1045
J. Med. Chem. 1982,25, 1045-1050
New Anticancer Agents: Synthesis of 1,2-Dihydropyrido[3,4-bIpyrazines (l-Deaza-7,8-dihydropteridines) Carroll Temple, Jr.,* Glynn P. Wheeler, Robert D. Elliott, Jerry D. Rose, Conrad L. Kussner, Robert N. Comber, and John A. Montgomery Kettering-Meyer Laboratory, Southern Research Institute, Birmingham, Alabama 35255. Received February 25, 1982
Reaction of a-aminoacetophenone oximes (2) with ethyl 6-amino-4-chloro-5-nitropyridine-2-carbamate (1) gave ethyl oximes (3), which were hydrolyzed under acidic 6-amino-5-nitro-4- [(2-0~0-2-phenylethyl)amino]pyridine-2-carbamate conditions to give the corresponding ketones (4). Related pyridines substituted with a keto side chain were prepared from 1 and 1,3-diaminopropanone oximes and by oxidation of the side-chain hydroxy group of ethyl 6-amino-4[ [ 3-(N-methyl-N-phenylamino)-2-hydroxypropyl]amino]-5-nitropyridine-7-c~bamates (6). Catalytic hydrogenation (7). of the nitro group of 4 over Raney nickel in a large volume of ethanol gave the l-deaza-7,8-dihydropteridines Several of the oximes 3 were successfully hydrogenated to give 7 directly. The resulting l-deaza-7,8-dihydropteridines showed potent cytotoxicity against cultured L1210 cells and significant anticancer activity against lymphocytic leukemia P-388 in mice. These biological activities are attributed to the accumulation of cells a t mitosis.
Steady progress has been made in cancer chemotherapy primarily because of the development of new agents, combinations of agents, and new approaches for the treatment of cancers. Despite those advances, some cancers do not respond to drug therapy, and some cancers that respond initially lose their responsiveness after prolonged drug therapy. The principal cause for these conditions has been attributed, respectively, to “natural” resistance and to the development of acquired resistance. The latter has been demonstrated in animal tumor systems in which the most resistant cells are steadily increasing-I One approach to meet the challenge of drug resistance is the development of new classes of agents, which can be used alone or in combination with conventional agents. Recently, we reported that ethyl 5-amino-1,2-dihydro3-[ (N-methylanilino)methyl]pyrido[ 3,441pyrazine-7-carbamate [7,R = C6H5N(Me)CH2]was active against experimental neoplasms, including L1210 leukemia, P-388 leukemia, and intraperitoneally implanted murine colon tumor 26 and had borderline activity against a number of other tumors.2 The mechanism of action of this compound was identified as the accumulation of cells at mitosis with both cultured cells and ascites cancer cells in vivo. In this paper we report the synthesis and biological activity of 7 [R = C6H,N(Me)CH,] and related 1,2-dihydropyrido[3,4blpyrazines (l-deaza-7,8-dihydropteridines). The development of procedures for the preparation of 1-deazapteridineshas been carried out in these laboratories for a number of years.3 These processes are modifications of the Boon and Leigh procedure for the synthesis of pteridines from chloropyrimidines and a-amino ketone^.^ The oximes of a-amino ketones (Table I) were prepared by several routes. Reaction of a-bromoacetophenones with hexamethylenetetramine gave the corresponding iminium salts, which were hydrolyzed by ethanolic hydrochloric acid to give a-aminoacetophenone hydrochloride^.^^^ The (1) Schabel, Jr., F. M.; Skipper, H. E.; Trader, M. W.; Laster, Jr.,
(2) (3)
(4) (5)
W. R.; Corbett, T. H.; Griswold, Jr., D. P.; in “Breast Cancer. Experimental and Clinical Aspects”, Mouridsen, H. T.; Palshof, T., Eds.; Pergamon Press: Oxford, 1980; p 199. Wheeler, G. P.; Bowdon, B. J.; Werline, J. A.; Adamson, D. J.; Temple, Jr., C. Cancer Res. 1982, 42, 791. (a) Montgomery, J. A.; Wood, N. F. J. Org. Chem. 1964, 29, 734. (b) Elliott, R. D.; Temple, Jr., C.; Montgomery, J. A. J . Org. Chem. 1968, 33, 533. (c) Elliott, R. D.; Temple, Jr., C.; Frye, J. L.; Montgomery, J. A. J . Org. Chem. 1971, 36, 2818. (d) Elliott, R. D.; Temple, Jr., C.; Montgomery, J. A. J. Med. Chem. 1974,17, 553. Boon, W. R.; Leigh, T. J . Chem. SOC.1951, 1497. Long, L. M.; Troutman, H. D. J . Am. Chem. SOC.1949, 71,
2473. (6) Sen, A. B.; Mukerji, D. D. J. Indian Chem. SOC.1951,28,401.
