158 Journal of Medicinal Chemistry, 1975, Vol. 18, No. 2
the aromatic ring and the iodoacetamide moiety in these reagents did not affect the first-order rate constants for inactivation of COMT. Also, the simple observation that modification of this amino acid moiety results in loss of enzymatic activity would imply a crucial role for this functional group in catalysis or binding. Our earlier observation4 t h a t inclusion of catechol substrate in the preincubation mixture protects the enzyme from inactivation by the N-iodoacetyl derivative 4 provides further evidence that an active site amino acid is being modified and that these compounds are specific active-site-directed alkylating reagents. Using the information obtained in this study, we are now carrying out incorporation studies and attempting to isolate and characterize the modified amino acid residues. Acknowledgment. The authors gratefully acknowledge support of this project by a research grant from the National Institutes of Neurological Diseases and Stroke (NS-10918) and a Grant-in-Aid from the American Heart Association. Partial support was also provided by a University of Kansas General Research Grant. The excellent technical assistance of Mrs. Bi-Shia Wu is also gratefully acknowledged.
Ross, Jamieson
References (1) R. Nikodejevic, S . Senoh, .I. W . Daly. and C . R. Creveling. J . Pharmacol. Exp. Ther., 171,83 [ 1970). ( 2 ) E. Shaw in "The Enzymes." Voi. 1. t'. D. Royer. Ed.. Academic Press. New York, Ti.\'.,1970. p 91. (9) S. J . Singer, Adt an. Protein C h ~ m .22. . 1 (1967). (41 R. T. Borchardt and D. Thakker. Hiochem. Biophys. R p r . ('ommun.. 54,1233 11973). ( 5 ) R. T.Borchardt. J . Med. Chem , 16, 377. 382. 387. 581 ( 19731. (6) C. F. H . Allen and G. W. Leubner in "Organic Syntheses," Collect. Vol. IV. N. Rahjohn. Ed.. Wilev. Tew York, N.Y.. 1963, p 866. (7) F. A . Ramirez and A. Burger, .J. rlmer. ('hem. .Sot., 72, 2781 (1950). (8)F. Renington. K. I). Morin, and L. C . Clarke, .J Amcr Chem. Soc., 76,5555 (1954). (9)P. H. Petra, Biochemist/?.. 10,3163 (1971). (10) .J. S.Buck and W . Y. Ide in "Organic Syntheses." Collect. 1943. p Vol. 11. A . H. Rlart, Ed.. Wiley, IVew York. N.Y.. 62%.
(11) .I.Axeirod and H. Tomchick. J . Bioi. C'hem., 234, 702 (1958~. (121 N. D . Xorris, F. McNeal, and C. R. Creveling, Abstracts of the 8th Middle Atlantic Regional Meeting of the American Chemical Society. Washington, D.C., Jan 1973, p 60. ( 1 3 ) R. T. Borchardt. Mol. Pharmacol., submitted for publicat ion.
Antiparasitic Nitroimidazoles. 8. Derivatives of 2-(4-Formylstyryl)-5-nitro-1-vinylimidazole William J . Ross* and William B. Jamieson Ld/> Research Centre Limited, Erl Wood Manor, Windlesham, Surrei, England Reteiued Ma\ 29, l W 4 A series of 33 thioacetals and hydrazones of 2-(4-formylstyryl)-5-nitro-l-vinylim1dazole was prepared and examined for antitrypanosomal properties. The thioacetals were inactive as antitrypanosomal agents but three hydrazones derived from .V-aminoguanidine, pyridylacetohydrazide chloride (Girard reagent P). and dimethylaminoacetohy-
drazide (Girard reagent D) displayed good activity against Trypanosoma rhodesiense In part 3' we described the antitrypanosomal properties of 2-(4-carboxystyryl)-5-nitro-l-vinylimidazole ( l a ) and a number of related compounds including the aldehyde lb.
CH=CHL la. R=COOH b. R = CHO C.
