Note: Descriptions are shown in the official language in which they were submitted.
t
v
-1-
1,2,4-BENZOTRIAZINE OXIDES AS
RADIOSENSITIZERS AND SELECTIVE CYTOTOXIC AGENTS
Technical Field
The invention relates to cytotoxic agents and
radiotherapy effective against hypoxic cells. More
specifically, the invention relates to certain novel
1,2,4-benzotriazine oxides, to methods of selectively
killing tumor' cells and/or sensitizing tumor cells to
radiation using selected 1,2,4-benzotriazine oxides, and
to novel synthetic methods.
25
~i~f~~.~3 _2_
Background Art
Hypoxic cell radiosensitizers are compounds
that selectively increase the sensitivity of hypoxic
cells to destructive radiation. Cytotoxins which have
enhanced activity under hypoxic conditions also provide
a means for selective destruction of cells under low
oxygen pressure. This specificity for hypoxic cells is
important because it is tumors that are typically
characterized by such cells. Virtually all tumors which
are present as solid masses contain these cells, while
normal cells generally have an adequate supply of
oxygen. Accordingly, anti-tumor agents can be made
selective fo;c tumors by virtue of high activity under
hypoxic conditions, and radiation can be employed more
effectively in the presence of these sensitizers.
Of course, the use of radiation treatment to
destroy tumor cells is only practical if damage to the
surrounding normal tissue can be minimized or avoided.
The effects of radiation are enhanced by the presence of
oxygen, and it is established that as the dose of
radiation is increased, the effectiveness of the
radiation in destroying target cells is enhanced most
dramatically when oxygen is present. Therefore,
selectivity for tumor cells toward radiation is
difficult to achieve -- normal cells, in view of their
oxygen supply, are generally more susceptible to
radiation than the target tumor cells. It is therefore
desirable to provide a means of sensitizing tumor cells,
but not.the surrounding tissue, to radiation treatment.
One solution would be to increase the supply of oxygen
to these tumor cells. This, however, has proved
difficult to do.
Various heterocyclic compounds and in particular
those with oxidized nitrogen moieties, have been used to
radiosensitize hypoxic tumor cells. Indeed, it has been
postulated that the oxidized nitrogen functionality is
responsible for this activity. Nitroimidazoles,
particularly mison_idazole (MIS) and metronidazole have been
studied extensively, and MIS is commonly used as a standard
in in vitro and in vivo tests for radiosensitizing
activity. (See, e.g., Asquith, et al, Radiation Res (1974)
60:108-118; Hall, et al, Brit J Cancer (1978) 37: 567-569;
Brown, et al, Radi<~tion Res (1980) 82: 171-190; and U.S.
patent 4,371,540. The radiosensitizing activities of
certain 1-substituted 3(5)-nitro-s-triazoles and of various
quinoxaline-1,4-dioxide derivatives have also been
disclosed.
In addition WO 86/06628 published November 20,
1986 assigned to the same assignee discloses a group of
radiosensitizers that do not contain oxidized nitrogen--the
substituted benzam:ides and nicotinamides and their thio
analogs. These compounds, nevertheless, are
radiosensitizers. It is important to distinguish the
ability to sensitize hypoxic cells selectively, for
instance, by enhancing their oxygen supply, from another
mechanism commonly encountered for "sensitizing" cells:
inhibition of the enzyme poly(ADP-ribose)polymerase, which
is believed to be essential in the repair of irradiated
cells after radiation. This repair mechanism is operative
in both hypoxic tumor cells and in normal cells. Hence,
administration of "radiosensitizers" which operate
according to this latter mechanism does
- 3 -
'':
t:
~Q~~.~~~ 4
not accomplish the desired purpose of selectively
sensitizing t:he target tumor cells.
A croup of compounds which has not previously
been suggestead for use in either selectively killing
hypoxic cells or in radiosensitizing such cells is
3-amino-1,2,96-benzotriazine 1,4-di-N-oxide and related
compounds. Related US patents 3,980,779; 3,868,371; and
4,001,410 disclose the preparation of a group of these
compounds and their use as anti-microbial agents,
particularly by addition of these materials to livestock
fodder. U.S. Patent Nos. 3,991,189 and 3,957,799
disclose derivatives of these compounds bearing
substituents on the nitrogen of the 3-amino group.
These compounds also have anti-microbial activity.
They present invention provides additional
compounds which specifically radiosensitize hypoxic
cells and which, furthermore, are directly cytotoxic to
hypoxic cellsc both in vitro and in vivo. Therefore,
administration of these compounds prior to or following
radiation treatment of tumors selectively kills the
. hypoxic (tumor) cells which survive the radiation dose.
Both the abi7.ity of these compounds to radiosensitize
hypoxic cells; and especially their ability to
selectively hall hypoxic cells directly are unexpected
properties of these compounds.
they invention also provides novel
1,2,4-benzotriazine oxides useful as radiosensitizers
and/or selective cytotoxic agents; methods of
synthesizing the compounds; and methods of administering
the compound's to achieve radiosensitization and/or
selective ce7.1 killing.
. _g-
Disclosure oi: the Invention
Then invention provides a valuable addition to
the group of compounds currently available as selective
radiosensiti:.ers and selective cytotoxic agents for
hypoxic tumor cells. Some of the compounds now newly
shown to be useful in this regard are known compounds.
Others are themselves novel.
Accordingly, one aspect of the invention is a
method of ra<iiosensitizing hypoxic tumor cells by
administering to these cells a compound of the formula:
O
T
y1 N \
N
y2 (I)
N X
O
n
wherein X is H; hydrocarbyl (1-4C);
hYdrocarbyl (1-4C) substituted with OH, NH2, NHR or NRR;
halogen; OH; alkoxy (1-4C); NH2; NHR or NRR; wherein
the various iEt groups are independently selected from
lower alkyl (1-4C) and lower acyl (1-4C) and the R's may
themselves bye substituted with OH, NH2, alkyl (1-4C)
secondary and dialkyl (1-4C) tertiary amino groups,
alkoxy (1-4C) or halogen. In the case of NRR, the two
R's can be linked together directly or through a bridge
oxygen into a morpholino ring, pyrrolidino ring or
piperidino ring;
n is 0 or 1; and
Y1 and Y2 are independently either H; nitro;
halogen; hydrocarbyl (1-14C) including cyclic and
unsaturated hydrocarbyl, optionally substituted with 1
or 2 substituents selected from the group consisting of
.
~~~~.~C~~ -s_
halogen, hydroxy, epoxy, alkoxy (1-4C), alkylthio
(1-4C), primary amino (NH2), alkyl (1-4C) secondary
amino, dialkyl (1-4C) tertiary amino, dialkyl (1-4C)
tertiary amino where the two alkyls are linked together
to produce a morpholino, pyrrolidino or piperidino,
acyloxy (1-4C:), acylamido (1-4C) and thio analogs
thereof, acet:ylaminoalkyl (1-4C), carboxy,
alkoxycarbonyl (1-4C), carbamyl, alkylcarbamyl (1-4C),
alkylsulfony7. (1-4C) or alkylphosphonyl (1-4C), wherein
the hydrocarbyl can optionally be interrupted by a
single ether (-O-) linkage; or wherein Yl and Y2 are
independently either morpholino, pyrrolidino,
piperidino, rtH2, NHR,', NR'R' 0(CO)R', NH(CO)R', 0(SO)R',
or 0(POR')R' in which R' is a hydrocarbyl (1-4C) which
may be substituted with OH, NH2, alkyl (1-4C) secondary
amino, dialkyl (1-4C) tertiary amino, morpholino,
pyrrolidino,~piperidino, alkoxy (1-4C), or halogen
substitutents, or pharmacologically acceptable salts of
said compound.
In another aspect, the invention provides an
improved method of fractionated radiotherapy which
involves treating the cells requiring radiotherapy with
a 1,2,4-benzc>triazine oxide of Formula (I), as just
defined, before or after subjecting the treated cells to
a plurality of distinct radiation doses over an extended
period of time, each of the radiation doses being less
than about 5 Gy.
The! compounds useful in conjunction with the
presently disclosed radiosensitizing methods, therefore,
are the mono-~ or dioxides of optionally substituted
1.2,4-benzotriazine which may contain a hydrocarbyl
(1-4C), hydroxyl., alkoxy or amino group, either
substituted or unsubstituted, in the 3-position, and
-7-
their pharmacologically acceptable salts as set forth in
Formula I
Then invention also provides a method for
selectively killing hypoxic tumor cells using certain of
these 1,2,4-benzotriazine oxides. The compounds which
are useful as selective cytotoxic agents are a subset of
the above-dei:ined compounds useful as radiosensitizers.
That is, while all of the compounds defined by Formula
(I) are generally effective as radiosensitizers, only
those compounds unsubstituted at the 3-position or
having a 3-annino or 3-hydrocarbyl (1-4C) substituent
(i.e., X=H, hydrocarbyl (1-4C), NH2, NHR or NRR with
each R as dei'ined above) and which are di-N-oxides (n=1)
are effective cytotaxic agents. In this aspect, the
invention provides a method of selectively killing
hypoxic tumor cells by administering one or more of
these compounds (or its salts) to the hypoxic tumor
cells.
Certain of the compounds encompassed by
Formula (I) are already known in the art as being useful
for other purposes; other compounds are novel. The
novel compounds encompassed by the present invention and
which may be prepared by methods disclosed herein
include compounds represented by Formula (I), in which
the substitu~ents fall into the following three classes:
I. X is OH, alkoxy (1-4C), NHR or NRR where
each R is independently an alkyl of 1-4 carbon atoms, yr
acyl of l-4 carbon atoms, or where the two R groups are
alkyls linked together to form a pyrrolidino or
piperidino ring or linked through an oxygen to form a
morpholino ring, and the R groups may be further
substituted with OH, NH2, alkyl (1-4C) secondary amino,
dialkyl (1-4C) tertiary amino, pyrrolidino, piperidino,
alkoxy (1-4C), or halogen substitutents;
n i s 1; and
Yl and Y2 are independently either H; nitro;
halogen; hyd:rocarbyl. (1-14C) including cyclic and
unsaturated lzydrocarbyl, optionally substituted with 1
or 2 substituents selected from the group consisting of
halogen, hyd:roxy, epoxy, alkoxy (1-4C), alkylthio
(1-4C), primary amino (NH2), alkyl (1-4C) secondary
amino, dialk:yl (1-4C) tertiary amino, dialkyl tertiary
amino where 'the two alkyls are linked together to
produce a mo:rpholino, pyrrolidino or piperidino, acyloxy
(1-4C), acylamido (l.-4C) and thio analogs thereof,
acetylaminoalkyl (1-4C), carboxy, alkoxycarbonyl (1-4C),
carbamyl, al'.kylcarbamyl (1-4C), alkylsulfonyl (1-4C) or
alkylphosphonyl (1-4C), wherein the hydrocarbyl can
optionally be interrupted by a single ether (-0-)
linkage; or wherein Y1 and Y2 are independently either
morpholino, ~ayrrolidino, piperidino, NH2, NHR', NR'R'
0(CO)R', NH(CO)R', 0(SO)R', or 0(POR')R' in which R' is
a hydrocarbyl (1-4C) which may be substituted with OH,
NH2, alkyl (:1-4C) secondary amino, dialkyl (1-4C)
tertiary amino, morpholino, pyrrolidino, piperidino,
alkoxy (1-4C), or halogen substitutents.
Pharmacologically acceptable salts of these compounds
are also included in this class of compounds.
