Note: Descriptions are shown in the official language in which they were submitted.
W 0 93/07$~8 21 2 1 9 ~ O PCT/US91/0890~
ADENOISE DIPHOSPHOKIBOSE POLYMERASE BINDING NITROSO AROMATIC `~
COMPOUNDS USEFUL AS ANTI-TUMOR AND ANTI-RETROVIRAL AGENTS
Fi~ld of ~he I~io.n
The present invention relates generally ts the ~:~
field of retroviral therapeutic agents and their :~
use in treating viral infections and can~ers.
More specifically it xelates to those
therapeutic agents which inhibit A~P-ribose
transferas~, and in particular variou~ nitroso-
benzopyron~s, nitroso-isoquinolinones and
ni~roso-benzamides. ;~
'.
BackqrQund of ~he Invention
The enzyme ADP-ribose transfera~e (ADPRT)
(E.C.4.2.30) is a chromatin-bou~d enzyme located `~
in the nucleus of mo~t eukaryotic cells. ~he
enzyme catalyæes the polymerization of ~he ~DP~
ribose moiety o ni~otinamide adenine
di~ucleotide ~(NAD~) to form poly lADP-ribo~e~
The polymes is co~alently attached to variou~
nuclear protein~, includi.ng the polymerase
i-tself.
~.
; : The many varied roles that ADP-ribosylation
plays in cellular metabolism ha~e made ~DPRT :a `:~
target for drugs essentia11y use~ful~ for ~'
combating-~ neoplasla ~and viral lnfections.
Numerous physiological ~activi~îes have been
detected for compounds that : inhibit the
30 ~ polymerase activity of ADP ~ . ~Such a~:iviti~S
include a~cell cyc1e depend~nt prevention of ,
carcinogen-induced ma1ignant t~ansformation of
: human :fibroblasts (Kun, E., ~irsten, E.) Milo,
.E. Kurian:~, P. and Kumari, H. L. (1983)~Qç~ .
N~l. Ac~;. S~i. U~ ~Q:7219-7223), con~erring
also carcinogen;resistance (Milo, G.E., Kurian,
~P., Kirsten, E. and Kun, E. (198~3 E~ L~-
332-336), inhibition of malignant
trans~ormation in hamster embryo and mou~e
: : ''
W~93/07~68 PCT/US91/~902
212~0
--2--
C3HlOTl/2 cell cultures tBorek, C., Morgan,
W.F., Ong, A. and Cleaver, J.E. ( 1984) ~QQ,
N~tl . ~ad, SC~ ;A 81: 243-247 ~, deletion C: f
transfected oncogenes from NIH 3T3 cell~
(Nakayashu, M., Shima, H., Aonuma, S., Nakagama
H., Naga~. M. and Sugi.mara, T. (1988)
Na~ a~ ~sci. U~ ~5:9066-9070), suppression
of the mitog~nic stlmulation of tumor promoter~
(Romano, P., Menapace, L. and Armato, V. (1983)
10Çarci.nQ~nesls 9:~147-2154), inhibition of
illegitimate DNA recombinations (Waldman, B.C.
and Waldman, ~. (1990~ ~L~ L8 - 18:59~
5g88) and int~gration (Farzaneh, F., Panayotou,
G.N., Bowler, L.D., Harda~, B.D., Broom, ~.,
Walther, C. and Shall, S. (1988~ ~çl~
Res. 1~: 11319-11326), :induction of sister
ohromati~ ~xchange ~ ~:Ikushima, T. ~1990)
Çh~nmnzcr~ ~:360-364~ and the loss of certain
: ampli~fi~d oncogenes (Grosso, L.E. and Pitot,
~0~ H:.C:. (1984)~ Bio~he~. .Bi~ hys. Res. ommun-
473-480; Shima, H.,~Nakayasu, M., Aonums,
S ., Sugimura ,~ T . and Nagao,; M. :~198~) ~LQg~
N~tl ! ~Acad. Sci U$~ 6:7~42-7445).
25 :~ : Compounds~nown to:inhibit AD~PRT polymerase
:: a~t:iv~ty ~in~lude~benzamide (Kun~ ~E., ~lrs~en,
; E., Milo~ G~.E.~ Kuri~an, P. and;Eumari~, H~.~ L.
1983) ~ 80:7~19-7~:23
substituted benzamid~s (Borek, C~., Morgan,~W.F.,:
3b~ Ong, A. and ~Cl~aver, ~.E. i(l984~ ~Y5L_l~a~
~~L:243-247i Romano~, F., Menapace,
:~ ~ L.~:and Armato, V. (1983)~Carcino~enesiQ 9; 2147-
: ~ 2154; Farzaneh,~ F., Panayotou,;~:~G.N.~Bowler,
L.D.~, Nardas, B.D.,~Bro~om, T., Walther, C.~and
. 35~ ; Shall, ~ c1988) ~D~o ~si~hL ~ :11319
~ 11326.; Grosso,~ L.E. and Pitot, H.C. ~1984)
: ~ ; :iQQb~m. BiQ4hy~ Res.~ Com~n. 11~:473-480,
..
~ ~ '
:: :::: ;::~
W~93J~7868 PCT/~S91/0~9~2
2l2l~a
:
-3-
Shima, H., Nakayasu, M., Aonums, S., Sugimura,
T. and Nagao, M. (1989~ Pxo~.. Natl. ~cad Sci.
~&~ 86:7442-7445), 3-aminonaphthylhydrazide ~:
(Waldman, B.C. and Waldman, A. (1990) ~
Acid~ _~e~ 5981-5988), isoquinoline,
~uercetin, and coumarin (1,2-benzopyrone~ (Milo,
G~E., Kurian, P., Kirsten, E. and Kun, B. (1985)
EE~Lhç~. 17~: 332-336). The anti-trans~iorming
and anti-n~oplastic effect of 1,2 benzopyrone
were d~monstxated i~ Yi~Q and La Vi~Q (T6ang,
et al., tl987)
Other known ADPRT polymera~e activity
inhibitors include 5-iodo-6-amino-1,2- :
benzopyrone as d~scribecl in U.S. Patent
applioation Serial No. 600,S93, filed October ~
19, 1990 entitl2d "Novel 5-Iodo-6-Amino-1,2- :::
senæopyrone~:and thelr Metabolites Useful as
Cystostatic and Anti-Viral Agents^' for use as
~ anti-tumor and anti-viral agents. The cited
: patent discusses the possibility of using 5-
iodo-6-nitroso-1,2-benzopyrone as an anti-tumor ~i~
: or anti-viral ag~n:t. ::
~,.
