Note: Descriptions are shown in the official language in which they were submitted.
This is a divisional application of copending application,
; , Serial No. 492,727,, filed Octob~r 10, 1985.
EDZ-1 -2-
BACKGROUND OF THE INrVE~TION
8-Aminoguanine, a compound known since the turn
of the century, has been reported to have PNP-activity
by R. ParXs, et. al., in Biochem. Pharm., 31 (2), 163
(1982).
~-(2-Furfuryl)guanine is a known compound
described in ~. Am. C~.e~. Soc., 81, 3046 (1959) having
no disclosed utility. The present invention is
related to novel purine derivatives not obvious to an
ordinarily skilled artisan, particularly, 9-heteroaryl
guanines as having PNP-inhibiting activity.
8-Amino-9-benzylguanine was discussed at the
16th Annual Graduate Student Meetiny in Medicinal
Chemistry, University of Michigan, Ann Arbor, Michigan.
However, the present compounds are not obvious from
either the synthesis or biological activity of
8-amino-9-~enzylquanine discussed.
With regard to various novel processes of the
present invention Ji-Wang Chern, et al, describe "A
Convenient Synthesis of 2-N-methoxycarbonyl-
aminooxazolo[5,4-d]pyrimidines" in_J. Het. Chem. 21,
1245~6 (1984). A similar synthesis is described by
S. Ram, et al, in "A Synt~esis of Carbamic
AcidCImidazo-Heteroaromatic]Methyl Ester Derivatives
Using Methoxycar~onyl Isothiocynate," eterocycles,
Vol. 22, ~o. 8, 1984, pp 1789-90, in which methoxy-
carbonyl isothiocyanate is ~sed in a one pot reagent
for the ring closure of an o-diaminopyrimidine
derivativs to afford a purine derivative possessins
the metho~ycarbonylamino unctionali~y at position
eight. -Fl~rther, the mechanism of these two syn-t-nesis
is disc~ssed ~y Ji-Wang C~e-n, et al, in "The ~ovel
Rins Opening of an Oxa~olo~5,4-d]Pyrimidine and
Subsequent Rearr~ngemen~ to ~orm an Imidazo~4,5-d]
35 Pyrimidine," ~eter?cycles, Vol. 22, ~o. 11, 1984,
pp 2439-2441.
2~ B
~DZ-l -3~
None of the disclosures include a disclosure of
reaction conditions, or an Ar as a heteroaryl or
substituted heteroaryl, substituent defined hereinafter
for the compound of Formula I prepared by the novel
processes of the present invention. That i5 t
corresponding Ar groups as defined hereinafter for
each of the novel intermediates III, II, and I to be
heteroaryl or substituted heteroaryl are not included
in the ab~V~ references and furthermore are not obvious
variants thereof.
SU~MARY OF THr Ii~VEI~TIOL~
The present invention relates to a compound of
the formula
Rl
¦ I
J~
wherein Rl is ~H or sa; R2 is hydrogen, ~HR in
whicn ~ is hydrogen or ~OR6-where R6 is al~yl of
one to four carbon atoms~ aryl or-arylalKyli ~3 is
hydrogen, hydroxyl, mercaoto, bromine or ~H~ whe-e K
is hydrogen or COR6 wherein ~6 is as defined above;
n is zero or one; m is zero, one, two, or three, with
tne proviso that m or n is at least one; ~ and Rs
are each independently hydrogen, alkyl of one to four
carbon atoms, hydroxyal~yl of one to four carbo~.
atoms, aryl, arylal~yl or cycloal~yl of three to six
carbon atoms, and Ar is heteroaryl or hetero~ryl
, ~2~
~Dz-~l
substituted by alkyl, alkoxy of one to four carbon
atoms, -C=C-C=C- attached to adjacent carbons so as
to form a benzo radical, or halogen; or a
pharmaceutically acceptable acid or base addition salt
thereof, excluding the compound wherein ~1 is OH, R2
is amino, ~3 is hydrogenf n is zero, m is one, and
Ar is 2-furanyl, i.e., 9-(2-furanylmethyl)guanine.
The present invention inclucles a method of
manufacture and a pharmaceutical composition
comprising an effective amount of a compound of the
For~ula I with a pharmaceutically acceptable carrier,
as well as a method of treatment of autoimmune
diseases such as arthritis, systemic lupus
erythematosus, inflammatory bo~el diseases, multi~le
lS sclerosis, juvenile diabetes, as well as
transplantation, viral infections and cancer by
administering an effective amount of a compound of the
Formula I in unit dosage form to a host of the disease.
That is, the amount is the amount effective for
2~ treating each of the autoimmune diseases. It is
understood, an ordinarily skilled physician would
begin with a less t~an effective amo4~t for treatment
and increase the dose until the desired effect is
obtained exercising care to administer an amount less
than the amount toxic to t~e host of the disease.
Both the above phar~aceutical composition and
metnod of treat~ent include as active ingredient
9-(2-furfuryl)guanine.
The present inventicn also includes the novel
intermediates as lollows:
(1) A compound of ~ormula III wherein R6 is alKyl
of one to rour car~on atoms, aryl, or arylal~yl, n is
zero or one; m is zero, one, t-~o, or three, ~ith tne
proviso that 1~ or n is at least one; ~4 and ~5 are
each independently hydrogen, al~yl of one to four
carbon atoms, aryl, arylal~yl, or cycloalkyl of three
to six carbon atoms, hydroxyal~yl of one to four
`` ' 12~
EDZ -I -5 -
carbon atoms, aryl, arylalkyl, or cycloalkyl of three
to six carbon atoms, hydroxyalkyl of one to four
carbon atoms, and Ar is heteroaryl or heteroaryl
substituted by alkyl of one to four carbon atoms,
alkoxy of one to four carbon atoms or halogen;
(2) a compound of Formula II wherein R6, n,
m, R4, Rs, and Ar are as defined above; a~d
(3) a compound of Formula IV wherein n, m, R4,
Rs, and Ar are as defined above.
lQ Additionally, the novel processes of the
present invention are as follows:
(A) A novel process for the preparation of a
compound of Formula I, wherein Rl is OH or SH,
R2 is NHR wherein R is as defined above, R3 is
hydrogen, hydroxyl, mercapto, bromine, or NHR where R
is hydrogen or COR6 wherein R6 is as defined above,
and n, m, R4, Rs, and Ar are also as defined above
which comprises heating a compound of the Formula III
wherein R6, n, m, R4, Rs, and Ar are as defined
above to obtain the compound of Formula I wherein R
is OH and R3' is NHCOOR6 wherein R6 is as defined
above, and if desired, converting sai~ compound to a
compound where Rl is S or SH by methods analogous to
those known in the art, and if further desired, where
R3' is N~COOR6 converting the compound to a compound
where R3 is hydrogen, or NHR where R is hydrogen or
COR6 wherein R6 is as defined above also by known
methods.
Particularly, the above orocess is for the pre-
paration of 8-amino-9-[(2-thienyl)methyl]quanine.
(~) A process for the preparation of a compound
of the formula Rl
a ~ N ~-- T
- 12~9~38
EDZ-~ -6-
wherein Rl, R2, R, R3, R4, Rs, m, n, and Ar are as
defined above, with the proviso that R3 is not Br,
and R3 is not NHR; which comprises reacting a compound
of the formula O
. ~
Hf ~ IV
~ N~ NH
Ar--~cH2)m ~ R4)
~ n
with formic acid and formamide at elevated tempera-
tures, to obtain a compound of the formula
O
21~N
R N
Ar----tCH2 ~ 5) n-
and, if desired, converting by methods analogous to
those known in the art the compound into a compound of
Formula I wherein Rl is SH and/or R3 is hydroxyl,
mercapto, or NHR wherein R is as defined above or a
pharmaceutically acceptable acid addition or base sal'
thereof.
(C) A process for the preparation of a
compound of the formula
o
~ Br
R2 ~ N
Ar- ~CH
r ~ 9~9L38
EDz-i -7-
which comprises treating a compound of the formula
Ar----~C~2 ~ ~) n
with i~-bromosuccinimide in an organic solvent,
and, if desired, converting by methods ~nown in the
S art the resulting compound into a pharmaceutically
acceptable acid addition or base salt thereof.
(D) A process for the preparation of a
compound of the Formula I wherein R3 is `.~R
wherein R is as defined above; whicA comprises
reacting a compound of the formula
~ (ca2~4 J
Rs n
~herein Xl, R2, R4, ~5, n, m, and Ar is as
defined above; witn hydrazine at elevated
temperatures and, optionally, ;~ith ~aney nic~el in
l_ an alcohol solvent, and, if desir~d, converting
the resulting compound where R is hydro~en to a
compound where R is C0~6 with an al~anoyl halide,
aroyl halide, or arylal~anoyl halide in tne
presence of an organic base, and, if desired,
2~ whers Rl is.O or 0~, conv~rting said compound
,
~2~
EDZ-l -8-
to a compound where Rl i5 S or SH by known methods,
and, if further desired, converting the resulting
compound by methods analogous to those known in the
art to a pharmaceutically acceptable acid addition or
base salt thereo.
(~) A novel process for the preparation of a
compound of Formula III wherein R6, n, m, R4, Rs,
and Ar are as defined above which comprises contacting
a compound of the Formula II wherein R6, n, m, R4,
Rs, and Ar are as defined above, with a coupling
agent in the presence of a solvent to obtain the
compound of Formula III.
The coupling agent of the process is preferably
N,~'-dicyclohexylcarbodiimide. The preferred
solvent is anhydrous dimethylformamide.
