N, 25.63. Found: C, 43.g7; H, 4.97; N, 25.58.
1 0 ~
Example 7
Svnthesis of 2-1sobutyrylamino-g-~5-o-
.Durin-6t~ Q~e (9a).
To a suspension of 8 (1.8 g, 6.73 mmoles~ in
pyridine (30 ml) was added trimethylsilyl chloride (7.61
ml, 60 mmole~) at O C. Aftex stirring the reaction
mixture for 2 h at room temperature, the reaction flask
was cooled in an ice bath and isobutyryl chloride (0.85
ml, 8.07 ~moles) was added. The reaction was allowed to
continue at room:temperature for an additional 4 h, after
which water ~lO ~ was addedl followed by CH2Clz (200
ml):. The organic:~layer was separated~and evaporated
; under reduced~pressure at 40~C. The resulting residue
2:5 ~wa :~az~otroped with ~oluenz (5 x 25 ml) to remove the
last ~race~ of pyridine, and puriSied by chromatography
over a ~ilica gel column using MeOH:CH2Cl2 (1:3, v/v) as
the eluent. ~ha ho~ogeneous fractions were pooled and
evaporated to yield 1.75 g (77%) o~ pure
N2-isobutyryl-2'-daoxy-~-guanosine.
A solution of the above N2-isobutyryl-2'-deoxy-
~-guanosine (1.5 g, 4.45 ~mole~) and dimethoxytrityl
chloride (l.81 g, 5.34 m~oles) in pyridine (20 ~1) was
stirred at room temperature for 2.5 h. The reaction
,~
W092/21690 PCT/US92/M79~ .
2~8~7~7 ~
-20-
mixture was diluted with cH~c12 t200 ml) and the organic
layer was washed with saturated aqueous sodium
bicarbonate solution (2 x 50 ml). The dried (over Na2SO4)
organic layer was evaporated under reduced pressure and
the residue was purified on a silica gel column using a
gradient of CH2Cl2- MeOH (0-4~) as the eluent. The
homogeneous fractions were collected and evaporated under
reduced pressure to yield 2.01 g (70.5%) of the pure
title compound; mp 152-154C (dec.). IR (KBr): ~max 1710
(C=O), 3100-3400 (OH, NH) c~ ; W (~eOH): lmax 238 nm
(~26,565), 262 nm (6}4,600): H NMR (DMSO d6) : 8 1.12 [d,
6 H, C(C~3)2], 2.30 - 2.45 (m, 1 H, c 2,H), 2.80 (m, 1 H,
C2~), 3.0-3.25 (m, 2 E, Cs~C~z); 3.7S (s, 6 H, 2 OCH3),
4.31 (br s, 2 H, C3,H and C4,0, 5.54 (d, 1 H, ~3,0~), 6.27
(dd, 1 H, J = 4.0 Hz, Cl,~), 6.89 7.41 (m, 13 H, DMT),
8.28 (s, 1 H, C~), 11.67 (br s, 1 H, N1~), 12.10 (br s,
1 H, N~). Anal~ Calcd. for C3sH3~50~ : C, 65.71; H, 5.83;
N, 10.95. Found: C, 65.32; H, 5.94; N, 10.61.
'' .'. `'
Exa~ple 8
SYnthesis, ~
cy3~oethylL~ N-~iisop~o~yl.aminQphosp~o~midi~e-5-o~
dim~thoxyt~ityl~2-deoxy-Q-.~-e~ythro- .nto~u~osyll-
,~u~in-611H)-one (LOa).
25. To a solution of ~ (0.75 5, 1.17 mmoles) and
N,N-diisopropylethylamine (0.82 ml, 4.69 mmoles) in
anhydrous dichloromethane (4 ml) was added 2-cyanoethyl-
N,N-diisopropylaminochlorophosphine (0.39 ml, 1.76
mmoles) under an argon atmosphere. A~ter stirring for 30
min at room temperature, the reaction mixture was diluted
with 10% NEt3 in EtOAc solution (75 ml). The organic
pha~e was washed with ~aturated aqueous sodium
bicarbonate solution (20 ml) and dried over anhydrous
sodium sulfate. Evaporation of the sol~nt under xeduced
' ''': '
.' ' '
-" "
~092/2~690 ~T/US92/04795
8~787
.:; , ,:.":
-21- -~
pressure and purification of the residue on a silica gel
column using EtOAc:CH2Cl2:NEt3 t45:45:10) solution as the
eluent gave 0.71g (7~.1%) of analytically pure produ~t.
31P NMR tCDCl~ 149.20 and 150.28; H N~ (CDCl3): ~ 1.0
- 1.40 [m, 21 H, N(CHC 3)2 and COCH(CH3)2], 3.80 (s, 6 H, :
2 OC~3), 6.30 ~m, 1 H, C~,H), 8.12, 8~16 (2s, C~ of the
isomers). Anal. Calcd. for C44Hs4N708P. H20 : C, 61.60; H,
6.58; N, 11.43; P, 3.61. Found: C, 61.26; H, 6.69; N,
11.47; P, 3.76.
: ' ~:'
: Example 9
Syn~h~sis o~ 2-Isobutyryl~lao-9-!5-Q.-
nu~:ine~6L~ thione (9b). ~ ~
: To a suspension of 7 (4~.0 g, 14.12 ~mol~s) in ~ . -
pyridine (40 ~1) was added trimethylsilyl chloride t17.1
~1, 140 m~oles) at OC! A~ter stirring the reaction
mixture for 2 h at room te~perature, the reaction flask
was cooled in an ice bath and isobutyryl chloride (1.7~ ~
ml, 16.95 mmoles) was added. The reaction was allowed to ~ -
continue at~room temperature for an additional 3 h, after
which water (40 ~1) was added, foll:owed by
dichloromethane; t200 ml). The oxganic phase was
separated, dried over Na2S04 and evaporated under reduced ;~
pressure at 40C.:The~resulting r~sidue was redissolved
: in methanol :(50~ml)~:and evaporated to dryness under
reduced pressure. The r~sidue wa3 dissolved in C~2C12,
filtered and the filtrate was used directly in the next
step without ~urther purification.
Evaporation of the solvant gave a residue, which was
dried by co-evaporation with anhydrous pyridine (2 x 20
ml), redi~sol~ed the resulting residue in pyridine (40
~1) to which dimethoxytrityl chloride (4.88 g, 14 ~ol~s)
was added. After stlrring the reaction nixtur~ at roo~
.
WO92J21~90 PCT/US92~04795 :
20887~7 ~ ~
:
-22-
temperature for 3.5 h, the mixture was diluted with water
(50 ml). The aqueous mixture was extracted wi~h CH2Cl2(3
x 100 ml) and the combined organic phase was dried over
anhydrous Na2SO~. Evaporation of the solv~nt at reduced
pressure gave a residue, which was p~rified on a silica
gel column using a gradient of CH2Cl2- MeOH (0 - 5~) as
the eluent. The homogeneous fractions were collected and
evaporated under reduced pressure to yield 6.02 g (65%,
for 2 steps) of pure title compound; mp 139~ C (dec.).
IR (KBr): umax 1690 (C=o), 3100-3400 (OH, NH) ~m ; W
(MeON): A~ax 234 nm (~22,300)~ 332 nm (620,300); lH NMR
(DMSO-d~ 1.13 [d, 6 H, C(C~3)2], 2.30 - 2.45 (m, l H,
C2~), 2.80 (~, 2 H, C2b~ and CO-C~), 3.0 - 3.30 (m, 2 H,
C5,~2), 3.74 (s,:6 H, 2 OC~3), 4.33 (br s, 2 H, C3,_ and
C~,H), 5.53 (d,~l H, C~,O~), 6.29 ~dd, 1 H, J = 3.8 Hz,
Cl,~), 6.89-7.37 (m, 13 H, DMT), ~.42 (s, 1 H, C~), 12.14
(s, 1 ~, N1H), 12,64 (br s, 1 H, N~ a31- Calcd. for
C3sH3~N5O6S. 1l3 CH3QH : C, 63.68; H, 5.79; N, 10.51, S,
~ 4.81. Found: ~ C, 63.34; ~, 5.59; N, 10.36; S, 4.64
: 20 : Example 10
2YnthÇ~L~L~_2-Isobu~yrylJEi~o-9- r 3-o-!B-
cvanoethyI)-~N,N-diisoDropvlamlno~hosphora~idi~e-5-O- :
thox~trityl-2-deoxy-~-D-ery~hro-pento~furanosvll-
; : Pu~ine-5f1H~-thione~ 0b).