The 6-nitroso benzopyrones have ~Q~ b~en -
~:
hitherto known or descri~ed. The only ~emotely
related compounds found in the litexature are 6-
nitro-1,2-~enzopyrone and 6 amino-1~2-
benz~pyrone (6-ABP3 (~ ~L__f~ _ Jao
~ 615 (1923)) for which, only scarce medicinal
~;
evaluation~ has been reported. In particular,
tes~ing was done for sedative and hypnotic
effects (J. Pharm. _Soc. ~apan, 13:351 (1953);
~ 271 (19S4)), hypothermal action
(:Yak~q~ Za~shi, 78:491 (1958)), and
antipyretic, hypnotic, hypoten~ive and
::
W0~3/07~6~ P~T/US9l/08902
2 1 2 1 ~ O O
adrenolytic action (Ibid) 83:1124 (1963)). No
significant application for any of these
compounds has been de~cribed except for 6 ABP.
2-nitrosobenzamide (Irne-Ra~a, K.M. and
Koubek, E. (1963) ~ _5~L_I~b~m. 28:3240-32~1),
and 4-nitrosobenzamide (Wubbels, G.G., Kalhorn,
T.F., Johnson, D.E. and Campbell, D. (lg82~ .J.
Q~ _ÇhQm. 47:4664-4670), have been reported in
the chemical literature, but no commercial use
of these i omer~ i^e known. Neither of these
articles ~u~g~st the use of ~itrosobenzamid~s as
ADPRT inhibitors.
The anti-viral ana ant~-tumorig~nic actions of
substituted and unsub~tituted 6-amino-1,2-
benzopyrone and 5-iodo-6-amino-1,2-benzopyrone
is the ~ubject of copending U.S. patent
applications Serial No. 5B5,231 ;filed on
20~ September ~ 21, 1990 entitle~ "6-Amlno-1~2-
~enzopyrones U~eful for Treatmen~ of Viral
Diseases" :and Serial No. 600,593 filed on
October l9,:1g90:entitled i'Novel 5-Iodo-6-Amino-
1,2~-Benz~opyrones and Thelr M~tabolites~U~eful~as
: : ~ 25 ~ Cytostatic ~and AntiviraI Agents"~ which are
: ~ ~ incorporated hereln by refer~nce. ;
: : : ThP. precursoL moleculs, 1,2-benzop~rone
: :~coumarinj, was ~hown to he an:inhibitory ligand
~0 of l adenosinediphosphor~bosyl t~rans~era~le
(ADPRT), a DNA-binding ~uclear protein present
:
: in all mammalian:cells (Ts~ng,:~ al~.,;(lg37)
PrQ~. Nat.~A~ad _S~i USA, 84:1107-llll).: ~`
; ; ~; ~ ~ :',
3$ ~aka~, et ~ .~ ~EBS~ ., 21~:7~ (1987) has :
:sh~own that 6-amino~ benzopyrone (6-ABP) ~i.nd~ :
speciii~ally to ADRPT at the site that also
. .
W093/~7868 PCT/~S91/08~02 ;
2121900
!
--5--
binds to DNA, indicating that both 6-ABP and DNA
compete for the same site on ADPRT. Synthetic
ligands of ADPRT inhibit DNA proliferation,
particularly in tumorigenic cells, (Kirsten, et
5~l-, (1991) ~ _ Çell. R~ 4~-
Subsequently, these ligands were found to
i~hibit viral replication and are the subject of
the copending U.S. patent application entitle~
"6-Amino-1-2-Benzopyrones useful for Tr~atment
of Viral Disea~es," Serial No. 585,~31, filsd on
September 21, 1990 which îs hereby incorporatea
by refere~ce.
Thus it is of int~re~t to provide ADPRT
polymerase activity inhibitors or use~as anti-
viral a~d anti-tumor agents. The-: sub~eet
invention provide~ for novel nitroso-1,2-
benzopyrone, nitr~oso-benz;~mide and nitroso-
;; isoquinolinone::~ compounds, and Yarious20 ~: structural~ly related o~her nitroso compounds for
e 8 anti-viral and anti-tumor therapeutic
~agents. The compounds taught for use:~in the
subject ~ nvention ~are believed to~ be
gnifi~can~ly: l:ess toxic ~and far more (500 to
25~ ; 1000 :fold)~potent: than structurally analogous
amino compounds. ~ `
Sum~ t~ IDV en
Th~ sub~ect invention:proYides fsr ~ovel anti~
3~0 ~ tumo~ and anti-viral compounds. These compounds
:in~lude ~ 6-ni.tro~o-1~ : 2-~enzopyro~é, 3-
itr~osobenza~mide, 5-ni~troso-~1:(2H)~
isoqulnol~inone,~7-nitroso-1~(2H)-isoquinolinone,
8-nit~o~o-1(2H)-isoquinolinone.~: Ths invention~ :
;~35 ~ :a1~o provides for compo~itions containing one or
: ~~ more:o~ t~e compounds, and for methods o~
.
: :: treating viral in~ections and cancer with these
::::~ ::
.:~
W0~3~07868 PCT/US91/08902
2121~00 ~
-6- -~
compounds and compos.itions.
Also provided for ar~ methods of treating
cancer and viral infections with 2- --
nitro~obenæamide and 4-~itrosobenzamide.
Composition containing one or more of the~e
compounds is al~o provid~d for.
D~ riptio.n cf Figur~6 ~.
Figure 1 i~ a graph comparing the degr~e of
AD~PT polymeras~ activity ~ADPRP) inactiv~tlon
exhibited by different concentrations of 6-
~itroso 1~2-benzopyrone, 3-nitroso be~zam~de,
and nitro o-1[2H)-isoquinQlinones (~OQ) (a
mixture of the 5 and 7 nitroso i~omers).
: , .