(F) A novel process for the preparation of a
compound of Formula II wherein R6, n, m, R4, Rs,
and Ar is as defined abové which comprises refluxing
a compound of Formula IV wherein n, m, R4, Rs, and
Ar are as defined above in anhydrous methanol wlth
anhydrous HCl then basified and treated with lower
alkoxy carbonyl isothiocyanate to Qbtain the compound
of Formula II.
(G) A novel process for the preparation of a
compound of Formula IV ~herein n, m, R4, Rs, and Ar
are as defined above which comprises:
step (1) reacting 2-amino-6-chloro-4-pyrimidinol
in methoxyethanol with arylalkylæmine in the presen^e
of triethylamine;
step (2) then treating the product of step (1)
with aqueous ~odium nitrite, step (3) reducin~ ~he
product of step (2) with sodium dithionite in formamide
and 90% formic acid to obtain the compound of
Formula IV.
~2~
EDZ-l~ -9-
Under certain circumstances it is necessary to
protect either the N or O of intermediates in the
above noted process with suitable protecting groups
which are known. Introduction and removal of such
suitable oxygen and nitrogen protecting groups are
well known in the art of organic chemistry; see fo~
example, (1~ "Protective Groups in Organic Chemistry,"
J. F. W. McOmie, ed., (New York, 1973), pp 43ff,
95ff; (2) J. F. W. McOmie, Advances in Organic
Chemistry, Vol. 3, 191-281 (1963); (3) R. A.
Borssonas, Advances in Organic ~lemistry, Vol. 3,
159-190 (1963), and (4) J. F. W. McCmie, Chem. &
Ind., 603 (1979).
Examples of suitable oxygen protecting groups
are benzyl, t-butyldimethylsilyl, methyl, isopropyl,
ethyl, tertiary butyl, ethoxyethyl, and the like.
Protection of an N-H containing moiety is necessary
for some of the processes described herein for the
preparation of compounds of this invention. Suitable
nitrogen protecting groups are benzyl, triphenyl-
methyl, trialkylsilyl, trichloroethylcarbamate,
trichloroethoxycarbonyl, vinyloxycarbamate, and the
lik-.
Under certain circumstances it is necessary
to protect two different oxygens wi~h dissimilar
protecting groups such that one can be selectively
removed while leaving the other in place. The ~enzyl
and t-butyldimethylsilyl groups are used in this way
either is removal~l2 in the presence of the other,
benzyl being removed by catalytic hydrogenolysis, and
t-butyldimethylsilyl being removed by reaction with,
for example, tetra-n butylammonium fluoride.
In the process described herein for the prepara-
tion of compounds of this invention the requirements
for protective groups are senerally well recognized
by one skilled in the art of organic chemistry, and
-. ' ".,
:' . .
.. . ~L~
EDZ-l -10-
accordingly the use of appropriate protecting groups
i5 necessarily implied by the processes of the charts
herein, although not expressly illustrated.
The products of the reactions described herein
are isolated by conventional means such as extraction,
distillation, chromatography, and the like.
The salts of compounds of Formula I described
above are prepared by reacting the appropriata base
with a stoichometric equivalent of the acid compounds
of Formula I to obtain pharmacologically acceptable
salts thereof.
The compounds of this invention may also exist
in hydrated or solvated forms.
The above novel processes beginning with (G) and
proceeding through ~F), and (E), or to and including
(A) to obtain the compound of Formula I wherein Rl is
OH and R3 is ~HCOOR6 wherein R6 is as defined above
may be conducted in a one pot reaction.
Further, the lower alkoxy carbonylisothiocyanate
may itself be added in the novel process (F) above or
prepared in situ by suspending potassium thiocyanate
in acetonitrile and adding methyl chloroformate to the
suqpension for a mixture which is heated at reflux in
the presence of the basified hydrochloride salt of
Formula I in the above process (F).
DET~ILED DESCRIPTION
The compounds of Formula I and intermediates of
Formula II and IV or the present invention exist in
tautomeric forms as purines or suanines as illustrated
below. Both forms ara included as part o~ tne inven-
tion and are indiscriminately described in t'ne
specificaticn.
3~3
EDZ - ' -11-
.
~1 Rl
R3 ~R3
~2N N ~ 2N~N
Ar ~CX2t~ ~) Ar ~C~I2)m ~< )
Guanine Purine
- I
~ CHCNHCOOR6 "
HN ~ _ _ N~ CXCNHCOOR6
l ~ 1 11
}I2N~N/ ~NH H2NJ~N/~ NH
Ar ~C~2) m~ 4 ) A~cH2t~R4\
II II
o OEI
HNJ~N~CHO ~ ~IHCXO
2~\N~ ~ H2~N
Ar (CX2~ 5 ) n ~<R
IV IV
. .
~ .
:~2~ L3~
DZ-l -12-
The term "alKyl of one to four carbon atoms"
means a straight or branched hydrocarbon chain up to
four carbon atoms such as, for example, methyl, ethyl,
propyl, isopropyl, butyl, isobutyl, sec-butyl or
tertiarybutyl. "~ydroxyalkyl of one to four carbon
atoms" means the same alkyl radic:al with a terminal
hydroxyl group.
The term "aryl n in_ludes an unsubstituted or
substituted aromàtic ring such as, phenyl or phenyl
substituted by halogen, e.g., fluorine, chlorine,
bromine, or iodine, alkyl of one to four carbon atoms,
such as methyl or ethyl, hydroxy, alkoxy of one to
four carbon atoms, such as methoxy or ethoxy, or
trifluoromethyl.
The ter~n "arylal~yl~ means an aromatic ring
attached to an al~yl chain of up to four carbon atoms,
~uch as unsubstituted or suDstituted phenylethyl or
benzyl where the substituents on the aromatic ring ma~
be the same as defined above.
2~ The te-rm "heteroaryl" means five- or six-.nembered
aromatic ring containing one or more heteroatoms, such
as nitrogen, oxygen and sulfur. Preferred radicals
are the 2- or 3-furanyl; 2- or 3-thienyl; the ~
or ~-pyridyl; or 2-, 4-, or 5-thiazolyl radicals.
Pharmaceutically acceptable acid addition salts
are those derived from inorganic acids such as
hydrochloricr sulfuric and the liXe, as well as organic
acids such as nethanesulfonic, toluenesulfonic,
tartaric acid, and the like. These salts may also be
3~ prepared by standard methods known in the art.
Pharnaceutically acceptable base salt3 are those
derived from inorganic bases such as sodium hydroxide,
potassium hydroxide or ammoniun hydroxide or organic
bases such as ar~inine, ~-methyl glucamine, lysine
and the like. These salts ~ay also be prepared oy
standard Inethods kno~n in the art.
8~
- EDZ-l -13-
A preferred embodiment of the present invention
is a compound of Formula 1 wherein Rl is OH or SH;
R2 is hydrogen or N~2; R3 is hydrogen, bromine or
N~2; n is zero or one; m is zero or one, where n or m
must be one; R4 and Rs are each independently
hydrogen, alkyl of 1-4 carbon atoms or hydroxyalkyl of
one to four carbon atoms, and Ar is Z- or 3-furanyl,
2- or 3-thienyl, or 2-, 3- or 4-pyridyl, oir 2- or
3-furanyl, 2-, 4-, or_~-thiazolyl, 2- or 3-thienyl,
or 2-, 3-, or 4-pyridyl substituted by alkyl of one to
four carbon atoms, or a pharmaceutically acceptable
acid addition base salt.
Another preferred embodiment of the present
invention is a compound o~ Formula 1 wherein Rl is
~; R2 is NH2; R3 is hydrogen, bromine or N~2; n
is 0 or 1; m is 0 or 1, where n or m mus~ be 1, and
Ar is 2- or 3-furanyl, 2- or 3-thienyl, ~-, 4-, or
5-thiazolyl, or 2-, 3- or 4-pyridyl, or 2- or
3-furanyl, ~- or 3-thienyl, 2-, 4-, or 5-thiazolyl,
or 2-~ 3-, or 4-pyridyl substituted by methyl or
etnyl, or a pharmaceutically acceptable acid addition
or base salt.
Particular embodiments of--the present invention
include:
g-[(3-pyridyl)methyl]guanine;
9-(2-thenyl~guanine;
9-[(2-pyridyl)methyl]guanine;
9-[(5-ethyl-2-thienyl)methyl]guanine;
-8-bromo-9-(2-thienylmethyl)guanine;
8-bromo-9-(5-ethyl-2-thenyl)guanine;
8-bromo-9-(2-furfuryl)guanine,
8-bromo-9-[(3-pyridyl)methyljguanine;
8-amino-9-(5-ethyl-2-thenyl)guanine;
8-amino-9-(2-thienylmethyl)guanine, and
8-amino-[9-(3-pyridyl)methyl]guanine.
"` '
8~13~
EDZ-l -14-
The most preferred compound is 8-amino-9-(2-
thienylmethyl)guanine.
The 8-bromo compounds are not only useful pharma-
cologically but are also useful as intermediates for
preparing certain compounds of the present invention.
The compounds of Formula I may be prepared
according to l~ethods B, A, and/or C as shown in the
following Schemes 1, 2, and 3, respectively.
Generally, Method A is preferred.