2 ~ To a solution~ of 9b (0.65 g, 1 mmolq) and
: ~ : N,N-dii~opropylethylamine (0.7 ml, 4 ~moles) in anhydrous
dichloromethane (8 ml~) was added 2-cyanoethyl-
N,N-diisopropylaminochlorophosphine (O.29 ml, 1.3 m~oles)
under an argon atmosphere. After stirring for 20 min at
. 30 room temperature an additional 0.29 ml of phosphorylating
agent was added, stirred for further 30 min and the
reaction~ wa terminated by ~he addition of anhydrous
methanol (20 ~il3. After stirring for 5 min, the xeaction
-
,
,
W0~2/21~90 PCT/U~92/0479~
~s 2V~7~7
23-
~ixture was partitioned betw~en 10% NEt3 in EtOAc and
satur~ted aqueous NaHC03 solution (50 ml). The organic
phase was separated, dried over anhydrous Na2SO4 and
evaporated to dryness under reduced preSsure. The
resulting residue was purlfied on a silica gel column
using EtOAc:CH2C12:NEt3 ~45:45:10) solution as t~ eluent
to give 0.678 g (79.5%) of the title compound as
colorless amorphous sslid. P NMR ~CDC13): ~ 149.15 and
150.6; 1H NMR (CDC13): ~ 0.9 - 1.4 [m, 12 H, N(CHCH3)2 and
COC~(C~3)2], 3.80 (s, 6 H, 20C~3), 6.25 (m, 1 H, C1,~
8.23 and 8.26 (2s, C~ of ~he isomer~ . Calcd. for
C~4N707PS : C, 61.74; H, 6.36; N, 11.45; P, 3.62.
Found: C, 61.50; H, 6.59; N, L1.12; P, 3.710
Example 11 ~.
Sy~.thesis_Q~ 3'.5'-0~- ~ methylsilyl-2'-
deo~y~6-thiog~anosi~e (~,).
To a suspen~ion of 2'-deoxy 6-thioguanosine (11) ~R.
H. Iwa~oto, E. M. Ac.ton and L. Goodman, J. Med. Chem., 6,
684 (1963)] (1.8 - g, 6.3 mmoles) in anhydrous
N,N-dimethyl-fo~mamide (25 ml) was added
t-butyldime~hylsilyl chloride (3.35 g, 22 m~oles),
followed by dry:imidazole :(2.98 g, 41.9 ~moles). The
ra?ction mixture was protect~d from moi~ture and stirred
Z5 at room temperature for 24 h. The sQlvent was evaporated
under reduced~ press~re at 40C to give a white solid.
The solid was purified by chr~matography over a silica
gel column using a gradient o~ CH2Cl2~ ~eOH (O ~ 5~) as
the eluent. The appropriate homog~neous fractions were
pooled and evaporated under reduced pressure to yield 2.4
g (73.6%) of pure title compound; mp ~ 300C (dec). IR
(KBr): vmax 1257 (C=S), 3100-3400 (NH, NH2) cm1; W
~MeOH) : Amax 226 nm (e5,500), 270 nm (~3,500), 344 nm
(11,800); H NMR (D~SO-d6) : ~ 0.07 [d, 6 H,
... , . ,- :. ~
W092/216~ PCT/US92/04795
2088787
-24-
t-Bu-Si(C~)2], 0.88 (d, 18 H, t-Bu-5iMe2), 2.20-2.73 (m,
2 H, C2,H ~nd C2"H), 3.60 - 3.85 (m, 3 H, C~,H and C5,CH2),
4.48 (d, 1 H, C3,~), 6.10 (t, 1 H, Cl,H), 6.83 (s, 2 H,
N~2), 8.0 (s, 1 H, C~) and 11.97 (s, 1 H, Nl~)- Anal.
Calcd- for C2~H41N5O35Si2: C, 51.61; H, 8.07; N, 13.68; S,
6.2~; Si, 10.97. Found: C, 51.36; H, 8.05; N, 13.65; S,
6.27; Si, 10.99.
~ . .
Example 12
Synthesis_of 2-Isobu~5y~yL2~1no-6-isobutyrylthio-9-
~S9~9~C~ (13).
To an ice-cold solution 0~ 1~ (2.2 g, 4.2 ~moles) in
anhydrous pyridine (100 ~1) was added isobutyryl chlori~e
(1.37 ml, 12,8 mmoles). The reaction mixture was stirred
at room temperature for 3 h be~ore it was poured into
: saturated aqueous sodium bicarbonate solution (300 ml)
containing crushed ic~ (100 g) and ethyl acetate (400
ml). The aqueous mixture was stirred for 15 min. The
organic phase was separated, washed with water (2 x 50
~: ml-) and dried over anhydrous sodiu~ sulfate. Evaporation
of the solvent under~reduced pressure gav~ a residue,
:which~ was co-evaporated with toluene (3 x 20 ml) to
remove:last traces:of pyridine. The residual pale yellow
~: : 25 oil was puriP:ied by~chromato~raphy over silica gel using
~: ~ 2% ~eOH in CH2C~2 as the eluent to giva 2.1 g (75%) of
pure title compound; mp 85C. IR (K~r) : ~ma~ 1690
(C=O), 3100-3400 (NH)cm1; W (~eOH): Amax 226 nm
(~9,700), 332 nm (~11,500); lH NMR (DMSO-d~ 0.08 ~d,
6 H, t-Bu-Si(C~3)2], 0.88 (d, 18 H, ~-Bu-SiMe2), 1.14 ~d,
12 H, 2CO-CHtC~)2], 2.28 - 2.89 (m, 3 H, C2,~, C2~; and
C~Me2), 3.61 - 3.89 (mj 3 H, C4,~ and C5,CH2), 4.52 (s, 1
H, C3,~), 6.20 ~t, 1 H, C1,~), 8.38 ts, 1 H, C~O and
11.93 (br s, 1 H, NH).
;: -
~092/21~0 P~T/US92/04795
, ~,
2~887~7
-25- :~
- .; '
Example 13
Synthesis of ~-Isobutyry-Lamino-6-i-s-obuty~ t-hi
s-f2-deoxy-~ D-e~x~ro-pento~uranosyl)pyri~e (16~-
To a solution of 13 (2.1 g, 3.6 Dmoles) in anhydrous
tetrahydroSuran (2g ~1) was added tetra-n-butylammonium
fluoride (17.5 ml, 17.5 ~moles, lM solution in THF) at
ooc. A~ter stirring at room temperature for 3 h, the
solvent was evaporated under reduced pressure and the ~ .
residual brown colored solid was puri~ied by
chromatography over a silica gel column using a gradient
of CH2Cl2- MeOH ~0-10%) as the eluent. The ho~ogeneous ~:~
frac~ions were collected and Qvaporated under reduced
pressure to give 1.15 g (84.2%) of the title compound; mp
~ 3000C. IR (XBr): ~max 1700 (C-O), 3100-3600 (NH, OH)
cm-1; W (MeO~): lmax 232 nm (e8,600), 246 nm (~11,500),
294 nm (~7,400); lH N~R (DMSO-d6): ~ 1.12 ~d, 12 H,
2COCH(CH3)2], 2.28 -:2.96 (m, 3 H, C2,H, C2~ and C~Me2),
3.53 - 3.86 (~, 3 H, C4,H and Cs,C~2)-, 4.43-(s, 1 H, C3,~),
4.94 (t, 1 H, c5,o~j ~ 5.36 (d, 1 H, C3,0H), 6.35 (t, 1 H,
C1,~3, 8~.61 (s, l H, C~ and 10.63 (s, 1 H, N~
: Example 14
. .
Synthgsis of 2-Isobutyrylamlno-6-isobutyry~thio=s-
f5-o-dime~hPxytrityl-2-deoxy-B-~-erythro-pento~uran
~urin~ (~0 -
Compound 1~ (1.0 g, 2.36 mmoles) was dried by
repeated co-evaporation with anhydrous pyridine (S x 20
ml), and redissoIved in dry pyridine (10 ml), to which
was added 4,4'-dimethoxytrityl chloride (1.15 g, 3.4
mmoles). After stirring for 2.5 h at room t~p~rature,
the reaction mixture was diluted with dichloromethane
(200 ml). The organic phase was washed eguentially with
water [100 ml), saturated NaHCO~ solution (lOo ml) and
.:; ;
W0~2J~l690 ~CT/USg2/04795
~' , : . .
2~g8787
-2~
then ~ith water (100 ml), before it was dried over . ::.
anhydrous Na2SO4. The sol~ent was evaporated under . ~-.
reduced pressure and the residue was purified by ;;~
chromatography over a silica gel column using 2% MeOH in
~HzCl2 as the eluent. The homogeneous fractions we~e
pooled and ~vaporated to dryness to give a white solid. .