Fig~re 2 is a compositei of graphs di~playi~g :~:
the inhlbi~ory effeots of the ADRPT ligands on
) 855 2 ells la cell ~line~of human~B-cell ~,
20~ linèage acute lymphobl~stic leu~emla~, (B) N9
cells~(a c:ell line of human T-oell lineage acute
: ly~pho~lastic;leukemia~, (C) HL-60 cells (a oell .~; 1ine~of ~uman ac~t~ nonlympho~lastic~leu~emia~
and~(D)~K562~oells~(a oell;~line~.~of human~chronio
25~ ;:: myelogenou;6 le:ukemia3~ These~ ~c~lls~;~werc :
: culture~ whil:e:un~er ~he influen~e of th~ ~rowth
Eaotors~in~ 0%~fetal;~ovlne ~rum (~FCS~), whereas:~
: : :~ ;:~in (E) and~(F) thc 855-2 oe:11s were:cultured~ln~
the presence of autocrine growth actor actiYit
i 30~ (A~F) or low molecular weight B-cell growth~
faotor ~(BCGF~, a T-oell derived lymphokinc)
resp2CtiVQly ~ s.7
~ ~ ~ : Figure 3 ~i~s ~a graph showing the inhlbition of
: ;35~ creasing lcvels~of :leukemio cell growth (ln:
~ :response to~inoreaslng concentrations of FCS) of .
:~ .
i
W~93/07868 PCT/US91/089~2
2121~9ûD
I
-7-
855-2 cells by 6-nitroso~ -benzopyrone (NOBP~ -
and 3-~itrosobenzamide ~NOBA).
Figure 4 is a graph showing that NOBP and NOBA :~
inhibit the ability of human leukemic cells
(855-2 and HL-60) to form colonies (CFU) from
single c lls in a semi-solid medium.
Figure 5 shows graph of the r01ative
inhibitory effects of anti-leukemic dosss of
ADRPT ligands on the ability of (A3 normal ~:
rhesus bone marrow stem cells or (B~ human
peripheral blood stem cells to form coloni~s ln
soft agar. Note that the NOBP and NOBA had
lS minimal effect on normal cells. :~
Figure 6 shows graph~; displaying the
inhibi~ory ef~ects of NOBP, NOBA and NOQ on f~ur
human brain ~umor cell line~
20:: Figure 7 is a graph comparing t~e
e~fe~tiveness:of NOBP with vincri6~ine. :~
'~.;`
Figure ~ is a g:raph displaying t~e effects of
NO~P, NOBA and MOQ on human breast tumor cel~
: 2s li~e M~A 468. ~ -.
: Figure 9 is a graph displayinq the ef~eots of
NOBP, NOBA and NOQ on murine leukema ~ell line
L 1210. ` :
~ De6~ri~ipn_cf SpQcifi~ ~mbQdimQn~
, .
The ~ubject in~ention pro~ides for ~everal
: :: nitroso aom~ounds that are ADPRT pol~mer~6e
:: activity inhibitors. These a~pounds find use
a6~anti-tumor and antl-viral compounds.
~:
~;
'..
WO93/07B68 P~T/US91/08902
2121900
~8- :~
Compound (I) has the following formulaO -~
~4--y~
R3 }~
S wherein ~, ~, ~, R~, ~, and ~ a~ selected
from th~ ~roup con~isting of hydrogen and
nitroso, and only;one of ~ , R4, ~, a~d
is a nitroso group.
~ ':`
A preferred embodiment of compound I i5 where
R4 is the nitroso gxoup, i.e., the molecule 6
nitro~o-1,2-be~zopyrone.
Compound II~has~the formula~
15;~: :(}I~
0;~ ~ ~wherein~ 2~:and ~are ~selected~rom the
group~consisting of~ hy~rogen~:and:nitroso,:~and~
only on~of~ , and~RJ:~Is:a~D~troso~gro~p.
2~5~Compound~ has the~formula~
R5
:.
~;
WC~93/07B68 PCT/US91/08gO2
2121~00
! -:
_g_ :
wherein ~, ~, R3, R~, and ~ are selected from
the group consisting of hydrogen and nitro60 t
and only one of R1, ~, ~, R4, and R5 is a
nitroso group.
Preferred embodim~nt~ of compound III are
where either R~ or ~ is the ni~roso group, i.~
5-nitroso-1(2H)-isoquinolinone and 7-nitroso-
1(2H~-isoquin41inone, re~pectively.
The disclosed synthPsis for 5-nitroso-1(2H~
isoquinolinone may produc~ 2 closely related
structural isomers, 7-nitroso-1(2H~
isoquinolinone and 8-nitroso-1(2H~-
isoqui~olinon~. Although experiments testing
the biological activity of 5-nitro~o-1(2H)-
iso~uinolino~ may ha~e contained significant
quantities of 8-nitroso-1(2H)-isoquinolinone or
20: ~ 7-nitro~o-1(2H)-isoquinolinone, all three
isomers are b~lieved to:possess similar anti~
tumor and anti-viral activity on the basis of
their cIose ~tructural :similarity. This
: : : hypQ~hesi~ may b~ ~ conveni~ntly ~ted by
;~~25: ~ s parating~ the isomers by thin layer
chromatography or similar methods, and comparing
the anti-tumor and~anti-~iral:activities of the
: : separated compounds.
3~ Detail~zd synthesis of 6-nitrofio-1~2
benzopyrone:, 3-nitroso-benzamide, 5-nitr~so~
1(2H~- isoqui-nolinQne ~ 7-nitroso-~1(2H):-
i~oquinol1nonez, and 8-nitroso-1(2H)-
iso~uinolinone~ are providea in the ex~mple
: 35 section bslow.
,.
W~93/0786~ PCT/US91/O~gO~ .
2121~00
-10-
In general, the nitroso compounds of the
subje~t of invention may be synthesized by
oxidizing a corre~pondlng amino compound to~a
compound of the ~ubject invPntion by oxidation
S with 3-chloroperoxy~enzoic acid (or other
pexoxyacids) in ethyl acetate or a halocarbon
solvent. Syntheses of thes~ precursor amino
compounds are described in the chemi~ l
literature and some of the compounds are
commercially available. Some precur~or amino
compound~ for oxidation to nitroso compounds of
the subject inven~ion are as follows: 3-amino-
1,2-b~æopyrone tSpectrum Ch~mic~l Mfg. Corp. t
Gardena~ CA 902483; 4-amino-1,2-b~nzopyrone
(Aldrich, Rare Che~i¢al Catalog3; 5-ami~o-1,2-
benzopyro~e (by reduction of 5-nitro-1,2-
benzopyrone, Çh~=L~ E~ ~7 16536d (1952)3; 7-
amino-1,2-benzopyrone ~Gottlieb, ~ al.,
~hem. Soc.~ ~P~rkin~_~r~ns. I 435 (~1979)); 8-
amino-1,2-benzopyrone lby reduction of 8-amino-
-benzopyrone, ~bdel~Megid, e~ al-, ~gy~~
~hQm~ 20:453-462~(1977)), and. 4-amlno-1(2H)-
i~quinolinone, by redu~tion of:~ the
: : correspond~i~ng~4:~nitro analog (Horning~
~1971) Ca~ J. .~hem. 49::2785-2796).