3~
EDZ -1 -15-
I. Scheme 1 - ~lethod B
HNJ13[No2 1~t ) Or~t n-e ~ o
H NJ~N Cl ~C~2~10 ~ N2tl N NH,
2 Ar
As
O ' ,0
R~IL3~ b iSrtO 2 ~ H~
E~2NJ~N Cl H2tl H tt pa2520J
6 ~n~)n ~Drr~i~ acld
~CFt21 ID
Ar
O O
tt ~o~nic dcid HNJ~3[NHC~lO
N ~o~amio~R Et 2N N NN
)n
~2) ~ tc~t2
Ar Ar
¦N aS /ACOH
HN~C ~>_8r _ H~ 3C ~ H ~r~ H~ N H2
t2N IN 2 H2
Ç~)n S~lR~)n S~ S~5
I CH 2 ~ H 2 ~ H 2 )~
r I ~ ~ ~ c
no~-n ar:
H 9N~ NHCON~
n
r
;ct
~.2~
EDZ~
II. Method a-~iscussion
Compounds of Formula 8 above may also be used
as starting materials and may be prepared by reacting
2-amino-6-chloro-4-hydroxy-5-nitropyrimidine, the
compound of formula 2 described in J. Chem. ~oc.,
1962, p. 4186, with the appropriate heteroaryl (al~yl)
amine of formula 3 in tne presence of an organic ~ase
at elevated temperatures. The resulting compound of
the formula 4 is then treated with sodium dithionite
-
and formic acid followed by further treatment with
formic acid and formamide at elevated temperatures to
afford the compound of Formula 8.
Alternative, starting materials of Formula 8
may be prepared according to a modified method of
C. ~. ~oelland, X. K. Robins in J. ;~ed. ~hem., 5, 55~,
(1962) starting with a compound of the Formula 5 whicA
is reacted with an ap~ropriate heteroaryl alkyl
amine of Formula 3, then with nitr~us acid to or.
the 5-nitrosopyrimidine, 6, which is reduced and rin~
closed by treatment with sodium dithionite, formic
acid and formamide as described above.
The heteroaryl (alkyl) amines of Formula 3 are
either commercially available or may be prepared by
known methods.
Treatnent of a compound of Formula 8 with
~-bromosuccinimide in acetic acid, dimethyl~ormamide
or ~ethanol ~roduces a compound of Formula la wAlcn
when treated with hydrazine hydrate gives the
hydrazine or directly the 8-amino derivative of
Fornula lb. The reaction of the 8-bromo compound with
hydrazine may or ~ay not proceed entirely to the
8-amino compound ~hus when the 8-hydrazine compound
is obtained, it may be fur~her reacted with ~aney
nicKel to allow tne red~lction to go to completion and
. .
~L~2~
EDZ-~ -17-
afford the desired 8-amino compound. Compounds of
formula _ may be further converted by known
methods to provide R6 substituents of formula ld ar,
where Rl is 0, converting said compound to a compound
of formula lc where Rl is S by reacting the said
compound with P2Ss in presence of a base such a~
pyridine (examples given).
.: ~. , :
~z~
EDZ--1 --18--
III. Scheme 2 - Method A
~F 'L"2
NH~3~ ~22)n tEtlN R~
82N N CL CH~O~OH, ~ tl25 C) 112N N Nll
r~ f lux; 1 8h ~ ~r ---~CN 2~R ,~
IINO~ ¦ H256 2~ 2 ~ SJ n
. Aq N~ O r. t .
A~OH ~-5h; 2
CN30N¦ ~R~) N-~520~;, HCOOH, 2
R n ¦bo~110~ 100-140 1
HCOOH H2NJ~N
J~r~CHl~ ~)n 4 ~R~)n
_"_.. , .. , ~
CH3oNlHcl ~H) ~902
O s)
IV~ NNJ~r 2-2HC~ NJkNN2
112N~NH '~q N2r~NN2 112Nl N hll
2+~< 5)n 6 ~RS)n
' ~Jhorr 51~-lt lif~
¦ R600C:ICS
O--tHNC0046 t tRf, -- C~3 or Zt)
III H H~ t DCC ~NI i iHCOOli
~ C~ R5)n
(n~d n~t l~ol~t-~ (n-~d noC t-ol~t~)
"~ ~N ~--OH/IHCl IIN~N
pllcoo1lo ~ I I \~112~11
N2)~N~H ~ llth~ llzl~N~
~r ICHl~R45)n ._ . R5)r~
. .,
9L2~3~3
~DZ--1 --19--
IV. Scheme 3 - Method C
Cl Ar Br
N NJ~
~ ,
2 3
N~ H2N~
Ar~CH 2~RS)n Ar~C~ 2~R5) n
1) Chromato- ¦ 2) HCl
graph lc
separaeion ~ ~ .
O O O
HNJ~ ~\ N~S HN~ ~ AqNH2N112 HNJ~
¦ I ~> ~ l l l ~ B r -~ H 2
H2N~N--~N~ H N~N~N~ R N~N~Nf
Ar- ~C~2~ ) Ar (CE~2~ 4) Ar~cN2+~Rs) n
6 ~ 8
~L~ 8S33L3~
EDZ-l -20-
GeneraLly, the processes of the present inven-
tion as shown in Scheme 2 above are as follows.
A 2-amino-6-chloro-4-pyrimidinol, that may be
in the monohydrate form, is suspended in methoxy-
ethanol, in the presence of excess amine or an organicbase such as triethylamine. The compound of Formula
XXX having n, m, R4, Rs, and Ar as defined above is
added to the suspension and heated optimally to a
temperature at which the suspension refluxes.
Refluxing is continued until thin layer
chromatography, for example, with 20% methanol in
methylene chloride, shows the reaction producing a
compound of Formula XX wherein n, m, R4, Rs, and Ar
are as dsfined above i9 complete.
The reaction mixture naving the compound of
Formula XX is then diluted with water and treated with
sodium nitrite in the presence of acetic acid. A
nitroso of the Formula X again having n, m, R4, Rs,
and Ar as defined above is obtained from the treatment
by the nitrite as evidenced by a color change and
precipitate. The temp~rature of the treatment is at
about room temperature.
The treatment mixture having therein the nitroso
of Formula X i5 contacted with sodium dithionite in
a solvent mixture such as formamide, formic acid,
at a temperature of from 6C-90C, preferably from 70-
80C. m e tempera~ure is then raised to the boiling
point of the solvent mixture, approximately 130-140C
for up to an hour, ~referably at least 20 minutes, or
when the color of the nitroso containing mixture
described above disappears and an inorganic salt
precipitates. The product of this contact is ~
compound or t~e Formula IV wherein n, m, R4, R5, and
Ar are as defined above.
3'`~ -
EDZ-l ~21-
Subsequently, the compound of Formula IV is dried
and suspended in an anhydrous solvent such as methanol,
ethanol, and the like. The suspension is then treated
with a dry acid such as HCl, to form the acid salt
shown as Formula IVa, wherein n, m, R4, Rs, and Ar
are as defined above, or ~alt corresponding to the
acid used for the treatment.
The salt IVa is basified with a concentrated
mixture of NH40H~and 97% hydrazine to obtain a base
of the Formula IVb wherein n, m, R4, Rs, and Ar are
a~ defined above. The base is unstable, how~ever, i9
dried, for example in a vacuum over P20s.
The dried free base IVb is added to a solution
of R600C~CS wherein R6 is as defined above. The
solution of R600CNCS may be prepared by suspending
potassium thiocyanate in a solvent suçh as
acetonitrile and treating with slightly less than an
equivalent of ClCOOR6 wherein R6 is as defined above
at reflux for about one hour, cooled, then stirred to
insure all of the alkylchloroformate is reacted
before the base IVb is added. The reaction of dried
free base IVb with the R6OOCNCS is mQ~itored to
completion with thin-layer chromatography using silica
in 20% methanol in methylene chloride to obtain a
compound of Formula II wherein R6, n, m, R4, Rs,
and Ar is as defined above.
A mixture of the compound or For~ula II and a
coupling agent such as N,N'-dicyclohexylcarbodiimide,
in a solvent such as anhvd~ous dimethylformamide, is
stirred at about room t~mperature until completion of
t~e reaction is shown by thin layer chromatograph to
yield a compound of Formula III wherein R6, n, m,
R4, Rs, and Ar are as defined above.
39~L3~
FDZ-l -22-
The reaction mixture having the compound of
Formula III and anhydrous potassium carbonate are
suspended in a solvent such as anhydrous methanol,
and refluxed until thin layer chromatography shows the
reaction producing a compound of Formula I wherein R
is O or O~, R3' is NHCOOR6, wherein R6 is as
defined above and n, m, R4, Rs, and Ar are as
defined above.
The compound of Formula I wherein Rl, is O
and OH and R3' is NHCOOR6 may then, if desired be
used to produce by known methods a compound of
Formula I wherein R3 is NHR wherein R is as defined
above other than CR6-
Likewise, a compound of Formula I wherein R
is S or SH may be prepared by known methods fromthe compounds of Formula I wherein Rl is O or OH.
The preparation of compounds IV, II, III, and I
may be carried out in one pot. dowever, separation
and purification of each of the compounds IV, II, III,
or I may be effected by conventional methods, if
desired.
Generally the processes of the present inven-
tion as shown in Scheme 3 - Method C above ara as
follows:
A mixture of 2-amino-6-chloropurine, potassium
carbonate, and the starting material of the formula
shown as 2 in Scheme 3 - Method C, that is generally
commercially available or can be pre~ared by methods
analogous to those known in the art, are stirred under
nitrogen for from about 2 to 48 hours. A mixture of
7- and 9- substituted chloropurines shown as Formula 4
and Formula 5 in Scheme 3 - Method C are obtained.
