J.Med. Chem. 1981,24,559-567 K. Patterson, Jr. (The Samuel Roberts Noble Foundation, Inc.,
Ardmore, OK 734011, and ASase was isolated as Previously described.s1 The enzyme fraction used in the inhibition studies had a Specific activity of 0.51 "01 of asPar&e Synthesized per mi&'" Of protein in 30 min. L-hpartiC-"c acid was incubated with L-glutamine, ASase, and other needed cofactors, and the Lasparae;ine-14c synthesa was isolated 88 previously d-W.31 The inhibitors were preincubated with ASase and necessary cofactors and then substrate aspartic acid was added, as outlined in paper 4 of this series.la Acknowledgment. This work was supported by grants
from the American Cancer Society (CI-96) and the Na-
559
tional Cancer Institute (CA-11714) for which the authors express their appreciation. The authors thank Dr. M.K. Patterson, Jr. (Samuel Roberts Noble Foundation), for supplying the frozen tissue from which the enzyme was isolated. The inhibition studies were by M ~ ~ . Beth L. Trend, to whom the authors are grateful. Special thanks we accorded Timothy Stukus and James F- Bagaglio for Preparation of starting materials. Supplementary Material Available: Comparison infrared spectra of 2a and 2b (Figure 1) (1 page). Ordering information is given on any current masthead page.
Methotrexate Analogues. 13. Chemical and Pharmacological Studies on Amide, Hydrazide, and Hydroxamic Acid Derivatives of the Glutamate Side Chain Andre Rosowsky,* Cheng-Sein Yu, Jack Uren, Herbert Lazarus, and Michael Wick Sidney Farber Cancer Institute and the Departments of Pharmacology and Dermatology, Harvard Medical School, Boston, Massachusetts 02115. Received December 15, 1980
Carbodiimide-mediated condensation of 4-amino-4-deoxy-M0-methylpteroic acid (APA) with several alkyl, aralkyl, and aryl amines, in the presence or absence of N-hydroxysuccinimide, was employed in order to prepare new lipid-soluble bis(amide) derivatives of methotrexate (MTX) as potential prodrugs. MTX dianilide was likewise prepared, in comparable yield, from APA and L-glutamic acid dianilide via the mixed carboxylic-carbonicanhydride method. Dihydrazide and bis(N-methylhydrazide)derivatives of MTX were formed readily from MTX diethyl ester. However, reaction with hydroxylamine led to MTX y-monohydroxamic acid as the sole isolated product. The bis adduct appears to form, but is unstable during workup. The identity of the product was confirmed by independent mixed anhydride synthesis from APA and the y-monohydroxamateof bglutamic acid. Treatment of MTX dimethyl ester with NJV-dimethylhydrazineunexpectedly yielded MTX y-monomethyl ester. MTX dianilide was active against LE10 leukemia in mice, with a +155% increase in life span a t a dose of 160 mg/kg given ip in 10% Tween 80 on a q3d X 3 schedule. The bis(p-chlorobenzylamide), bis(p-methoxybenzylamide),and dihydrazide were also active against L1210 leukemia in vivo, but to a lesser extent than the dianilide. The y-monohydroxamic acid derivative showed activity (+Ill% ILS a t 40 mg/kg) similar to that of MTX and was found to bind to a partially purified dihydrofolate reductase preparation from L1210 cells with an IDSOof 0.005 pM as compared to 0.007pM for MTX. In vivo experiments in mice indicated that the pharmacokinetic properties of this compound and of MTX are similar but failed to demonstrate any advantage over MTX in terms of selective uptake into tumor (sc implanted P388 leukemia) or improved penetration of the central nervous system. The activities of the dianilide, bis(benzylamide), and dihydrazide derivatives in vivo are of interest in view of their low toxicity relative to MTX against cells in culture, which suggests that these derivatives are probably acting as prodrugs in the intact animal.