In ~ddltion to compounds (l) to (III), the~
subject in~ention contemplates various
structurally related compounds that have similar
` ~ : 30 carcino~tatic and/or antl-viral activities
These ~tru~turally related compounds could~be~
c~nv~nien~ly scre~n~d ~on the ~basis of: their
: highly pot~ent~lnhibitory~ ef~ect on ~ADPRT
~ polyme*ase activity. Structurally related
compounds o~ ;int~rest include derivatives
: : substituted by: additional nitroso groups and
small, e.g., ~C1-C3 alkyl groups. A1BO of
:
W~93/0786g PCTJUS91/08~02
~21~)0
interest are various nitroso substituted
stxucturally related heterocyclic ring~ such as
3 , 4 - d i h y d r o - 1 ( 2 H ~ - i 5 o q u i n o 1 i n o n e s ,
nicotinamides, pthalhydrazide~, and 1,~-
benzoxazine-2,4-diones.
Another aspect of the compound~ of th~ ~ubject
invention are the ea~e with which they permeate
cell membranes and their relative absence of
lQ non spe~ific binding to protelns and nucl~ic
acid.
In practiae, the ADPRT polymeras~ i~hibitors
of this inventio~, namely compounds (I) to
(III), and any of their pharmaceuti~ally
acceptable salts, may b~ adminis~tered in
amount~, either~alone or in combination with
each other, and in the ph rmaceutical form which
will be suf f ic ient and effective to 1nhibit ~ ~ 20 neoplastl~ gro~ h or v:iral replication or
prevent ~he development of the cancerous growth
or vlr~1 inection in the mammalian ~o~t.
:.
Admi~istration of the active compounds~and
6alts described herein can be Yl~ any of the
: accepted modes;of admlnistra~lon for therapeutic
agent~. These methods include s:ys:temic or looal
: administration: such a~ oral, parenter~
tran~dermal, subcutaneous, or topi~al
administration modes. The preferr~d method of
administxation o these drugs is intravenous,~
: except in~ tho~ cases :where th~ subjec~ has
.
topiaa~ ~:umor~ or lesions, ~where the topi~al
~ ~ admin~tration~ may : be : proper. In other
~ a~oes, it may be ~ecessary to adminl~ter the
oompo3ition in other parenteral or~even oral ~`
forms.
'
'~
W093/07~68 PCTJVS~1/08902
2121900 ~ :
-12
Depending on the intended mode, the
compositions may be in the solid, se~i-solid ox
liquid dosage form, such as, for example,
injectables, tablets, suppositorles, pills,
time-release capsules~ powders, liquid~,
suspensions, or the like, preferably in unit
do~a~es. ~he compositions will include an
effecti~e ~mount of at least one of compound~
(I) to (III), or pharmaceutically acceptabl~
~alts t~ereof, and in addition it may i~clude
any conventional pharmaceutical excipientæ and
other medicinal or pharmaceutical drugs or
agents, carr~ers, adjuvants, diluents, etc., as
cuætomary in the pharmacautical sciences.
'".
~or solid compositionæ, in addition to the
oompouna~ tI) to (III), such exGipientæ as~ for
: exampl~, pharmaceutical gradss of mannitol,
:~ 20: laatose, s:tarch, magnesium stearate, sodium
: saccharin, talcum, cellulose, glucose, ~ucrose,
, ~
magnssium:carb~na~e, and the like may~be used.
~he compounds of:th~e subject invention may be
als~ formuIate~ ~as:; suppositories using, for
~25 example,~ polyalkylene glycols~ for example,
propylene glycol, as the carrier. ~ ~ ~s
:: Liquid, partioularly in~ectable compo~itions
can,:for example, be prepared by dissolving,
~30 dis`persing, etc., at least one of act~è
compounas (~ o ~III) ~in a pharmaceutical ~;
solution such~ as, for example,~water, saline,
aqueous dextrose, glycerol, ethanol,~DMS0 and
the like, ~to thereby form the in~ctable ` ~-~
3~ : solution or ~uspension.
~'~
~:
:
W093/07~8 PCT/U~9l/08902
2121~
-13-
If desiredO the pharmaceutical composition to
be administered may also contain ~inor amou~ts
of nontoxic auxiliary substances such as wetting
or emulsifying agents, pH buff~ring agent~, and
other substances such as, for example, ~odium
acetate, triethanolamine oleate, etc.
.
Parenteral injectable administration is
generally used for subcutaneou~, intramu~cular
or intravenous injections and infu ions.
Injec~ables can be prepar~d in convsntional
~orms, either as liquid solutions or suspensions
or solid forms suitable for dissol~ing in liquid
prior to injection.
A more recently devi.sed approach for
parenteral administration employs the
implan~atlon of a slow-release or sustained-
r~lease systems, which assure~ that a constant
level of d~sage ls maintained, accoxding to U.S.
~Patent No. 3,710j795, which is incorporated
herein by reference.
:
Any of the above pharmaceutical composition~
: may contain 0.1-99%, preferably 1-70% o~ the
: ~ 25~ active ingredient.:
: Actual method~ of preparing such dosage forms
are known, ox will be apparent to those skill~d
' in this art, and ~are ~described in detail in~
R~mina~Q 'Q ~h~rmacçu~i~al~ ien~, : Mack
Publishing~Company, Easton, Pennsylvania, 17th
Edition, 19~5. The ~omposition or ~ormulation
to be adm1nistered will, in any event, contain
such quantity of the active compound(s) that
will ~ssure: ~hat a therapeutically effective
amou~t will be delive~red to a patient.
W093/07868 PCT/US91/08902 ~
21 21!~0~
-14- -
therapeutically effective amount mean~ an amount
effective to prevent development of or to :-~
alleviate the existing symptoms of the subject
being txeated. ~;
The amount of active compound administered
will, of course, be dependent on the subject ~-
being trea~ed, on the subj~ct's weight, the
~everity of the affliction, the mann~r of
administration and the judgm~t of the -~
~0 prescribing physician. HoweverO an effective --.
dos~ge may b~ in the range of 1 to 12 mg/kgJday,
preferably 1 to 5 mg/kg/day, given o~ly for 1- -
2 days at one treatm~nt cycle. Generally, the
upper limit for the drug dos~e determination~
it~ ~fficacy ~ balanced with its pos~ible
toxicity.