The desired compound of Formula 4 is separated and
treated with an aqueous acid such as dCl followed
by addition of a weak solution of a base such as ~aOH.
The mixture is heated to assure it is neutralized
~ 28~
- EDZ-l -23-
followed by conventional separation of the desired
product of Fonmula I wherein R3 is hydrogen.
Subsequently, reactions to produce compo~mds of
Formuls 7 and Formula 8 as shown in Scheme 3 -
Method C are described above for corresponding stepsin Scheme I - Method B.
The compounds of the present invention have been
~hown to exhibit significant enzyme inhibition
activity and cytotoxic activity. In the purine
nucleoside phosphorylase (P~P-4) enzyme assay, total
inhibition was achieved at a concentration less than
about 300 micromoles on certain compound~ of the
present invention. P~P-4 activity was measured
radiochemically by measuring the formation of
~14-C]-hypoxanthine from ~14-C~inosine
CBiomedicine, 33, 39 (1980)] using human erythrocyte
as the enzyme source. The same compounds also were
found by a standard test (HTBA-l) CScience, 214, 1137,
(1981)] to be selectively cytotoxic for T-cells in the
presence of 2'-deoxyguanosine at a sLmilar
concentration range and nontoxic to B-cell in the
presence of the same amount of 2'-deoxyguanosine.
Representative examples are shown in the activity
ta~le.
.
.. ~ ~ , . .
,
~2~;3`~
EDZ-l -24-
.
Activity Table
Example Arl ¦ Method ¦ PNP-4 ¦ T-Cell ~dGuo
~umber ¦ LpreparationlICsO(~M)I(10 ~M) IC50 (~M
1 ¦3-PY ¦ B ¦ 21.9 ¦ 54.1
2 or 9a¦2-Th ¦ A or B ¦ 0.17 ¦0.83
3 ¦2-Th-5-Et ¦ B ¦ 0.93 ¦4.15
9b ¦2-FU ¦ B or A ¦ 0.25 ¦2.57
9c ¦3-Th ' ¦ A, B, or C¦ 0.085 ¦ 0.49
10 9d ¦2-Th-3-CH3¦ ~ ¦ 4.05 ¦ 8.6
16Ad ¦3-TH-2-CH3¦ A ¦ 1.72 ¦ 18.2
16Ac ¦CH2-2-Th ¦ A ¦ 6.25 ¦ 17.6
16Ak ¦3-TH-5-CH3l A ¦ 0.63 ¦ 2.8
16As l l A ¦ 8.45 ¦ ~12~5
1~ 1 1 1
15 9e ¦ ~ ¦ B ¦140
_
16Ae ¦2-Th-5-Me ¦ A
16Af ¦2-Py ¦ A ¦ 4.6
lpy = pyridine, Th = ~iophPne, Fu = furan
EDZ-l- -25-
Since T-cells play a central role in immune
response, use of the compounds of the invention is
contemplated for the immunoregulation of autoimmune
disease such as rheumatoid arthritis, systemic lupus
erythematosus, inflammatory bowel disease, multiple
sclerosis, myasthemia gravis, transplantation,
juvenile diabetes, cancer, and viral diseases. The
present invention thus includes composi~ions
containing a compound of Formula I in treating disease
such as autoimmune disease characterized by abnormal
immune response in warmblooded animals. According to
this aspect of the invention, the properties of the
compounds of the invention are utilized by
administering to a warmblooded animal an effective
amount of a pharmaceutical composition containing as
the active ingredient at least about 0.1 percent by
weight, based on the total weight of the composition
of at ieast one such compound of the invention.
Pharmaceutical compositions of the invention can
be formulated in any suitable way, preferably with an
inert carrier for administration orally, parenterally,
ophthalmically, topically, or by suppository.
For example, the compounds o the present
invention are formulated into dosage forms such as
tablets or syrups by blending with an inert pharma-
ceutical carrier such as lactose or simple syrup by
methods well known in the art. For injec~able
dosage forms, they are formulated with vehicles such
as water, propylene glycol, peanut oil, sesame oil,
and the like. I~ these dosage forms? the active
ingredient is from about 0.05 grams to 0.5 grams per
dosage unit.
The present invention is further illustrated by
way of the ollowing examples.
, ' ~
':
3~1
EDZ-l -26-
EXAMPLE 1
9-[(3-Pyridinyl)methyl]guanine
3-Pyridylmethylamine (15.8 ml; 0.1517 mole~ was
added to a suspension of 2-amino-6-chloro-4-hydroxy-
5 5-nitropyrimidine (14.45 g; 0.0758 mol) in isopro
panol (600 ml). The mixture was heated under reflux
for two hours and then stirred overnight at room
temperature when the product, 2-am~no-4-hydroxy-6-
[(3-pyridyl)methylamino]-5-nitropyrimidine,
crystallized out~ The product was fil~ered, washed
with water, and air dried.
The crude nitropyrimidine (25.32 g) from above
was suspended in formamide (150 ml) and 90% formic
acid (5~ ml) and the suspension was warmed to 70C
in a water bath. Sodium dithionite was carefully
added to the ~arm suspension and then boiled for
15-2U minutesO The reaction mixture was diluted with
hot water (300 ml), treated with charcoal and then
boiled for an additional 20-25 minutes, filtered
through celite, cooled and concentrated under
reduced pressure to give formamido-~yrimidine which
was collected by filtration, washed with- acetone,
and dried under vacuum at 56C.
The above product was resuspended in formamide
(100 ml) and formic acid (8 ml) and was heated under
rerlux for 3.5 hours, poured onto 4~ ml ice-~ater
and then filtered. Two crystallization from boiling
water gave the analytical samyle of the desired
product (4.5 g) mp >3uOC.
3~ EX~PLE 2
The procedure described in Example 1 was repeated
to prepare the following 9-(heteroaryl or substitu~ed
~2~
EDZ-l -27-
heteroaryl)methyl guanines, starting from appropriate
hetaroaryl or substituted heteroaryl methylamines~
9-(2-Thienylmethyl)guanine, mp >300~.
9-[~2-Pyridinyl)methyl~guanine, mp >3~0C.
9-(~-Furanylmethyl)guanine, mp 296-299C, dec.
(~nown Compound: J. Am. Chem. ~oc., 1959, ~1:3U46.)
9-[(3-methyl-2-thienyl)methyl]guanine, mp >290C (dec).
9-[(2-methyl-3-~hienyl)methyl]guanine, mp >270~ (dec).
9-[(benzo[blthie`n-3-yl)methyl]guaIline~ mp >3~0C (dec).
9-(3-thienylmethyl)guanine, mp 320-3~2C (dec).
EXAMPLE 2A
2-Amino-9-[(2-thienyl)meth~l]-~-chloro~urine
A mixture of 2-amino-6-chloropurine ~Aldrich
Chemical Co.) (7.~7 g; 0.44 mol), potassium
car~onate (6.64 g; 0.~48 mol), and 3-thenylbromide
(see ~S Patent number 3,74b,72~) (7.~ g; 0.~4~ mol)
in ~F (200 ml) was stirred under nitrogen at room
temperature for 4~ hours. The mixture was filtered
and the filtrate evaporated to dryness under vacuum,
et~yl ether was added and t~e precip1tate was
collected by filtration to give a mixture oE 7- and
9-suDstituted chloropurines. A sample of ~ure 9isomer
~as prepared by chromatogra~hy on silica gel ~ith 5~
methanol/me~nylene chloride as the eluting solvent to
separate it from the 7-isomer. Analytical sample -~as
obtained by crystallization froIn acetonemethanol-
mixture, yield 2.36 g, mp softens at 185C (dec) and
then melts at 203-204C (dec).
~X~IYLE 2~
rhe procedure descri~ed in 2xample 'A was
repeated to ;~re~ara the following 2-~mi~o-9-~(hetero-
aryl or su~stitu.ei heteroaryl)metnylJ-6-cnloro~urines,
~2893L~3~
~DZ-l -2~-
startiong from appro~riate heteroaryl or substit~ted
heteroaryl ~ethylhalides.
2-Amino-9-[(;2,5-dimet~yl-3-thienyl)metn~11-6-
chloro~urine, mp 190-192C (starting material 2,5-
dimethyl-3-thenyl chloride was prepared according to
the lit. proce~ure; ~uu-Hoi and Nguyen-~oan, ~ec.
Trav. ~him, 194~, 68:5).
2-Amino-9-(3-~uran~lmetnyl)-~-chLoropurine
(starting material 3-furfurylchloride was prepared
according to lit. procedure; S. P. Tanis, Tet. Letts.,
1982, 23:3115).
EX~MPL~ 2C
9-[(2,5-dimethyl-3-thienyl)meth~l]~uanine
A mixture of Z-amino-9-[(2,5-dimetnyl 3-
thienyl)~ethyl]-6-chloropurine (3.8 g, ~.0129 mol)
and 2N ~Cl ~as heated on a steambath for 3.0 hours
and then heated under refl~x for another hour. At
the end of this time 1~ i~aO~ solution was added to
the solution till basic and the mixt~re was heated
2U for another five minutes. The mixture was then
acidi~ied with acetic acid, cooled, and ~iltered to
give 3.6 g, of the product. ~n analyticai sample was
obtained by chromatography over silica gel using 10
metAanol/chloroform as eluting solvent, mp >3UuC
(dec).
~XAi~PL~ 2D
The procedure descriked in ~xample 2C was
re2eated to orepared 9-(3-tnenyl)guanine, m~ 3Z0-
322~ (dec).