Several previous reports from this laboratory have dealt with the chemical synthesis and biological evaluation of prodrug derivatives of methotrexate (4-amino-4-deoxyNO-methylpteroyl-L-glutamicacid, MTX). Classes of compounds which have been studied include diesters,la* b i s ( a m i d e ~ )a, ~ and ~ ~ y-glutamyl conjugates; and more recently a series of m o n o e ~ t e r s . In ~ ~this ~ ~ paper we de(a) A. Rosowsky, J. Med. Chem., 16,1190 (1973); (b) G.A. Curt, J. S. Tobias, R. A. Kramer, A. Rosowsky, L. M. Parker, and M. H. N. Tattersall,Biochem. PharmacoZ.,25,1943(1976); ( c ) A. Rosowsky and C.-S. Yu, in "Chemistry and Biology of Pteridines", R. L. Kisliuk and G. M. Brown, Eds., Elsevier/ North Holland, New York, 1979, pp 377-381; (d) G. P. Beardsley,A. Rosowsky, R. P. McCaffrey, and H. T. Abelson, Biochem. PharmacoZ.,28, 3069 (1979); (e) A. Rosowsky, H. Lazarus, G. C. Yuan, W. R. Beltz, L. Mangini, H. T. Abelson, E. J. Modest, and E. Frei 111, ibid., 29,648 (1980); ( f ) G.P. Beardsley and A. Rosowsky, AACR Proc., 21,264 (1980). A. Rosowsky, W.D. Ensminger, H. Lazarus, and C.-S. Yu, J. Med. Chem., 20, 925 (1977). For related work on bis(amides) of MTX, see the following papers: (a) J. R. Piper and J. A. Montgomery, in "Chemistry and Biology of Pteridines", R. L. Kisliuk and G. M. Brown, Eds., Elsevier/North Holland, New York, 1979,pp 261-265; (b) F. M. Sirotnak, P. L. Chello, J. R. Piper, J. A. Montgomery, and J. I. DeGraw, ibid., pp 597-602. A. Rosowsky and C.-S. Yu, J. Med. Chem., 21, 170 (1978). 0022-2623/81/1824-0559$01.25/0
scribe several additional examples of the amide type, whose structures (1-11) and methods of synthesis are shown in COR
I I (CH2 I COR
Pter-NH
Pter -NHCH
)2
1-11
roH H
I (CH2)2 I CONHOH 12
Pter = 4-amin0-4-deoxy-N~~-methylpteroyl
Table I. The monohydroxamic acid 12, a heretofore unknown MTX analogue differing only in the replacement of the y-COOH group by yCONHOH, was also prepared. Compound 12 was a good inhibitor of dihydrofolate reductase, was moderately toxic to human and mouse leukemic cells in culture, and showed in vivo antitumor activity comparable to that of MTX against L1210 leukemia in mice. Interest in this compound stemmed from the fact that, while the y-CONHOH group is structurally very (5) (a) A. Rosowsky, G. P. Beardsley, W. D. Ensminger, H. Lazarus, and C.3. Yu, J. Med. Chem., 21,380 (1978); (b) H.T. Abelson, G. P. Beardsley, W. D. Ensminger, E. J. Modest, and A. Rosowsky, AACR Proc., 21, 265 (1980). 0 1981 American Chemical Society
Rosowsky et al.
560 Journal of Medicinal Chemistry, 1981, Vol. 24, No. 5
In CO I+
0 (D
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rl
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8
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Methotrexate Derivatives
(0
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Journal of Medicinal Chemistry, 1981, Vol. 24, No. 5 561
* c o o O
N
I
?
cr) (0
rl
Q, rl
rl
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a h l
similar to the T-COOH group, it should not be amenable to polyglutamate formation. Chemistry. The reaction of MTX diethyl or dimethyl ester which was previously used to obtain bis(amides)2 proceeded smoothly with ammonia, simple straight-chain N-alkylamides, or pyrrolidine but gave low yields (or failed entirely) with more hindered aliphatic amines such as 1-aminoadamantane, N,N-di-n-butylamine, or piperidine. When piperidine was used, the major products appeared to be y-monoesters, whose formation was presumably due to the presence of residual moisture in the reactants or On the other hand, treatment of MTX with 2 molar equiv each of piperidine and N,N-dicyclohexylcarbodiimide (DCC) in dry DMF at room temperature for about 3 days resulted in a 35% yield of the desired bis(amide) 2, after remaval of a small amount of acylurea impurity (10% yield) by silica gel column chromatography. Acylurea side productse have been observed in this laboratory previously in DCC-mediated coupling reactions involving MTX4 and appear to be general for this series. That compound 2 was a bis(amide) rather than mono(amide) derivative was apparent from its insolubility in aqueous base and its infrared spectrum, which showed no ester absorption in the 1740-cm-' region. DCC coupling was also employed to obtain several bis(benzylamide) analogues from MTX. The best results (Table I) were obtained with N-benzyl-N-methylamine, which gave a 67% yield of the bis(amide) 5. There appeared to be an inverse correlation between the yields of the bis(amides) in these reactions and the tendency to form acylureas. Thus, with 1-phenylethylaminethe yield of the bis(amide) 7 was only 16%, whereas the acylurea product was obtained in 42% yield. On the basis of infrared and NMR spectral evidence, as well as microchemical data, the side product is probably a monoamide monoacylurea, though a definitive choice between structures 13a and 13b
'-0
CONCH(Me)Ph
CONCONH
I Pter-NHCH I (CH2)2 I CONH CH(Me) Ph
I Pter-NHCH I b
m ._ N
N 0
9 0
*0.,.