The i~vsntion having been descri~bed, the
ollo~in~ examples are off~red to illustra~e the
~20~ subJect invention by way of illustration, not ~y ~.-
way o limita~ion.
: EX~YPL S;:
~ ~ lU5~ J
An example of a~method fsr the preparatlon:of
6 nitroso-1,2-benzopyr~n~s ~is provided as~
: ~ollows~
30~ To a stirred s:olution~ of :6-amino~ 2-
~b~enzopyrone~hydrochloride (~.00 g, 20 mmol) in
water~(40~ml) ~at ~2'C was: added a æo1ution o~
: ~odium tung~ate (5.93 ~, 20 mm~1) in water ~(20
: ~ : m1~):f~110wed by 30% a~u~ous hydrogen peroxide (5
;~35~: ml)~and s~irring~was continued for 1.5 hours.
: The oxida~ion product was extracted rom the ~
~ :'
~93/~7868 PC~/V~91/OX902
2121L900 '.:
-15-
green-colored mixture with two lOO ml volumes of .-~
ethyl acetate, the combined extracts washed with
O.l N HCl (50 ml) and than water (lOO ml). ~he
ethyl acetate was removed by rot~ry evaporation
and the residue recrystallized rom warm ethanol
(250 ml~
Analysi~ of Rea~iQn P~oduc~
The green crystals obtained from the ~`
recrystallization ~t~p (l.48 g, 42~ yield)
displayecl light a~sorption at 750 nm
characteri~tic of monomeric arylnitroso
compounds. Mass: ~pectrum: m/z (~elative .:
intensity): 175 (M+, ~loo?, 1~1 ( 16.88), 145
(33.77), 133 110.38), 117 (S6.09), 89 (7~.71), ,
63 (57.13). High re~olution data for the M+
peak: calculated for C~H5NO~: l7500268; ~ound:
l75.0271 (de~iation - i.l ppm). 1H-NMR (CDCl3,
: ~: 300:MHz3 ~ (pp~) from TMS:~doublet (6.S72 and -. 6.604) H-4 split:by H~3; ~doub1~t ~7.~472 aDd
:~ : 7.5013 H-8 split by H-7; doublet of doublet~ . `
(7.860/7.866 and ~7.8B9/7.798) H-7 split by H-8~ j`.
and ~inely~ ~pl1t ~by~H:-5; double~ (7.91~0 and
; 7.9~423 H-3 split by~H-:4; doublet (8~.308 and ~
25~ 8~.315)~H~5~fine1y ~plit by H-7. ~V/VIS spectrum ~ ~ d
: in e~hanol, ~:max (~)~: 75~ nm (46)j 316 nm 1~.96
x:103), 27~ nm (2.24~x lO4):. Melting Poin~:~ The
:compound polymerizes a~ove I60-;C, blacken~ and~
melts in the range of 325-340 C.
~ 30: This nitroso-compound:may also be prepared by~
react1ng 6-~amino-1,2-benzopyrone (as the fxee :
:ba~e) wi~h 3-chloroperoxybenzoic acid in ethyl
aceta~e or halocarbo~ solvents.
: ~ .
~.
~ 3/0786~ PCI~/~Sgl/0~902.
2 1 2 1 9 0 0 ! ~
-16
II. Synthe~is of 3-nitrosobenzamide
To a stirred solution of 3-aminobenzamide
(Aldrich Chemical Co.) (0.476 g, 3.50 mmol) in
ethyl a~etate (50 mL) at ambient temperature wa~
added 1.208 g of 3-chloroperoxybenzoic aaid
(commercial grade, 50-60% purity, Aldrich~,
whereupon the solution turn~d green. After 10
minutes the mixture was extracted with 0.14M
aqueous sodium bicarbonate (58 mL), washed with
three succ~ sive 40-mL portions of water, dri~d
over sodium sulfate, then reduced in voluma to
20 mL by rotary evaporation and placed in the
freezer t-20~C), whereupon the product slowly
deposiked a~ a iight y~llow solid during a
period of 72 hours ~0.180 g, 34% yield~
Th~ 2-nitro~obenzamide and 4-nitrosobenzamide
i~omers may be similarly prepared by p~rforming
the abo~e:oxidat~on on 2-aminobenzamid0 and 4-
20~ aminobenzamide, respectiYely.
Melting poi:nt: The substance darken~ above~
13~5 ~, softens~and apparently polymeri~es in:the
:: 25~ range::lS0-1~60 ~, and melts at 240-250^C (with
decompo~ition3. In solution~th~ compound ~ 1B
:: gre~n-blue. ~ Ma~s: spectrum: m/z ~relative
: in~ensity): 150 (M+,lOOj, ~36 (10.9), 12Q
. ~ I
(77.2), I03 (31.6)j 92 (46.5), 85 ~:2~.83, 71
:~ 3b (33~3). Hi~h resolution data~or the M~peak: :
:~ : calcula~ed~fr~C7H6~22: 150.042928; found:
~1$0.042gOO;~deviation = 0.2 ppm~. NMR~spectrum~
,.
1H-NMR t~MSO-d6,~300 MHæ) ~ (ppm:) from TMS: broad :
: ~ sin~l~t~(7.737) N-H; t (7.824, 7.850, 7.~75) H- :
~ ~5 sp~it ~y H-4 and~H-6; d (8.059 and 8.086~
6 split:by H-5;~d~(B.357 and 8.383) H-4 split by
:H-S:;: s (8.472) H-2. The single~ at 7.737
: :
:
.