.
- EDZ--1 --29--
E ,YA.~IP LE 2 r'
9-(3-furanylmethyl)~uanine
The crude 2-amino-9-~3-furfuryl)-6-chloropurine
(4.74 g, 0.019 mol) was suspended in methanol
(175 ml) and a solution of sodiu.~ methoxide, prepared
from sodium metal (1.75 g; 0.07~ g atom), and methanol
(75 ml), was slowly added to the suspension, followed
by 2-mercaptoethanol (6.1 ml = 6.8 g; 0.0~7 mol) and
water (0.35 ~1). The reaction mixture was heated
lU under reflux (N2 atm) for two hours, when an
additional amount of sodium methoxide from 1.14 g
sodium (0.05 g atom) and 25 ml methanol was added.
After an additional 2~5 hours reflux, the reac~ion
mixture was concentrated under vacuum to 75 ml and
then diluted with water (20~ ml), and acidified with
acetic acid (pd 5.5). The white ppt. was filtered,
washed with water, and dried, yield 4.05 g; mp
308-310~.
EX~ LE 3
9-~(5-Ethyl-2-thienyl)methyl~quanine
2-~mino-6-chloro-4-~yrimidinol, monohidrate
(2~.96 g; 0.1193 mole) was suspended in methoxyethanol
(3~0 ml) and 5-ethyl-2-thenylamine (16.5~ 9;
O.li53 mole) prepdred from 2-ethyltnio~nene accocdlng
to the Lit. Procedure, (J~S, 1948, 70:4018) t~as added
- to the suspension. The resulting solution was heated
under reflux for one hour and tAen 16.~ ml of
triethylamine was added and the refluxing continued
for an additional 18 noura~ The reaction mixture was
~oured into ice ~ater (bOO ml), diluted wit:~ acetic
acid (100 ml) and then tredted with a solution of
., .
EDZ-l ~30-
sodium nitrite (16 g) in ~ater (1~ ml). The mixture
was stlrred at room temperature for 1.5 hours and the
resulting salmon colored nitroso compound was
collected by filtration and washed wit~ water.
The crude nitrosopyriJnidine was then reduced
with sodium dithionite in formamide (200 ml) and
90~ formic acid (1~0 ml) at 70C and then boiled
for 20 minutes. The reaction mixture was diluted
with water ~300 ml) and the boiling continued for
an additional 30 minutes, filtered hot, and then
allowed to crystallize in the refrigerator. The
crude N-formyl derivative (28 g) was collected by
filtration, washed with ~ater and air dried and then
cyclized witb formic acid (1~ ml) and formamide
(10~ ml) at reflux te~perature for four hours. The
hot reaction mixture was ~oured into 500 ml of
ice water to give t:~e cr~de quanine which was then
purified by dissolving in boiling 1.5 ~ treatin~
with charcoal and thesl precipitatin~ with alnmonium
hydroxide. rhe crude yuanine was t:~en redissolved
in hot 1 ~ .~a~ solution, treated witn cnarcoal,
filtered and the filtrate acidified witn acetic
acid to give the desired product ~hich was used
in tAe next step without ~urther purification.
EX~PL~ ~
The procedure descrioed in ~xample 3 ~as re2ea~ed
to prepare 9-~2-thenyl)guanine, m~ >30~~ starting
from 2-al~ino-6-chloro-~-pyrimidinol and 2-thenyla~ine.
~23~ 3~
~DZ-l -31-
2X~PLE S
8-Bromo-9-(2-thienylmethyl)quanine
N-3romosuccinimide (2.~2 g; 1~.7 mmol) was
added to a cold (0C) suspension of 9-~2-thenyl)
guanine (3,5 g; 14.1 mmol) in DMF (1~0 ml) and
the mixture was stirred ~or 30 minutes at 0C
and then at room temperature for 24 hours. The
reaction mixture was then diluted witn 75 ml of
water and filtered. Recrystalllzation of the
lU product from D~F gave the analytical sample,
yield 3.1 g; mp ~9~-295C (dec).
EX~MPLE 6
The procedure described in 2xample 5 was re~eated
to prepare the following 8-~romo-9-[(substituted
heteroaryl)methyl]guanines, starting from appropriate
9-L(substituted heteroaryl)methyl]guanines in each
case.
8-oromo-9-~5-e hyl-2-thienylmethyl)guanine
8-bromo-9-(2-furanylmethyl)guanine, mp >3~0C.
8-bromo-9-[(2-methyl-3-thienyl)methyl]guanine, mp
>280C (dec).
3-bromo-9-~(o2nzo[blthien-3-yl)methyl]guanine, mp 25~-
2~0C (dec).
~XA.~PL2 7
8-Bromo-9-[(3-pyridin~)methyl]guanlne
N-Bromosuccinimide (1.59 g; 8.95 ~mol) was addea
to a suspension of 9-[(3-pyridyl)~ethyl~guanii~e
(2.0 g; 8.14 i~mol) in glacial acetic acid (20 ml) ~n~
the mixture was stirred at room emperature LOr
3U 3.5 Aours. The reaction ~ ture ~as concentrated
,
~2~L3~1 -
EDZ-l -32-
under reduced pressure and then diluted with water
and fil~ered. The crude product was triturated with
water, filtered, and washed with water and dried.
Yield 1.91 g, mp ~300C.
EX~PLE 8
.
8-Amino-9-(5-ethyl-2-thien~lmeth l)guanine
A mixture o~ 8-bromo-9-(5-ethyl-2-~enyl)guanine
(6.5 g; 18.3 mmol) and 60% aqueous hydrazine ~200 ml)
was h~ated to reflux under nitrogen atmosphere for
20 hours. 2-Methoxyethanol (50 Ml) was added and the
refluxing continued for an additional 48 hour~ in the
open air. The orange-brown solution was cooled,
diluted with water (150 ml) and allowed to crystallize
in the refrigerator overnight. The crude product thus
obtained was converted to the hydrochloride salt by
recrystallizing from boiling isopropanol and 1 N HCl.
Yield, 0.49 g; mp 215-218C, dec.
~MPLE 9
The procedure described in E~ample 8 was repeated
to prepare the followiny 8-amino-9-[(substituted
heteroaryl)methyl)guanines, starting ~rom appropriate
8-bromo-9-~(substituted heteroaryl)methyl]guanines:
8-Amino-9-~(3-pyridyl)methyl]guanine, mp >300C and
additionally, the following compounds a through e
were prepared.
a. 8-Amino-9-(2-thenyl)guanine or 8-aminc-9-[(2-
thienyl)methyl]guanine as hydrochloride sal',
0 5 ~2~ mp 223-226C (dec).
b. 8-Amino-9-(2-furanylmethyl)guanine monohydro-
chloride, 1.0 H20, m~ 197-19~C (dec).
c. 8-Amino-9-[(3-thienyl)metnyl]suanine, monohydro-
chloride, monohydrate, mp 275-278C ~dec).
~28~
rDZ~~ -3~-
d. ~-Amino-9-[(3-methyl-2-thienyl)methyl]guanine,
0.25 ~O, mp 290C (dec).
e. 8-Amino-9-[(benzo [D] thien-3-~l)methyl]guanine, 0.5
H2O, mp >300C (dec).
Starting materials, such as 2-, 3~, or 4-
pyridylmethylamines, 2-thenylamine also called 2-
(aminomethyl)thiophene or ~-thiophene methylamine, and
2-furfurylamine are com;nercially availa~le (for
_ example, Aldrich ~hemical Company). The suDstituted
thenylamines were synthesized from the su~stituted
thiophenes using a general literature procedure.
(~. D. ~artough and S. L. Meisel, J. Am. ~hem. Soc.,
19~8, 70:4018).
2-Amino-6-chloro-4-hydroxy-5-nitroQyrimidine
lS was synthesized according to a method described
in the literature (A. Stuart and H. C. ~. ~ood,
J. Chem. ~oc., 1963:11~6),
2-~nino-6-chloro-4-~yrimidinol monohydrate
was purchased from Aldrich Chemical ~ompany.
~ PLE l~
2-A~ino-4[~(2-thienyl)meth~l]amino~ (formamido)-
6-~yrimidinol ta compound of ~cheme 2 Formula I'J
wherein n is one, m is zero, R~ and R~ are hvdroqen,
Ar is ~-thien~
2-~nino-6-chloro-4-pyrimidinol, monohydrate (85~,
100.0 g, 0.5191 mole) was suspended in metho~yethanol
(700 ml) and 2-thiophenemetAylamine (9~, 61.3 g,
0.~1~7 mole) is added to the suspension. The mi~ture
was heated under reflux for two hours and then 73 ml
3~ (d = 0.725, ~.52 mole) of triethylamine ~as added and
the refluxing continued for an additional 13 hours.
(The reaction was followed by TL~: 20~ metnanol-
C~13.) The reation Inix_ure was roured lnto ice water
(1~00 ml), diluted with acetic acid (~00 ml), and tnen
- ~8~
EDZ-l -34-
treated with a solution of sodium nitrite (80 g,
1.16 mole~ in water (300 ml). The mixture was stirred
at room temperature for four hours, and the resulting
reddish colored nitroso compound (X) was collected by
filtration and washed ~ith water (the reaction was
followed by observing the color change in the forma-
tion of the precipitate).
The crude nitrosopyrimidine of Formula X,
(of Scheme 2, Formula X, wherein n is one, m is zero,
R4 and Rs are each hydrogen and Ar is 2-thienyl)
prepared above was divided into two batches and each
in turn was then reduced with sodium dithionite (>90~
70 g, 0.36 mole) in formamide (300 ml) and 90~ formic
acid (300 ml) at aooc and then boiled for 20 minutes.