II. X is NH2;
n is 1; and
Y1 and Y2 are chosen such that one but not
both may be hydrogen and one or both may independently
be either vitro, saturated or unsaturated hydrocarbyl of
~-14C, or unsaturated hydrocarbyl of 2-6C, optionally
~1~:~.°~~
substituted with 1 or 2 substituents selected from the
group consisting of halogen, hydroxy, epoxy, alkoxy
(1-4C), alkylth.io (1-4C), primary amino (NH2), alkyl
(1-4C) secondary amino, dialkyl (1-4C) tertiary amino,
dialkyl tertiary amino where the two alkyls are linked
together to produce a morpholino, pyrrolidino or
piperidino, acyloxy (1-4C), acylamido (1-4C) and thio
analogs thereof, acetylaminoalkyl (1-4C), carboxy,
alkoxycarbonyl (1-4C), carbamyl, alkylcarbamyl (1-4C),
alkylsulfonyl (1-4C) and alkylphosphonyl (1-4C), wherein
the hydrocarbyl. can optionally be interrupted by a
single ether (-~0-) linkage; or wherein Y1 and Y2 are
independently either morpholino, pyrrolidino,
piperidino, NH2;, NHR', NR'R' 0(CO)R', NH(CO)R', O(SO)R',
or 0(POR')R' in which R' is a hydrocarbyl (1-4C) which
may be substituted with OH, NH2, alkyl (1-4C) secondary
amino, dialkyl (1-4C) tertiary amino, morpholino,
pyrrolidino, pi.peridino, alkoxy (1-4C), or halogen
substitutents. Pharmacologically acceptable salts of
these compound's are also included in this class of
compounds.
III. X is hydrogen or hydrocarbyl (2-4C)
optionally substituted with OH, NH2, alkoxy (1-4C) or
halogen substit:uents;
n is 1; and
Yl and Y2 are independently either H; vitro;
halogen; hydroc:arbyl (1-14C) including cyclic and
unsaturated hyctrocarbyl, optionally substituted with 1
or 2 substituents selected from the group consisting of
halogen, hydroxy, epoxy, alkoxy (1-4C), alkylthio
(1-4C), primary amino (NH2), alkyl (1-4C) secondary
amino, dialkyl (1-4C) tertiary amino, dialkyl tertiary
-10-
amino where t:he two alkyls are linked together to
produce a morpholino, pyrrolidino or piperidino, acyloxy
(1-4C), acylamido (1-4C) and thio analogs thereof,
acetylaminoa7.ky1 (1-4C), carboxy, alkoxycarbonyl (1-4C),
carbamyl, al~;ylcarbamyl (1-4C), alkylsulfonyl (1-4C) or
alkylphosphonyl (1-4C), wherein the hydrocarbyl can
optionally beg interrupted by a single ether (-O-)
linkage; or urherein Yl and Y2 are independently either
morpholino, pyrrolidino, piperidino, NH2, NHR', NR'R'
0(CO)R', NH(C:0)R', 0(SO)R', or 0(POR')R' in which R' is
a hydrocarby7. (1-4C) which may be substituted with OH,
NH2, alkyl (7.-4C) secondary amino, dialkyl (1-4C)
tertiary amino, morpholino, pyrrolidino, piperidino,
alkoxy (1-4C), or halogen substitutents.
Pharmacologically acceptable salts of these compounds
are also inc7.uded in this class of compounds.
The' invention also provides a straightforward,
one-step synthesis for preparing 1,2,4-benzotriazine
oxides unsub:ctituted at the 3-position (i.e., the
compounds of Formula (I) wherein X=H) by treating the
corresponding 3-amino-1,.2,4-benzotriazine oxide with a
lower alkyl nitrite under reductive deaminating
conditions.
2~ Brief Descrix~tion of the Drawin4s
Figures 1A, 1B and 1C show the selective
cytotoxicity of 3-amino-1,2,4-benzotriazine 1,4-dioxide
for hypoxic cells derived from hamster, mouse and human
tissues.
Figure 2 shows the in vivo efficacy of
3-amino-1,2,56-benzotriazine 1,4-dioxide in enhancing the
killing of tumor cells when combined with radiation.
, , _11-
Figure 3 shows the killing of tumor cells in
vivo by 3-amino-1,2,4-benzotriazine 1,4-dioxide when the
tumor has been made hypoxic by the intraperitoneal
administration of the antihypertensive drug hydralazine.
Figure 4 is a graph illustrating aerobic
radi.osensitiz:ation of CHO cells by hypoxic pretreatment
or post-treatment with 3-amino-1,2,4-benzotriazine
1,4-dioxide, as described in Example 22.
Figure 5 illustrates tumor cell survival of
SCCVII tumor irradiated with 8x2.5 Gy in four days, also
as described in Example 22.
Figure 6 graphically illustrates the growth
delay of SCCVII tumors irradiated with 8x2.5 Gy in four
days, also asp described in Example 22.
Figure 7 shows in graph form the average skin
reaction of normal mouse skin irradiated with 8
fractions (every 12 hours) of between 3 and 6 Gy per
fraction.
Modes of Carryinct Out the Invention
A. The Compounds Useful in the Invention
They compounds useful as radiosensitizers and
selective cyt:otoxic agents as described herein are
derivatives of 1,2,4-benzotriazine oxide represented by
Formula (I).
These compounds, as shown in the Formula (I),
contain a group X in their 3 position. X will vary
specifically as set forth above, depending upon the
activity desired. Subject to the above-recited
selection criteria, X is chosen in general from
hydrogen; un~~ubstituted hydrocarbyls (1-4C) such as
methyl, ethyl., s-butyl and the like; hydroxy; alkoxy
-12-
(1-4C) such as methoxy, ethoxy, propoxy, t-butoxy and
the like; primary amino (NH2); secondary amino (NHR)
where R is are alkyl or acyl of 1 to 4 carbons, such as
methylamino, ethylamino and the like; tertiary amino
(NRR) where each of the R groups is an alkyl or acyl of
1 to 4 carbons, for example diethylamino and the like,
or the two R's join to form a morpholino, pyrrolidino or
piperidino ring. In the case of the various alkyl and
acyl R group:., they can be further substituted with OH,
NH2, lower a:~kyl (i-4C) secondary amino and dialkyl
(1-4C) tertiary amino, morpholino pyrrolidino,
piperidino, alkoxy (1-4C) or halogen (fluoro, chloro,
bromo or iodo) substituents.
In the case of the hydrocarbyl X groups, they
can be furthe r substituted with OH, NH2, alkyl secondary
amino, dialk~~l tertiary amino, alkoxy (1-4C) or halogen
(fluoro, chloro, bromo or iodo) substituents.
The compounds of Formula (I) additionally
contain groups Yl and Y2. These groups are selected
specifically according to the criteria set forth above,
depending upon the utility desired.
Subject to these criteria, Yl and Y2 may be
selected from hydrogen; vitro; halogen (e. g. fluoro,
chloro, bromo or iodo); or hydrocarbyl (1-14C). When
hydrocarbyl, Y1 or Y2 may be saturated or unsaturated,
cyclic or-ac:yclic, and may optionally be interrupted by
a single ether linkage. Thus, the unsubstituted
hydrocarbyl forms of Yl or Y2 can be, for example,
methyl, ethyl, n-propyl, s-butyl, n-hexyl, 2-methyl-n-
pentyl, 2-et:hoxyethyl, 3-(n-propoxy)-n-propyl,
4-methoxybutyl, cyclohexyl, tetrahydrofurfuryl,
furfuryl, cyclohexenyl, 3-(n-decyloxy)-n-propyl,
4-methyloctyl, 4,7-dimethyloctyl, or the like.
. ~ -13-
The hydrocarbyl Y1 and Y2 groups may
optionally be substituted with 1 or 2 substituents
selected from halogen such as fluoro, chloro, bromo or
iodo; hydrox;~r; epoxy; alkoxy (1-4C) such as, for
example, metlzoxy, n-~propoxy and t-butoxy; alkyl thio;
(1-4C) primary amino (NH2); morpholino; pyrrolidino;
piperidino; secondary amino (NHR') where R' is a 1-4C
alkyl, such as methylamino, propylamino and the like;
tertiary amino (NR'R'); acyloxy and acylamido groups
represented by R'C00- and R'CONH-, respectively, and
their thiol analogs represented by R'CSO- and R'CSNH-
respectively;; carboxy (-C(0)OH); alkoxycarbonyl
(-C(0)OR'): carbamyl (-C(0)NH2); alkylcarbamyl (1-4C)
(-C(0)NHR'); alkylsulfonyl (1-4C) (R'SOZ-); and alkyl
phosphonyl (:l-4C) ( R'P(OR')0-).
In addition Y1 and Y2 can each independently
be -NH2, -NHI~', -NR'R', -OCOR', -NH(CO)R', -0(SO)R' or
-0(POR')R' in which the various R' groups are lower
alkyls (1-4C) which themselves may be substituted with
OH, NH2, alkyl secondary and tertiary amino,
pyrrolidino, piperidino, alkoxy (1-4C), or halogen
substitutenta.
A particularly promising class of compounds
for use both as radiosensitizers and selective cytotoxic
agents include those represented by the following
structural Formula (II):
0
y1 ~ N ~
~~ N
(II)
Y ~ N ~yH ~ (CH ) ~ NR R
2 m 1 2
0
-14-
~~~~1.~'~
In Formula (II), one of Y1 and Y2 is H, the other an
electron-withdrawing group (e. g., vitro, carboxy,
alkoxycarbonyl, alkylsulfonyl), R1 and R2 are
independently selected from the group consisting of
hydrogen and lower alkyl, or the R1 and R2 groups may be
linked to form a piperidino or pyrrolidino ring, and m
is an integer from a-4 inclusive, preferably 1 or 2.
Where X is OH, of course, the compounds may
also be prepared and used as the pharmaceutically
acceptable salts foamed from inorganic bases, such as
sodium, potassium, or calcium hydroxide, or from organic
bases, such as caffeine, ethylamine, and lysine.
When X is NH2, NHR, or NRR, e.g., NH-CH2-
(CH2)m-CH2NR1R2 as in Formula (II), pharmaceutically
l~ acceptable acid addition salts may be used. These salts
are those with inorganic acids such as hydrochloric,
hydrobromic or phosphoric acids or organic acids such as
acetic acid, pyruvic acid, succinic acid, mandelic acid,
p-toluene sulfonic acid, and so forth. (Amino
20 substituents on the hydrocarbyl side chain can also, of
course, be converted to salts.)
The 1,2,4--benzotriazines may be usedin the
practice of this invention as the mono- or dioxides;
i.e. either the 1-nitrogen of the triazino ring may be
25 oxidized, or both the 1-and 4-nitrogens may be oxidized.
