J. B. CHYLIfiSKA, T . U R B A ~ K A NID, M. hIORDARSK1
484
Vol. G
Dihydro-1,3-oxazine Derivatives and their Antitumor Activity J. B. CHYLI~~SKA, T. U R B A ~ S K I , Institute of Organic Chemistry, Polish Academy of Sciences, Vnrszawa 10, Poland AND
ILL MORDARSKI
Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Wroclaw, Poland Received October 10,1962
In continuation of earlier on the carcinostatic activity of 1,3-oxazine derivatives, a number of dihydro-1,3-oxazine compounds condensed in the 5,6-positions with aromatic rings were prepared. They showed cytotoxic activity in vitro against amobarbital ascites sarcoma and Ehrlich ascites carcinoma. Some of the compounds show activity in vivo against the latter tumor.
Two recent papers2v3 on dihydro-1,3-oxazines as antitumor agents have prompted us to report our own results on the subject, in continuation of previous work. A number of phenols of the general formula I, pnaphthol derivatives (11), 2,7-dihydroxynaphthol, 4and 5-hydroxyhydrindenes, and 2-hydroxyacridine have been condensed with formaldehyde and a number of
OH
R' I1
T
I
primary amines as described in the l i t e r a t ~ r e . ~The formulas of dihydro-l,3-oxazine derivatives, with some experimental data such as reaction time, yields, melting points, and analyses of the products are given, in part, in Tables I-V. Some 3,4-dihydro-1,3-benzoxazines (111) derived from certain phenols were used for preparing 2-thiono derivatives (IV) .
IV
The properties of these compounds with some experimentd details are collected in Table IT.
Experimental Biological Methods.-Cytotoxic
activity in vitro was estimated
(1) (a) T. Urbadski, D. Giirne, 2. Eokstein, and S. Slopek. Bull. Acad. Polon. Sei., Cl.111, 8 , 395 (1955); (b) T. Urbadski, Cr. Radrikowski, Z. Led6chowski, and W. Czarnocki, Nature, 178, 1351 (1956); ( 0 ) T.Urbadski, D. Game, S. glopek, H. Mordsrsks, and M. Mordarski, Nature, 187, 426 (1960); (d) J. B. Chylidska and T. Urbadski, Bdl. Acad. Polon. Sei., Ser. Chim., 7 , 635 (1999); (e) T.Urbadski, D. GUrne, S. glopek, H . Mordarska, and M . Mordarski. Nature, 187, 426 (1960); (f) 9. Slopek, H. Mordarska, M . Mordarski, T. Urbabski. and D. Giirne, Bdl. Acad. Polon. Sei., Ser. Sci. Chim., Oeol. Geograph., 6, 361 (1958); (g) Z. Eokstein, P. Gluzinski. E. Grochowaki, M. Mordarski. and T. Urbabski, Bull. Acad. Polon. Sei., Ser. Chim., 10, 331 (1062); (h) T. Urbadski, Nature, 168, 562 (1951). (2) M. E. Kuahne and E. A . Konopks, J . Med. Pharm. Chem., 6 , 257 (1962). (3) M. E. Kuehne, E. A . Konopka, and B. F. Lambert, ibid., 6, 281 (1962). (4) W.J. Burke, J . A m . Chem. Soc., 71, 609 (1949).