~0~3~(~7X6X PCT/US91~90~
2 1 2 ~
corresponds to 1 proton; th0 second N-H proton,
spectrally non-equivalent in this compound, is
overlaid by the doublet of H-4. This doublet
integrates to 2 protons and can be re~olved by
addition of D20 to ths DMS0 solution. W-VIS
absorption spectrum in absolute ethanol, ~max
~ 750nm ~37.6), 304nm ~5.35 x 103) and
218nm (1.50 x 104~. An absorptlon maximum at
750nm i5 charac~eristic of monomeric aryl~itroso
compound~
III.
l(~H)-I~oquinolinone (isocarbostyril)
(Aldrich) was nitrated using a general m~thod
for i~oquinoline compounds (C.G. ~e~evre and
R.J.W. LeFevre, ~. Chçm. ~Q~. 1470 (1935)). The
nitration product ~a mixture of the 5-nitro and
7-nitro isomers, as assig~ed by Y. ~awazoe and
Y. Yoshioka, Çh~m__~hg~DL_~yll. (Tokyo3 16:715-
720 (196B), althou~h one:of the isomers could be
the 8-nitro isom rj was then reduced to the
: : corre~ponding amino-1(2H)-isoqui~olinones using
a combination of potassium borohydrid~ and
~ palladium-on-carbon catalyst in aqueous
: m~thanol.~ To ~ the resultant amino-1(2H)~
~ isoquinolinones (as free ba~ses) (0.560 g, 3.50
: mmol) in ethyl acetate :(175 mL) a~ 30 C was
.~
3.0 1 added 1.208 g of 3-chloroperoxybenzoic:a~id
~Aldrich). The mixture became cloudy and after ~ -
20 ~inute~ it was ~iltere~, extracted with 0.14M
sodium bicarbonate (5~8 ~L), washed with two 50-
mL portio~ of water, and dried o~er sodium ;.
: 35 sulfate. The volume of the solution was reduced
to 50 mL by rot~ry evaporation and then pl~ced ~.
in the freezer (-20 C), w~ereupon an orange ~;
solid product was deposited (0.102 g).
.
:~;
W093/~7X6X PCT~VS91/08902.
2l2lsno 1 ,-.,
-18- ~
Analysis of_R~action Product ~;
Meltiny point: substance darkens above 175-C, -~
softens, blackens and ~pparently polymerizes
above lg5'C, and finally melts in the range 310-
335 C. NMR analysis: lH-NMR (DMSO-d6/D20, 300
MH2) ~ ~ppm~ from TMS: m (6.723, 6.741, 6.752); ~
m (7.511, 7.518, 7.533, 7.539, 7.~47, 7.559, ,
7.577, 7.585); m 17.663, 7.674, 7.686. 7.698,
7.707); d (7.818, 7.846). In the absenc~ of
D20, the compound also di~pl~ys a b~oad 8ingl~t
at 11.90 ppm. The i~omeric components were
analytically resolved by thin-lay~r
chromatog~aphy (silica gel plates, ethyl acetat~
solvent), givi~g two bands, ~ 0.82 and ~ 0~72.
Mass spectrum for ~ ~ 0.82: m/~ (relative
intensity): 174 tM~, 100), 160 ~2~.8), 144
: ~93.0), l17 ~9;0;.8~, 97 l(2~.91~ 89 (96~1)J 71
~24.1). Hi~h resolution data for the M+ peak: ~`
calculated fo~ C~H6N2O2- 174004292B; ~ound:
174.043200 :(deviation =~-0.3 ppm). For the :;~
: component ~having ~ 0.72, M+, calculated fox
: CgH6N2O2 174.04~928;~Found: 174.043200 (devia~ion
= -1.6 ppm).~ ~These data confirm that the
compounds ar~ mono-nitroso isomers.
I~. ADPRT I~active S~udi~
The compound~ of the subject inv~ntion were
: tested for their ability to inactivate the
~ 30 polymerae~ activity of adenosinediphosphori~o~yl;~
:~: tran~ferage ~AD-PRT~). Assays were: performed
according~ ~to the~:method of Buki an~d Xun,
ÇÇhÇm.~27:5990-5995 (19883, using cal~ thymus
ADPRT. The assay result~ as ~i~en in ~able I
::~ :: 35~ provide the I~ (the co~centration of the
: compound ~hat inhibits enzyme activity 50%j ~.
: values for ADPRT of the nitroso precursox (6-
: : ~: :
~-93/07B68 2 1 219 O ~ PCT/US91/08902
-19- ' ~'
amino-1,2-benzopyrone) and the mor~ potent 5-
iodo-derivative (Table I, compounds 1 and 2,
respectively). The nitxoso compounds (3,4,5 in
Table I) are all hi~hly active as anti-tumor and
anti-HIV molecules (as ~hown in later s~ctions)
and are effective ~ven after expo~ure of cells
for a period as ~hort as 30 minutes. 5-I-6~
nitro~o-1,2-benæopyrons (compound 6) in these
studies has been shown to b~ a relatively poor
inhibitor of ADRPT (It is believed that ~he iodo
subst~ tution deactivat~s th~ NO group as an
electrophile) and it~ biological action i~ 10 :~:
times wea}c~r than that o~ 6-MO-1,2-benzopyrone.
For these reasons, the compositions of the
present invention are believed to be superior to ::
5-I-6-nitroso-1,2 benzopyxone, which has be~n
shown to be a p~or permeant molecule.
~ '
I50 data for aromatic inhibitors of ADPRT ~:
No . IIlhi~i~r
6-NH2- 1, 2-benzopyrone* 370
2 5-I-6:-NH2-1,2~benzopyrone* 41 ::
3 3-NO-benzamide 15
4 5(7)-nitroso-~2H)-isoquinolinone** 13
6 NO-1,2-benzopyron~ 40
`, ~ !
6 5-I~6-NO-1,2-benzopyron~ ~ 400
*biochemic~l precursor of nitroso compounds 5
and 6
**a mixture of the 5- and 7-nitroso compounds
Assay condition~: ADPRT~ 0.4 ~g; coDNA~ 4 ~g; ~'
inhibitor diluted betw~en 0.8 and 600 ~Mj in 50 :-
~l of 50 mM Tri~-HC-l/ gO mM KÇl, 5 mM 2-
mercaptoethanol, 0.5 mM EDTA, 0.1 mM NAD (~32~
P)-labelled~, pH 7.5. Polymerization at 25 C
for 4 minutes.
;
. .,
WV93/0786X PCT/U~91/08902
2 1 2 1 9 0 0
-20-
Figure 1 illustrates the % inactivati~n of
ADPRT polymerase activity observed after 2 hour~ :
of incubation with the nitroso-compound
inhibitors at s~veral concentrations. ~:~
Additional experiments in~olving the
equilibration between 65Zllt2 and ADPRT-bound ~n~2
suggest that the ADPRT inhibition activi~y of
the hitroso compounds appears to act by ~;~
destabili~ing the protein through the ejecting
of Zn~2. (Buki K.G,, Bauer PoT~ I Mendeleyev, F.;
Hakam, H. and Kun E. (1991) FEB$ L~. 290~
185). The above m~chani~m of action for ADPRT
inhibitor~ is speculative and does n~t
constitute any limitatio~ on claimed subject
matter.