The solid was dissolved in minimum volume of CH2Cl2 (- 5
ml) and reprecipitated by the addition to pentane (200
ml) to yield 0.15:g (85~) of the pure title compound, mp
108C (dec.). IR (XBr): ~max 1700 (C=O), 3200-36~0 ~NH,
OH) cm ; W (MeOH): Amax 236 nm (~15,700), 296 nm
(~6,400); H NMR (DMSO-d6): ~ 1.09 ~q, 12 H, 2 :~:
COC~(CH3)2], 2.30 - 2.90 (m, 4 H~ C2,H, Cz~, 2 C~e23, 3.70
(s, 6 H, 2 OC~3), 3.98 (d, 1 H, C4,~ .55 (m, 1 H, C3,~),
5.33 (d, 1 H, C3,O~), 6.40 ~t, 1 H, Cl,~), 6.70 - 7.28 (m,
13 H, DMT), 8.49 (s, 1 H, C~) and 10.56 (s, 1 H, N~
Example 15
Svnthesis of 2-Isobuty~lam~o-6-isobytyrylthio-9-
r 3-O~ cya~o thyl)-N~N-diisoproE~la~i~ophosp~o~a
dite-5-o-~L~et~Q~y~ L~L~leoxy-B-D-ery~hl3a3~EI~e~
uranosvll~uri~e (14). ~ ;
To a solution~ of 15 (0.~0 g, 0.42 mmoles) and ::
N,N-diisopropylethylamine (0.29 ml, 1~68 mmoles) in ..
anhydrous dichloromethane (5 ml) was added with stirring : ;
2-cyanoethyl-N,N-diisopropylchlorophosphoramidite (0.19 :.
~1, 0.84 mmoles) under an argon atmo~phere. The reaction
mixture was stirred at room temperature for 1 h, after .
which ethyl acetate (45 ml) and NEt3 (5 ml) were added.
The mixture was partitioned between cold (0 - 5C~
saturated aqueous NaHCO3 solution (50 ml). The organic
phas~ was dried over anhydrou~ Na2SO4and evaporated undar
reduced pre~sure. The re~idual oil was puri~ied by
chromatography over a silica gel colu=n using
:
;.
wos2~l6so ~T/US92/~795
~? 2~g87g~
W27
EtOAc:CH2Cl2:NEt~ (45:45:10, v/v) as the eluent. The
homogeneous fractions were combined and evaporated under
reduced pressure to yield 0.18 g (47~i) of the titl~
compound as a~orphous white solid. tH NMR (DMSO-d6)~
1.34 Em, 24 H, 2cOcH(C~3)2 and McH(c~3)2~, 205 - 4.0 (m,
8 H, C2,~, C2~, OCH2C~2CN, 2cOc~)~ 3.71 (s, 6 H, 2 OC~3),
3.88 - 4.12 (m, 3 H, C6~H and C5,CN2), 4.83 (m, 1 ~, C3~H),
6.43 (t, 1 H, C~), 6.69 - 7.29 (m, 13 H, DMT), ~.52 ts,
1 Hj C~) and 10.5 (br s, 1 H, N~); 31P NMR (DMSO-d~
134 and 144.
:
Example 16
~ , ~.erythro-pen~Q~ an.o~ 9r~ L1~l~9~ (13~
`15 To a suspenslon of 2'~deoxy-6-thioguano~ine (1?) ~N.
B. Hanna, K. Ra~asamy, R. K. Robins, and G. R. Revankar,
J. Het~rocycl . Chem., 25, 1899 (1988)~ (2.0 g, 7.1
` mmol~s) in anhydxous pyridine (25 ml) was added
trimethylsilyl ,chloride (10.8 ml, 71 ~moles) at 0C.
After stirring the reaction mixture for 2 h at room
temperature, the ~eaction flask was cooled in an ice bath
and isobutyryl:chloride (0.9~ ml, 8.6 mmoles1 was added.
. The reaction~as~allowed to continue at 0C ~or 30 min
and~or 3 h at room:temperature. Water (40 ml) was added
~; ~ 25 :to the reaction~mixture, followed by dichloro~ethane (200
: ml). After shaking for 5 min, the organic layer was
separated and evaporated under reduced pressure at 40C.
The resulting residuo was azeotroped with toluene ~5 x 25
ml) to remove last traces o~ pyridine, and puri~ied by
chromatography over a silica gel column using 5% NeOH in
CH2Clz as the eluent. The homogeneous fractions were
combined and evaporated to yield 1.6 g (63%) of the title
compound, mp > 240C (dec.). IR (K~r): vmax 1290 (C=S),
1690 (C=ol, 3150-3250 (NH, OH) cm~; W (MeOH): Amax
,:
. '
W~92/21~9~ P~/VS92/047~5
;~ , :, -
2~378 ~' :
-28-
232 ~m (68,100), 330 nm (~10,700); 1H NMR (DMSO-d6): 8
1.}4 [d, 6 H, CH(CH3)2], 2.24-2.86 (m, 3 H, C2,H, C2"H,
CH~), 3.53 (m, 2 H, Cs,CH2), 3.85 (m, 1 ~, C4,H), 4.38 (s,
1 H, C3,H), 4.98 (t, 1 H, Cs,O~)I 5.33 (t, 1 H, C3,0H), -~
6.21 (t, 1 H, Cl,~) , 8.42 (s, 1 H, C~), 12.0 (br s, 1 H,
NlH) and 13.30 (br s, ~ H, NHCO). ~a~l- C~lcd. for ~ .
C14Hl~N50~S.H20: C,45.27; H,5.69, N,18.85; S,8.63. Pound: ~ .
C,45.46; H,5.41; N,18.54; S,8.97.
~', '.
Example 17
$vnthesis o~ 2-IsobutyFylamino 9-~5-O-
dimethoxytri'cyl-2-deoxy~ D-e~ythro-pentofu.ranosyl~- , .,,::.
(20). ` ~ `
A solution of 1~ ~1.5 g, 4.2 mmoles) in anhydrous
pyridine was evaporated several ti~e~ (4 x 25 ml) under
diminished pressure to obtain a dry residue, which was
redissolved in dry pyridine ~20 ml). ~o the pyridine .
solution was added 4,4'-di~ethoxytrityl chloride (1.53 g,
4.5 mmoles) ~nd sti~red at room temperature for 4 h. The
reaction mixture was partitioned between CH2Cl2 (200 ml)
~nd water (200 ml). The organic layer ~as separated,
dried over anhydrous Na2SO4 and evaporated under reduced
pressure. The residue was azeotroped with toluene (4 x
50 ml)~to re~ove last trac~s o~ pyridine, and purified by
chr~tography over a silica gel column using a gradient
of C~2Cl2- ~eOH (O ~ 4%3 as the eluent. The homogeneous
fractions were pooled and evaporàted under reduced
pressure to giva a~soIid. The white solid was dis$olved
in minimum volume o~ CH2C12 (3 ml) and reprecipitated by
the addition to pentane (~100 ml) to yield 1.12 g (67~)
o~ the title compound; ~p 146C (dec.). IR (KBr): ~max
1250:(C=S), 1690 (C=O), 3100-3600 (NH, OH); W (XeOH)~
Amax 236 ~ (617,700), 286 nm (~5,200), 332 nm (~14,300~;
H NMR (DNSO-do): ~ 1.14 Ed, 6 H, CH(~3)z] 1 2.15 - 2.86
.
. '
" ''
W092/216~0 r~/usg2/047ss
2o88787
-29-
(m, 3 H, C2,H, C2"H and CH~, 3.72 (s, 6 H, 2 OCH3), 3.96
(m, l H, C4,H), 4.43 (t, 1 H, C3,H), 5.37 (d, 1 ~, C3,0H), -~
6.27 (t, 1 H, Cl,~), 6.76 - 7.30 (m, 13 H, DMT), 8.90 (s,
1 H, C~), 11.93 (s, 1 H, NH) and 13.43 (s, 1 H, NHCO~
~nal. Calcd. for C35H37N506S. 1/2 CH30H: C,63.47; H,5.85;
N,10.42; S,4.77. Found: C,63.20; H,5.69; N,10.33;
S,4.78.
Example 18
Synthesis of 2-Isobuty~3~ ino 9~ Q=f~
_ _ ~
~ `,',.
To a solution of 20 (0.35 g, 0.53 mmoles) and
: N,N-diisopropylethylamin~ (0.37 ml, 2.1 m~oles) in
anhydrous dichloromethane (5 ml) was added with stirring
: 2-cyanoethyl N,N-diisopropylchlorophosphoramidit2 (0.25
ml, 1.06 mmoles) under an argon atmosphere. The reaction
mixture was stirred at room temperature for 50 min.