Specific particularly preferred compounds
which are useful in the radiosensitization and cytotoxic
procedures of the invention include
3-hydroxy-1,2,4-benzotriazine 1-oxide;
30 3-hydroxy-1,2,4-benzotriazine 1,4-dioxide;
3-amino-1,2,4-benzotriazine 1-oxide;
3-amino-1,2,4-benzotriazine 1,4-di-oxide;
6(7)-methoxy-3-hydroxy-1,2,4-benzotriazine 1-oxide;
-15-
6(7)-methoxy--3-hydroxy-1,2,4-benzotriazine 1,4-dioxide;
6(7)-methoxy--3-amino-1,2,4-benzotriazine 1-oxide;
6(7)-methoxy--3-amino-1,2,4-benzotriazine 1,4-dioxide;
6(7)-ethoxy-3-hydroxy-1,2,4-benzotriazine 1-oxide;
6(7)-ethoxy-'.3-hydroxy-1,2,4-benzotriazine 1,4-dioxide;
6(7)-ethoxy-'.3-amino-1,2,4-benzotriazine 1-oxide;
6(7)-ethoxy-".3-amino-1,2,4-benzotriazine 1,4-dioxide;
6(7)-[4-acetamido-n-butanoxy]-3-hydroxy-1,2,4-
benzotriazine 1-oxide;
6(7)-[4-acetamido-n-butanoxy]-3-hydroxy-1,2,4- .
benzotriazine 1,4-dioxide;
6(7)-[4-acet<imido-n-butanoxy]-3-amino-1,2,4-
benzotriazine 1-oxide;
6(7)-[4-acetamido-n-butanoxy]-3-amino-1,2,4-
benzotriazine 1,4-dioxide;
6(7)-[1-(2,3--dihydroxy)propoxy]-3-hydroxy-1,2,4-
benzotriazine 1-oxide;
6(7)-[1-(2,3-dihydroxy)propoxy]-3-hydroxy-1,2,4-
benzotriazine 1,4-dioxide;
6(7)-[1-(2,3-~dihydroxy)propoxy]-3-amino-1,2,4-
benzotriazine 1-oxide;
6(7)-[1-(2,3-~dihydroxy)propoxy]-3-amino-1,2,4-
benzotriazine 1,4-dioxide;
6(7)-[(2-furyl)methylamino]-3-hydroxy-1,2,4-
benzotriazine 1-oxide;
6(7)-[(2-furyl)methylamino]-3-hydroxy-1,2,4-
ber~zotriazine 1,4-dioxide;
6(7)-[(2-furyl)methylamino]-3-amino°1,2,4-
benzotriazine 1-oxide;
6(7)-[(2-furyl)methylamino]-3-amino-1,2,4-benzotriazine
1,9:-dioxide;
6(7)-(2-methoxyethylamino)-3-hydroxy-1,2,4
benzotriazine 1-oxide;
-16-
6(7)-(2-methoxyethylamino)-3-hydroxy-1,2,4-benzotriazine
1, 96-dioxide;
6(7)-(2-methoxyethylamino)-3-amino-1,2,4-benzotriazine
1-oxide;
6(7)-(2-methoxyethylamino)-3-amino-1,2,4-benzotriazine
1, 96-dioxide;
6(7)-carbethoxymethoxy-3-hydroxy-1,2,4-benzotriazine
1-oxide;
6(7)-carbethoxymethoxy-3-hydraxy-1,2,4-benzotriazine
1, 9b-dioxide;
6(7)-carbethoxymethoxy-3-amino-1,2,4-benzotriazine
1-oxide;
6(7)-carbethoxymethoxy-3-amino-1,2,4-benzotriazine
1, 96-dioxide;
6(7)-[(2-methoxyethyl)carbamylmethoxy]-3-hydroxy-1,2,4-
benzotriazine 1-oxide;
6(7)-[(2-methoxyethyl)carbamylmethoxy]-3-hydroxy-1,2,4-
benzotriazine 1,4-dioxide;
6(7)-[(2-methoxyethyl)carbamylmethoxy]-3-amino-1,2,4-
benzotriazine 1-oxide;
6(~)-[(2-methoxyethyl)carbamylmethoxy]-3-amino-1,2,4
benzotriazine 1,4-dioxide;
6(7)-[(2-hydroxyethyl)carbamylmethoxy]-3-hydroxy-1,2,4-
benzotriazine 1-oxide;
6(7)-[(2-hydroxyethyl)carbamylmethoxy]-3-hydroxy-1,2,4-
benzotriazine 1,4-dioxide;
6(7)-[(2-hydroxyethyl)carbamylmethoxy]-3-amino-1,2,4-
benzotriazine 1-oxide;
6(7)-[(2.-hydroxyethyl)carbamylmethoxy]-3-amino-1,2,4-
benzotriazine 1,4-dioxide;
6(~)-[1-(2-h~rdroxy-3-morpholino)propoxy]-3-hydroxy-
1,:?,4-benzotriazine 1-oxide;
-17-
6(7)-[1-(2-h;Ydroxy-3-morpholino)propoxy]-3-hydroxy
l,;Z,4-benzotriazine 1,4-dioxide;
6(7)-[1-(2-hydroxy-3-morpholino)propoxy]-3-amino-1,2,4
benzotriazine 1-oxide;
6(7)-[1-(2-h;Ydroxy-3-morpholino)propoxy]-3-amino-1,2,4
benzotriazine 1,4-dioxide;
6(7)-[3-amino-n-propoxy]-3-hydroxy-1,2,4-benzotriazine
1-oxide;
6(7)-[3-amino-n-propoxy]-3-hydroxy-1,2,4-benzotriazine
1,4-dioxide;
6(7)-[3-amino-n-propoxy]-3-amino-1,2,4-benzotriazine
1-oxide;
6(7)-[3-amino-n-propoxy]-3-amino-1,2,4-benzotriazine
1,4-dioxide;
6(7)-[2,3-epoxypropoxy]-3-hydroxy-1,2,4-benzotriazine
1-oxide;
6(7)-[2,3-epoxypropoxy]-3-hydroxy-1,2,4-benzotriazine
1,4-dioxide;
6(7)-[2,3-epoxypropoxy]-3-amino-1,2,4-benzotriazine
1-oxide;
6(7)-[2,3-epoxypropoxy]-3-amino-1,2,4-benzotriazine
1,4-dioxide;
6(7)-[3-methoxy-2-hydroxy-n-propoxy]-3-hydroxy-1,2,4-
benzotriazine 1-oxide;
6(7)-[3-methoxy-2-hydroxy-n-propoxy]-3-hydroxy-1,2,4-
benzotriazine 1,4-dioxide;
6(7)-[3-methoxy-2-hydroxy-n-propoxy]-3-amino-1,2,4-
benzotriazine 1-oxide;
6i7)-[3-methoxy-2-hydroxy-n-propoxy]-3-amino-1,2,4-
benzotriazine 1,4-dioxide;
6(7)-[4-ethosKy-3-hydroxy-n-butoxy]-3-hydroxy-1,2,4-
benzotriazine 1-oxide;
6(7)-[4-ethos~y-3-hydroxy-n-butoxy]-3-hydroxy-1,2,4-
~~~~~ ~~;~ -la-
benzotriazine 1,4-dioxide;
6(7)-[4-ethos:y-3-hydroxy-n-butoxy]-3-amino-1,2,4-
benzotriazine 1-oxide;
6(7)-[4-ethos:y-3-hydroxy-n-butoxy]-3-amino-1,2,4-
benzotriazine 1,4-dioxide;
6(7)-[3,4-dihydroxy-n-butoxy]-3-hydroxy-1,2,4-
benzotriazine 1-oxide;
6(7)-[3,4-dihydroxy-n-butoxy]-3-hydroxy-1,2,4-
benzotriazine 1,4-dioxide;
6(7)-[3,4-dihydroxy-n-butoxy]-3-amino-1,2,4-
benzotriazine 1-oxide;
6(7)-(3,4-dihydroxy-n-butoxy]-3-amino-1,2,4-
benzotriazine 1,4-dioxide;
6(7)-methyl-3-hydroxy-1,2,4-benzotriazine 1-oxide;
6(7)-methyl-3-hydroxy-1,2,4-benzotriazine 1,4-dioxide;
6(7)-methyl-3-amino-1,2,4-benzotriazine 1-oxide;
6(7)-methyl-3-amino-1,2,4-benzotriazine 1,4-dioxide;
6(7)-ethyl-3-hydroxy-1,2,4-benzotriazine 1-oxide;
6(7)-ethyl-3-hydroxy-1,2,4-benzotriazine 1,4-dioxide;
6(7)-ethyl-3-amino-1,2,4-benzotriazine 1-oxide;
6(7)-ethyl-3-amino-1,2,4-benzotriazine 1,4-dioxide;
6(7)-chloroac.etamido-3-hydroxy-1,2,4-benzotriazine
1-oxide;
6(7)-chloroacetamido-3-hydroxy-1,2,4-benzotriazine
1,4-dioxide;
6(7)-chloroacetamido-3-amino-1,2,4-benzotriazine
1-oxide;
6(7)-chloroacetamido-3-amino-1,2,4-benzotriazine
1,4-dioxide;
6(7)-[(2-hydroxyethyloxy)acetamido]-3-hydroxy-1,2,4-
ben.zotriazine 1-oxide;
6(7)-[(2-hydroxyethyloxy)acetamido]-3-hydroxy-1,2,4-
benzotriazine 1,4-dioxide; .