Scheme I
E t 0 2 C H N ’ -cl
‘NHCh$R
EtOZCHN-
II
1
X
3, X = NOH 4,X=O
R
I
HzNCHaCHOH
6
/
I
7
condensation of the latter with hydroxylamine hydrochloride in a refluxing mixture of ethanol and pyridine gave the a-aminoacetophenone oxime^.^ Several a-aminoacetophenone oximes were prepared by alkylation of phthalimide with a-bromoacetophenones, followed by conversion of the product to the oxime with hydroxylamine and removal of the phthaloyl protecting group with hyd r a ~ i n e . ~ , *In another approach, 1-bromo-3-phthalimidopropanonewas reacted with N-methylmilines to give 1-(N-methyl-N-phenylamino)-3-phthalimidopropanones. The latter were condensed with hydroxylamine to give the corresponding oximes from which the phthaloyl blocking group was removed with hydrazine to give l-amino-&(Nmethyl-N-pheny1amino)propanoneoxime^.^ Several 1amino-3-(N-methyl-N-phenylamino)-2-propanols were also prepared by alkylation of N-methylanilines with epichlorohydrin and amination of the resulting epoxide with ammonia.ll Alkylation of a-aminoacetophenone oximes (2) (Table I) with ethyl 6-amino-4-chloro-5-nitropyridine-2-carbamate ( 7 ) (a) Elliott, R. D.; Temple, Jr., C.; Montgomery, J. A. J. Org. Chem. 1970,35,1676. (b) Gnichtel, H. Chem. Ber. 1970,103, 3442. (c) Okac, A.; Jokl, V. Chem. Listy 1953, 47, 534. (8) Schweiker, G. C. Diss. Abstr. 1953, 21, 464. (9) Gnichtel, H. Chem. Ber. 1965, 98, 567. (10) Chen, S.-S.; Jonsson, S.; Semeniuk, F. T. J . Pharrn. Sci. 1962,
.
51. 108.
(11) Eisleb, 0. German Patent 473 219, 1926; Chem. Zentr. 1929, l O O ( I I ) , 350.
0022-2623/82/1825-1045$01.25/00 1982 American Chemical Society
1046 Journal of Medicinal Chemistry, 1982, Vol. 25, No. 9
Temple et al.
Table I. u-Amino Ketone Oximes (2) and a-Amino Alcohols ( 5 ) yield,a no.
R
method
mp, "C
%
formula
anal.
I 56 b C ,,H N ,O ,O. 33H ,O c , H, N 2 C6HSN(Me)CH2 2 4-MeOC6H,N(Me)CH, I 28 b C , H 17N,O .O. 37 H *O c , H, N I11 40 96-1 10 C,H,,N 2 0 2 C,H' c, H, N 2 4-MeC,H4 I1 34 127-129 C,H ,,N20.0.13C,H,O c , H, N 2 2,4-Me,C6H, I1 64 e C,,H,,N,O f 2 3,4-Me,C6H,2 I1 34 115-118 C ,,H 14N20 .0.64HC1 c , H, N h 2 $-FC,H, I11 70 142-143 C,H ,FN ,O 2 4-C1C,Hq I11 37 129-1303 C ,H,ClN ,O .HCl c, H, N I1 22 2 2,4-Cl2C,H, 115-120k C,H ,C1,N ,O .0 .17H ,O c, H, N 2 3,4-C1,C6H, I1 91 123-127 C,H ,Cl,N,O c , H, N 2 3-MeOC,H4 I1 77 1 e C,H,,N,O, 2 4-MeOC,H4 I11 6 137-139 C,H,,N,O~HC1~O.31H2O C, H, N 2 3,4,5-(MeO),C,H, I1 70 134-137 C,,H1,N,O;O.16C,H,O c , H, N I11 20 2 