Ethyl acetate:(45 ml):and triethylamine (5 m}) were added
20: to: the reaction mixturz and the resulting solution was
partitioned b~tween ice-cold saturated aqueous NaHCO3
solution (50~ml)~ The organic phase was separated, dried
over anhydrous~ Na2SO4 and evaporat d under reduced
pressure:~to give:an:~oil. The residual oil was dissolved
25 : i~ C~C12 (3:ml~) and purifièd by chromatography on a
:: silica gel column using EtOAc:CH2Cl2:NEt3(45:45:10, v~v)
as the elu2nt. The appropriate homogeneous fractions
were pooled and evaporated to give 0.43 g (94~) Or 1~ as
amorphous white solid. 31p NMR~CDCl3). ~ 148.01 and
149.08; 1H NNR~(CDCl3): ~ 0.84-1.35 ~m, la H, CH(C~3)2 and
2 NCHÇC~3)z~ 2.5-4.0 (m, 8 H, Cz~ C2~, 2C~- and
O~zC~2CN), 3.72 (d, 6 H, 20C~3), 4.1-4.85 ~m, 3 H, C4,H
and C5,C~2), 6.18 (t, 1 H, C1,~), 6.73-7.46 ~m, 13 Hj DMT),
' . !, ~ ~
.: . : . ' .
~92~2t~90 PCT/Us92/04795 ":
20~8~7 .`.-.. `
-30-
7.90 (d, 1 H, C~), 8.13 (d, 1 H, NH) a~d 8.55 (s, 1 H,
N~). Anal. Calcd. for C~H54N~O7SP. 1.5 H20: C,59.85;
H,6.50; N,ll.lo; S,3.36; P,3.50. Found: C,60.01; H,6.79;
N,11.23; S,3.26, P,4.01.
Example 19
Svnthesis of 2-Isobutvrylamino=7-~-D-ribo~uranosyl-
v~rolo r 3 . 2 -d l pyrimldin-4 ( 3~ o e (~) .
To a suspension of 2-amino-7-~-D-ribofuranosyl~
pyrrolo-~3,2-d]pyrimidin-4(3~,5H~-one ~ N. S.~Girgis,
M. A. Michael; ;D.~F. Smee, Ho~A~ Alaghamandan, R. R.
Robins and H. B.~ Cottam, J. Med. Chem., 33, 2750 (1990)]
(2.0 g, 7.1:mmoles)~ in anhy~rous pyridine ~20 ml) was
added trimethylsilyl~chloride (9.0 ml, 71 mmoles) at 0C.
~ 15 After stirring the reaction ~ixture for 2 h at room
:~ temperature, the reaction fIask was cooled in the
ice-bath and isobutyryl chloride tO.90 ml, 8.5 mmoles)
was added. The reactio~ was allowed to continue at room
:~ temperature for an additional 4 h, after which cold water
(20~ml) was addedj followed by CH2Cl2 t150 ml). The
: : organic phas~ wa~ separated and evaporated under reduced
~pressure at~40C.~ The~rQ~ultinq residu~:was azeotroped
with: toluene~ 4 x~25 ~ml) to re~ove last~ traces of
pyridine,: and tr~itur2ted with dry CH~Clz.: The~colorless
25~ solid~ that~separated~ was collected by filtration and
dried~.~ The filtrate~which contain~ a smal} amount of the
~: product wa~ chromatographed on a 8ilica gel column using
: 5% MeOH: in CHzCl2 as the eluent. The homogeneous
fraction3 were collected, evaporated to dryne~s in vacuo
and combined with the above product to yield 1.90 g (76%~
o~ ~he title compound; mp 222-224C.- IR (RBrj: vmax 1680
(C=O), 305~0-3500~ (OH, NH~ c~l; W (MeOH): A~ax.246 nm
~(el5,400), 274 nm (~10,700); H N~R (DMSO~ 8 1.10 [~
6 H~ C(C~3)2], 2-75 (m, 1 H, COC~), 3.30 - 3.80 (m, 3 H,
, -:
-
~/0 9'~/~1690 PCr/U$92/04795
~0~787
C4,~1, C5,C~j2) ~ 3.90 (t~ 1 H~ C3~H) I 4.09 (t~ 1 H~ C2~
4.81 (d~ l H~ J = 6.0 Hz~ Cl,H)~ 7.42 (d~ 1 H~ J~= 2.8 HZ,
CoH) ~ 11.35 (br s, 1 H, N~3) and 11.98 (br s, 2 H, N}~;and
N~j-iBu). ~nal. Calcd. ~or C~5H20N406. 1/2 MeOH : C, 50.54;
H, 6.02; N, 15.21. Found: C, 50.81; H~ 5.70; N, 14.83.
Example 20 . -~
tetraisop~o~xl-1,3-disiloxanediyLL~D~Ei~uranos~11-
~ ~ 123). ,:
To a solution of 22 (2.68 g, 7.61 ~oles) in :
anhydrous ~ p~yr~idi~ ( 3 0 ml) was added
1,3-dichloro-l,1,3,3,-tetraisopropyldisiloxàne (2.91 ~l,
~ ~9.13: m~ol~s).~ After stirring the reaction ~ixture for
: 2.5 h at room ~temperature, it was partitioned ~etween
dichloromethane (100 ml) and water (100 ~ h- organic ~.
: phase was separated, dried over anhydrous Na2SO4 and
evaporated under reduced pre~sure. The reisidue was
purified by~chromatography over a silica gel colD
: 20 using 2S MeOH in dichloro~ethane as the eluent. The
: appropriate ~ho~ogenèous fractions were pooled, ~ol~ent
evaporated under reduc~d preRsure to yield 3.07 g (67.8%)
of the titl~ compound, ~p.148-150:C. IR (KBr): 1680 ~ :
(C-O),~ 3QOO -;3500 (OH~, NX) cm ; W (MeOH)~ A~ax 244 nm
2:5 : ~ (~c30,400), 276~nm~ 12,600); lH NMR (D~SO-d~): S 0.90 - : :
1.2;0:~[m, 34 H, ~OCH(C~ , and 0-si(c~xi2)2-o-si~(c~Lei2)2]~ ~
2.75 (m, 1 H, COC~Me2), 3.75-4.30 (m, 5 ~, C2,~, C3,~, C4,~ ~-
and C5,C~2), 4.92 (d, 1 ~, J = 2.6 Hz, Cl,~), 7.28 (s, 1 H,
C~j), 11.40 ~s,: 1 H, N5~ and 12.00 (br s, 2 H, N~1 and ~ ~-
N~-iBu). ~ Calcd. for C2~N40~Si2. 1/3 MeOH : C,
54.22; H, 7.88; N, 9.25. Found: C, 54.62; H, 7.93; N, :~
8.83.
.:
'' :'
:: ~ ; .:,''' ::
. . .: .
.'-'.'' ~ .
' ; !' . ; .:': ' ! ' :
WO 9~/21690 PCT/~S92~04795
2 0 ~ P ~ 8 r~
-32- ~ :
Example 21 : -
Synthesis of 2-Isobuty~y~lamino-7- r 35-0-~1,1~3,3
tetraisopropyl-l L3-disiloxanediyl~-2-phenoxythio- `~
car~cnyi~Q=~sibo~uranosyl]py~rolo~3 2-d~sL~ailin~
4(3~l~s~ne (Z6~. :
To a suspension o ~3 (0.80 g, 1.34 mmoles) and
4-dimethylami~opyrldine t0.33 g, 2.7 mmoles) in anhydrous -.
acetonitrile (10 ml) was added phenoxythiocarbonyl
chloride (0.20 ml, 1.47 mmoles) under an argon
atmosphere. After stirring at room temperature far 6 h,
the reaction mixture was diluted with CH2Cl2 (75 ml). The
organic phase was separated, washed with water (2 x 25
ml) and dried over anhydrous ~a2SO~. The solvent was
evaporated under reduced pressure. The r~sidue thus
obtained was~purlfied by chromatography over silica gel -::
: column using 1.5% MeOH :in CH2C12 as the eluent. The
appropriate homogeneous fractions were pooled and ; .
evaporated to yield 0.71 g ~72.5%) of the title compound,
mp 98-99C. W (MeOH): Amax 262 nm (~26,000), 332 nm :.:
(~12,500); 1H~ NMR (D~SO-d6): ~ 0.80 - 1.30 [m, 34 H,
~:' COCH(C~3)2 and O-Si(C~2)2-O-Si~C~)2], 2.80 (m, 1~
COCHMe2), 3.80 -~4.20 (m, 4 ~, ~,H, C~,~ and C5,C;~2), 4.92 ~:
(br s, ~ C2,~ 5.21 (d, 1 H, J = 3.8 Hz, C~,H), 7.10
- 7.60 (m, 5~H, OC~5), 8.03 (s, 1 H, C~1), 11.?9 (br s,
1 H, N~) and~12.:24;:(br s, 2 H, N~ and N~-iBu).
.' :' '
:
, Example 22
Syntbesi~_of 2-Isobutyrylaml~Q-7-t~,$-~ .3-
tetra~o~ropyl-1.3-disil~x~e~iyl~-2-~eoxY-3-D-ery~h~o- :
~entof:ux~osyl]pyrrolo r 3~,2-dlpYrimi~La-4f3H.~-Qne (25). ~ :
: To~ a- solution~of 26 (0.65 g, 0.845 mmoles) and
,-azobisisobutyronitrile (50 mg) in anhydrous toluene
(25 ml) was added tr1-n-butyltin hydride (1.36 ml, 5.1
,:
..