0
z.CIJ
m
0
0 0
0
0
m
u
h U
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13b
13a
cannot be made. That steric effects probably play a strong adverse role in the coupling reaction is indicated by the very low yield (6.7%) of the bis(amide) 1 from l-aminoadamantane. In some instances coupling was performed via the Nhydroxysuccinimideroute (Table I, method B), which has been reported to be superior to DCC alone for the formation of peptide bonds! When 4-chlorobenzylamine was condensed with MTX free acid in the presence of DCC, a 19% yield of the bis(amide) 3 was obtained; however, when the intermediate N-hydroxysuccinimide ester of MTX was prepared, the yield of compound 3 increased to 62%. Similar favorable results were observed when MTX (6) D. H. Rich and J. Singh, in "The Peptides: Analysis, Synthesis, and Biology", Vol. 1, E. Gross and J. Meienhofer, Eds., Academic Press, New York, 1979, Chapter 5.
Rosowsky et al.
562 Journal of Medicinal Chemistry,1981, Vol. 24, No. 5
was condensed with 4-methoxybenzylamine in order to prepare the bis(amide) 4 (11and 66% yields, respectively). The dianilide derivative 8 was accessible directly from MTX via the DCC/N-hydroxysuccinimide approach (19% yield); however, in view of previous indications that some side-chain racemization might occur during MTX reactions involving DCC,4we chose to also prepare compound 8 via mixed anhydride condensation (Table I, method C). Reaction of N-carbobenzoxy-L-glutamicacid with aniline in the presence of DCC and N-hydroxysuccinimide,followed by removal of the Cbz group by catalytic hydrogenolysis and coupling of the dianilide with the mixed anhydride from isobutyl chloroformate and 4-amino-4-deoxy-WOmethylpteroic acid, led to the desired product 8. The yield of 8 in the final step was 21%, and a substantial amount acid was recovered of 4-amino-4-deoxy-WO-methylpteroic unchanged, as is customary in this r e a c t i ~ n . ~ ~ ~ ~ The dihydrazide derivatives 9 and 10 were obtained from MTX diethyl ester on reaction with hydrazine and Nmethylhydrazine, respectively (Table I, method D). After only 30 min at room temperature, compound 9 was formed from hydrazine in almost quantitative yield. The reaction rate with N-methylhydrazine was slower; even after 48 h, the yield of compound 10 was only 28% and at least two other products were detected by TLC. The structure of 10 was assigned on the basis of a published study7 reporting that acylation of N-methylhydrazine by esters occurs predominantly on the NHp group and is favored when the acyl group is large. When MTX dimethyl ester was heated with N,N-dimethylhydrazine (48 h under reflux), none of the desired dihydrazide could be obtained: instead, a 57% yield of MTX y-monomethyl ester was isolated. Similar cleavage of diesters to monoesters in the presence of N,N-dimethylhydrazine has been reported previou~ly.~" The monohydroxamic acid analogue 12 was obtained directly from MTX diethyl ester and hydroxylamine in the presence of base. When a methanol solution containing MTX diethyl ester and an excess of hydroxylamine sodium salt was stirred at room temperature for 2 days and the reaction was terminated by adding ether, a TLC-homogeneous solid (Rr0.43, cellulose, pH 7.4) was isolated which gave a positive hydroxamate test (violet color with FeClJ and whose IR spectrum showed no ester C = O absorption in the 1740-cm-' region. On passage through a DEAEcellulose column, the initial product underwent transformation to a new TLC-homogeneous compound (R, 0.61, cellulose, pH 7.4) which still gave a positive FeC1, test for the hydroxamic acid function. Though definitive evidence is lacking, it appears that the initial product (Rf0.41) may be a bis(adduct) which loses a molecule of hydroxylamine rapidly during ion-exchange chromatography in alkaline solution. That the final product (Rf0.61) is the y-monosubstituted derivative 12 is clear, however, from the fact that this compound was also obtained from 4-amino-4deoxy-N1O-methylpteroic acid and L-glutamic y-monohydroxamic acid via the mixed anhydride route (Scheme I). Mixed anhydride coupling of APA to unprotected amino acids in the presence of 1,1,3,3-tetramethylguanidine has been used previously in this laboratory to prepare MTX y-monoethyl and has other precedents in the peptide literature.8 Bioassay. Most of the compounds synthesized during this investigation were tested as inhibitors of the growth (7) R. L. Hinman and D. Fulton, J. Am. C h e n . SOC.,80, 1895 (1958). (8) D. S. Kemp, S.-W. Wang, J. Rebek, Jr., R. C. Mollan, C. Ban-
quer, and G. Subramanyan, Tetrahedron, 30, 3955 (1974).