~ ~`
V. ~i515~Uh~LL An~i-C~ncer Ac~ivi~i~ of ~:
Ni~osQb~nzoeyr~es, Ni~;rQs~2k~amisl~
and NitrosQ~ uinolin~-ne~
: :
:.. ~:
Experiments~ were perfomed in which various
human~ leukemia cell~ lines were exposed to
increasing concentrations of 6-amino-1,2
:~ benzopyrone ~:~ABP), 5~-iodo-6-amino 1,2-
~ benzopyrone (~IABPj, 6-nitro~ -benzopyrone
: ~ ~ 25 (NO2BP), 6~nitroso-1,2-benzopyrone :(NOBP)~ 3-
nitrosobPnzamide (NOB~? or 5~7)-nitroso-1(~H)~
isoquinolinone (NOQ) (a mixture of the 5-
nitroso and 7 nitroso ieomer~ nd the level of
3H], thymidine~ uptake ~wasi determin~d as a
measure of oellular proliferation. ~B shown in
: ~igure 2, ~or ea:ch of the cell lines tssted
(855-2 ~ells, Fig.~2A; H9 cells, Fig.~2B; H~-
- .;
60 c lls, Fig. 2C; K562 cells, Fig. 2D~ the ~`;
nitroso-containi~g ligands ~NOBP, NOBA, NOQ)
were ~able to inhibit 3H-thymidine uptake in
lower molar concentrati~ns than the other
compounds. NOBP, NOBA and NOQ powerfully
~V~93/07868 PCT/U~91/O~gO2
2121~0~ ~
-21-
inhibited 3H-thymidine uptake ~t a concentration
of 1~ ~M, a concentration at which the other
compounds exhibited compara tively slight ~:.
inhibitory ef :f ectE~ .
Experiments with H9 cells grown in 10% fetal
bovine ~erum (FCS) (Fig. 2B~ found NOQ to be the
most pot~nt inhibitor, demonstrating al~o~t
complete inhi~ition a~ 10 ~M levels. NOBP
demon~trated about a 30% decrease in thy~idi~e
uptake at 10 ~M, and an almost complste
inhibition of uptake at 100 ~M. NOBA
demonstrated ab~ut 75% level of inhibition at 10
pM, about 85% inhibition at 100 ~M, and almoBt :.
complete inhibition at 250 ~M. The remaini~g
amino and nitro compounds were ~ignificantly
le~ potent and did not display complst~
inhibition until concentration~ of 100:0 ~M were
reached. : : ~
',,
;20 Experiments with ~562 cel:Ls grown in;1~% f~tal
~ bo~ine serum (Fig. 2~) fou~d NO~ and NOBP to be
:: the most pot~nt inhibitors of cell gr~wth. Both
~ ~ ~OQ and:NOBP~resulted in the almost~complcte
: ~ : inhibition~at ~on~entratlons of 10 ~M.~ NOBP was:
; ~5 almost: as potent~as NO~ and prsduced~about 90#
: : inhibition at a concentration~ of;~10 :pM, a~d
almos~ complete inhib~ition at a concentration of
100 ~M. ~Th~ other 3 compounds test~d were
signifid~ntly less potent.
.
: ~,
Exp~riments with 855-2 cells grown in 10%
fetal bovine~serum:(Fig. 2A~ found that ~OQ,~and
NOBP producea almost ~omplete lnhibl~t~ion at a
~concentratlon of lO ~M. At a concentration of 1
~ ~M, ~ produced some~hat more inhibition than
: 35 NOBP, and NOBP produced ~omewhat more inhibition
.
.. . . .. ..... ............ . . . . .... ... . .... .. . . ..... . . . . . . . . ... . .. . .... .
WO ~3/07~68 P~/US91/089~2 .
~12~!30Q
-22-
than NOBA. Experiments using HL-60 cells ~Fig.
2C) provided similar corlclusions. The other 3
compounds tested wsre significantly less potent.
The effect of differ~nt growth factor~ OI~ the
growth inhibitory effect~ of NOBP was te~ted. -~.
855-2 cells that were grown in media with ( 1
10% fetal bovine serum, (2~ autocrine growth ~:~
factor (At;F) and (3) low mol~cular weigh1~-BCGF
(a T cell derived lymphokir~e) wl~re expo8~d to
increa~ing conc~ntrations of the ADRPT ligands.
The results are provided in Figure 2 (~, E, F) .
Cells grown in each o the growth faotox~ we~
all potently inhibited by the nitroso containing
compounds, with concentrations of 5 to 10 ~M
resultin~ in 100% inhibit:ion, Thus, NOBP, NOBA
and NOQ ex~rt potent inhibitory effect~
regardless of the sourt~e of growth factor ~-
activity. ;:
~: ~ 20 ~ In order to ex~lude the possibility that NOBP :~:
and NOBA manifest their growth inhibitor~ ;
: effects through inactivation of gxowth factors,
the effects of lO ~M NOBP or NOBA (constant
: ~ concentration)~on 855-2 cell~s in the presenae of
~25 : increasing concentrations~:of:~et:aI bovine ~erum
FCS) were tes~d (FCS) contains growth factors
or 855 2 cells). The data are pro~ided ~in
: Figure 3. Growth arrest occurs irrespective of~
the csncentration of FCS. Thus, the mode of
~30 action of :NOBP, does not ~appear to be~ by
antagoni~m -~f ~rowth factors but at ADPRT si~es
: ~ related to DNA-repllcation. ~;
umor cell inhibitory concentratio~s of NOBP
~ and NOBA were shown not to a~~ct adver~ely the
viability ~f normal cells. Experiments w2re ~
~,
:`
~ 3/078~s PCr/US91/~8902
2 ~ 2 ~L ~
-23-
performed in which the functions of various
cancer cells ( 855-2 and HL-60 leukemia cells,
D32, D37 and CRL 7712 glioblastoma c ll lin~s,
185 medulla tumor cell line, L1210 murine
leukemia cell line, MDA-468 human breast tumor
cell line) and normal cells (neutrophil
leukocytes and bon~ marro~ or peripheal blood
stream cells) wexe asseæ~ed in the absence or
presence of the compounds. The results are
~hown in Figures 4-9. Together, the data
indicate that a concentration of 10 ~M o~ the
n$troso-containing ligands effectively
suppressed cancer cell grow~h but demonstrated
only modest effects the functions on normal
cells.