The temperature was approximately 130-140C at this
point. The reaction was complete when the red color
completely disaQpears and inorganic salt precipitates.
The reaction mixture was diluted with water (300 ml)
and the boiling continued for an additional 30 minutes,
filtered hot, and then allowed to crystallize
in the refrigerator. The reaction was monitored to
completion by TLC (sio2; 20~ C~3O~ in C~C13. The
crude ~-formyl derivative,
2-amino-4~(2-thienyl)methyl]amino~-5-(formamido)-6-
pyrimidinol, (100 g) was collected by filtration,washed with water, and dried and used in ~he next step
without further purification in most cases.
E~MPLE lOA
~he procedure dessribed in ~xample 10 was
repeated to Qrepare 2 ~mino-[~(3-thienyl)-
methyl~amino]-5-~formamido)-6-pyrimidinol starting
from 3-thiophene methylamine and 2-amino-6-chloro-4-
~yrimidinol.
~. ; .
.:.
- ~L2~
EDZ-l ~35~
E.YAL~PLE 11
~-Amino-4-[[~2-furanvlmethvl)amino]-5-(formamido)-6-
pvrimïdinol
A mixture of 2-amino-6-chloro-5-ni~ro-4-pyrilni-
dinol (J. CHem. ~oc., 1962, p 4186) (31.5 9;
0.15 mol) methanol (1~00 ml) and Eurfurylamine
(2~.1 g; u.3 mol) was stirred and heated under
reflux (i~ atm) for six hours. The reaction mixture
was cooled, filtered, washed with water, and air
dried to give 34.43 g of yellow solid, mp 286-2~gC
(dec), which was used in the next reaction.
The crude nitropyrimidlne (33.9 g; 0.135 mol) was
suspended in formamide (2g~ ml) and 88-~ formic acid
(145 ml), and then warmed up to 80~. Sodlum
ditAionite (~7 g; 0.327 mol) was slo~ly added to the
war~ (8b-850C) susoension over a period of 50 ~inutes,
maintained at the tem~erature (~ 85C) lor another
3~ minutes, then diluted with boiling water (12~ ml),
and heated the mixture around 850r for ano~her
~u minutes when tan colored crystals were ~ormed.
The product was filtered off, washed ~it~ ~ater, and
dried over P20s under vacuum ~vernight. ~ield,
23.~ g, mp 24~-247C (dec). In most cases tnese
compounds ~ere carried through the reaction se~uences
2~ witAout characterization.
E~2LL~IPL~ 11~j
~ he procedure descriL~ed ir. ~xample 11 was
repeated to pcepare the ~ollowing 2-amino-4Ll(heter
aryl or substituted heteroaryl)methyl]amino]-~-
3u (f~rmamido)-6-pyrimidinols, (~'a~ie 1) starting Crom
appropriate heteroaryl or substituted neteroaryl
methylamines and ~-amino-6-chloro-~-nitro-4-
pyrimidinol.
- ~21!~
EDZ-l -36-
T~L~ 1
NHCHO
~N / ~
N--~N~H
Ar
Ar or Ar
2-Thienyl
S
3-Thienyl
'' ' ~,3 "'
3-Furanyl
~9
1~ (2-thienyl)methyl
,~
--E2C S
3-;~e-2-thienyl
H3C
~ e-3-t~ienyl
,~3
EI3 C S
,
.D~.-l ~37~
TA3~E 1 (C3NT'~)
~r or Ar
5-1~le-2-thienyl
~ S CH3
5 2-Pyri~ inyl
Bel~zoLb~thien-2-yl
2-tniazolyl
1~ _ N
/(~S~
4-thiazolyl
N/
(~-thienyl)methyl
H2C ~
15 5-Me-3-thienyl Ps ~ c~3
12~
EDZ-l -38-
TA~LE 1 (CO~
Ar or Ar
5-l~e-2-furanyl
C~3
4-i~e-3-thienyl CX3
S
4-~e-2-thienyl CH3
/~S
EX~MPL~ 12
2,5-Diamlno-4-[(2-thienylmeth~l)amin~]Qyrimidin~6-ol,
dih~drochloride (a com~ound of ~chelne 2, r~?nnula I~la,
wherein n is one, ~ is zero, R4 and Rs are edch
hydrogen, and ~r is 2-thienyl)
~he crude N-formyl derivative as prepare~3 in
Fxam~le 1~ aDove (40 g, D.1508 .-nole) ~as ~uspended
in anhydrous methanol (500 ml) and a stream of dry
~Cl (g) was passed tnrough the solution wnile r.ea~ing
th- mixture at reflux. The reaction was continued foc
2.5 hour when a clear solution was forme~ fo110wed by
2u a crystalline precipitate. The mixt~re was cooled
i!l all ice bath and then filtered to give tile salt,
2,5-diamino-4-[~2-thienyl.~e~nyl)amin~]~yriinidin-6-ol,
dihydrochloride, (~.6 ~). Concentration of the
mothec li~uor gave an addl~-onal amount of tne sal~
.
:-
~DZ-l -39-
(8.65 g). Total yield, 37.25 g (79~). The material
was carried on without further purification.
Alternatively, the N-formyl derivative was
refluxed with 5~ methanolic-~Cl (g) to give the desired
diamine- 2 HCl salt.
EXA.~PLE 12A
The procedure described in ~xample l~ w~-~epeated
to prepare the ~ollowing 2,5-diamino-4-L[(heteroaryl
or substituted heteroaryl)methyl]amino~æyrimidin-6-
ol, as dihydrochloride salt (Table ~), starting fromappropriate 2-amino-4- L L (heteroaryl or substituted
heteroaryl)methyl]amino~-5-(formamido)-b-pyrimidinol
(Table 1).
TABL~ 2
0
HN ~ 2
HCl
~2N ~ N NL
Ar
Ar or ~r
3-Thienyl
2-Furanyl
o
3-~uranyl
~3
,
- lZ~L3~ -
- ~DZ-l ~40-
TA~LE 2 (CO~T'D)
Ar or Ar
(2-thienyl)methyl
~¢~
--H2C S
5 3-Me-2-thienyl H3C
~3
2-Me-3-thienyl
~\
H3C ~ S
5-Me-2-thienyl
~
~/~
2-Pyridinyl
1~N - )J
s2nzo[~]thien-2-yL
~5r
2-thid7olyl i~
~S~
. .
.
.~ .,
: -,
" ', ~ '' : '
.
~28~
- ~.DZ-l -41-
TA~L~ 2 (CONT'~)
Ar or Ar
4-thiazolyl N
S
(3-thienyl)~ethyl
--H2
S-!~le-3-thienyl
~ '
S CH3
5-:~e-2-furanyl
O CX3
4-~e-3-thienyl
~ H3
4-~e-2-thienyl
~3
S~
,
~, ~"' .
: "
:'
EDZ-~ 4~
EX~PL~ 13
~ethyl [[[2-Amino-1,6-dihYdro-6-oxo-~-[(2-thien~l-
methyl)amino]-5-e-yrimidinylJamino]thioxome~h~1]-
carDamate (A cor~lpound of Schene 2, Formula II
wherein Rh is meth~l, n is one, M iS zero, R4 and
R~ are eacn hvdro~en and Ar is 2-thienyl~
The crude di;lydrochloride salt as prepared
in ~xample 12 above ~37.2 g, ~ mole) was suspended
in water (300 ml) and then basified witn a mixture
l~ of concentrated N~40~ and 97% hydrazine ~3:1)
(40 ml) to give the free base which was dried ovar
vacuum over P20s for 20 hours. Yield 26.1 g (975i)
of the base shown as compound of ~cheme II,
Formula IV~ wherein n, m, X4, ~5, and Ar are as
defined above. This free base was unstable.
A suspension of potassium thiocyana~e (l~
O.i~6 mole) in acetonitrile (25~ ml) was treated
with methyl chlorofor~ate (99~i) (13.8 ml, 0,177 mole)
and the mixture ~as heated at reflux for one Aour,
cooled, and then filtered to remove inorgallic s21ts.
The oright yellow colored filtrate is~ 3tirred overni~ht
at room te.n~erature under nitrogen. Care should oe
taXen so that all of the methylchlorofor~ate has
reacted '~efore proceeding. The dry base (26.1 ~) ~as
added to the solution of methoxycarbonyl isothiocyanate
and the stirring continued for 36 nours at room
temperat~re under nitroyen. ~he reaction was moni-
tored to completion hy TL~ (si~2; ~ C~3~ in
CHCl3). The product was filtered of_ and -~asned
3~ ~ith ;nethanol to give th~ thiourea derivative, metAyl
[[[~-amino-1,6-dihydro-6-oxo-4-[(?-tnienyl.nethvl)-
amino]-5-pyrimidinyl~alnino]thioxomethyl]car3ainate.
Yield 37.4 g (96~), mp 225-2~C; (~ ure by
HPLC).
- . ~28~3~
EDZ-l ~43
Alternatively, the nitro or nitrosopyrimidines
were ca~alytically reduced and rea~ted i~nediately
with ethoxycarbonyl isothiocyanate to give ~he thlo-
ursa derivative.
This material was carried on to the next step
without further purification.
.
E~AMPLE 13A
. ._ _ ~ . . .