"1
R = -CH \
0
The latter compound was equiactive with la against T y p a n o s o m a rhodesiense infections in mice when dosed ip b u t was considerably less active when dosed orally.' However, it was shown2 that l b is rapidly metabolized in the mouse to la and excreted as its g l u ~ u r o n i d e We . ~ considered that if l b could be suitably derivatized we might accomplish two things: ( a ) increase the intrinsic activity of the compounds against trypanosomes; ( b ) prevent rapid metabolism and excretion of the compound. Chemistry. It was already known' t h a t IC had reasonable activity against a number of T Q p a n o s o n a species so we prepared by standard methods (see Experimental Section) a number of thioacetals (2-7) derived from l b which we considered would be metabolically and chemically
more stable t h a n IC. The hydrazones 8-19 were prepared by analogy with the many hydrazones derived from 5 n i trofurfuraldehyde and related compounds.4.5 In particular, compounds 10, 11, and 12 were prepared as analogs of nitrofurazone, nitrofurantoin, and guanofuracin, respectively. Xitrofurazone has been shown to be effective against T r y p a n o s o m a g a m biense infections in guinea pigs6 and t o inhibit a T o p a n o s o m a cruzi infection in mice.7 Compounds 1:3-16 can be considered as analogs of nifurtimox,8 a promising compound for the treatment of acute and chronic Chagas disease (Table I). The quaternary acylhydrazones 20 and 24 were prepared in a n attempt to impart some water solubility to the compounds and also as analogs of 5-nitro-2-furfuraldehydetrimethylammonium acetylhydrazone chloride which has been shown to inhibit inections of T . cruzi in mice.' Biological Results. All the compounds were tested against infections of T . rhodesiense in mice using the procedures described by Hawking.9 If the compounds showed activity in this test they were tested against T . cruzi, T. gambiense, and T r y p a n o s o m a congolense by a similar procedure. Only compounds active against one or more of the above organisms are listed in Table 11. None of the thioacetals 2-7 showed activity comparable to IC and this series was abandoned. The simple hydrazones 8 and 9 were both inactive as was 10, the analog of nitrofurazone. The nitrofurantoin analog 11 exhibited marginal activity against T . rhodesiense, T . g a m biense, and T. congolense but was inactive against T . cruzi. The guanofuracin ana-
Journul of Medicinal Chemistry, 1975, Vol. 18, No. 2 159
Antiparasitic Nitroimidazoles
Table I OIN J N 1L C H + CH=CHZ Yield,
%
R
Compd
Crystn solvent
MP, “C
2
-Ci;O-J ‘S
74
EtOH
143-1 44
3
-d ‘S -J
78
EtOH
1 46-1 47
62
EtOH
114-1 15
73
CHCI3-pet. ether
221-222
58
CHCl,-pet.
169-1 7 0
64
CHCl,-MeOH
2 55-2 5 6
95 86 64
EtOH AcOH Dioxane
2 16-2 17 298-299 dec 261-262 dec
79
DMF
283-284 dec
81 90 86 84 76 82 90 72 53 55 76 62 66 66 52 49 58 70 70 75 68 52 71
DMF HzO CHC13 CHC1,-pet. ether MezC0 EtOAc EtOAc CHC13-pet. ether (CH20Me), M e C e H 20 D M F-H, 0 DM F-HzO DMF-H, 0 MezC0-H, 0 CHCl,-pet. ether EtOAc EtOAc EtOAc CHC1,-pet. ether CHCl,-pet. ether CHC13-pet. ether DM F-Et OH DMF-H20 DMF
248-250 dec 196-1 9 7 201-202 268-269 2 15-2 16 19 1-1 92 187-188 175-176 2 74-2 7 6 192-193 248-2 5 0 225-266 dec 2 3 5-23 7 190-191 155-157 153-154 dec 183-1 84 2 14-21 5 214-215 219-220 2 53-2 5 4 270-272 dec 2 49-2 50
4 5
- C‘S G S 2
6
-CH
/S\
(CH l4
‘S’
7 8 9 10
-CH =NNHC 6H5 - C H =NNH-2,4 - (No, )zC,H3 -CH= NNHCONH, CONH
\
11
-CH=?iN’
12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34
-CH=NNHC (=NH)NH2 -CH=N-C -N(CH,CH, ),o -CH=N-c -N(CH,CH, ),S -CH=N-C -N(CH,CH, ),SO, -CH=N-C-N(CH~)~ -CH=N -C - (CH,CH, 1, NCH,CH,OH -CH=NN(CH,CH,OH 1, -CH=NNHC H, COZ Et -CH=NNHCOCH, -c-NC,H, C1-CH=NNHCOCH, -2-Py -CH=NNHCOCHZ -3-Py -CH=NNHCOCH2 -4 -Py -CH=NNHCOCH,N+(CH,), C1-CH=NNHCOCH,N(C H& -CH=NNHCOCH,N(C~H, ), -CH=NNHC OC H, NHCH 3 -CH=NNHCOCH,NHC2H, -CH=NNHCOCH, - c - N C ~ H ~ -CH=NNHCOCH, -C -NC,HI, -CH=NNHCOCH, -c-N(CH~CHZ)@ -CH=NNHCOC H, -C -N(CH2CH2)2S0, -CH=NNHCOCH, - C H =NNHCOP h
‘CH
CO
+
ether
FormulaQ 1
5N303S
C24H23N302S2 c Z 0H1?N5% c2 0H15N?06
1SH14N603
Ci,H,,N,O4 ‘ 0.5HzO 15
5 N7
O2 ’
C18H19N503 C18H15N5%S 18H19N504S
cl gH2 IN5O2
C20H24N603 1BH1BNBo4
C18H19N504
C21H,,ClN,O,
2H2O
c2 lN 8N603 l
C21H18N603 cZ lH1
C1gH13ClNG03 2H2O ON603
C20H24N603 lTHl s N 6 0 3 Ci8H20N603
C20H22N603 C20H22N603
C20H2ZN604 C20H22N605S
c1
sHf 5N5 O3
C21H17N503
aAll compounds were analyzed for C, H, and N .