-19-
6(7)-[(2-hydroxyethyloxy)acetamido]-3-amino-1,2,4-
benzotriazine 1-oxide;
6(7)-[(2-hydroxyethyloxy)acetamido]-3-amino-1,2,4-
benzotriazine 1,4-dioxide;
6,7-dimethox;~r-3-hydroxy-1,2,4-benzotriazine 1-oxide;
6,7-dimethoxy-3-hydroxy-1,2,4-benzotriazine 1,4-dioxide;
6,7-dimethoxy-3-amino-1,2,4-benzotriazine 1-oxide;
6,7-dimethoxy-3-amino-1,2,4-benzotriazine 1,4-dioxide;
6,7-diethoxy-3-hydroxy-1,2,4-benzotriazine 1-oxide;
6,7-diethoxy-3-hydroxy-1,2,4-benzotriazine 1,4-dioxide;
6,7-diethoxy~-3-amino-1,2,4-benzotriazine 1-oxide;
6,7-diethoxy-3-amin~-1,2,4-benzotriazine 1,4-dioxide;
6(7)-propionyl-3-hydroxy-1,2,4-benzotriazine 1-oxide;
6(7)-propionyl-3-hydroxy-1,2,4-benzotriazine
1,4-dioxide;
6(7)-propion:Y1-3-amino-1,2,4-benzotriazine 1-oxide;
6(7)-propion;Y1-3-amino-1,2,4-benzotriazine 1,4-dioxide;
6(7)-(2-acetoxyethoxy)-3-hydroxy-1,2,4-benzotriazine
1-oxide;
6(7)-(2-acetoxyethoxy)-3-hydroxy-1,2,4-benzotriazine
1, 4-dioxide;
6(7)-(2-acetoxyethoxy)-3-amino-1,2,4-benzotriazine
1-oxide;
6(7)-(2-acetoxyethoxy)-3-amino-1,2,4-benzotriazine
1, ~4-dioxide;
6(7)-n-hexyloxy-3-hydroxy-1,2,4-benzotriazine 1-oxide;
6(7)-n-hexyloxy-3-hydroxy-1,2,4-benzotriazine
1, ~4-dioxide;
6(7)-n-hexyloxy-3-amino-1,2,4-benzotriazine 1-oxide;
6(7)-n-hexyloxy-3-amino-1,2,4-benzotriazine 1,4-dioxide;
6(7)-ethylamino-3-hydroxy-1,2,4-benzotriazine 1-oxide;
6(7)-ethylamino-3-hydroxy-1,2,4-benzotriazine
1,4-dioxide;
-20-
6(7)-ethylamino-3-amino-1,2,4-benzotriazine 1-oxide;
6(7)-ethylamino-3-amino-1,2,4-benzotriazine 1,4-dioxide;
6(7)-(2-methoxyethoxy)-3-hydroxy-1,2,4-benzotriazine
1-oxide;
6(7)-(2-methoxyethoxy)-3-hydroxy-1,2,4-benzotriazine
1, ~4-dioxide;
6(7)-(2-methoxyethoxy)-3-amino-1,2,4-benzotriazine
1-oxide;
6(7)-(Z-methoxyethoxy)-3-amino-1,2,4-benzotriazine
1, 4-dioxide;
6(7)-(aminoacetamido)-3-hydroxy-1,2,4-benzotriazine
1-oxide;
6(7)-(aminoacetamido)-3-hydroxy-1,2,4-benzotriazine
1, 4-dioxide;
6(7)-(aminoacetamido)-3-amino-1,2,4-benzotriazine -
1-ox ide;
6(7)-(aminoacetamido)-3-amino-1,2,4-benzotriazine
1,4-dioxide;
6(7)-(carbam;Ylmethoxy)-3-hydroxy-1,2,4-benzotriazine
1-oxide;
6(7)-(carbamylmethoxy)-3-hydroxy-1,2,4-benzotriazine
1,4-dioxide;
6(7)-(carbamylmethoxy)-3-amino-1,2,4-benzotriazine
1-ox ide;
6(7)-(carbamylmethoxy)-3-amino-1,2,4-benzotriazine
1,4-dioxide;
6(7)-(carboxyrmethoxy)-3-hydroxy-1,2,4-benzotriazine
1-ox ide;
6(7)-(carbox;ymethoxy)-3-hydroxy-1,2,4-benzotriazine
1,4-dioxide;
6(7)-(carbox~ymethoxy)-3-amino-1,2,4-benzotriazine
1-oxide;
-21-
2pp1903
6(7)-(carboxymethoxy)-3-amino-1,2,4-benzotriazine
1,4-dioxide;
6(7)-[1,2-dih,ydroxyethyl]-3-amino-1,2,4-benzotriazine
1,4-dioxide;
6(7)-[1-(3-ethylamino-2-hydroxypropoxy)]-3-amino-1,2,4-
benzotriazine 1,4-dioxide;
6(7)-[2-ethylamino-1-hydroxyethyl]-3-amino-1,2,4-
benzotriazine 1,4-dioxide;
6(7) -[2-hydroxyethyl]-3-amino-1,2,4-benzotriazine
1,4-dioxide;
6(7)-[1-hydro~xyethyl]-3-amino-1,2,4-benzotriazine
1,4-dioxide;
3-(2-hydroxye~thylamino)-1,2,4-benzotriazine 1-oxide;
3-(2-hydroxye~thylamino)-1,2,4-benzotriazine
1,4-dioxide;
6(7)-chloro-3-(2-hydroxyethylamino)-1,2,4-benzotriazine
1-oxide;
6(7)-chloro-3-(2-hydroxyethylamino)-1,2,4-benzotriazine
1,4-dioxide;
3-(1-hydroxye~thylamino)-1,2,4-benzotriazine 1-oxide;
3-(1-hydroxye~thylamino)-1,2,4-benzotriazine
1,4-dioxide;
1,2,4-benzotriazine 1-oxide;
1,2,4-benzotriazine 1,4-dioxide;
3-methyl-1,2,4-benzotriazine 1,4-dioxide;
3-ethyl-1,2,4-benzotriazine 1,4-dioxide;
3-propyl-1,2,4-benzotriazine 1,4-dioxide;
6(7)-amino-1,2,4-benzotriazine 1,4-dioxide;
6(7)-ami.no-3-methyl-1,2,4-benzotriazine 1,4-dioxide;
6(7)-amino-3-ethyl-1,2,4-benzotriazine 1,4-dioxide;
6(7)-methoxy-1,2,4-benzotriazine 1,4-dioxide;
6(7)-methoxy-~3-methyl-1,2,4-benzotriazine 1,4-dioxide;
6(7)-[1-(2,3-~dihydroxypropoxy]-1,2,4-benzotriazine
-22- 200190
1, 9~-dioxide;'
6(7)-[1,2-dihydroxyethyl]-1,2,4-benzotriazine
1, 9~-dioxide;
6(7)-[1-(3-et:hylamino-2-hydroxypropoxy)]-1,2,4-
benzotriazine 1,4-dioxide;
6(7)-[2-ethyl.amino-1-hydroxyethyl]-1,2,4-benzotriazine
1-9r dioxide;
6(7)-chloro-1.,2,4-benzotriazine 1,4-dioxide;
6(7)-[2-hydroxyethyl]=1,2,4-benzotriazine 1,4-dioxide;
6(7)-[1-hydroxyethyl]-1,2,4-benzotriazine 1,4-dioxide;
6(7)-vitro-3-amino-1,2,4-benzotriazine 1,4-dioxide;
3-(3-N,N-diet:hylaminopropylamino)-1,2,4-benzotriazine
1, 96-dioxide;
6(7)-vitro-3--(2-N,N-diethylaminoethylamino)-1,2,4-
benzotriazine 1,4-dioxide
and their pharmaceutically acceptable salts and the
thioamide analogs of the foregoing list of compounds. It
should be noted that the Yl or Y2 substituents set forth
in most of the above compounds as present in either the
6 or 7 positiions (designated ~6(7)") or in both the 6
and 7 positions (designated "6,7") may also be present
at the 5 and~~or 8 ring positions.
30
_23_
B. Preoaratio~n of the Compounds of the Invention
General methods for preparing some 3-amino
derivatives are found in the above-referenced patents to
Ley et al., for example US 3,980,779. The compounds are
prepared from benzofuroxan of the formula:
0
'f
N ,~
~ ~ /O
by reaction with a salt of cyanamide, followed by
acidification of the reaction mixture. The benzofuroxan
starting material is not symmetric with respect to its
own 5 and 6 positions (which are the 6 and 7 positions
of the resulting 3-amino benzotriazine oxide).
Therefore, a nuixture of the 6- and 7-substituted
materials may result. If desired, this mixture can be
separated using conventional means into individual
components having a substituent in either the 6 or 7
position.
The dioxide may also be prepared from the
parent monoxidle or 1,2,4-benzotriazine by peracid
oxidation (seep Bobbins et al, J Chem Soc 3186 (1957) and
Mason et al, J' Chem Soc B 911 (1970)).
30
4
In addition, the monoxide may be prepared by:
(1) cyclization of a 1-vitro-2-aminobenzene
compound using H2NCN~2HC1;
(2) oxidation of the parent compound given by
the structure
s~ N -~ ~J
or by controlled reduction of the corresponding dioxide
(see Mason, aupra, and Wolf et al, J Am Chem Soc 76:355
(1954)).
They 1,2,4-benzotriazines may be prepared by
cyclization of formazan precursors using BF3/AcOH (see
Scheme I and Atallah and Nazer, Tetrahedron _38:1793
(1982)).
3-amino-1,2,4-benzotriazines may be prepared
either by cyc;lization of a parent compound (see Scheme
II and Arndt,. Chem. Ber. 3522 (1913)) or by reduction of
the monoxide or dioxide as above.
They 3-hydroxy-1,2,4-benzotriazine oxides may
be prepared using peroxide and sodium tungstate (Scheme
III), a novel. synthetic procedure for making the
3-hydroxy-1,9~-dioxide compound, or concentrated sulfuric
acid and sodium nitrate (Scheme IV).
30
-25-
~ll~'~t.~'~~
N'N BF~/AcOH
N ~L,~ X
N~ X
NH
0
Scheme I
H
NH SMe O N
O . 2 . ---~. 0 0
~N=c-N=IN-c=NH N
2
Scheme II
0 0
YL_ ~N 1 N
~N
~ N H202 Y
2 (/1 ~ 2
Y s Y N~\ OH
Na2WOQ 2H20
2
> 50°C p
Scheme III
0
f
YL N L _ NWN
WN H ~ Y
2 4
2 ,
Y= Y N/ \ 0 H
N ~ NdNO
2 2
-26-
goo ~ 90
The invention also encompasses a novel method
of preparing 1,2,4-benzotriazine oxides unsubstituted at
the 3 position (sometimes referred to herein as the
"3-desamino" compounds). The novel synthesis involves
reductive deaminatian of the corresponding 3-amino
structure. In contrast to prior methods of synthesizing
3-desamino-1,2,4-benzotriazine oxides, the present
method enables a simple, straightforward one-step method
which gives 'the desired product in a high yield. The
method invol~~es treating a 1,2,4-benzotriazine oxide of
Formula (I), wherein X is NH2, with a lower alkyl
nitrite under reductive deaminating conditions. By
"reductive deaminating conditions" is meant reaction
conditions which will give rise to at least about 10%,
preferably at least about 50%, of the desired
3-unsubstituted reaction product. A preferred lower
alkyl nitrite for use in said method is t-butyl nitrite.
Exemplary re<3uctive deaminating conditions involve
reaction in a compatible solvent, e.g., dimethyl
formamide, at a temperature of at least about 60°C,
typically at a temperature in the range of 60-65°C.
This reaction is illustrated generally at Scheme V, and
is exemplified in Examples 12-15 herein.
O O
1 N ?
\ \
y I N t-BuN02.. ~ y1 N
y ~ DIdF y
~ N ' "N'H 60-65oC
2
On Scheme V On
_27_
C. Formulation and Administration
As demonstrated below, the oxidized
benzotriazine~s of the invention may be used to
radiosensitiz;e or selectively kill hypoxic tumor cells
in warm-bloodied animal hosts. A way in which they may
be used is ir,~ conjunction with agents known to
selectively create hypoxia in tumors. Such methods
include the use of antihypertensive drugs such as
hydralazine, or agents which affect the amount of oxygen
carried by tree blood. While these compounds will
typically be used in cancer therapy of human patients,
they may be used to kill hypoxic tumor cells in other
warm blooded animal species such as other primates, farm
animals such as cattle, and sports animals and pets such
as horses, dogs, and cats.
Hypoxia is believed to be associated with all
types of solid malignant neoplasms. The compounds of
the invention may, therefore, be used to radiosensitize
or to kill ne~oplastic epithelial cells, endothelial
cells, connective tissue cells, bone cells, muscle
cells, nerve cells, and brain cells. Examples of
carcinomas and sarcomas include carcinomas such as
epithelial cell, acidic cell, alveolar cell, basal cell,
basal squamous cell, cervical, renal, liver, Hurthle,
30
~~1.~~~
-28-
Lucke, mucinous and Walker, and sarcomas such as
Abernathy's, alveolar soft part, angiolithic, botyroid,
encephaloid, endometria stroma, Ewing's fascicular,
giant cell, 7.ymphatic, Jensen's, juxtacortical
osteogenic, y:aposi's, medullary, and synovial. Specific
examples of tumors that have been sensitized with other
radiosensiti::ers are reported in Adams, G.E., Cancer: A
ComprehensivEa Treatise (F. Hecker, Ed) vol 6, pp
181-2Z3, Plenum, New York, 1977.
The compounds may be administered to patients
orally or parenterally (intravenously, subcutaneously,
intramuscularly, intraspinally, intraperitoneally, and
the like). When administered parenterally the compounds
will normally be formulated in a unit dosage injectable
form (solution, suspension, emulsion) with a
pharmaceutically acceptable vehicle. Such vehicles are
typically nontoxic and nontherapeutic. Examples of such
vehicles are water, aqueous vehicles such as saline,
Ringer's solution, dextrose solution, and Hank's
solution and nonaqueous vehicles such as fixed oils
(e. g., corn, cottonseed, peanut, and sesame), ethyl
oleate, and isopropyl myristate. Sterile saline is a
preferred velhicle and the compounds are sufficiently
water soluble to provide a solution for all foreseeable
needs. The 'vehicle may contain minor amounts of
additives such as substances that enhance solubility,
isotonicity, and chemical stability, e.g., antioxidants,
buffers, and preservatives. When administered orally
(or rectally) the compounds will usually be formulated
into a unit dosage form such as a tablet, capsule,
suppository .or cachet. Such formulations typically
include a solid, semisolid or liquid carrier or diluent.