4-O2NC,H, 124-125 C,H9N,0;0.28HC1 c , H, N 2 4-CF3C,H, I1 31 129-130 C9H9F3N20 c , H, N 2 4-C, H ,C, H, I11 73 b C ,,H ,,N20 ,0.33HC1 c , H, N IV 23 7 1-72 5 C,HSP 5 4-C1C6H,N(Me)CH, IV 32 117-1194 C,,H,,C1N,O c , H, N 5 4-MeOC, H,N( Me)CH , IV 17 163 c IIHl,N,O, c , H, N a Overall yield. Indefinite melting point. Reference 9. Reference 7b. e Oil. f m / e 178 (M+). Phthalimide m / e 168 (M+). Hexamethylenetetramine salt., Free base. Presoftening at 76-81 "C. m / e 180 interrnediate.l" (M+). rn Amino ketone intermediate.' Phthalimide intermediate.8 P Reference 11. 4 Resolidified and remelted 123 "C. J
Table 11. Ethyl 4-(Substitu ted-amino)-6-amino-5-nitro-2-pyridinecarbamates (3-6) reaction yield, no. R method time, h % mp, "C 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 4 4
C,H,N(Me)CH, 4-MeOC,H,N(Me)CH2 C,H' 4-MeC6H, 2,4-(Me),C,H, 3,4-(Me),C,H, 4-FC,H4 4-C1C,Hq 2,4-C1,C6H, 3,4-C1,C,H3 3-MeOC,Hq 4-MeOC,H4 3,4,5-(MeO),C,H, 4-0,NC,H4 4-CF,C,H4 4-C6H,C,H, C,H,N(Me)CH, 4-CIC,H4N(Me)CH, C6HS
4 4
4 4 4 4 4 4 4 4 4
4-MeC,H4 3,4-Me2C,H, 4-FC6H, 4-C1C,H4 3,4-C1,C,H3 3-MeOC,Hq 4-MeOC6H, 3,4,5-(MeO),C,H, 4-0,NC,H4 4-CF,C,H4 4-C,H5C,H,
C6HS
6 6
4-C1C6H,N(Me)CH, 4-MeOC,H,N(Me)CH2
V V V V v V V
v
V V
v v v
v V VI1 VI1 VI VI VI VI VI VI VI VI VI VI VI VI VI11 VI11 VI11
24 15= 8 2 2 1.5 2 3 2.5 1.5 3 4
1 2 2 48 2.5 20 2 3
4 24 18 6 5 20 2.5 24 2
1 18 18 18
84 75 52 87 18 82 86 73 45 64 56 55 37 44 47 48c 80 20 59 73 80 48 59 69 81 53 80 65 31 80 73 64 79
192-193 190 245-246 234-235 198-200 23 6-2 37 240-242 224-225 177-179 214-216 223-227 218-219 205-208 207-208 224-225 -130dec
dec dec dec
dec dec dec d.ec dec dec dec
177-178 184-185 196-197 dec 204-205 180-181 dec 191-192 >171-172 dec 194 dec 192-193 dec 204-205 dec 166.5 - 16 7.5
f
C,,H,,N,O, c 19H2,N 7 0 6 C,,H,,N,O, C,,H,,N60,~0.22HC1 C18H22N605
dec
-123-125 80d
88-90 181 108 dec
formula
C 18H,,N,O ,. 0.2 5H,O C 16H,,FN,O;O. 13HC1 C,,H,,C1N,Os~l.1H,O C,,H,,CI,N,n, C ,,Hl,C1,N60,~0.54C,H,0 C,,H,,N,O, C,,H,,N,O,~O.12HCl C,,H,,N,O,~O.BHCl C,,H,,N,O, C,,H,,F,N,O,
( 1)12 under nitrogen in refluxing ethanol containing triethylamine as an acid acceptor gave the ethyl 6-amino-5nitro-4- [ (2-0~0-2-phenylethyl)amino]pyridine-2-carbamate oximes (3) listed in Table 11. (Scheme I) Treatment of these oximes with a 1:l mixture of 1N hydrochloric acid and dioxane at 60 "Chydrolyzed the oxime function to give (12) Elliott, R. D.; Temple, Jr., C.; Montgomery, J. A. J. Org. Chem. 1966, 31, 1890.