log 12 showed good activity ip against T . rhodesiense and T. cruzi b u t was less effective PO. T h e nifurtimox analogs 13-16 proved active ip but were generally less active than the parent compound Ib. In a n attempt t o improve water solubility t h e hydroxyethyl compounds 17 and 18 were prepared. Compound 17 proved reasonably active but 18, the open-chain analog of 13, was inactive as was the ester 19. We next considered the preparation of acylhydrazones derived from t h e Girard “T” and “P” reagents; such derivatives would serve the dual purpose of being water soluble and be analogs of a n active compound (uide supra). Compound 20 was active against T. rhodesiense, T. g a m biense, and T. congolense
but we were unable to determine the dose which would give a parasitological cure against T. cruzi due to toxicity problems. However, t h e activity of 20 against T. rhodesiense, T. gambiense, a n d T. congolense warrents special comment as it approaches that of t h e standard drugs (see Table 11). However, 20 is somewhat toxic (LD50 -50 mg/kg ip in mice) and this limits its usefulness. As we considered the toxicity of 20 to be due t o t h e presence of t h e quaternary function, the pyridylacethydrazones 21-23 were prepared as nonquaternary analogs but all were inactive. T h e trimethylammonium analog of 24 was less active t h a n 20 but t h e Girard “D” derivative 25 proved t o have twice t h e activity of la and l b against T.rhodesiense but was inac-
160 Journal ofMedicinal Chemistry, 1975, Vol. 18, No. 2
Ross, Jarnieson
Table 11. Minimum Dose Level in mg/kg Shown to Be 100%Effective against Trypanosoma1 Infections in Mice T. T . rhodesiense
__
Compd
la lb
PO
2 3 4
25 25 50 50 50 25
6
50
11
200
IC
25 200 100 200 ND Inactd at 25 Inact" at 50 ND
colzgoleme, iP P O iP iP ___-__-_______~.._I______ T . c m -___ zib
1'. qnmbieizse, a
100 500 200 200 100 80
200 500 500 ND NDC ND
12.5 25 25 ND ND ND
25 50 100 100 ND ND
Inact at 50 Inact at
ND
ND
ND
ND
100
200
Inact at 100 Inact at 100 Inact at 100 Inact at 200
ND
25
25
200
25
100
200
200
ND
ND
100
100
ND
ND
ND
5
10
ND
10
10
ND
50
ND
ND
ND
ND ND
ND ND
ND
5
ND
5
Inact at 5 5
ND
5
10
ND
0.75
Inact at 2
200
12
12.5
100
50
13
100
100
14
100
15
200
16
200
Inact at 500 Inact at 100 Inact at 200 ND
17
50
100
18
2 00
Inact at
3
200 10
20
100 200 Inact at 200 Inact at 50 Inact at 200 Inact at 10
24
10
10
Inact at 10
25
12.5
12.5
26
25
25
29 31
25 25
25 25
ND
Suramin
1
Pentamidine Diminazene Melarsoprol
1.25
Inact at 1 Inact at 1.25 Inact at 1 0.5
Inact at 5 Inact at 5 Inact at
1 0.75
Inact at 50 Inact at 25 Inact at 25
10
Inact at 2
I _ _ _ _ _
aMice were dosed for four consecutive days, commencing on the day of infection. 100% efficacy is equivalent to 30-day post-infection survival with negative parasitemia. bMicewere dosed for five consecutive days, commencing on the day of infection. 100% efficacy is equivalent to 60-day post-infection survival with negative parasitemia. CND = not done. dCompounds were defined as inactive if they failed to prolong the mean survival time (MST) of infected mice to the time scale defined in footnotes a and b. MST for control animals were as follows: T . rhodesiense and T . garnbiense. 4 days; T . cruzi. 13-14 days; 7'. congolense, 7-10 days. tive against T . cruzi. A detailed description of the activity of 25 is given in Table III. A number of congeners of 25 were prepared in order to define the requirements for activity. The N-diethyl analog 26 was somewhat less active than 25 while the secondary amines 27 and 28 were both inactive. Introduction of a pyrrolidine function to give 29 reduced activity while the presence of a piperidine 30 function resulted in loss of activity. The morpholino compound 31 had similar activity to 29 b u t t h e thiomorpholine dioxide 32 was completely inactive. Replacement of the tertiary amine function in 25 by a methyl 33 or phenyl 34 groups gave inactive compounds.