Exemplary diluents and vehicles are lactose, dextrose,
-29-
sucrose, sorbitol, mannitol, starches, gum acacia,
calcium phosphate, mineral oil, cocoa butter, oil of
theobroma, alginates, tragacanth, gelatin,
methylcellulo;se, polyoxyethylene sorbitan monolaurate,
methyl hydrox:ybenzoate, propyl hydroxybenzoate, talc,
and magnesium stearate.
The amount of compound administered to the
subject is su:Eficient to radiosensitize or to produce
cytotoxicity :in the malignant neoplasm to be treated but
below that which may elicit toxic effects to normal
tissue. This amount will depend upon the type of tumor,
the species o1: the subject being treated, the indicated
dosage intended and the weight or body surface of the
subject. The radiation may be administered to humans in
a variety of different fractionation regimes, i.e., the
total radiation dose is given in portions over a period
of several days to several weeks. These are most likely
to vary from daily (i.e., five times per week) doses for
up to six weeha, to once weekly doses for four to six
weeks. An individual dose of the benzotriazine will be
given before or after each radiation treatment and is
likely to be i,n the range of 0.01 to 20 mmol/kg and
usually in they range of 0.1 to 2 mmol/kg. In these
treatment regimens, each radiation dose is typically 1-5
Gy, preferably, less than 2.5 Gy and more preferably
2-2.5Gy. Typically, one dose of radiation is
administered pier day although 2 or more might be used if
tolerated by the patient
It has now been found that the compounds
disclosed herein as radiosensitizers, particularly
3-amino-1,2,4-benzotriazine 1,4-dioxide, both sensitize
o' -30-
tumors to radiation without increasing the sensitivity
of normal skin, and work in highly fractionated
radiation regimens. As demonstrated in Example 22
herein, pre- or post-irradiation treatment of cells
with, for example, 3-amino-1,2,4-benzotriazine
1,4-dioxide, under hypoxic conditions, radiosensitizes
cells even when the drug is not present during the
radiation exposure and the cells are aerobic.
For use as selective cytotoxic agents., the
compounds of the invention can be administered alone,
~~rith radiation or other cancer cytotoxic agents, with
vasoactive drugs (e. g., hydralazine), or under other
conditions which reduce the amount of available oxygen
carried by tile blood such as anemia or drugs which
increase the binding of oxygen to hemoglobin, all of
which can enhance selectively the degree of hypoxia in
the tumor.
Examples
The following examples further illustrate the
compounds of the invention and methods for synthesizing
and using them, and are not intended to limit the
invention in any manner.
Experimental: All reactions were carried out
i~ flame-dried glassware and under a blanket of Argon.
t-butyl nitrite (90%) was purchased from the Aldrich
CtfEemicai Company. ~imethylformamide was distilled from
calcium hydride. 7-Nitro-3-amino-1,2,4-benzotriazine-
3-amine 1-oxide was purchased from Parish Chemical
Company, trifluoroacetic anhydride, N,N-diethylethylene-
diamine, N,N~-diethylpropylenediamine and sodium..
tungstate di,hydrate were purchased from Aldrich Chemical
Company and 70% hydrogen peroxide was a gift from
~~~~~~~ -31-
Interox America. All reactants were used without
further purification. Flash chromatography was carried
out on E. Merck 230-400 mesh silica gel under a positive
pressure of argon. NMR spectra were obtained on a
Varian XL-400 or Jeol FX90Q spectrometer and in
d6-acetone, d4-methanol, or d6-dimethyl sulfoxide, as
indicated, and are reported relative to the central peak
in the appropriate multiplet (2.04, 3.30, and 2.49 ppm,
respectively), W spectra were obtained on a
Perkin-Elmer 552 spectrophotometer in 95% ethanol, mass
spectra were obtained on an LKH 9000 mass spectrometer,
and elemental analyses were carried out by Desert
Analytics, Tucson, AZ.
Example 1: Preparation of 3-Hydroxv-1,2.4-Benzotriazine
1,4-Dioxide
0
T
I~~ t
~"''~~ N''1' Nl,li
1
A stirred mixture of 1.50g (9.25 mmole) of
3-amino-1,2,4-benzotriazine 1-oxide (1), 100.0 ml acetic
acid, and 30,.0 ml of 30% hydrogen peroxide was treated
with 3.05 g 1;9.25 mmole) of Na2W04 2Fi20. The mixture
was stirred in an oil bath at 60°C for 4 days. The
Yellowish orange mixture was cooled to about 30° and
filtered to remove a light yellow non-W absorbing
solid. The orange solution of hydrogen peroxide in
~~Q~~.~~~ -32-
acetic acid was evaporated to semi-dryness carefully
with several. additions of water and acetic acid to
remove most of the peroxide. The eoncentrated solution
was allowed to stand at room temperature to afford four
crops of an orange solid, 0.87g (42% yield of the sodium
salt of 2). Wmax (20% CH30H/H20): 262.2 (e 39,460);
477 (E 7,030). IR (neat): 3530u, 3150u, 2650u, 2180u
and 1635u. Anal. (calculated for the sodium salt):
C7H4N303Na 1..25H20, 223.64: C,37.6; H,2.93; N, 18.79.
_fund: C, ?'.7.8; H,2.75; N,18.65.
0
T
N
(~~,~Nf)~
~ O~
O
2
Examflle 2: Preparation of 3-Amino-7-Trifluoromethvl-
1,2.4-Henzot:riazine 1-Oxide:
0
r
F3C N
o ~~'
2
3
A mixture of 4-chloro-3-nitrobenzotrifluoride
(Aldrich, 2..70 g, I2.9 mmole) and eyanamide
dihydrochloride (2.75 g, 24 mmole) (previously prepared
-33-
by treating am ether solution of cyanamide with HC1 gas
and collecting the precipitated solid) was heated at
140°C for 1 h~,. The residwe was treated with 2N NaOH (45
ml), heated for a further 5 min, and then allowed to
cool. The precipitate was collected, washed with H20,
dried, and triturated with acetone-toluene to yield I.32
g (45%) of 3 as a light yellow solid M.P. 301-302°,
TLC: Rf 0.60 (9:1 methylene chloride: methanol on
silica gel plates). Mass. Spec.: M+=230 (q = 100).
Example 3: Pretaaration of 3-amino-7-Decyl-1,2.4-
Benzotriazine~ 1-Oxide
1 S ~LOH21
~ ~~J~
N NH2
4
Preparation of 4-(1-decyl)-2-nitroaniline:
Acetic anhydride (400 ml) was added over a 30-minute
period to a stirred solution of 4-decylaniline (Aldrich,
80g, 0.34 mole) in hexanes (2.41). After stirring for
Ih, the mixture was cooled and treated over 30 min. at
5-10°C with T0% nitric acid (34 ml). Stirring was
continued at 5-10°C for 1h and at 25°C for 16h. The
mixture was dliluted with H20 (11), stirred for 5h,
poured,into am open dish and allowed to stand for 16h.
After further' dilution with H20 (1.51), the solid was
collected andl recrystallized from an 85% ethanol
solution (in water) to give 92g (84%) of the
intermediate as an orange solid, m.p. 64°C.
5
-34-
A solution (100 ml) of 85% KOH (19g, 0.288
mole) in H20 was combined with a suspension of
4-(1-decyl)-2;-nitroaniline (89g, 0.28 mole), prepared
above, in methanol (900 ml). The mixture was stirred
for 6h, neutralized to pH 7-8 with concentrated HC1, and
evaporated iru vacuo to near dryness. After dilution
with H20 (400 ml), the solid was collected and air-dried
to give i7g (I00%) of the intermediate as an orange
solid, m.p. ~~9°C.
l.C~g (8.7 mmole) of cyanamide dihydrochloride
(previously F~repared for use by treating an ether
solution of c:yanamide with HC1 gas and collecting the
precipitated solid) was added portionwise over 10 min to
a preheated melt (190°C) of 4-(1-decyl)-2-nitroaniline
prepared in the preceding step (500 mg, 1.8 mmole). The
reaction mixture was heated at 190°C for 5 min, cooled
to 25°C, treated with 6N KOH (10 ml), and heated at
90-95°C for 1.h. After cooling to 25°C, the solid was
collected, waished with H20 and ethanol and air-dried to
give 0.258 (9:6%) of compound 4 as a light yellow solid,
m.p. 177°C (clec). Mass. spec. M+=285 (q=100), 302
(q=13).
Example 4: F~reparation of 3-Amino-7-Carbamyl-1.2,4-
Benzotriazine~ 1-~Yide
0
r
', -35-
~~U~1.~~
Preparation of 4-chloro-3-nitrobenzamide:
2~r2g (0.1 mole) of 4-chloro-3-nitrobenzoic acid
(Aldrich) and thionyl chloride (20 ml) were combined,
allowed to stand for 16h, and refluxed for 4h to give a
clear red solution. The solution was evaporated in
vacuo and aze~otroped with benzene. The residue was
dissolved in acetonitrile (20 ml) and added over 30 min
to cold (-10"C) concentrated ammonium hydroxide (100
ml). After 3h at -10°C and 16h at 25°C the mixture was
poured into an open dish and allowed to evaporate to
dryness. The' residue was slurried in H20 and the solid
was collected and air-dried to give 19.8g (98%) of the
intermediate as a light yellow solid, m.p. 153°C.
A solution of Na (3.45g, 0.15 mole ) in
ethanol (75 ml) was added to a solution of guanidine
I5 hydrochloride' (15.88, 0.165 mole) in ethanol (75 ml).
After 1h the mixture was filtered and the filtrate was
combined with a suspension of 4-chloro-3-nitrobenzamide
(10g, 0.05 merle) prepared above, in ethanol (50 ml). The
mixture was stirred and refluxed for 16h, cooled to
0-5°C, and acidified with concentrated HC1 (8 ml). The
collected solid was combined with K2CO3 (28g, 0.2 mole)
and H20 (40 ml) and the mixture was stirred and heated
at 100°C for 8h. After cooling to 25°C, the solid was
collected, washed with H20, and air-dried. The solid
was suspendedt in boiling ethyl acetate, collected and
washed with hoot ethyl acetate. The solid was repeatedly
suspended in boiling dioxane and collected (6x100m1).
TFi~ combined filtrate was evaporated in vacuo to a
solid. The solid was suspended in 95% ethanol,
collected andl air-dried to give 0.44g (4.3%) of compound
5 as a light yellow solid, m.p. 300°C. TLC: Rf=0.23
' -36-
(methylene chloride: acetone of 2:1, silica gel plates).
Mass. Spec.: M+ 205 (q~ 100).
Example 5: 1?reparation of 7-Acetyl-3-Amino-1.2.4-
Benzotriazine 1-Oxide Oxime
0
H c'"~ . H.,
CD C~~
1O ' N ~2
6
A combined mixture of 7-acetyl-3-amino-
12.4-benzotriazine 1-oxide (prepared in Example 5;
50 mg, 0.25 rnmole), hydroxylamine hydrochloride (200 mg,
2.88 mmole), pyridine (1 ml) , and ethanol (1 ml) was
heated at 90--95°C for 1h and then cooled to 25°C. The
mixture was <iiluted with 95% ethanol (5 ml) and the
Solid was collected and air-dried to give 30 mg (56%) of
compound 6 a:a a light yellow solid, m.p. 278°C (dec).
TLC: Rf=0.60 (9:1 methylene chloride: methanol). Mass
Spec . : M+=2719 ( q=100 ) .