c , H, N c, H, N c , H, N c, H, N c , H, N c , H, N c , H, N c , H, N
;:
;b
c , H, N c, H, N c, H , N c, H, N c , H, N
C,,H,,N60,~H,0~0.5(CH,),S0 C, H, N
C 18H,,ClN,O ,'0.5H,O
c, H , N
C,,H,,N,O' C,,H,,N,O,
c, H, N
C1,H,,N,0;0.66C,H,0,~0.l6H2O C, H, N H, N ; Ce
c , H, N c, H, N c , H, N c, H, N 17H 19N506 c , H, N C,,H,,N50,~2.2H,0~0.1C4H,02 C, H, N C,,H,,N,O,~HCl~H,O c , H, N C ,,H 16 F ,N ,O 5 , 0.7 5C ,H ,O c, H, N C 22H21 N ,O ;HC1 .H,O c, H, N c ,,H,N,O, c, H , N c 18H,,ClN 6 0 5 C, H,N l,H%N606 c, H , N 1bH 16 FNSo5
C,,H,,ClN,O, C,,H,,C1,N,0;0.25C,H80~ C,,H1,N,O,~0.8H,O
Solvent, methanol. H: calcd, 4.10; found, 3.59. Crude yield, used directly to prepare ketone. softening. e C: calcd, 54.69; found, 54.26. f Indefinite. a
anal.
With pre-
the ethyl 6-amino-5-nitro-4-[(2-oxo-2-phenylethy1)aminolpyridine-2-carbamates(4) listed in Table 11. Similarly, reaction of 1 with the 1,3-diminopropanone oximes (Table I), followed by hydrolysis of the resulting oximes (Table 11), gave the ethyl 6-amino-4-[[3-(N-methyl-Nphenylamino)-2-oxopropyl]amino]-5-nitropyridine-2-carbamates listed in Table 11. Another approach for the Preparation of the Pyridines containing the keto side chain involved the alkylation of l-amino-3-(N-methyl-N-
Journal of Medicinal Chemistry, 1982, Vol. 25, No. 9 1047
1,d-Dihydropyrido[3,4- blpyrazines
Table 111. Ethyl 3-Substituted-5-amino-l,2-d ihydropyrido[ 3,4-b]pyrazine-7-carbamates (7 ) re ac tion time, yield, no. R method h % mp, "C formula ~~~
IX
12
63
165b
X X
42 20
32 16
goe
4-MeOC6H,N(Me)CH, C6HS 4-MeC6H, 2,4-Me2C,H3
IX X X IX
3 6
5 5
73 89 83 87
187 >180dec 270-280dec 158-160
7 7 7 7 7
3,4-Me,C6H3 4-FC6H, 4-C1C,H4 2,4-C1,C,H3 3,4-C12C,H3
X X X IX Xi
5 12 19 24 40
83 78 77 64 57
190-192dec 296-300dec >270 dec 203-205dec 299-302dec
7
3-MeOC6H,
X
15
70
174-176
7
4-MeOC6H,
X
6
71
>170dec
7
3,4,5-(MeO)3C6H,
X
24
70
7
4-H2NC,H,
X
6
17
7
C6HSN(Me)CH2
7 7
C,H,N(Me)CH, 4-ClC,H,N(Me)CH2
7 7 7 7
C18H22N602'
0.3H,0.0.15C,H,0C C ,,H,,N60,~0.5C'2H60c c ,,H,IClN6O,~ 0.5H,O .O. 5C ,H60C
f
250-255 dec >300dec
anal. C, N ; H~
354 (M') 388 (M') 384 (M')
C19H24N603
311 (M')
16H1,N502
325 (M') 339 (M')
17H C18H21N502'
0.1C,H60~H,0C C,~H,,N~02~0.7C,H,0C 16H ,6FN
mle a 354 (M')
5'
2
16H16C1N50Z
C HI,C1,N ,O ,. 0.8H ,O C 16H, , C 1 2 N ~ 0 2 ~ 0 . 6 H 2 0 ~ C,H,OC C17H19N503' 0.3C2H,0.O .2H,O CI7Hl,N,O,~ 2. 1H,O .O .64C ,H60 C ,9H,3N,0,.HCl . 0.5H ,O
339 (M') 329 (M') 345 (M') 379 (M') 379 (M') 341 (M') 341 (M') 401 (M')
C16H18N602'
326 (M')
Cl,H16F3N50Z'
379 (M')
2.5HC1 .O .5C2H60
7
7
4-CF3C6H, 4-C6HsC6H,
X Xj
48 48
56 86
>300 dec d
0.2H,0~0.15HCli C,,H,,N,O;HCl~ 0.23C,H9NOC
387 (M')
Prior sintering. The presence of reaction Mass spectral data were determined with a Varian MAT 311A instrument. solvent and water of hydration in these compounds was confirmed by the 'H NMR spectra, which were determined in (CD,),SO solutions with a Varian XL-100-15 spectrometer operating at 100 MHz [internal (CH,),Si]: H 2 0 ,6 3.33-3.35 H: calcd, 6.46; found, 7.05. e Sintering. (br s); CH,CH,OH, 6 1.06 t, 3.45 q ; (CH,),NCOCH,, 1.96, 2.79, 2.94 (3 s). f Indefinite. C: calcd 55.57; found. 56.11. H : calcd, 6.68; found, 6.16. C1: calcd, 1.41; found, 1.41. Solvent, N,N-dimethylacetamide. J