. .
.
;
W09~ 90 P~T/~S~2/047g~
~`ii 2~$7
:.: '.
-33
mmoles) under an argon atmosphere. The mixture was
heated with stirrin~ at 65C for 2.5 h, a~ter which the
solvent was evaporated to dryness under reduced pressure.
The residue thus obtained was purified by chromatography
over a silica gel ~olumn using 2% MeOH in CH2C12 as the
eluent. The appropriate homogeneous fractions w~re
collected and evaporated to yield 0.30 g (61.2%) of the
pure title compound, mp 93-95C. IR (KBr~: ~max 1680
(C-O), 3100-3500 (NH) cm1; W (MeOH): Amax 248 nm
(~24,200), 282 nm (~10,600); 1H NMR (DMSO-d6): ~ 0.80
- 1.20 ~m, 34 ~, COCH(CH3)z and 0-Si(CHMe2)2-0-Si(CHMez~2],
2.75 - 3.10 (m, 3 H, COCEMe2, C2,~ and C2~), 3.75 - 4.30
(m, 4 ~ , C4,~ and C5,C~2), 4.92 (dd, 1 ~, Cl,H), 7.24
(s, 1 H, ~O, 11.41 (s, 1 H, N~) and 11.93 (s, 1 H,
N~-iBu) . ~31- Calcd. for C2~H~N6O6Si2-MeOH : C, 55.06;
R, 8.24; N, 9.16. Found: C, 55.36; H, 8.06; N, 8.46.
Seleotlve alkylation of a solution o~ 25 in dry
acetonitrile with isobutyryl chloride i~ the presence of
NaH will give the.N-5 isobutyrylated derivative (24).
: 20 When the diisobutyryl derivative ~24) is treated with a
molar so}ution of tetra-n-butylammonium flouride in THF,
deprotection o~ the 3',5'-hydroxyl ~roups will occur to
give 2-isobutyrylamino-N5-isobutyryl-7-~-D-erythro-
pentofuranosyl)pyrroloti,2-d~pyrimidin-4t3H)-one (27~
. 25 Selective protection of the 5'-hydroxyl group of 27 with
4,4'-di~ethoxytrityl~ether will ~ollow the conventional
protocol to obtain the 5'-ODMT derivative ~). The
3 '-hydroxyl group o~ ~ wiIl be converted into the
phosphora~idite (29) by the reaction with 2-cyanoethyl-
N,N-diisopropylchlorophosphor~midite in the presence of
diisopropylethylamine in anhydrous dichloromethane.
' .''-
.
,. -:
.
Wt~ ~2~21~igO PCr~US92~04795 ~ ~ :
~ ''~' ' .'
20~37~7
-34-
Example 23
Stressed triPlet formation of sites o~ TA and GC
inversion.
In normal anti-parallel triplex formation the TFO
binds to duplex DNA by ~eans of a GGC or TAT triplet base
within the major helix groove. In this class of triple
helix structure the third strand selectively binds anti-
parallel with respect to the mora purine duplex strand.
However, occ~sionally there are sites within these
o tripl~xes where ~he purine rich strand is interrupted by
pyrimidine. Such a disruption constitutes the for~a~ion
o~ an altered bindiny site and is related to the
polypurine reference isomer by inversion about a locial
base pair diad. Preliminary molecular modeling suggested
that T and G bases in the third strand oan ~orm a TAT ànd
GGC triplet at sites-of TA and CG inversion but only upon
substan~ial distortion of the duplex and TF0 backbone.
In that instance duplex sites of TA and CG inversion
develop a locally altered duplex structure of a kind
which could be recognized by a structured specific probe.
To measure this altered duplex structure mapping of
the~che~ical cleavage which results fro~ binding of
copper phenanthroline, (Cu(OP)z) a duplex DNA
intercalator was used as a structural probe. Copper
phenanthroline ~s~ the capacity to cleave DNA near its
binding site. Mapping analysis o~ triplexes which form
at two sites within the EGFR pro~oter region were
examined. As seen in Figure 3 upon binding of the
correspcnding anti parallel TF0, the duplex develops high
Cu~OP)z sensitivity relative to uncomplexed DNA only at
sites of TA or CG inversion. The Cu(OP) 2 is generally
excluded from binding elsewhere within the triple helix
as would be expected fro~ simple co~petition between the
, ,.
.
"
.
. , . .. - . , . . , - . ... , ;. : - :- ~
` W~92/2~690 PCT/US92/~79~
,, 20~87~7
.
35- ;
two classes of bound ligand. The ~GFR sites are shown in
Table 1.
: ~ ~
~: ,
~'~
'
Table 1 shows a detailed: map o~ the 3'A-EG36ap and
3'A-EG4ap complexes. Controlled TFOs 3'A-~lV38p and~
5 ; 3'AEG31ap are also shown. This is a detailed map of the
; EGFR promoter region near -352 from the start codon. The
low resolution structure of the 3'AEG4ap and 3'AEG36ap
trlplex is presented. Bracketed numbers refer to
distance from the radio-labeled Hind III terminus shown
~10 in Figure 3. Controlled TFOs 3'-AHIV38ap and 3'A-EG31ap
do ~ not~ bind.;~ Sltes are underIined where a
polypurine/polypyrimidlne stretch is interrupted by TA or
; CG~inversions~within~the triplex binding~site. Note that
in both complexes Cu(QP)z hypersensiti~ity is induced at
15~ ~ the inverslon~sites-~by;~slte selective triplex forma~ion.
This e~fect was not detected in the uncomplexed duplex or
upon adding control oligonucleotides. Although this
mapping data cannot define the details of distortion
which is detected, it does provide evidence of
hypersensitivity and ~:uggests t~.at the bound TFO may not
lay passively as~a spacer over such sites. Instead, the
; lnversion site forms triplet H bonds which require
substantial compensatory distortion of the duplex binding
site. Local narrowing of the major groove necessarily
:
, .
SUBSTITU~E SHEET : ~
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WO9~/21690 PCT/US92/04795
` 2~887g7
-36- ~ :
accompanied by expansion of the minor groove presents a
specific structuxal possibility.
Example 24 ~ :;
and 5~G_¦~versi.Q
one structural hypothesis to explain the-stretched ..
triplet formation at sites of TA and GC inversion is ~ -
presented in Figure 1 which is based upon a reverse
Hoogsteentantl~RH/a) model for a stable triplet formation
: 10 within adjoining polypurine runs. In Figure 1 the :
triplet schemes have been presented in a schematic form .
to emphasize~sym~etry and ~ bonding relationships. .In
the process:of standard GGC tri~l~t for~ation, a third -.. - :
9trand guanosine~ donates two H-bonds to G of the - .:
underlyin~ duplex. Similarly, T donates one and accepts
one ~-bond from A~in the u~derlying dupl~x. In both
: ~ instances, G ;and T possess a pseudo two~old axis of
sy~metry and consta~ntly can engage in both Hoogsteen and
reverse Hoog~een type triplets. The top half of
Figure 1 is a sche~atic repre~entation of the reverse ~
Hoogsteen triplets. ~he thick bar associated with the -` ~ :
third~ strand G :or T~reQresents the position assu~ed by
the~furan~se plane ~end-o~ and i~ projection) in an anti- ;
confirmation. ~ Such a rever~e~ Hoogsteen anti(Rh/a)
:25 configuration places~the third strand anti-parallel with
respect to ~he:purine rich re~erence strand o~ the duplex
target as required by low resolution structure
det~rmination. The re~erenced strand o~ the underlying
duplex is positioned to the left in Figure 1. The
symmetry among the three strands which comprise such . : .
triplets is pre~ented graphically with the arrows. .
The bsttom half of Figure l d~pic~-~ the
corresponding si~uation resulting fro~ duplex sites with
CG (left) or T~ (right) inversion about the base diad.
,
:.::
....
..
.' ~':
. . ~ : " ,~ .
~09~l~1690 PcT/us~2/o47s5
20~878 ~ ~
. .
-37-
This produces a dislocation within a
polypurine/polypyrimidin~ target site. If a base triplet
is to be f~rmed at such a defect, continuity must be
maintained with flanking regions of the triplex. To
satisfy that connectivity reguire~ent, the TFo strand at
the site of CG or TA inversion ~ust retain an overall
antiparallel orientation with respect to the purine rich
reference strand of the duplex target. This is
equivalent tv the requirement that triplets must switch
to a parallel strand orient tion relative to the purine
containing strand at the site o~ TA or CG inversion.
Such a change of symmetry is d2picted by arrows in the
: lower portion of Figure 1.