Scheme Ia CONHOH~
COzEt
I
I
COZEI
12
-
r
INno 1 Pter-OH
Pter = 4-amin0-4-deoxy-N'~-methylpteroyl. Table 11. Activity of MTX Amides against Leukemic Cells in Culture
compd
MTX
human lymphoblastic leukemia (CEM)
ID5a,aPM murine leukemia (L1210)
rat basophilic leukemia (RBL
0.01 0.003 3.4 1.6 > 10 >10 (29%) 6.6 >10(44%) 8.2 6.4 9.4 7.6 0.69 7 >10(25%) >10 8 3.3 0.41 9 I. 5 0.95 0.96 12 0.25 0.062 a See ref 16 for details of the assay procedure. Values given in parentheses are percent inhibitions of cell growth (48h) at a drug concentration of 10 which was the highest dose tested. Results are averages of triplicate experiments and have a standard deviation of +lo%. b See ref 2. 1 2 3 4 5 6
0.003 >10 (38%)
a,
of CEM human lymphoblastic leukemia cells and L1210 mouse leukemia cells in vitro (Table 11). Against CEM cells, slight activity was shown by the dianilide 8 (IDm = 3.3 pM).A low level of activity was likewise observed with the bis(N-benzyl-N-methylamide) and bis(N,iV-dibenzylamide) derivatives (compounds 5 and 61, but the addition of a methyl group on the benzylic carbon (compound 7) reversed this effect. The bis(pchlorobenzy1amide) and bisb-methoxybenzylamide) derivatives (compounds 3 and 4) were less active than the previously reported bis(benzy1) analogue? Against L1210 cells, the most active bis(amide) was once again the dianilide 8, which had an ID5,, value of 0.41 pM. Also active below 1 pM were the bis(N,Ndibenzylamide) 6 and the dihydrazide 9, but not the bis(N-benzyl-N-methylamide)5. The other bis(amides) were less active, though it may be noted that, for the most part, their IDm values were lower against L1210 cells than against CEM cells. In several instances (compounds 6 , 8 , and 9), this difference in activity was approximately 10fold. Two compounds were also tested against cultured rat basophilic leukemia cells (RBL)2and were found to have greater activity than against CEM cells. The dihydrazide 9 had an ID, value of 7.5 pM against CEM cells but only 0.96 pM against RBL cells. This 8-fold increase in activity against the RBL line was similar to our earlier
Methotrexate Derivatives Table 111. Binding of MTX Amides to Dihydrofolate Reductase ID,, PM L1210 enzyme L. casei comDd (ligand-binding assay) (kinetic assay) MTX 0.0068 f 0.0022' 0.021 f 0.008' 1 3.1 6.3 2 0.012 0.25 3 0.18 2.3 4 0.13 0.40 5 0.0 26 0.44 6 0.53 0.86 7 0.011 0.31 8 0.074 1.1 9 0.020 0.36 12 0.0048 0.03 0 Mean 2 SD from three separate experiments are reported for MTX.