VI. ~9~iLJ ~ Y~
The cytotoxicity of Oj 2 ~M, 4 ~M, 8 ~M and 10
~M NOBP wa~ maasured by examining the effect of
: 20 the compound on the colony f~rmation (CFU-GM) of.
normal human stem:~ells (PBSC~. The results of
: ~ the experiment~ are pro~ided i~ figure 5B.
Toxicity was not detected,~e~en though levels of
: NOBP suf~ficient to ~ : block fl55-2~ cell
~: 25 :~ proliferatlon completely were tested.
: ' ~ ::
~similar ~FU :stem cell toxicity assay ~was
: pe~formed in which~comparisons were made betw~en
(A~P) 6-amino-1,2-benzopyrone l mM; (~ABP)~ 5
I~6~amino-1,2-benzop~xone 250 ~M, (NO2BP) 6
:
nitro-1,2-benzopyrone ~weakly active) 250 ~M,
NOBP 10 ~M, and NOBA lO:~M. The results of:the
experiments axe provided in figure ~A. ~Whereas
the 6-amino~ enzopyrone, 5-I~6-amino-1,2-
benzopyrone and the 6-~itro deri~ative were
~oxic at the tested given doses, the almost
inefeative (again~t t~mor cells) 6-nitro
::
WO93/0786B PCT/US91/08902
21219UO "
I
-24-
derivative and the highly effective (against
tumor cells) NOBP and NOBA were non-toxic.
The effects of lO~m Nosp and NO~A on
superoxide generation by normal human peripheral
blood neutrophil leukocytes was te~ted. The
results are provided in tabla X I . Only minor
reductions in sup~roxide generation were
observed.
1 0 ~gkL~
Effects of 10 ~M NOBP and NOBA on the Generation
of Superoxide by Human Neutrophils
nmol 02/hr/105 cells
I~ LLi
105 PMN ~ PMA: 5S.9 ~ 7.7
+10 ~M NOBP 34.1 ~ 14.1
+10 ~M NOBA 44.4 ~ 10.0
~20 VII. '
Vincristine, a highly toxic chemetherapeutic
compound, is the currently:used in the treiatment
o~ leukemia and other malignancies. Studie:s
~5 were :performed i~ order to dete;rmine the
: ~ concentration of vincristine that produ¢es the
same level of growth inhibition a~ lO ~N NOBP,:~ :
when a~sayed on: 855-~ leukemia:cells grown i~
Vi~rQ. Vincristine was~tested~in doses of ~.l,
:30 : ~1, 10 and lOO~M. ~s shown in Fig~e~:7. 100 ~M
of vincristine (a highly toxi~ co~cent~ation)
:~ was re~uired~ to produce the~:~ame level of
: inhibition a~ 10 ~M o NOBP, thus NO~P is about
: 10 times more potent than an e~ual ~ioncentration
o~ vincristine, and is ~t toxic to normal
cells. :;
; Thus certain aromatic nitroso molecules tha~
are also inhibitors of ADPRT polymerase activity
~0~3/07~68 PCT/US9l/OB902
2 :1219 ~ U
-25- ;~
may be useful chemotherapeutic cytostatic agents ~:~ because of their effactiveness combined wi~h low
toxicity.
,'",,~
VIII. Anti-HIV_action of_NOBP/ NOBA and NOO ~n
The ability of NOBP (6-nitroso-1,2-
benzopyrone3 and NOBA (3-nitrosobenæamide~ to
inhibit HIV infections wer~ tested using the
methods described in the ~9~ L___
Immu~l~iç~l M~hod~ 76:171-183 (1985).
Exposure to the two drugs was only for 30
minutes at the commencement of viral inf~ction,
and dxugs were never re-added. The results
given in ~able III pro~ide t:he ID~ of HIV ti~er
10 days after infection of cell cultures with
HIV. The data in Table III demonstrate that
10 ~M of the ~itroso-containinS ligands cause~
a three log decrease in ~the HIV-1 infectivity
~: ti~er.
: :
~ ~ ABLE 111
~ç~=~am~ Virus Titer lloq ID ~L~ Q~ ~
: Virus ~lone~ 5~25 :~.
500 ~M ABP ~ : 4.50 ~ ~
250 ~M:IABP ~ 4.66 ::.
~ ~250 ~M NO~BP 4.93
+10 ~M NO~P : 2.01
:: : +10 ~M NO~ 1.05
; ~ +10 ~M~NOQ : ~ 1.73
: 35 IX. ~ : : ~ ,
:~ Exp rimetns were performed to determine i~the ~:
:inhibition~of prollferation o~ 855-2 ce;lls seen
4;0 ~: in cu1ture~nd in soft agar is due to the
~ : cytostatic or cytocidal effect of the nitroso
: ~ : compounds NOBP, :NOBA, and NOQ. Cells at
~.
:
W093/0786~ PCT/~S91/08902.
2 1 ~ 1 ~3 0 û
- 2 6 - ~
lxlO5/ml (concentration used in bone marrow
assay) were treated with NOBP, NOBA and NOQ at
1, 2.5, 5 and lO~m fox 2 hours then ~timulated :
with 10% fetal calf serum a~d incubated for 24
hours. MTT ~3-[4,5-Dimethyl-2-yl~-2,5-
diphenyltetrazolium bromide) at 1 mg/ml was then
added for 16 hours. ~he absorbance of the
pellete~ cell was then measured at 550nm after
adding DMSO to solubilze the cells
Results: With lO~M NOBP, NOBA and NOQ,
complete killing was observed in 85S-2 cell~ at
lOO,OOO/ml.
..;
All publications, patents, and patent
applications cited abo~e are herein incorporated
by reference.
The foregoing writt~n specification i5 ~.
~20 co:nsidered to be su~ficient to enable one
;: skilled in~ the art to pract~ice the invention.
ndeed, various modifi:~ations of the above-
de~cribed modes~for carrying:out the invention
which are obvlou6 to tho~e killed :in the ~ield
:: 25 o~ pharmaceutical ~ormul~ation or rel~ated fields : ~.
are intended to be within the scope of the ~ ~ .
following claims. ~ ~
: ~ : :
.:
~:,
.':