For an u~modified ~ase, there are two degrees of
fr~edom which can be exsrcised to achieve such local
reversal o~ triplet sy~metry:
1. conver ion from Hoogsteen to reverse Hoogsteen
H-bonding, or
2.: anti/syn glycosidic rotatioh.
we have displayed the Hoogsteen to reverse Hoogsteen
degree o~ fresdom in:the lower por~ion o~ Fi~ure 1. In
this configuration the glycosidic bond is kept anti to
produce H~a triplets~a~ the sites of inversion.
: ~: Because of ths pseudo two fold sym~etry o~ G and T,
:~ 2s there is little ~difference in bond strength ~or the
Hoogsteen and reverse Hoogsteen triplets. Therefore, in
: evaluating the possibi.lity that the H/a triplet scheme
can occur at a TA or CG dislocation, the principal
concern is not H-bond strength or strand orientation per
seJ but is instead the substantial transverse dislocation
of the third strand backbone required to acco~modate the
defect. That dislocation is identified by horizontal
arrows at the top of Figure 1 and amounts to
approximately a 3-5A displacement. Alteration of local
.
W~9~/2~90 P~T/US92/0479~ -
~`i 2088787 ~ ~
-38~
base stacking interaction is also associated with such a
dislocation, which could also contribute a local change
in triplex stàbility.
The triple helix structure and thermodynamics can be
evaluated-in the context of the model in Figure 1. The
3-5A dislocation and attendant stacking change might be
difficult to accommodate into an orderly triplex,:thereby
resulting in local reduction of base stacXing and triplet
~ H-bonding energy, relative to the stable interactions
within polypurine/polypyrimidine runs. The destabilizing
e~ect of such defects upon triplex stability is
therefore consistent with the model.
The model in Figure 1 suggests that, although the
triplets are i~per~ect, they can form at sites of CG and
TA inversion and may hav~ a net stabilizing influence on
triple helix formation. ~his conclusion from the model
is consitent with the Cu(OP)2 hypersensitivity assay that
the duplex becomes deformed at these sites in the
triplex. This deformation may help facilitate ~ormation
of the stressed triplets.
Example 2 5
2'-Deo}y~ c~ odçl to L~prove _tr~plet
fo~matioa at sites o~G_~d ~ nvers on.
If triplet formation is made stable at sites of TA
and C5 invsrsion,~ oligonucleotides can be designed
against any duplex site, rather than just at purine rich
targets. Eased upon the model in Figure 2, one solution
to the binding problem at CG sites is to alter the
,position of the two hydrog~n bond donors of guanosine,
relative to the site of glycosidic bond attachment to the
furanose. This solution is displayed in Figure 2. As
can be seen the result i~ a reduction o~ the transverse
dislocation required to acco~modate triplet H/a bonding
at sites of CG inversLon.
.
""".,~
~,, . . ~
W~92t~690PCT/US9~/~7~5
. ~J
2088~87
-39-
Figure 2 displays an approximation to that proposed
al~Pration, employing 2 deoxyformycin as the
corresponding G homologue. Formycin (l, Figure 5) is a
naturally occurring nucleoside antibiotic, with
5structural similarity to adenosine. However, due to its :-:
unusual c-C glycosidic bond, the aglycon moie~y becomes
more aromatic (abs. max shifts to 310nM) and its state of
protonation is altered relative to A. At neutral pH, the
formycin ring is protonated a~ sites N1 and N2 ~Figure 5).
10~s such, the ring is capable of dona~ing 2 H-bonds to an
acceptabl~ donor a~d is therefore homologous to G, with
respect to triplet for~ation. , ~.
Thus, by substit~ting 2'-~eoxy~or~ycin for G, the ~ ~:
third trand backbone beco~es less distended at sites of
15CG inversion.: No e also, the improved si~ilarity of
~uranose placement, relative to a GGc triplet at sites of
CG inversion (lower part o~ Eigure 2).
Example 26 ..
Int~Qductio~ o~ ~F int Q a Proqesterone response~element
20A duplex DNA target is presented in TabIe 2 which
corresponds to ~ha binding domain for progest~rone .
response ele~ents in a progesterone response~ promoter. . : .
The two protein binding ~ites are underlined. The TF0
specific for thi~ site~is presented in the preferred
antiparallel orientation.
The dF-DMT-phosphoramidite (~) was introduced into
the TFO 3'A-PRE2ap. As an unmodified TFO, the
progesterone responsa element PRE2ap binds sQlectively to
the purine rich target ~ite described in Tabls 2.
TA~LE 2 A q~ CONTAINING TFO: 3'A-PRE2Fap (SEQ. ID.
No. l).
Si- _ = C TT~oo~c~ Tcr~ n~T~IG-3l TARG2T
3 ' -U:;ASGT~G~C~ G~WC~TFTG~A~AG-5 ' TA;~lOE.T
5~-~rGTGrTGTGGGG>o;;~TGr~ sG~-3~:3~A-pRE2ap
5'-G50~FTSCTGGG&GG~ C ~ G~Gr~TTGT-3l 'A-PRE2F~p ~ -
~ .:
WO 9~/~1690 PCr/U~;92/~4795
~ ~o~8787 ~ ~
-40~
. :. :
36BP site, 3ll36=87% purine, 60~ 5C, 3 sites of dE
substitutio~.
.: .
The ~orresponding dF derivative (3'A-PRE2Fap) was
. synthesized by the standard solid phase ~ethod. The TFOs
are prepared employing solid-phase synthesis using ~he
CPG method and B-cyanoethylphosphoromidite chemistry on
a Milligen 8700 synthesizer. The TFOs are modified at
the 3'-ter~inus as primary amine~ i.e. 3'-O~2CHOHCH2N
which is introduced as part o~ the link to the CPG
support: 1-3'-amino modifier" provided by Glenn Research.
In all ins~ances, T~Os are purified by C~EPLC, followed
by detritylation, organic extraction, then
rechro~atography over a G25 molecular sieve. The purity
of tAe resulting material is analyzed by high resolution
electrophoresis and by HP~C.
Example 27
CompariSQn of St~D~3~Ll~5-with ~ F
The binding o~ 3' A-PRE2ap and its 2'-d~oxyformycin
homologue were monitored at 37C by the band shift
method. The standard binding buffer: l0mM Tris/HCl,
10~ Mg~12, p~ ~7.~8~was used. In this experiment, the
; ~ radiolabelled duplex target fragment of Table 2 is
titratQd~with increasing concentration of unlabeled TFO.
Binding is then detected by the characteristic
electrophoretic mobility difference between duplex and
triplex on a native 10% acrylamide gel.
In this assay the 2~-deoxyformycin containing TFO
displayed approximately l0-~old increase in affinity as
compared to its unmodi~ied counterpart (Figure 4).
3~ Th~re is an apparent titration midpoint decrease from
l0~M to l0'~ in~TFO equivalents. Thi~ signiSlcant
''~'''
~,
~092~1690 PCT/~S92/04795
2 ~ ~387~ ~ . '' ''' '
-41~
enhancement of bidning affinity resultin~ from dF
substitution provides a method to accommodate sites of CG
(and TA) inversion within target sites for antiparallel
triplex ~ormation.
Example 2~
Introduction_of dF into an insulin rece~tor site
TFo.
A purine rich domain within the ~ouse insulin receptor
gene has been identified (Table 3). Its TFO compelment
3'A-IR2ap is presented bound in an antiparallPl
orientation. 3'A-I~2ap is presented in the orientation
which would resul~ if it were to bind so as to for~ GGC
~nd TAT triplats in a antiparallel orientation.
Numbering ref~rs:to th~ principal capping site described
by Sibley, et al. ~Proc. Natl. Acad. Sci. USA 86:9732
(}989)].
TABLE 3 THE ~R, SITE 2 ~PSTREAM REGION WITH
TC REPEATS, IN XUMAN AND MOUSE (IR2Fap; SEQ. ID. No. 2)
-13~ _99
_____ > __ ~ >
5'-CC5C~:~C~G~5eTGC~G~rr~K~XX~:XXX~ W-3' r~RGET
GGO~:U~=XXX~G-5' SITE-
5~_ ~ 3' Ig2ap
5: ~ 3' IR2Fap
38BP site, ~4~ purine, 76% CG, 5 sites of dF
subs~itution.
,
Example 29
Introductio~_o~ dF into the ~F ~Fomot-ex- :.
A purine rich do~ain within tha EGFR promoter has been
identified,~ along with its antiparallel TFO complement
wos2/21~9o PCT/US92~047~5
20~8787
. .
-42- ~ :
3'A-EG36ap (Table 4). Numbering refers to the position
relative to the start codon.
TABLE 4 THE EGFR,EG36 SITE TARGET FUNCTION: .
Spl BINDING SITE #4 (EGFR36Fap; SEQ. ID. No. 3). ..
.~ . .