30
i ' _37_
Eaauaule 6: ~?reparation of 3-Amino-6(7)-Decvl-1,2,4-
Henzotriazine 1,4-Dioxide
0
T
N
~~aH2i
~NHz
0
7
5-i;l-decyl)-benzofuroxan: A combined mixture
of 4-(1-decy7L)-2-nitroaniline (779, 0.28 mole), 5.25%-
Na0C1 in H20 (4769, 0.34 mole), 85% KOH (20.38, 0.31.
mole), nBu4NHS04 (4.79, 0.014 mole), and CH2C12 (2.28 1)
was stirred rapidly for 6h and diluted with H20 (500 ml)
and CH2C12 (7. 1). The separated organic phase was
washed successively- with 1N HC1 (1 1) and brine (2 x 1
1)), dried (Ma2S04), and concentrated in vacuo to yield
a red oil, 70 g (92%).
A :solution of 5-(1-decyl)-benzofuroxan as
prepared above (10 g, 0.036 mole) and benzyltriethyl
ammonium chloride (0.36 g, 0.0016 mole) in DMSO (180 ml)
was treated gradually over several hours with cyanamide
(13.0 g, 0.37. mole) and K2C03 (36.8 g, 0.27 mole). The
mix~ure~-waa atirred for 48h and filtered. The filtrate
wa~..diluted with H20 (6 1) and glacial acetic acid (40
ml~ and extracted with CH2C12 (4 x 500 ml). The
combined organic solution was washed successively with
5% NaHC03 solution (1 x 500 ml) and brine (2 x 500 ml),
dried (Na2S09~), and evaporated in vacuo to dryness. The
crude product: was purified by chromatography on silica
-38-
geZ using CH2C1.2: methanol (98:2) tv give 1.8g (16%) of
compound 7 as a~ red solid, m.p. 155°C (dec). Mass.
Spec.: M+=318 (q=4), 285 (q=100).
Example 7: Pre~~aration of 1.2.4-Benzotriazine
1.4-Dioxide
0
H2O2 NON
of ~ -~ 0 0
X13
0
8 9
A mi~;ture of 1.80 g (13.73 mmole) of 8, 90$
H202 (9 ml), trifluoroacetic anhydride (13.5 ml) and
Na2W04.2H20 (li!.50g, 38 mmole) in CHC13 (170 ml) was
stirred at room temperature for 5 days. The reaction
mixture was diluted with H20 (100 ml) and extracted with
CHC13 (100 ml). The organic layer was washed with H20
(50 ml), dried (Na2S04), and the solvent removed in
vacuo. The residue was chromatographed on silica gel
using EtOAc-CH~!C12 (1:1) to give 0.30 g (I3.4%) of
compound 9 as a~ yellow solid, m.p. 204-205°C. Anal.
Calc'd. for C7~t5N302 (163.13): C, 51.5; H, 3.09; N,
25.76. Found: C, 51.6; H, 3.36; N, 26.01. Mass Spec.
M+=I63 (q~100), 147 (q=50). TLC: Rf=0.27
(E~t~-CH2C12, 1:1, silica gel plates). IR (nujol):
160~'p, 1460u, 1.300u, 1230u. wmax (H20): 227
22,900) 252 (E 12,950), 392 (E 4,080).
-39-
Eaamflle 8: F~reparation of 7-Chloro-3-Hvdroxv-1.2~4-
HenzotriazinEe 1,4-Dioxide
0
T
--: ~
O O~
~2
a 0
~L ~ T
CL
O ON ~ O O 1N
OH ~NH2
10 ~ 1
0 0
11 12
A mixture of 1.50 g (7.63 mmole) of 10 in 100
ml acetic acid was treated with 2.52 g (7.63 mmole) of
Na2W04~2H20 and 30 ml of 30% H202. The mixture was
stirred and heated for 6 days at 50~C, then slowly
evaporated to dryness to remove H202. The residue was
boiled in 250 ml H2C and filtered to remove about 25 mg
of starting nnaterial 12. The aqueous solutions were
then extracted with 2 x 250 ml portions of ethyl
acetate. A deep red crystalline material that was
characterized as 12 by TLC and Mass. Spec. analysis
formed in the partitioning mixture above and was
collected by filtration to afford 60.0 mg of a yellowish
orange solid (3.7% yield), characterized as follows as
1~?x which showed good solubility in a mixture of hot
isopropyl alcohol and water. Mass. Spec.: M+=212
(q=100)(compound 10); TLC: Rf= 0.34 (acetone, silica
gel plates).
The ethyl acetate solutions above, separated
from the H20 layer after the filtration to remove 12,
-40-
2001903
wire evaporated to dryness. The residue was then
treated with isopropyl alcohol at room temperature to
afford a dull orange solid, 0.41g (25% yield) of 11.
Mass. Spec.: M+=213 (q=70); TLC: Rf=0.22 (acetone,
silica gel plates). Compound 11 was characterized as
the ammonium salt, C7H4C1N303~NH3, m.w. 230.61, as
follows. The free acid 11 was dissolved in concentrated
NH40H and then chilled in ice and filtered to remove a
trace of insoluble 12. The red filtrate and washings
were evaporated to dryness, leaving a reddish-orange
solid. The solid was treated with 50 ml of boiling
1,2-dimethoxyethane, collected on a filter and washed
with an additional 25 ml of hot 1,2-dimethyl ether.. The
solid was dried over P205 at 56°C/1.0 mm, leaving 0.244
g (87% yield) of 13 ,
°
c i .~./w
G ON
-l~.o
b a
0
13
Anal. Calc'd. for C7H4C1N303 NH3 (230.61): C, 36.5; H,
3.06; N, 24.30. Found: C, 36.5; H, 3.07; N, 23.94.
Wmax (HBO): 219 (~ 12,580); 265.4 (e 40,000); 4830486
(~ 6,640).
-41-
200j90~
Example 9: 1?reparation of 7-Nitro-3-Amino-1,2,4-
Benzotriazine 1,4-Dioxide
O
T
T
--~ DiN -'~
~'' ' ~.W N ~ ~
~'N~NHCOCF3
n
l5
0
T
Oy N
-'!
'~.~' L NOON
c
r6
7-Nitro-3-trifluoroacetamido-1,2,4-
benzotriazine 1-oxide (15): A solution of 7-vitro-3-
amino-1,2,4-benzotriazine 1-oxide (14) (4.00g, 19.3
mmol; Parish Chemical Co.), CHC13 (125 ml) and
trifluoroacet:ic anhydride (12.0 ml, 85.0 mmol) was
stirred at room temperature for 44 hr. The resultant
light yellow solid was filtered, washed with CHC13 (50
ml) and dried to give 5.35g (91% yield) of the product
as a yellow :3olid. Anal. Calc'd, for C9H4F3N504: C,
35.7; H, 1.3:3; N, 23.10. Found: C, 35.7; H, 1.23; N,
23.06.
7-Nitro-3-amino-1,2,4-benzotriazine 1,4-oxide
(16): To a stirred solution of 7-vitro-3-
trifluoroacet~amido-1,2,4-benzotriazine 1-oxide prepared
above (15) (:Z.50g, 8.25 mmol) in CHC13 (200 ml) was
S
42 2UU 1 ~Q~
added Na2W04.2H20 (90 mg, 0.273 mmol) followed by 70%
H20~ (10 ml). After 15 min the solution was treated
with trifluoroacetic anhydride (8.0 ml, 56.7 mmol) and
stirring was continued at room temperature for 64 hr.
The reaction mixture was chromatographed (EtOAc, 20%
MeOH/acetone, and finally 20% DMF/acetone) then
recrystallizeld in acetone to give 1.20g (65% yield) of
the product (16) as an orange solid, mp 286-28S°C
(dec.). UV: a 259, 300, 345, 387, 472. Anal. Calc'd.
for C7H5N504: C, 37.70; H, 2.26; N, 31.39. Found: C,
37.70; H, 2.1.3; N, 30.94.
Example 10: Preparation of
3-(3-N,N-Diet.hylaminopropvlamino)-1,2,4-Benzotriazine
1,4-Dioxide
O
N''"N
.--~, '~ ~ "~ N
O 1D ,
ek
t8
0
1'
,,.., N .., ~
~'''~'~~ R,~-w N NEty
N
V
lea.
3-(,3-N,N-diethylaminopropylamino)-1,2,4-
benzotriazine! 1-oxide (18): A solution of 3-chloro-
~~~1.:~~ i -43-
1,2,4-benzotriazine 1-oxide (17) (3.0g, 16.5 mmol)
(produced by the method of Sasse et al., U.S. Patent
4,288,771) in CH2C12 (100 ml) was treated with
N,N-diethylpropylenediamine (9.5 rnl, 88.3 mmol). After
20 hr at room temperature the mixture was diluted with
1,2-dichloros~thane (50 ml) and washed successively with
Na2C03 and H;~O. The yellow solution was dried (Na2S04),
filtered and evaporated in vacuo to give 3.938 (87%
yield) of the product as a yellow solid.
Recrystallizartion (ether/petroleum ether) yielded pure
material, mp 47-48°C. Anal Calc'd. for CI4H21N50 (18):
C, 61.10; H, 7.69; N, 25.44. Found: C, 61.30; H, 7.80;
N, 25.61.
3-1;3-N,N-diethylaminopropylamino)-1,2,4-
benzotriazine 1,4-dioxide (18a): To a stirred solution .
of 3-(3-N,N-cliethylaminopropylamino)-I,2,4-benzotriazine
1-oxide 18 prepared as above (1.608, 6.10 mmol) in CHC13
(50 ml) was added trifluoroacetic anhydride (22.0 ml).
After 15 min the mixture was cooled to -10°C, 70% H202
(10 ml) added and then stirred at room temperature for
20 days. The reaction mixture was dried (Na2S04),
filtered and evaporated to dryness. The residue was
dissolved in saturated NaHC03 solution (50 ml) and
extracted wii:h CH2C12 (3x150 ml). The organic layer was
dried (Na2S0~~),_filtered and evaporated to give the
product LBa, 0.518 (29% yield) as a red solid, mp
92-94°C. NM~t: d ( 400 t~Iz, CDC13 ) 1.11 ( 6H, t, J=7 .1
Hz, CH3), 1.84-1.90 (2H,. m, H-2'), 2.48-2.64 (4H, m,
NCH2CH3.and H-3'), 3.68 (2H, br t, J=5.5 Hz, H-1'), 7.46
(1H, ddd, J="7.1, 7.0 and 1.2 Hz, H-6), 7.84, ddd, J=7.0,
6.9 and 1.2 Hz, H-7), 8.31 (2H, m, H-5 and 8), 8.80 (1H,
br s, NH). (1V: 7~ 220, 270, 476. Anal. Calc'd. for
C14H21N502. (1/3 H20): C, 56.50; H, 7.34; N, 23.55.
Found: C, 56.90; H, 7.15; N, 23.40.
Example 11: Preparation of 7-Nitro-3-(2-N,N-_
Diethvlaminoethvlamino)-1,2.4-Henzotriazine
' ' Dioxide
0 0
r
Oi N v ~_
~ ' N ~ ~ N'- ~1 '~ .~~~ C~.