The procedure describ~d in Exampla 1~ was
repeated to prspare the fol}owing methyl ~or ethyl~
[LL2-anino-l~6-dihydro-6-oxo-4-cL(heteroaryl or
5 ubstituted heteroaryl)methyl~amino]-5-pyrimidinyl]-
amino]thioxo-nethyl]car'oamate (Table 3) stArting
from appropriate 2,5-diamino-4-C.(heteroaryl or
substituted heteroaryl)methyl~anino]pyr~nidin-6-oL,
dihydrochloride ~alt (Table 2).
. .
- ~28913~
EDZ -L ~44~
TABLE 3
HN ~ NH--C-NHCOO R6
H2N~NJ~ NH
AY'
Ar orAr R6
2-Thienyl ,~3 Et
S
3-Thienyl ~3~ CH3
2-Furanyl ~3 CH3
-
3-Furanyl ~ CH3
mp 246-249C
O ( dec)
~2 -thienyl ) methyl ~ C~I3
--H2C~l\S~ 234-239 C
2--~e- 3 -thi enyl ~ C~ 3
np 235-237C
C \S/ ( ~ec)
-, ~
'". :
: ' :
EDZ~ 5~
TABLE 3 (CONT'D)
Ar or Ar R6
5-Me-2-thienyl ~ CH3,
S ~ CH mp 227 230C
(dec)
2-Me-2-thienyl " , Et
2-Pyridinyl ~ CH3
Benzo[b]thien-2-yl ~ CH3
2-thiazolyl ~ ~ CH3
S mp 211-215C
15 4-thiazolyl ~ ~ CH3
mp 217-218C
(3-thienyl)methyl ~ CH3
20 5-Me-3-thienyl ~ c~3
S CH3
~2~g~3~
EDZ-l ~4~
TABLE .~ (CONT'~)
Ar or Ar R6
5-Me-2-furanyl CH3
~ mp 228-229C
\ O ~ CH3 (dec)
4-Me-3-thienyl ~ CH3 C'~3, Et
S
4-Me-2-t~ienyl ~ c~3 CH3, Et
S
. EXAMPL~ 14
M~thyl ~5~amino-7 [(2-thienylme nyl)~nino]-
oxazoloC5,4-dJ~yrimidi_-2-yl]carbamato (52e
Scheme 2, Formula III wherein R~ is methyl, n is
one, m is zero, R4 and Rs are each h~dro~en
and Ar is 2-thieny~)
A mixture of the thiourea derivative as
pre~ared in Example 13 ~35 g; 0.096 mole) and N,N'-
dicyclohexylcarbodiimide (DCC) (~9.4 g, 0.288 mole)
was suspended in dry DMF (1800 ml) and stirred at
room temperature for 24 hours. The course of tne
reaction was followed by TLC (SiO2, 20~, C~3O~
in CHC13). The DMF was compls~ely strippe-~ off
under vacu~m and the -esi~ue .-iturated twic~ with
., ~ . :
., .
: .
~2~9~3L3~3
ED~ 47-
C~2C12 to give tne desirad carbamate, methyl [5-
amino-7-[~2-thienylmethyl)amlno~oxazolo[5,4-d~-
pyrimidin-2-yl~carbamate. Yield 27.~ g (90~),
mp 00C. Purity 97.6~ (~PLC).
This material was carried on to the next step
without further purification.
E~MPLE 14A
The procedure described in Example 14 ~as
repeated to prepare the followin~ methyl (or ethyl)
1~ [5-amino-7-[~(heteroaryl or sub~tituted heteroaryl)-
methyl]amino]oxazolo~5,4-d]pyrimidin-2-yl]carbamate
(Table 4) st3rting from appropriate methyl (or ethyl)
~2-amino-1,6 dihydro-6-axo-4-[~(hateroaryl or
substituted heteroaryl)methyl]amino]-5-pyrimidinyl]-
lS amino]thioxomethyl~carbamate (Table 3).
- ~2~ 3~38
EDZ--l --4 8--
TABLE 4
~NI}COOR6
H 2NJ" L Ar
Ar ~ or Ar R6
2-Thienyl ~3 Et
S
3-Thienyl ~3 CH3
2-Faranyl CE13
3-F-~lranyl i CH3
mp 28~3-291 _
0 (dec)
1~ .
( 2 -thi ~nyl ) methyl CH 3
//~ mp ~270C
--H C/~S / ( dec )
23 2-Me-3-t;-1ienyl \~ C~3
~/ ~ mp >270C
H 3C \S ( dec )
. ~
EDZ-l -49-
TAB T .~ 4 (CONT'D)
Ar or Ar
5-Me-2 thienyl CH3, Et
~ p ~250C
~ \ S~~~C~ (dsc)
2-Pyridinyl ~ C~3
BenzoLbjthien-2-yl ~ I ~ CH3
.
2-tniazolyl N ~ CH3
/~S~ -
4-thiazolyl N ~ C'~3
~ ~
(3-thienyl)methyl CH3
---H2C~____
~S~'
5-L~e-3-thi2nyl CH3
~ S ~ C~3
~L2~9~L3~
EDZ-1 -50-
TABLE 4 (CONT'D)
Ar or Ar R6
5-Me-2-furanyl CH3
,~
/ \ O ~`CH
5 5-.~e-2-thi~nyl ~ C~3, ~t
S CH3
5-~e-3-tnienyl ~ ,CH3 C~3
~S~ '
4~e-2-thienyl c~3 Et
_ mp ~2~0C
E ~I~LE 15
~ethyl [2-amino-6,9-dihvdro-6-oxo-9-(2-thieny1methyl-
~ urin-8-yl]car~nate (See ~che~e 2, For~ula I
wherein R~ is NHCOOR6,~he~ Rs is ,neth~1, n is
one, In is zero, R4 and R~ are each hydro~en an
Ar is 2-thien~1)
.
A mixturs of the oxa~olocarbamate as prepared
in ~xalnple 14 (25 g, 0.078 mole) and anhydrous K2CO3
was su~pended in anhydrous ~ethanol and hea.3d to
reflux for eiynt hours. 'Fne course or t'ne reac~ion
9~3~3
EDZ-l -51-
is being followed by the TLC system mentioned above.The reaction mixture was then evaporated to dryness
under reduced pressure and the residue dissolved in
ammonium chloride solution (16.8 g; 0.312 mole in
200 ml of water). The resulting precipitate was
collected and dried giving 24.:39 g of the metnyl[2-
amino-6,9-dihydro-6-oxo-9-(2-thienylmethyl)-lH-pyrin-
8-yl]carbamate, somet mes contaminated with the
8-amino compound, i.2., in this example, 89.58~
carbamate and 9.54~ 8-amino compound, 8-amino-9[(2-
thienyl)methyl]guanine of Formula I wherein R3 is
NH2
m is material was carried on to the next step
without further purification.
EXAMPLE 15A
The procedure described in Example 15 was
repeated to prepare the following methyl (or ethyl)
[2-amino-6,9-dihydro-6-oxo-9-[(heteroaryl or
substituted heteroaryl)methyl-l~-purin-8-yl~-
carbamate (Table 5) starting from appropriate methyl(or ethyl) t5-amino-7-~(heteroaryl or substituted
heteroaryl)methyl]amino]oxazolo~5,4-d~pyrimidin-2-yl]-
carbamate (Table 4).
~, ..
39~L38
.
EDZ-l -52-
TABLE 5
N
HcooR6
H2N N N
Ar
Ar orAr ~
2-T~ienyl Et, ~np
~ >250C (dec)
S
3-ThienyL C~3
2-~uranyl C~3
' ~ ~ .np >300C
~ o / (dec~
3-~uranyl ~ CY.3
~p >270C
0 (dec)
(2-~ienyl)methyL c'~3
~2C S
. 2-~'12-3-t.liesly~ C~13
20,
~ C~ \S~
. . , , ' ,:
' , ' ` ~' '' " ''
: ' ' ' .
12~3~3L.3~
- ED'Z-l -53-
TABLE 4 ( CONT ' ;) )
Ar or Ar ~6
5-Me-2-thienyl CH3, Et
~/~ illp >250 C
~\S/\C~3 (dec)
2-Pyridinyl 1~ CH3
N
3enzo~b]thien- -yl [~ CL{3
2-t~i~zolyl N~ CH3
S
4-th~ azolyl N--\~ C~3
l~S/
(3-~nie4yL)~e:hYL --r42C~3 c~3
5-M2-3-t~ienyl ~ Cli3
S C~I3
.3~3
- EDZ-l ~54~
TABL2 4 (CONT'~)
Ar or Ar R6
5-Me-2-furanyl C~3
~ mp >250C
/ \ O ~ C~3 (dec~
4-.~e-3-thienyl~ -CH3 CH3, Et
S
4-Me-2-tnienyl~ CH3 C~3 Et
Il
/\S/
EXAMPLE 1~ -
8-.~ino-9-[(2-t~ienyl ? .-nethyl]~uanine (See Sche,ne 2
Forinula I ~herein R~ is ~, n is one, l~ is zero,
R4 and ~ are each hvdro~en and Ar i~ 2-~hienyl)
The crude carbamata as ~reparsd in Exa,nple 15
(a9.5~ t'ne carbama~e ;~lus 9.5~ the ~-a~-n~no colnpound
(20.39 ~; 0.064 nole) r~ the previous reac.ion is
suspended in isopropdnol (125 ml) and 1 U ~Cl ~125 ml;
0.125 mol~) anil the mixture heatad at reflux for -20
hours (the reaction is ,nonltore~ '~y TLC (SiO2:20~
~e'~H in C~C13, CH3C~:~.OAc:H2O ~:l:l)j, wnen a clear
solutio.n is for~e~. On _oolin~ the ?roduct
crystallizss ou~ 'ro~n tna solution as the
... ..