-3S2 ~317 -
3 1 -AAGAGG~ GGAGAGG.~s~sJGGC~AGGG~GGZLGG~5 t
5~ TTGTGGT~GGTGTs~GT~GTG&GGTTG>GGTsJG~3~ EGFR36~p :
s~-TTGTGGTGGTGGTGTFGT~5TGGDrrTG>GGTs~G-3l EGFR36F~p
,
,....
36BP site, 92~ purine, 67% GC, 2 sites of ~ substitution
Example 30
Int~oduction~3L~L/ i:~Q_hL~ S_
A target site wLthin the HIV-l LTR is presented, ;
corresponding to a series of adjacent Spl binding sites.
~umbering refers to position relative to the viral ~RNA
s*art site.
'rABLE S~PROPOSED Spl TRANSCRIPTION j ~
ACTIVATOR E3INDING SITE~4 (HIV38Fp; SEQ. ID. N~. 4). . ~;
-ao ~ 51 ~.
9476~ 950l . :-
S'-A = t;GCGTGe~TGGGCGGG~CTGGG~AGTGGCGa~-3' T~RGET
:~ 3'-T~C~TCCGC~C~GG~CCCGCCCTGAkC~C~C~CC$eTC-5' SITE -
5'-TGGaTG~ rGGGGTG~GGGGGTGTGGGGTGTG&G&TG-3' ~IV38p
: 5'-TG>GGFGTGGF~TG~GGG~FTGGBGTGTGBFG~G-3' ~IY38Fp ~:
;
38BP site, 77% GC, 74S porine, 6 sites of dF substitution ~ ~ .
':
''''': ':
` .
- -.
,:
.
' ` ,`~''',', ' ~ ' " .
~092~2~90 PCT/US9~t~479~ . .
2~8~
-43-
Example 31
Svnthesis of 7-N-benzoylamino-3- r 2-0-methyl-3-o-fB-
cyanoet~yl~-N,N-diiso~ro~ylamino~hosphoramidite-5-0-
di,~etho~rl.tyl ~ -ri}2~ur~nosy~y~z~o ~ 4,"_3~d l -
~rimi~ine ~35).
The synthesis of compound 35 follows the reaction
sequence shown in Figure 10. Treatment of a suspension
of formiycin ~ (1) in anhydrous pyridin~ with
trimethylsilyl chloride at 0C for 2 h, followed by the
addition of 1.1 molar equivalent of benzoyl chloride and
sub equent hydrolysis o~ the tri~thylsilyl protecting
groups with water yields N-7-benzoylformycin A ~30).
Protection of the ',5'-hydroxyl groups of 30 was
acco~plish~d by the r~action with 1,3-dichloro-1,1,3,3-
te~raisopropyldisiloxane to afford 7-N-benzoylamino-3-
~3,5-Q-(1,1,3,3 tetraisopropyl-1,3~disiloxanediyl-~-D-
ribofuranosyl]pyrazolo~4,3-~]pyrimidine (31). Selective
methylation of the 2'-hydroxyl group of 31 with either
diazo~ethane in the presence of 1 mM stannous chloride
~M.J. Robins, S.R. Naik and A.S.R. Lee, J. org. Chem.,
39: 1891 (1974)] or with m~thyI iodide in the presence of
freshly precipitated si}ver oxide ~E. Sochacka \and A.
Malkiewicz, Nucleosides Nucleotides, 9: 793 (~1990)]
yields 7-N-benzoylamino-3-~3,5-Q-(1,1,3,3-tetraisopropyl-
1 , 3 - d i s i~l o x;~a~n e d i y l - 2 - 0 - m e t h y l - ~ - D
ri~ofuranosyI]pyrazolot4~3-d]pyrimidine (34). Upon
:~: subs~quent deprotection of the 3',5'-0-protected silyl
gorups by the treatment with tetra-~-butylammonium
fluoride ~1~ solution in THF) at 0-5C for 2 h, the
desired key intermediate 2'-0-methyl-N-7-ben~oylformycin
A ~) is produced. Coupling of 33 with 1.1 molar
equivalent of 4,4'-dimethoxytrityl chloride in anhydrous
pyridine at ambient temperature for 4 h, yields 7-N-
benzoylamino-3-[2-0-methyl-5-0-di~iethoxytrityl-~-D-
W09~ 90 P~T/US~2tO479
;`,,. 2~ ,8
-44-
ribofuranosyl]pyrazolo~4,3-d]pyrimidine (32). With 2'-
and 5'-hydroxyl groups protected, the 3'-hydroxyl of 32
is now activated to ths phosphoramidite by the treatment
with 2-cyanoethyl-N ? N-diisopropylchlorophosphoramidite in
~he presence of N,N-diisopropylethyla~ine to afford the
t~rget compound 35. The product 35 can be purified on a
silca ~el column.
All patents and publications mentioned in this
specification are indicative of the levels of those
skilled in the art to which the invention pertains. All
paten~s and publications are herein incorpora~ed by
reference to:the same extent as if each individual
publicàtion was specifical}y and individually indicated
to be incorporated by ref~r~nce.
One skill2d in the art would readily appreciat~ the
present invention is well adapt~d to carry out the
obiect~ and obtain the~ends and advantages mentioned, as
well as those inherent therein. The compounds, methods,
procedures and techniques described herein are presently
repr~sentative of the~ pre~erred embodiments and are
intended to be examplary~and not intended as limitations
: on the s~ope. Chang~s~therein and other U~8g will occur
to:thosejskilled in the art which are encompassed within
the spirit of the~:inve~tion or defined by the scope of
25: the appe~ded claims.
~ :
'` "` '~
. ' ' .:
.
: ~ " '.
: `:', ' - '
'``' :'- '
. . .
.: ~. -. .
' .
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, ~:
W ~ ~2/~16~0 PC~/US92/047g5
2~7~ i ~
-45~
' :.
SEQUENCE LISTING
: . ~
(1) GENERAL INFORMATION:
(i) APPLICANT: R~vankar, Ganapathi R.
Hogan, Michaol
Rao, Takkellapati S.
S~hroff, Hite~h N.
~ii) TITLE OF ~NVENTION: 2'-DEOXYRIBONUCLEOSIDES, USE IN TRIPLEX
EQRMING OLIGONUCLEOTIDES ANU PROCESS FOR PREPARING T~E
SA~E ` ' '
, .
(iii) NU~BER OF SEQUENCES: 15 `~
~iv) CORRESPONDENCE ADDRESS:
(~) ADDRESSEE: Thomas D. Paul
(~) STREET: I301 ~Xinney, Suite 5100
(C) CITY: Houston
(Dj STATE: Tèxa~
(E) COUNTRY: U.S.A.
(F) ZIP: 77010-3095
~v) COMPUTER READABLE~FORM:
~A) MEDIUN TYPE~FlopFy disk
B) COMPUTER: IB~ PC compatible
(C) OPER~TING SYST~: PC-DOS/MS-DOS
(D) SOFTWAR~:~PatentIn R~l~ase #1.0, Veraion ~1.25
(vi) CURRENT APPLICATION DATA: ;,
(A) APPrICATION NUMBER: US
(8) EILING DATE:
(C) CLASSIFICATION:
(viLi) ATTORNEY/AGENT INFORMATION:
(A) NAME: Paul,~Tho~as~D.
(B) REGISTRATTON NUM8ER: 32,714
(C) REFE~ENCElDOCgET NUM8ER: D-5348-PCT
(ix) TEL~CONMUNICATION INFORMATION:
~A) TELEP~ONE: 713/651-5151
(B) TELEFAX:~713/651-5246
i(C) TELEX: 762829
(2) INFORMATION FOR SEQ ID NO~
(i) SEQUENCE CHAR~CTERISTICS:
(A) LENGTH: 36 base paira
(8) TYPE: nucleic acid
(C) STRANDEDNESS: sLngle
~D) TOPOLOGY: linear
55 ~ ~ii) NOLECULE TYPE: DNA~(genomic) ~ ;
` (iii) HYPOT~ETICAL: YES
: .
SUBSTI i UTE SHEEl-
.
: : ; ~
~0 92J~1690 PCr/l~S92/04795
" ` ~ J '': ,"
-46-
(xi) SEQUENCE DESCRIPTION: SEQ ID NO~
GTGTNTTGTG GGGGGTGGGG GTNTTGTGTG TNTTGT 36.~: -.
(2) INFORMATION FOR SEQ ID NO:2. .~.
(i) SEQU~NOE CHARACTERIST~CS:
(A) L~NGTH: 39 ba~e pairs
(B) TYY~: nucl~ic aeid :- .:
(C) STR~NDED~ESS: ~ingle .
(D) TOPOLOGY: linear
. .
(ii) MOLECULE TYPE: DNA (geno~ic)
(iii3 HYPOTHETICAL: Y~S
~xi) SEQUENCE DESCRIP~ION: SEQ ID NO:2: ::.