observations with other bidamide) derivatives of MTX, for which several possible explanations were advanced including the possibility that RBL cells, which are of myeloid rather than lymphoid origin, contain elevated levels of amidases. The present finding that the IDm values for compound 9 against the lymphoid L1210 line and myeloid RBL line are essentially the same appears to rule out such an interpretation, however, and suggests that the data are a reflection primarily of species differences (i.e., human vs. rodent). Of all the compounds tested in this work, the most cytotoxic proved to be the y-monohydroxamic acid ester 12, which had an IDWvalue of 0.25 pM against CEM cells. Against L1210 cells, compound 12 had an IDa value of only 0.062 pM and was thus only 6-fold less active than MTX itself, whose IDWagainst this cell line was 0.01 pM. The affinities of the MTX bis(amides) 1-9 and y-monohydroxamic acid 12 for partially purified bacterial (Lactobacillus casei) and mammalian (L1210 mouse leukemia) dihydrofolate reductase was likewise investigated. A competitive ligand-binding assafl"' was employed with the L1210 enzyme, whereas studies with the Z.casei enzyme involved a standard spectrophotometric assay.ll The competitive ligand-binding assay, which depends on the ability of a test compound to compete with [3H]MTX for binding to the enzyme, is convenient when limited amounts of enzyme are available and is known to agree well with kinetic assays based on spectrophometric measurement of the rate of conversion of NADPH to NADP. The IDM values obtained for MTX and the amide derivatives in these two assays are listed in Table 111. Under our assay conditions, MTX showed IDa values of approximately 0.02 pM against the L. casei enzyme (kinetic assay) and 0.007 p M against the L1210 enzyme (ligand-bindingassay). The y-monohydroxamic acid 12 was effective against both enzymes and, in fact, appeared to have a slightly higher affinity for the L1210 enzyme than MTX itself. The bis(4-chlorobenzylamide) (2), bis(N-benzyl-N-methylamide) (51, bis(1-phenylethylamide) (7), and dianilide (8) derivatives were likewise fairly active against L1210 dihydrofolate reductase, with IDMvalues of 0.01-0.1 pM. Activities against the L. casei enzyme followed a qualita(9) C. E. Myers, M. E. Lippman, H. M. Eliot, and B. A. Chabner, R o c . Natl. Acad. Sci. U.S.A., 72, 3683 (1975). (10) E. Arons, S.P. Rothenberg,M. da Costa, C. Fischer, and M. P. Iqbal, Cancer Res., 35, 2033 (1975). (11) L. E. Gunderson, R. B. Dunlap, N. G. L. Harding, J. H. Freisheim, F. Otting, and F. M. Huennekens, Biochemistry, 11, 1018 (1972).
Journal of Medicinal Chemistry, 1981, Vol. 24, No. 5 563 Table IV. Activity of MTX Derivatives against L1210 Leukemia in Mice' dose, no. mg/kg MTX of q3d equiv, ani- median compd 1, 4, 7 mg/kg mals T/C, days % ILS 80 65 5 11.0/9.0 t 22 130 5 13.0/9.0 160 t 44 64 5 11.0/9.0 80 t 22 128 6 15.0/9.0 160 t 66 30 5 13.0/9.0 40 t 44 80 60 5 19.Ol9.0 t 111 90 5 23.0/9.0 t 155c 120 9 expt 1 7.1 5 14.0/10.0 t 40 7.5 15 14.2 5 14.0/10.0 t 40 20 expt 2 18.8 5 16.0/9.0 t 77 40 37.6 5 12.0/9.0 t 33 12 expt 1 7.5 7.3 5 15.0/10.0 t 50 15 14.6 5 16.0/10.0 t 60 expt 2 20 19.4 5 17.0/9.0 t 89 40 38.8 5 19.0/9.0 t 111 MTX~ 15 20 15.5/9.0 t 72 30 20 16.5/9.0 t 83 60 20 18.019.0 t 100 ' Groups of five B6D2F, mice were injected ip with l o 5 L1210 cells on day 0. Test compounds were administered ip in 10%Tween 80 or 1 :1: 8 emulphor-ethanolwater suspension, and MTX was administered ip as the disodium salt in water. Data given for MTX represent median survivals from several experiments. The ILS was > 250% in two out of five animals at this dose.
tively similar trend, with 12 being the most active and 1 being the least active in the series. Examination of the data in Table I11 revealed that the y-monohydroxamic acid 12, which was the most active compound against the L1210 enzyme in the ligand-binding system, was also the most toxic to intact L1210 cells. Similarly, the dianilide 8 and dihydrazide 9, which had IDa values of