EDZ-l ~55~
hydrochloride sal~ of 8-amino-9[(2-thienyL)-
methyl~guanine. Yield 15.1 g (76~). Purity 98~ by
~PLC, mp 219-222~C (dec).
The hydrolysis was also c:arried out in 10~
methanolic sodiuln hydroxide solution under reflux
tenperature, followed by neutralization and recrystal-
lization from appropriate solvent.
EXAMPLE 16A
. , .
The procedura described in Example lo wa~
repeated to prepare the following 8-amino-9L(hetero-
aryl or substituted heteroaryl)methyl~guanines
(Table 6) starting f-om appropriate methyl (or ethyl)
[2-amino-o,9-dihydro-o-oxo-9-[(neteroaryl or
substitlted heteroaryl).nethyl~ purin-8-yl~-
carbamate (Table 5).
TABL~ 6
2 ~ `'H ~
Ar or X ,~pC
2-Fur~nyl ~ '~Cl 253-7
a. 3-~hianyl ~r--~ H~ 20 275-d
S
3~
E~Z-l -;6-
TA3L~ 6 (CONT'D)
Ar or Ar X mpC
b. 3-Furanyl ~ H l 293 4
~ ~ (d)
S O
. . _ . . .
c.(2-thienyl)methyl ~Cl H20 153-5
(d)
- H 2 S
10 d. 2-~e-3-t~ienyl ~ '.~Cl 26~-~
H3C ~ S
e. S-Me-2-t~ienyl -~Cl 0.25 ~260 -
* ;,~0
lS ~ S C~3
f. 2-Pyridinyl ~ 1.5 -HCl 270-2
N ~ - 0.25 J2 (-i)
~. 3enzolojthien-2-yl 0.25 ;~2 ~300
~ ~ (d)
. ; :
12891 38
EDZ--1 --5 7--
TA~LE 6 ( CO~T ' ;~ )
Ar or Ar X ~pC
h. 2-thiaz~lyl N 1.2 HCl >250
~3 1.2 ~2
S
i. 4-thiazolyl N ~ 1.1 HCl ~250
~ ~ 0. 3 ~2
j . ( 3 -thienyl ) methyl 0. 9 EICl ~ 177-~3
H20 (d)
~2C~,
k. 5-Me-3-t~ienyl HC1 0.5 H20 212-;
\ (d)
l; ~
- S /~C~
L. ;-~e-2-furanyl ~Cl-1.15 212-~
~ ~2 (d)
/ ~ o CX3
12~ 3~3
EDZ-l -5~-
T~3LE 6 (CONY"D)
Ar or Ar X mp C
m. 4-Me-3-thienyl 0.~ HCl ~240
~ C~3 1.25 H20 (d)
n. 4-Me- ~ ienyl ~
E~IPL~ 17
2,~-Dianino-l,9-dihydro-9-(2-thienyl~neth~1-6H-~urine-
6-t~ione
A mixture of P25s (2.4 g: 10.95 mmol),
pyridine (30 .~1) ~nd 8-amino-9~2-t;~ienyl)methyl~-
guanine (1.5 g; 4.a7 mmol) was heated under reflux
l; for 4.5 hours an~ then poured int~ 200 ml of boilin~
water and boilad for one nour. The mixture was
allowed to stand at room t2mperature overnight. The
precipitated solid ~as collected, diss31ved in 1 ~
NaOH, traated wl~h ac~ivated charcoal, filtered, and
2~ t;~en acidiried with ylacial acetic acid to pH 5.4.
ne precipitated solid was c~llected, ~is301ved in
1 ~ HCl, t~eated with ac_ivarad charcoal, filtered,
and .~eutralized wit~ NH4~H to pH 7.~7 to giv2
5~2 m~ o_ the deslred product, ~p >300C.
- - 12~9~38
EDZ-l -59~
EX~PLE 17A
The procedure described in Example 17 was
rapeated to prepare tne following 2,8-diamino-1,9-
dihydro-9-[(heteroaryl or substituted heteroaryl)-
methyl]-6H-purin-o-thione, starting from appropriata
8-amino-9[(heteroaryl or -ubstituted heteroaryl)alXyl]
guanine.
2,8-Di~ino-1,9-dihydro-9-(3-thienylmethyl)-6H-
purine-6-thione, 0-5 H2O~ mp 275~ (dec).
2,8-Diamino-1,9-dihy~ro-9-~2-(2-thienyl~ethyl]-
6a-purine~6-~ione, 0.25 H2O, mp >260C (dec).
EXAMPLE 1 a
2-~ino-?,9-dinydro-9-(2-thienylmetilyl)-lH ~urine-
6,~3-dione
lS ~ mixture of a-oro~no-9-~(2-t;~ienyl)methyL]-
guanina (see Exa~ple 5? (3.12 g; 9.56 mmol), acetic
anhydride (7; ml), glacial acetic acid (75 ml) an~
annydrous sodium acetate (14.9 g, 0.1816 mol) was
heated to reflux for 20 hours. The ~ark solution
which formed ~aJ then evaporated to dryness under
reduced pressura. ~he re-idue was -lissolved in
aqueous methylamine (150 ml), --tirred at room
tenperature for 4a hours and then heated to reflux for
2.5 hours. The methylamine -~as ~i~tilled off under
reduced prassure and the residue was racrystallize~
from ooilin~ methanol-~ater mixture to giv9 1. 23 g of
the analytical pr~duct, mp ~300C.
... . . ..
- ~L28~3~3
~DZ-l -60-
~X~MPLE l~
2-~ino-l,7,8,9-tetra'nydro-9-(2-thienylmet'nyl)-~-
thioxo-6~-~urin-6-one
A mixture of ~-bro~o-9-C(2-thienyL)methyl]
guanine (see Exarnple 5) ~2.0 g; 6.13 mrnol), ~MF
(250 ml), and thiourea (0.93 g; 12.26 mmoL) was ~neated
under r~flux for 20 hours dnd than the solvent was
evaporated to dryness under reduced ~ressure. The
residue was dissolved in ~J ~aOH, treated with
charcoal, filtered, and acidified with ~lacial AcOH to
yive a pala yellow solid. ~nalytical sample was
prepared by repeatin~ the ~urifica~ion ~roces~, yield
70~ mg; mp >280C.
EX~MPL~ 20
,~ 6,9-dih~ro-c,-oxo-9-~2-thianylmet'ny~ urin
2,8-di-yl]bis aceta.nide
.~ mixture of a-~ino-9-t(2-thianyl)methyl~-
guanine (0.5 g; l.88 r~ol), ~F (l0 ml), pyri~1ine
(5 ~1), and ace~ic anhydri~e (5 ml) was stirred at
~room te~nperature for 36 Aours. The mi.~ture was
~ilute~ with ether (50 ml) and filtered to ~ive
analytically pure product, Inp 243-~~'.
STARTI~G MAT~R~La
Starting material~ are prepared a~ 'ollows usi.~g
a known proce~1ure or following a ~ro~e~ure analogous
o that ~own in t~e art.
5-MethyL-2-~iienyl.net.lyla~ine, H. oar-~gh, et al,
J. .~n. Cnem. Soc., 19~, 70:~01~.
aenzo[b~t:'nio~en-2-yl-.methyl~nine, ~. S~.irley,
30 et ~L, J. A~. Chem. Soc., l9~2, 7~:664.
~2~3913~31
~ EDZ-l -61-
3-~ethyl-2-thienylmethylamine, H. Hartough, et al,
J. ~a; Chem. Soc., 194~, 70:~01~.
BenzoLb]thio~hen-3-yl-methylamine was prepare~
by Gabriel Synthesis from the correspondin~ chloro-
compound (W. King, et al, J. Org. Chem., 1948,3:635).
2-~ethyl-3-thienylmethyl~nine ~as prepared by
lithium alu.-ainum hydride (LAH) reduction of the
correspondin~ nitrile (M. Janda, et al, Coll. Czech.
Comm., 1974, 39:959).
2-(2-thienyl)ethyLamine and 2-(3-thienyl)ethyl-
amine were preparad by the lit. method of `i. Hertz, et
al, in J. ~a. Cnem. Soc., 1951, 73:351.
2-Methyl-4-thienyl methyla-aina was preparad by
~H reduction of 2-methyl-4-cyano-thiophene which was
oreyarad fro,n the correspondin~ 4-oromo compound
(~. Goldfarb, at al, Zh. Obs. ~nim. 1964, 34:969)
and CuCN.
3-Metnyl-4-tnienylmethylaraine was s~ilarly
orepared as follows:
Br ~ ~ x3 H2N-H2C ~ -C~3
~ -Met~yL-2-~ienylaethyl~-ai.le ~as ~reoared by
~d reluction or ~he corresoondl^.g aldox~e.
2- an~ 4- T~iazolylm6r yl ~aines were ~re~ared
~ccording to the iiterat~ ~ ~roce~ur@s (~. G. Jo..es,
e' al, J. ~m. Cnem. ~oc., 1950, 72:45~6).
r .~ 39~L3~
E.l~Z -1 -62-
F O RMULA
~;N
<4)
~N
E~2N N~NH , III
P~r ~C~2t~ ) n
~U~NHCNHCOOR6
H2~J ~N NH II
Ar (C~2~R4 )
5 n
o
J~NHCHO
2N `N~NH
.~CH2 51 n
,, . :.