by a modification of Miyamura's test.& Two types of cells were employed in the experiments: Ehrlich's ascites carcinoma and amobarbital ascites sarcoma. Cells from the peritoneal exudate in mice, 10-12 days after transplanation, were used. The peritoneal exudate (with 5 pg./ml. of heparin added) was centrifuged and the separated cells were washed with .067 M phosphate buffer of pH 7 . 2 and then resuspended in a volume of Tyrode's solution sufficient to give suspensions containing 2 X IO7 cells per ml. The cell suspensions were added to the medium (pH 7 . 2 ) containing 0.5% glucose, 0.25% NaCI, 0.3% K2HP04, 1% peptone, and 15% heparinized sheep serum. The density of the tumor cell suspensions in the medium was ca. 2.4 X I O e cells per ml. The cell-containing medium waa distributed in 1.5 ml. quantities in test tubes of 6 mm. diameter, and 0.5 ml. of the solution of the compound was added per tube; the same volume of physiological saline solution was added to the control tubes. The tubes were incubated for 6 hr. a t 37'. The cells were then separated by centrifuging and washed 3 times with physiological saline. The washed cells were suspended in 2 ml. of the same medium, but with addition of 1% agar and methylene blue (0.85 ml. of a 0.5% solution of methylene blue per 100 ml. of medium), and again incubated for 3 hr. a t 37'. During incubation in the control tubes containing physiological saline instead of solutions of the compound, methylene blue is reduced by the tumor cells, only 2-3 mm. of the upper layer of the agar retaining the color of the dye due to secondary oxidation by atmospheric oxygen. In the tubes containing test compounds, the indicator dye remained unaltered (complete inhibition), or was only partly decolorized (partial or weak inhibition), depending on the concentration and activity of the compound. A tube of medium with methylene blue but not inoculated with cells constituted an additional control showing whether the medium alone waa capable of reducing methylene blue. The results of these in vitro tests are collected in Table VI. Determination of the in vivo oncostatic activity of a few compounds was carried out with Ehrlich's ascites carcinoma model strain, in the form of subcutaneous tumor, instead of the usual exudative f o r m in the peritoneal cavity. The solid form of the tumors was produced by inoculating 106 exudate cells under the skin in male non-inbred albino mice (strain A or RIII)weighing 20-22 g. The mice with implanted subcutaneous tumors were inspected after 2 weeks when the tumors were already palpable and their dimensions could be measured. Mice in which the tumor did not take, or with excessively large tumors, were rejected; however, with a dose of 106 exudate cells both these events were encountered but rarely. The animals qualifying for the experiments were divided into 2 groups, the controls comprising 12-15 mice, and the treated groups 10 mice each. The test compounds were administered subcutaneously to the abdomen in doses of 500 pg./mouse during 6 days per week for 2 weeks. The solvent used for preparing the solution of the compound in question wm injected to the control groups. Forty-eight hours after the last injection of the compound, the mice were weighed, anesthetized with chloroform, the tumors were dissected and weighed separately. The experiments were repeated 4 times. Inhibition of growth of the tumors was expressed as percentage, according to theformula: yo inhibition = l O O [ ( P - M ) / P ] where P is the average tumor weight in the control group, and M the average (5) 8. Miyamura, Antibiot. Chemother., 6, 280 (1956).
September, 1963
485
CYTOSTATIC DIHYDRO-1,3-OXAZINES
PHYSICAL PROPERTIES OF
TABLEI DIRYDROBENBOWINE DERIVATIVES
R*
1 IC
CH3
R2 H B
2 2c 3 3c 4
CH3
H
Br
H
C2H;
H
Br
CsHiI C&ii CcHIi
H H
CH3 CH3
H
C6Hii
H
C1
C6Hii
H
So."
5~
G Gc 7 7c 8 8c 9 10 1oc
Rl
R3
11 CH2CcHj CH3 12 CH2CcHj CHs 13 CHiCsHj H 14 C H L C & , H 14c 15 CH2C~HsH 15c 16 C6H,Br-p H 17 C6HJh-p H 18 C6H,Br-p H 19 CsH,Br-p H 20 C&Br-p H Hydrochloridr of the
R6
H
H
r
1
50
Br
1.5
35
H
Br
1.5
30
H
I .5
25
H
H CH3 H
1
40
H
H
1
35
C6Hj H
H
1
55
CH3 H
CH3 CH3
H CHj
1 1
50 60
H H C6H5 Br
CH3 CrHj H H
H H H H
1 1 1 1
90 20 60 60
Br
H
Br
1 5
30
H
CH2CaHj H CH&sHs H
R4
Reaction Time, hr. at b.p. Yield, %
CH3 H H Br H H Br H Br C1 H H C1 H C1 corresponding base
40 65 60 1 45 2 40 denoted by c . 1
1 2
M.P., OC.