phenylamino)-2-propanolswith 1 to give ethyl 6-amino4- [ [3-(N-methyl-N-phenylamino)-2-hydroxypropyl]amino]-5-nitropyridine-2-carbamates (6). Oxidation of the side-chain alcohol function of 6 was successful with 6 [R = C6H5N(Me)CHzand 4-C1C6H,N(Me)CHz],but unsuccessful for the preparation of 6 [R = 4-MeOC6H4N(Me)CHzl. The catalytic hydrogenation of the a-amino ketones 4 with a 2- to 3-fold amount of Raney nickel in a large volume of ethanol at atmospheric pressure at room temperature or with intermittent warming with a water bath gave the intermediate 5-aminopyridines, which were cyclized in situ with the elimination of water to give the ethyl 3-substituted-&amino-1,2-dihydropyrido[3,4-b] pyrazine7-carbamates 7 listed in Table 111. Some of the pyridines containing oxime functions were unsuccessfully hydrolyzed to the ketones, and these oximes were hydrogenated directly to give the desired l-deaza-7,8-dihydropteridines. Biological Evaluation Previously, both 1-deaza- and 3-deazamethotrexate were prepared in our labor at or^.^^ Although 3-deazamethotrexatewas active against leukemia L1210 in mice and inhibited dihydrofolic reductase, 1deazamethotrexate showed only borderline activity, indicating that the 1-nitrogen of methotrexate was necessary for activity. However, an intermediate in the synthesis of 1-deazamethotrexate, ethyl 5-amino-1,2-dihydro-3-[[p(methoxycarbony1)-N-methylanilino]methyl] pyrido[ 3,4b]pyrazine-7-~arbamate,~~ showed cytotoxicity in the KB cell culture screen and activity against leukemia L1210 in mice. In contrast, this intermediate was a weak inhibitor of both Streptococcus faecium and dihydrofolic reductase (pigeon liver), and preliminary data from equilibrium dialysis experiments indicated that the compound was not
binding to DNA. These results led to the synthesis of a series of l-deaza-7,8-dihydropteridines and investigations into their mechanism of action. Recently, we reported that treatment of cultured L1210 cells with 7 [R = C6H5N(Me)CHz]at a concentration that prevented proliferation and colony formation of the cells resulted in a progressive increase with time of exposure in the number of cells in mitosis.2 This compound was as effective as vincristine in promoting the accumulation in mitosis of cultured L1210 cells. In addition, this compound caused the accumulation of L1210 cells in mitosis in mice bearing ascitic L1210 leukemia. Biological data on two series of l-deaza-7,8-dihydropteridines (7) are listed in Table IV. In the 6-(Nmethylani1ino)methyl series, the concentration of drug required for the inhibition of the proliferation of cultured L1210 cells (IDSO)was greater than that required for vincristine but similar to that of nocodazole.2 Within the series, the ID,, for the 4-methoxyphenyl compound resembles that of the parent phenyl compound, whereas the 4-chlorophenyl and 4-(methoxycarbony1)phenyl compounds had higher ID,'s. The mitotic indexes (MI) listed in Table IV show that each compound caused the accumulation of cells at mitosis.2 These values of the MI are the results of preliminary work but are probably minimum values (see below). Superimposition of the semilogarithmic plots of the inhibition of cell proliferation and the MI at various concentrations is shown for 7 [R = C6H,N(Me)CHz] in Figure 1. The similarity of the plots suggested that cytotoxicity can be attributed primarily to mitotic arrest. The decrease in the MI at the higher concentration is caused at least in part by lysis of some of the cells. Although each of these compounds has significant activity
1048 Journal of Medicinal Chemistry, 1982, Vol. 25, No. 9
Temple et al.
Table IV. Biological Data for l-Deaza-7,8-dihydropteridines
P-388' 10, tumor cell implant, ip mitotic indexb compd or R nocodazole" vincristine C,H ,N(Me)CH,
L1210 ID,,,a pM 27 x 10-3