~ ~t 19
r
U Z Al .v ~ M ~. ,~
< < [~ ( ---
"y er-''\N NE:Ei~ HCt
is
.10
0
1~ N ,.- N ,'
2 5 s ~ ~ JU ''~-' N ~' Ey a EI C t
1, ~
ai
7-nitro-3-(2-N,N-diethylaminoethylamino)-
1,2,4-benzotriazine 1-oxide hydrochloride (20): A
-45-
solution of 7-vitro-3-chloro-1,2,4-benzotriazine 1-oxide
(13~ (1.60g, 7.06 mmol) (prepared as generally shown in
Sasse et al., U.S. Patent 4,289,771, with (a) NaN02 and
H2S04 at 40°C, follawed by (b) chlorination with POC13
at 106°C) in CH2C12 (50 ml) was treated with
N,N-diethylethylenediamine (6.0 ml, 42.7 mmol). After
16 hr at room temperature the mixture was evaporated to
dryness under high ~racuum at 60°C. The yellow solid was
stirred in 20% iPrOH/ether (150 ml) for 5 hr, filtered,
washed with iPrOH then petroleum ether and dried
(80°C/1.0 mm:Eig) to give 1.808 (74% yield) of the product
20 as yellow needle crystals. NMR 8 (90 MHz,
d6-DMSO/d4-MeOH) 1.25 (6H, t, J=6.0 Hz, CH3), 3.25 (6H,
m, NCH2), 3.82 (2H, m, H-1'), 7.74 (1H, d, J=7.0 Hz,
15 H-5), 8.52 (1H, dd, J=7.0 and 2.O Hz, H-6), 8.91 (1H, d,.
J=2.0 Hz, H-8).
7-vitro-3-(2-N,N-diethylaminoethylamino)-
1,2,4-benzotriazine 1,4-dioxide hydrochloride (21). To
a stirred solution of 7-vitro-3-(2-N,N-diethylamino-
Zp ethylamino)-1,2,4-benzotriazine 1-oxide hydrochloride
(20; prepared as described above) (0.50g, 1.46 mmol) in
CHC13 (50 ml) at 0°C was added trifluoroacetic anhydride
(9.0 ml). After 30 min 70% H202 (4.0 ml) was added and
the mixture stirred at room temperature for 3 days, then
~5 dried (Na2S04), filtered, and evaporated in vacuo to
dryness to give the trifluoroacetate salt 0.678 (45%
yield?. This product was dissolved in saturated NaHC03
solution (30 ml) and extracted with CH2C12 (3x30 ml).
The dich_loromethane was washed with H20, dried (Na2S04),
3J filtered, saturated with gaseous HC1 and evaporated to
dryness to give 0.35g (63% yield, 28% overall) of the
product as a red solid, m.p. 194-195°C. UV: a 260, 306,
-46-
200190
388, 479. Anal. Calc'd. for C13H18N604~HC1: C, 43.50;
H, 5.34; N, 23.43. Found: C, 43.20; H, 5.37; N, 23.11.
The following Examples 12-15 are directed to
reductive de,amination reactions for preparing compounds
of Formula (I) which are unsubstituted at the
3-position, i.e.. wherein the substituent "X" is
hydrogen.
Example 12: Pretaaration of 1,2,4-8enzotriazine 1,4-
Dioxide by Reductive Deamination of 3-Amino-1.2.4-
Benzotriazin~e 1,4-Dioxide
o ~ o
T T
~ N '."' m n~ "'~ N
~~ 1 C3
'~-/ ~ ~ .-~ N N ~ ~ N i-
L ~ ~,
0 0
22 9
To a rapidly stirred solution of t-butyl
nitrite (867 mg, 1.0 ml, 8.41 mmol) in DMF (20 ml) at
60-65°C was .added 3-amino-1,2,4-benzotriazine
1,4-dioxide ("SR 4233") (500 mg, 2.81 mmol) (prepared by
the method of Seng et al., Anctew. Chem. internat. Edit.
11:11 (1972)) in small portions over 5 min. Following
the addition, and subsidence of the concomitant
effervescence (approx. 5 min), the solution was cooled
and reduced 'under high vacuum to a dark waxy solid.
Flash chromatography (30~ EtOAc/CH2C12) gave a yellow
solid, mp 188-189.5°C (dec.), which was recrystallized
, -47_
v Zp(~ 1 g 4~3
from methanol to give 195 mg (43% yield) of the product
9 as bright ~~ellow platelets, mp 192-194°C (dec.). NMR:
d (400 MHz, d6-acetone) 8.04 (1 H, ddd, J=8.5, 7, 1.5
Hz), 8.15 (1 H, ddd, J=8.5, 7, 1.5 Hz), 8.42 (1 H, dd,
J=8.5, 1.5 H:c), 8.43 (1 H, dd, J=8.5, 1.5 Hz) 9.05 (1 H,
s, H-3). W.: J~ 405, 300, 225. MS, m/z (relative
intensity) lfi4(9), 163(100, M'~), 147(13), 136(19),
90(7), 78(27;1, 76(26), 75(8), 64(9), 63(10), 52(12),
51(48), 50(2E3), 38(8), 37(5), 30(18), 28(6), 27(7).
Anal. Calc'd,. for C7H5N302: C, 51.54; H, 3.09; N,
25.76. Found: C, 51.42; H, 3.02; N, 25.66.
Example 13:- Preparation of 7-Allyloxy-1,2,4-
Benzotriazine 1,4-Dioxide Via Reductive Deamination
0
N~
C~ 1 ---~. ~ ~ ~ a t
~N/~N~ ~N/
23 24
7-Allyloxy-1,2,4-benzotriazine 1,4-dioxide 24:
To a stirred solution of t-butyl nitrite (271 mg, 0.312
ml, 2.63 mmo'.l) in DMF (15 ml) at 60-65°C was added
7-allyloxy-3--amino-1,2,4-benzotriazine- 1,4-dioxide 23
(205 mg, 0.875 mmol) in small portions over 5 min.
After 30 min additional t-butyl nitrite (271 mg, 0.312
ml, 2.63 mmo:l) was added, and shortly thereafter the
deep red solution effervesced and lightened appreciably
~1~~~.~~;~ -48-
in color over' a period of a few minutes. After an
additional 30 min the resultant orange solution was
reduced under' vacuum to a brown solid which was
sequentially flash chromatographed (10% EtOAc/CH2C12)
and crystallized (CH2C12/petroleum ether) to give 72 mg
(38% yield) of the product 24 as light orange crystals,
mp 147-148°C. NMR: d (400 MHz, d6-acetone) 4.89 (2 H,
ddd, H-1', J1,',2'=5.5, J1~,3'cis=Jl°,3'trans=1.5 Hz),
5.36 (1 H, dctd, H-3', J3~,2'cis=10.5, J3~,3~=3,
J3~,1~=1.5 H~:), 5.52 (1 H, ddd, H-3°, J3~,2'trans=17~5,
J3',3'=3~ J3'1'=1.5 Hz), 6.14 (1 H, ddt, H-2',
J2',3'cis=10.5, J2',1'=5.5 Hz), 7.70 (1 H, d, H-8,
Jg,6=2.5 Hz), 7.74 (1 H, dd, H-6, J6,5=9.5, J6,g=2,5
Hz), 8.33 (1 H, d, H-5, J5,6= 9.5 Hz), 8.93 (1 H, s,
H-3). W: a, 425, 410, 365, 355, 320, 245, 200. MS
m/z/ (relative intensity) 220(4), 219(34,M+), 103(4),
77(4), 75(4),. 63(13), 62(4), 42(3), 41(100), 39(16).
Anal. Calc'd. for ClpHgN303: C, 54.79; H, 4.14; N,
19.17. Found: C, 54.73; H, 4.16; N. 19.15.
Example 14: Preparation of 7-(3-N-Ethylacetamido-2-
acetoxvpropoxv)-1.2.4-Benzotriazine 1.4-Dioxide Via
Reductive Amination
OAc
( o
~/'~ ~, ~'J' N O ~ ~ N ~ N
l ~ ._..~,. ( ~ 0
a a Et w .J~
V G N la, ~" i
I~CN6'E~
25 26
i4~~'~.~~~ -49-
To a stirred solution of t-butyl nitrite (185
mg, 1.79 mmo:l) in DMF (5 ml) at 60°C was added via
syringe a solution of
7-(3-N-ethylacetamido-2-acetoxypropoxy)-3-amino-
1,2,4-benzotriazine 1,4-dioxide (25) (125 mg, 0.329
mmol) in DMF (5 ml) over a period of 1 min. After 5 min
additional t-butyl nitrite (217 mg, 2.10 mmol) was added
and an immediate reaction occurred, as evidenced by the
evolution of a gas and a change in color of the solution
from red to :light orange. After an additional 10 min
the solution was stripped to a yellow/brown solid and
eluted throu<~h silica gel with 5% MeOH/CH2C12 to give
119 mg of a yellow oil. Recrystallization from
CH2C12/ligroin gave 90 mg yellow solid (75% yield), mp
179-180.5°C. NMR: d (400 MHz, d4-methanol, mixture of
rotamers, ratio approx. 2:1) 1.12, 1.22 (t's, 1:2, 3 H
total, J=7 Ha), 2.06, 2.07 (s's, 2:1, 3 H total), 2.11,
2.17 (s's, 2.:1, 3 H total), 3.41-3.92 (m, 4 H),
4.34-4.48 (m" 2 H), 5.48-5.58 (m, 1 H), 7.76-7.86 (m, 2
H), 8.36-8.42 (m, 1 H), 9.04, 9.06 (s's, 2:1, 1 H
total). W: a 420, 405, 365, 350, 315, 240, 200. MS:
m/z (relativE~ intensity) 365(0.5), 364(1.4, M+),
349(0.5), 348(1.1), 347(0.5), 332(1.2), 331(3.6),
187(7), 186(ES6), 102(6), 100(21), 84(30), 63(6),
58(100), 56(8), 43(65), 42(9)~ 41(5), 30(14), 29(5),
28(8).
-50-
r~~~~~. ~~~
8:a~~le 15: Preparation of 7-Nitro-1.2.4-Benzotriazine
1t4~-Dioxide 'via Reductive Deamination
o C
r T
---~ ~z t'~ ~ d
~~ N~~N11 ~'~-~ /
i ~ N
O
14 27
To a stirred solution of t-butyl nitrite (88
mg, 0.85 mmo:1) in DMF (5 ml) at 60°C was added
7_vitro-3-amino-1,2,4-benzotriazine 1,4-dioxide (14) (38
mg, 0.17 mmo:l). After 30 min the addition of further
t-butyl nitrate (175 mg, 1.70 mmol) to the dark red
slurry was irnmediately followed by a change in
coloration and effervescence. After an additional 10
min the orange solution was reduced to a red solid _in
vacuo and chromatographed with 1% AcOH/CH2CI2 to give 3
mg of the product 27 as a yellow solid (10% yield). NMR
S (90 MHz, d6-dimethyl sulfoxide) 7.68 (d, 1H, J = 9.2
Hz), 7.92 (dd, 1H, J' = 9.2, 2.2 Hz), 8.10 (d, 1H, J =
2.2 Hzf, 8.65 (s, 1H)). W: a 420, 310, 240, 205.
M~:m~z (relai:ive intensity) 209 (9), 208 (100, M+), 192
f~'4~, I81 (1!E), 162 (16), 105 (9), 77 (28), 75 (52), 74
(2?~, G3~(21;1, 62 (16), 30 (77), 18 (26).
2001903
Example 16: In Vivo Assav for Activity in Combination with
Radiation
Compounds of the invention were tested in vivo
for activity by the assay of Brown, J.M., Radiation Res
(1975) 64:633-47. For this assay, SCCVII carcinomas in
female C3H mice weighing 20-25 g were used. These mice
were bred under specific pathogen-free conditions and were
3-4 months old at the beginning of each experiment. The
SCCVII tumor was grown intradermally in the flank from an
inoculation of 2 x 105 tumor cells taken from the 2nd-8th in
vitro passage of t:he tumor cells after removal from the
previous in vivo tumor. 7.'wo tumors per mouse were
implanted, and were used as subject tumors when they
reached a volume of approximately 100 ml. At this point
the tumors contained approximately 20o hypoxic cells.