Formula
87-89.5 185-187 77-79.5 169-1 72 57-60 171-174 30-32 192-195 89-92 240-243 97-98 148-1 51 70-72 135-137 86-88 71-73 150-153 94-97 40-43 89-90 85-87 182-184 74-76 175-175.5 82-85 109-1 1I 116-117.5 87-89 132-134
-Calcd.y C H N
CsHloBrNO 47.4 C9Hl1BrClS0 C9H9Br2N0 35.2 CsHloBr&lNO CloHl1BrzS0 37.4 CloH12Br2ClN0 CisHziYO C16H24CINO ClnHlsBrNO Cl4HI9BrCINO Ci4H1,CISO CinHiyCLSO CZOH~~NO CzcH2aCISO CiTHigNO Ci7HisNO C17H2,CINO 70.5 Ci7HiaSO CidLiNO C2iHigSO ClsHllBrSO CISH15BrC1?YI0 C15H13Br2S0 47.0 ClsHI4Br2C1SO ClsH14BrN0 59.2 ClaHllBrzSO 45.6 C14H10Br3N0 37.5 C14H11BrCIN0 51.7 Cl4Hl0BrCI2NO46.8
-Found? C H
N
Ref.
4.4 6 . 1 47.6 4 . 6 6.2 5.5 5.3 3 . 0 4.6 35.4 3.3 4 . 5 3 . 5 4.4 37.6 3 . 8 4 . 6
5.6 4.9 4.8
la 4.9 4 4, l a 4, l a 5.7 6 5.1 5.0 4
5.5
5.6
4.c,
Id Id 6 . 7 4.8 70.7 7 . 0 5 . 1 Id 5.5 5 . 8 Is 5.2 5 . 5 Id 4.7 4.8 la 4.1 4.2 l a 3 . 4 3 . 7 47.3 3 . 9 3 . 5 4.6 3.0 2.2 3.4 2.8
4.6 3.8 3.1 4.3 3.9
59.4 45.6 38.0 51.7 47.2
4.8 2.9 2.3 3.4 2.8
4.8 7 4.0 3.2 4.4 7 3.9
TABLEI1 PHYSICAL PROPERTIES OF SAPHTHOXAZINE DERIVATIVES
Reaction Time, hr. room Yield, SO."
R'
R2
R3
temp.
21 lb 2 1 ~ CH3 H H 22 CH, OH H 24 23 C2Ha OH H 24 24 CHsCsH6 OH H 24 H SO&a 24 25 CH2CsHs H H 24 26 C6HaBr-p 27 CsH4CHa-p H H 24 5 Hydrochloride of corresponding base denoted by
%
80 98 87 96 75 90 85 c. *
M.p.. OC.
67-68 185-187 210 190 139-140
CiaHisNO C13HirCINO CiaHiaNO, Ci4HisNOs C19H17N02 CloHlaNNaOaS 118-119 C1,H14BrN0 86-88 CigHi7NO At boiling point.
(6) R. H. Rigtering. U. S. Patents 2,806,031:2,811,523(1957). (7) N. Nods, J . Chem. Soc. Japan, 6 2 , 743,747 (1959); Chem. Zentr., 181.
7856, 7857 (1960).
Formula
--Calcd.C H
N
7.0
---FoundC H
N
Ref.
6.8
8.l b 6.1 6 . 5 73.0 6 . 3 6 . 5 Id 6.2 Id 6.1 78.3 5 . 9 4.8 78.6 5.9 4.9 Id 60.5 4 . 3 3 . 7 60.7 4.9 4.0 Id 9 5.1 4.9 9 72.9
(8) W.J. Burke, M. J. Kolbezen, and S. Wayne. J . A m . Chem. Soc.. 74, 3601 (1952). (9) W.J. Burke, K. C. Murdock and G. Ec, (bid.,76, 1677 (1954).