GGTGGGGNTN GTGTNGTNNG TTTGTGGGGG TGGGG4&GG 39
~ (2) }NFORMATION FOR~SEQ ID NO:3:
(i) SEQUENCE CaaRACTERISTICS:
(A):~ENGTH: 36 base pair~
. ~B) TYPB: nucleLc acid
(C) S~RANDEDNESS: ~inql~
~ (D) TOPOLOGY: linear
: 30
(ii) MOLECULE TYPE: DNA (genomic) ~:.
... : ,
HYPOTHET}CAL: YES
(xi) SEQUENCE~DESCR}PTION: SEQ ID NO:3: .!.;
: TTGTG&TGGT GGTGTNGTGG~TGGGNTTGG& TGGTGG 36 :
(2) INFORMATION FOR SEQ~ID NO:4: .
(i) SEQUENCE CHARACTE~I~5TICS: . -
: (A) LENGTH:~ 38:base pairs
: : ~ (B)~TYPE: nucleic~acid ~ -
(C) STRANDEDNESS:~single ~. ::
(D) TOPO~OGY: linear
(ii) MOr~CULE TYPE:: DNA (genomlc) : ~ -
(iii) HYPOTHETICAL: YES ~ . ;
.. .
. .
(xi) SEQUENCE DESCRIPTION: sEg ID NO:4:
,. :''
TGGGTGGNGT GGNNTGGGNG:GGTNTGGGGT GTGGNGTG 38 ...
. . .
(2) INFORMATION FOR SEQ ID NO:5: . ::
~.
,.
:
- ~: SUBS~ITUTE 5HEET ~ - ~
~VO 92~21~0 PCI/US92/04795
2 0 X ~
-47- :
(i) SEQUENCE CHARACTERISTICS:
(A~ LENGTH: 85 base pairs
(B) TYPE: nucleic acid
(C) ST~ANDEDNESS: double
(D) TOPOLOGY: linear ~.
(ii) MOL~C~E TYPE: DNA ~genomic)
(iii) HYPOT~ETICAL: NO ~
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:
GGGTCCCCGC TGCTGGTTCT CCTCCTCCTC CTCGCATTCT C TCCTCCTC TGCTCCTCCC 60
GATCCCTCCT CCGCCGCCTG GTCCC 85
~2) INFORMATION FOR SEQ ID NO:6:
(i) SEQUENCE CHARA~TERISTICS:
: (A) LENGT~: 36 bas~ pairs
(8) TYPE: nucIeic:acid
~C) STRANDEDNESS. ~in~le
: ~D) TOPOLOGY:~linear
~: (ii) NO~ECU~E TYPE: DNA:(genomic)
(iii) HYPOT B TICAL: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:
TTGTGGTGGT GGTGTGGTGG TGGGGTTGGG TGGTGG 36
:
(2) INFORMATION FOR SEQ ~ID NO:7O
i) SEQ~ENCE CEARACTERISTICS:
40:~ : (A) LENGTH: 38~ba~ pairs
(B) TYPE: nucleic acid
::~ (C) STRANDEDNESS: single
) TOPOLOGY:~linear
(iL~ MOLECULE TYPE: DNA Igenomic)
(iii) HYPOTÆ TICAL: NO
(xi) SEQVENCE DESCRIPTTON: SEQ ID NO:7:
TGGGTGGGGT GGGGTGGGGG GGTGTGGGGT GTGGGGTG 38
(2) INFORMATION FOR SEQ ID NO:8:
(i).SEQUENCE CHARACT~RISTICS:~
(A) LENGTH: 35 base pair~
(B) TYPE: nucleic acid
(C) STRANDEDNESS: sinqle
(D) TOPOLOGY: linear
:: j,
SUB~:~I i UTE: 5~E~ T
... .. ..... . .... . . . .... ... . .. . .. . ... . . . . .
YV~ 92~21~91) P~/US92/04795
i ~ g 7 ~ 7
-47a- :
(ii) ~OLECULE TYPE: DNA Igenomic)
tiii) HYPOTHETICAL: NO .:.~
. .
(xi) SEQUENCE DESCRIPTIONs SEQ ID NO:8: ~
TTGTGGTGGT GGTGGTG> TTGTGGTG&T G&TGT 35 ~ :
1 0
(2) INFORMATION FOR SEQ ID NO:9~
(i) SEQUENCE CHARaCTERISTICS:
(A) LENGTH: 26 ba e pair0 .. ,~
(B) TYPE: n~cleic~acid ,~
(C) STRANDEDNESS: single - . ~ :-
(D) TOPOLOGY: linear ~
(ii) MOLECULE TYPE: DNA ( genomiG) : :
,
(iii) HYPOTHET~CAE: NO
,
25 . (xi) SEQUENCE~DESCRIPTION: SEQ ID ~O:9: : ~ ~;
TGGGTGGTGG TGGGGGGGTG GGTG&4 2 6
(2) INFORMATION FOR SEQ ID NO:10: ;
: 30 ~ ~
(i) SEQUENCE CHARACTERIS~ICS:
(A) ~ENGTH: 39:base pair~
(B) TYP~: nucleic acid
~C) STRANDEDNESS: double
(D) TOPO~OGY: linear .
(iL) MO~ECULB~TYPE: DNA (~enomic) `.
~iii) HYPOTHETICAL: NO :.
:
(xi) SEQUENCE~DESCRIPTION: SEQ ID NO:10:
, . .:
45: CCTCCCCGAG CTCTGCTGGC: TTTCTCCCCC TCCCCCCCC 39
~2) INFORMATION FOR SEQ ID NO:Il: ::.-
~i) SEQUENCE~CHARACTERISTICS:
~A) LENGTH: 39 base palrs ~:
~) TYPE: nucleic acid
(C) STRANDEDNESS: aingle
(D) TOPOLOGY: linear
~ii) MOLECULE TYPE: DNA ~enomic)
~iii) HYPOTHETIC~L: NO
,: :
,
,
SuBsTlf~lTE SH~
~ .
WO 92~1690 PCI/US92/04795
~ 7 8 7 -47b~
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:
,,
GGTGGGGGTG GTGTGGTGGG TTTGTGGGGG TGGGGGGGG 39
(2) INFORMAT~ON FOR SEQ ID No:12:
~i) S~QUEiN OE CEARACTERISTICS: ~`~
~A) LENGTH: 47:ba~ pair~
~) TYP~: nudleic acid
(C) ST~ANDEDNESS: double
~D) TOF~LOGY: lin~ar
(ii) MOLECULE TYPE: DNA ~genomic)
~iii) ~YPOTHETICAL: NO
: ~:
~ (xi) SEQUE~OE VESCRIPTIONt SEQ ID NO:12:
20 `
GATCTGTACA CTCTGTTCTC CCCCCACC~CC CTGTACACTC TGTTCTG 47
~2) INFOR~ATION FOR SEQ~ ~D NO:13
(ij SEQUENCE cHaRAcTERIsTIcs:
(A) LENGTH: 36 baae pairs
(~) TYP~s nucleic acid
: (c) STRANDEDNESS: double
~: : (D) TOPOEOGY: linear
~:30
(ii) MOLECULE TYPE:~ DNA (genomic)
(iii) HYPOTHETICAL: No~
(xi) SEQUENCE~DESCRIPTION: SEQ ID NO:13:
TTCTCCTCCT CCTCTGCTCC~TCCCGATCCC TCCTCC 36
~2) INFORMATION FOR~SEQ~ID:NO:14:
(i)~SEQUENCE:CHARACTERISTICS:
::: (A) LENGTH::36:base pairs
(~3): TYPE:~ nucleic~acid
(C) STRANDEDNESS: ~in~:le
(D) TOPOL~GY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(lil) HYPOTHETICAL: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:
GTGTGTTGTG GGGGGTGGGG:GTGTTGTGTG TGTTGT 36
,.
SUBSrlTUTE SHEET
~ ~.
~, , ~ , , ! ' . . ; ' ` ' ' ' ~ ';
WO 92/21~90 ~ PCI'/US92/0479S
2, ~ 3 3 ~
--47c-- :
~2) INFORMATION FOR SEQ ID NO:15
~i) SEQUENCE CHARACTERISTICS~
(A) LENGT~: 38 baae pairs
~B) TYPE: nucleic acid
~C) STRANDEDNESS: double :
(D) TOPOIOGY: line~r
. ;
(ii) MOLBCULÆ TYPE~ DNA (genomic)
(iii) Hypo~HæTIcaL: NO
,
',.~ ;; .
~: 15 (xi) SEQU~NCE VESCRIPTION: SEQ ID NO:15: ::
AGGGAGGCGT GGCCTGGGCG GGACTGGGG~ GTGGCGAG 38 ~ .
. -'
:,
: '. `',~, ' :` ' '
, . .:
: . ...
: : .
~':
: ` ' ,
,~ .
' ~: '
. :
:.
S~,;JBS T I~U~E SHEFT: ::
: ` : :.:`:
.
:
,` . .