The test compound was tested at a fixed injected
does of either 5 m~nol/kg or 2/3 of the LDSO (whichever was
lower). Suitabl°e control of the test compound-injected but
non-irradiated and saline-injected and irradiated mice were
also included. A fixed radiation does of 20 Gy was applied
at variable intervals of 2 hr after to 3 hr before
injection of the drug. By using these intervals, the
results give an indication of both the optimum irradiation
time and the extent of extra cell killing compared to
radiation alone. 'The results of such time-course
experiments using :3-amino-1,2,4-benzotriazine 1,4-dioxide
are shown in Figure 2. They show enhanced cell killing
compared to radiation only, more than would have been
expected on the basis of additivity of the two individual
cytotoxicities. T:he similar increased cytotoxicity when
- 51 -
.....
iC:~!~~.~~~~s °52-
the4-drug is given before or after radiation indicates
serl~ctive:to:Kicity to the hypoxic cells rather than a
radiosensiti;sing effect of the benzotriazine dioxide.
Irradiation of the SCCVIi tumors was done by
irradiating nonanaesthetized tumor-bearing mice in a
Plexiglas bo:K. irradiation conditions were 250 kVp
X-rays, 15 mid, FSC 33 cm, added filtration of 0.35 mm
Cu, half value layer 1.3 mm Cu, and a dose rate of 317
rad/min.
The amount of cell killing was judged by
survival rate of dissected and cultured tumor cells as
follows. The: tumor-bearing mice were killed 24 hr after
irradiation, and tumors were dissected from the skin,
cut into several pieces, and made into a fine brei by
high-speed chopping with a razor blade attached to a
jigsaw. The brei was added to 30 ml of Hank's buffered
salt solution (HESS) containing 0.02% DNase, 0.05%
promase, and 0.02% collagenase. The suspension was
stirred for :30 min at 37°C, filtered, and centrifuged at
1,600 rmp for 10 min at 4°C. The cell pellet was
resuspended in complete Waymouth's medium plus 15% fetal
calf serum (FCS) and an aliquot mixed with trypan blue
and counted with the use of a hemacytometer. Suitable
dilutions of this serum plated into 60- or 100-mm
polystyrene petri dishes (Lux Scientific Corp) in 5 or
15 ml of med3~um. After incubation for 13 days, the
co~.anies were fixed and stained, and those containing 50
cl~~~i or mores were counted. The dilution yielding an
avesrage count: of 25-100 colonies in a 60 mm dish was
used im calculation of results.
~~~~.~~~ _53_
Example I7: Cvtotoxicitv Tests
Cytotoxici.ty tests were carried out using
3-amino-1,2,4-benzotriazine 1,4-dioxide and a variety of
aerobic and ;hypoxic cells in culture (human, mouse, and
hamster). T'.he cells in spinner flasks were gassed for
one hour at 37°C with either air or nitrogen containing
5% C02 prior to adding the specified amounts of the
drug. Figures 1A, 18 and 1C show the results for cell
survival of mouse, hamster and human cells at various
concentrations of 3-amino-1,2,4-benzotriazine 1,4-
dioxide. It was found that only 1 to 2% of the drug
concentration under aerobic conditions was required to
get equal c ell killing under hypoxia. This ratio of
selective hypoxic toxicity (50-100) is higher than that
for any compound so far reported in the literature.
Example 18: Determination of LD50
LD~~p is determined in BALB/c female mice
(weighing 20-25 g) following intraperitoneal (ip)
injection, unless the compound tested has low
lipophilicity and is very soluble, wherein intravenous
(iv) administration is used. LD50 values at 1, 2, 5,
and 60 days are determined by administering graded doses
of the drug dissolved in physiological saline
immediately prior to injection.
Example 19: Radiosensitivity in Vitro
The results of assays to determine the
concentration of drug necessary to produce a sensitizer
enhancement ratio of 1.6 of hypoxic cells in culture are
as follows:
-54-
Compound C1.6 ((mM))
7-chloro-3-amino-1,2;4-benzotriazine 3.3
1-oxide
6(7)-methoxy~-3-amino-1,2,4-benzotriazine
1,4-dio:Kide '1.0
3-hydroxy-:1,2,4-benzotriazine 1,4-dioxide '"2.0
Modifications of the above described modes for carrying
out the invenltion that are apparent to those of skill in
the chemical, pharmaceutical, medical, and related arts
are intended to be within the scope of the following
claims.
Example 20: Enhanced Tumor Cell Toxicitv Using
Hvdralazine
Hydralazine is an antihypertensive drug which
acts by rela:King the smooth muscle around blood vessels.
This has the effect of preferentially shunting blood
flow into normal tissues and away from tumors, which
process produces immediate hypoxia in the tumors. If
3-amino-1,2,n~-benzotriazine 1,4-dioxide is given in
conjunction with this agent, there is a massive increase
in tumor cell killing. In this experiment, neither
hYdralazine nor the aforementioned benzotriazine
compound produced any significant cell killing in the
SCCVII tumor,, whereas the combination of the two reduced
survival by a factor of 103 (i.e., only 1 cell in every
1000 was lefit viable). The experimental procedures are
the same as described in Example 9, and the results are
shown in Figure 3.
~:~~~~.~~~ -55-
Example 21: Physicochemical and Biolocrical Properties
of Some I,2,4-Benzotriazine 1,4-Dioxides
The i:ollowing table sets forth various
properties of compounds 22, 14, 18a, and 21 as
determined by t:he inventors herein:
Solubility El/2b
C.~,d. Mol. _ (mM) Log Pa (mV) RHTo HCRd LD50e
Wt.
22 178.2 13.50 -0.32 -332 1 100 0.50
14 223.2 2.00 -0.97 -133 2 140 1.00
18a 291.4 >181 -1.34 -348 3 100 0.25
21 358.9 96.40 -0.20 -140 3 211 0.40
a.) Log of the octanol-water partition
coefficient as measured by the method of Fujita et al.,
J. Amer. Chem. Soc. 86:5175 (1964), using pH 7.4
buffer.
b.) Polarographic half-wave reduction
potentials measured in Britton & Robinson pH 7.4 buffer
using a dropping mercury electrode.
c.) Relative Hypoxic Cytoxicity: Ratio of
equitoxic concentrations of 22: analog for HA-1 cells
attached, under, hypoxic conditions. Exponentially
growing cells were placed in suspension culture and
gassed for 90 nnin in nitrogen or air prior to addition
of drugs. Samples were removed periodically for a
survival determination, and the ratios determined from
a comparison oi: the resulting survival curves.
d.) Hypoxic Cytoxicity Ratio: Ratio of
equitoxic concentrations of each analog for HA-1 cells
-56-
atta~,ned under' hypoxic:aerobic conditions. Treatment
conditions as above.
e.) l3alb/c female mice 3-5 months of age were
used in the LDSp experiments. LDSp was evaluated as
described in Example 18.
As may be readily deduced from the table,
novel compounds 14, 1~8a,and 21 exhibit significantly
enhanced cytotoxicity against hypoxic HA-1 tumor cells
in vitro compared to 3-amino-1,2,4-benzotriazine
1,4-dioxide (22), while retai:~ing the high differential
cytotoxicity against hypoxic cells compared to aerobic
cells. These results suggest that these drugs will be
more tumor-specific and therefore more effective as
antitumor agents in vivo.
Example 22: Fractionated Radiotheratay Usina
3-Amino-1.2.4-Benzotriazine 1,4-Dioxide
The following experimental work establishes
that pre- or post-irradiation treatment of cells _in
vitro with compound 22 under hypoxic conditions
radiosensitizes the cells even when drug is not present
during radiation exposure and the cells are aerobic.
a.) Figure 4 shows the results of experiments
in which the survival of Chinese hamster ovary (CHO)
cells after graded doses of x-rays was determined
either with a hypoxic compound 22 exposure given before
or after irradiation. The preirradiation treatments
consisted of 20 uM drug for exposure times of 1.0 (D
in Figure 4), 1.5 (~), 2.0 (a) and 2.5 (Q) h
duration prior to cells being reaerated and irradiated.
These drug treatments alone reduced cell survival to
approximately 32, 19, 8 and 2~ respectively. The
~~~'~~.~~ -57-
postirradiation drug treatment consisted of 20 uM for
1.5 h (+), which alone reduced cell survival to 23%.
Compared to the survival curve for cells exposed to
pre- or post-irradiation hypoxia only (t ), treatment
with compound X22 sensitized cells to aerobic
irradiation. The sensitization was predominantly a
change in the :slope of the radiation survival curve.
Do decreased from 1.34-Gy (95% confidence limits:
1.09-1.76 Gy) 1:o 0.80 Gy (95% confidence limits:
0.73-0.88 Gy), based on least-squares regression
analysis of pooled data for the exponential portions of
the survival curves for control and treated cells.
However, sensitization at low radiation doses was also
readily apparent. Survival ratios for "no drug:drug-
treated" CHO csalls irradiated with doses of 1-3 Gy
averaged a factor of 3.6. The amount of the
radiosensitizat:ion produced did not vary with the
severity of them drug treatment.
b.) The data of section (a.) on
radiosensitizat:ion of aerobic cells at low radiation
doses by hypoxi.c activation led us to test the
feasibility of obtaining preferential
radiosensitizat:ion of tumors in vivo. Our protocol was
to use eight doses of 2.5 Gy/dose in four days
(irradiating 2 x/day). Because of the in vitro data
showing that ra~diosensitization could only be achieved
by hypoxia activation, either before or after
irradiation, and because we did not want to make the
tumors hypoxic :before irradiation (as this would make
them resistant), we made the tumors hypoxic after
~Q~~.~~~ 58
irradiationusing the vasoactive drug hydralazine (HDZ)
at the same time as injecting compound 22. We used two
different types of controls. First, compound 22 alone
before each dose of radiation, and second, the potent
hypoxic cell sensitizer SR 2508 (a 2-nitroimidazole,
DuPont, undergoing Phase III clinical trials in Europe)
before each dose. The effectiveness, or lack of
effectiveness, or SR 2508 indicates whether the
radiation response of the tumor is being governed by
hypoxic, or .aerobic, cells respectively. We assayed
the efficacies of the treatments using clonogenic cell
survival and also regrowth delay. Figures 5 and 6 show
the results.
As Figure 5 illustrates, there was no effect
of SR 2508 (:1000 mg/kg) given before each radiation
dose, but compound ~2 (0.08 mmole/kg) given alone or
with hydrala;zine (HDZ) produced a large enhancement of
the radiation response. Part of this is attributable
to an additive response (crosses), but the additional
cell killing is the result of radiosensitization of
aerobic cells.
In Figure 6, it may be seen that SR 2508 (1000
mg/kg) before each radiation dose had no
radiosensiti;zing effect, but compound 22 (0.08
mmole/kg) alone before irradiation or with HDZ after
irradiation produced a large increase in effect
compared to radiation or drug alone.
The major and unexpected result of these
experiments is the radiosensitization of the tumors by
compound 22 .given before each radiation dose without
the addition of hydralazine. This cannot be accounted
l ~ ~~~~~.~~~~ -59-
for by a radiosensitization of hypoxic cells, since SR
2508 (a hypoxic radiosensitizer) is ineffective. Thus,
it is a radiosensitization of aerobic tumor cells.
This would not be useful if it radiosensitized
aerobic normal cells. We tested this by performing the
same eight fraction protocol on the response of normal
mouse skin using a skin reaction scoring scale
previously used by us. Figure 7 shows the result.
There is no radiosensitization of normal skin.
In conclusion, the data show that the aerobic
cells of tumors can be radiosensitized in multifraction
regimes similar to those used in radiotherapy. The
radiosensiti;zation i.s tumor specific (i.e., does not
occur in nornnal cel7.s), and appears to be the result of
activation b;y hypoxic areas in the tumors.