Z'ol. t i
486
tumor weight in the treated group. The results of the zn tests are collected in Table 1'11.
PLNI
Results and Discussion Out of 49 compounds examined in vitro (38 bases and I 1 hydrochlorides) only 3 did not show any activity. Some of the active compounds. e . g . , KO. 6c. 8c, 9, 13, 13c, and 36, showed very strong activity agaiiwt both types of test tumor cells. Some of' them were highly active against only one species, e . q . , S o . 23 and 2'7
N o general conclusion about the relation hetn.ceii t l i v htructure of the compounds and their cancerostatic, activity could be drawn. Iri h o tests were carried out with compounds which showed strong or moderately strong in v i t i ~activity ~ and a t the same t h e were not highly toxic and possessed relatively good solubility in water or propylene glycol. Toxicity data will be published later. Compounds 6c aiid 1Oc show moderate (ca. 5Oq7,) i i i hihitioil of tumor growth. The strongest iuhibitioii of 5,j and i8Yc was obtained with compounds I-lc and
September, 1963
CYTOSTATIC
487
DIHYDRO-1,3-OXAZINES
TABLEVI CYTOTOXIC ACTIVITY OF DIHYDROOXAZINE DERIVATIVES
~ 0 . 5
1 le 2 2c 3 3c 4
5c 6 6c 7 7c 8 8c 9
10 1oc 11 12 13 14c
15 l5C 16 a
Cytostatic concentration Ehrlich ascites carcinoma Partial Complete or weak
1000 1000 100 1000 1000 1000 1000 100 1000 100 100 100 1000 100 100 100 100 1000 100 100 100 1000 100 100
producing inhibition Amobarbital ascites sarcoma Partial Complete or weak
Cytostatic concentration (Fg./ml.) producing inhibition Ehrlich Amobarbital ascites sarcoma ascites carcinoma Partial Partial Complete Complete or weak or weak
(pg./ml.)
1000 1000 100 1000 1000 1000 1000 1000 1000 100 1000 1000 1000 100 100 100 100
100
10 10 10 10 10
10
No?
17 18 19 20 21 21c 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38
10
100 100 10 100 10 10 10
100 100 100 1000 100 100
10 10 10 10
10 10 10
1000 1000 100 1000 100
10 100
1000 1000 100
100
100
100 100 1000 10 1000 10 100 1000 100 1000 100
0 1000
100 10 1000 1000 100 1000 1000 1000 100
100
1000 1000 10 1000
1000 1000 10 1000
10
100 10 10 100
100 10
Hydrochloride of corresponding base denoted by c.
TABLE VI1 In ViVO ACTIVITYAGAINST EHRLICH ASCITES CARCINO>CLIA
R5
x0.a
14c 9 10 1Pc 12
6: 7C
Daily dose, mg./mouse
R'
€I H
H H H H H
30
a Hydrochloride of corresponding base denoted by c. freedom.
% inhibition
1.0 1.0 1.0 1. 0 0.5 1.0 1.0
48 0 0 47 19 55 0
0 0 0.6 2.2 1.2 0.7
1.0
58
1.8
* Coefficient acrording to distribution of
30, respectively. These substances will be used for a more det?.iled study. The present experiments confirm our former results on the carcinostatic activity of benzhydro-1,3-oxazine
Weight reduction of mice, g.
Student.
c
tb
VC
2.5
20
3.4
20
4.0
20
3.6
20
rl'umber of degrees of
derivativeslb,' and tetrahydro-1,3-oxazine derivatives.le,f,g. This seems to support a general conclusion that 1,3-oxazine derivatives are compounds exhibiting not only a definite biological activitylh but some of them are carcinostatic.