I t would appear, therefore, that maximum antitrypanosoma1 activity for these acylhydrazones resides in compound 25 and any change in the structure of the hydrazone function is detrimental to activity. In conclusion, we were able to prepare a number of derivatives of lb ( e . g . , 12, 20, and 25) which are more active against T. rhodesiense than the parent compound or our previous best compound la. However, in general, the compounds had poor activity against T. cruzi and none were quite as active against T. rhodesiense as the standard drugs suramin, pentamidine, diminazene, or melarsoprol.
Journal of Medicinal Chemistry, 1975, Vol. 18, No. 2 161
Pyrazolo [3,4-clpyrimidinesand &razol0[3,4-blpyridines
T a b l e 111. Per Cent Activitya of Compound 25 against African Trypanosomes ~~~~~~
~
T . rhodesiense Dose, mg/k 12.5 X 4 10 x 4 50 X 1 25 X 1 2 x 4 5 x 1 a%
T . congolense
T . gambiense
Route
’% act.
iP
100 100 89
PO PO PO
sc SC
Dose, mg/kg 10 x 4 25 X 1
Route
(& act.
Dose, mg/kg
PO
100 100
50 x 4 25 x 4
PO
Route
% act.
iP
79
SC
100
97 100 80
activity calculated as on Table 11.
Experimental Section
solid which was filtered off. The bright yellow filtrate was carefully diluted with petroleum ether (bp 40-60”) and allowed to crystallize: yield 6.1 g (66%);mp 190-191”. Acknowledgments. We thank Mr. M. C. McCowen, Dr. M. D. Pittam, and Miss J. O’Brien for the biological results and Mr. G. Maciak of the Lilly Research Laboratories, Indianapolis, for the microanalytical data on the compounds described in this paper.
Melting points were taken on a Gallenkamp apparatus (Registered Design No. 889339) using capillaries and are uncorrected. All compounds were characterized by ir, uv, nmr, and elemental analyses (C, H, N) which were within 4=0.4% of the theoretical values. Acethydrazide, benzhydrazide, and Girard reagents “T” and “P” were purchased from BDH; Girard reagent “D” was obtained from Eastman-Kodak. All the other required hydrazides were prepared by literature methods.1° The hydrazines used for the preparation of compounds 13-18 References a n d Notes were prepared by literature methods11 and kindly supplied by W. J. Ross, W. B. Jamieson, and M. C. McCowen, J . Med. Mr. J. P . Verge. General Method for Thioacetals. The aldehyde l b (0.05 mol) Chem., 16,347 (1973). D. M. Morton, J . N. Green, and D. M. Fuller, unpublished was refluxed with the appropriate thiol (0.05 mol) in benzene and work from these laboratories. in the presence of p-toluenesulfonic acid (100 mg) for 2 hr, the D. M. Morton, J . N. Green, and D. M. Fuller, Xenobiotica, water from the reaction being removed via a Dean-Stark separa3,257 (1973). tor. After cooling, the benzene solution was washed with NaHC03 K. Miura and H. K . Reckendorf, Progr. Med. Chem., 5, 320 solution, followed by HzO, and then evaporated in U ~ C U Oto give a bright yellow solid which was crystallized from an appropriate (1967). British Patent No. 1,317,256 and 1,317,257 issued to Lilly Insolvent, e g., ethanol. dustries Ltd., May 16, 1973. General Method for Hydrazones. The aldehyde l b (0.025 mol) N. J. Giarman, J. Pharmacol. Exp. Ther., 102,185 (1951). and the hydrazine or hydrazide (0.025 mol) were refluxed in a solA. Packchanian, J. Parasitol., 38,30 (1952). vent such as EtOH or CHC13, with or without the addition of a M. Bock, A. Haberkorn, H. Herlinger, K. H. Mayer, and S. few drops of glacial acetic acid, for 2-4 hr. On cooling the resulPetersen, Arzneirn.-Forsch., 22, 1564 (1972). tant crystalline solid was filtered off and recrystallized from an F. Hawking, Exp. Chemother., 1,131 (1963). appropriate solvent. 2- (4-N,N-Dimethylglycinamidoiminomethinestyryl)-5-nitro(a) H. L. Yale, K. Losec, J . Martins, M. Holsing, F. Perry, 1-vinylimidazole (25). Aldehyde lb (6.7 g, 0.025 mol), N,N-diand J. Berstein, J. Amer. Chem. Soc., 75, 1933 (1933); (b) H. methylglycine hydrazide hydrochloride (3.8 g, 0.025 mol), and soZimmer and D. K. George, Chem. Ber., 89,2285 (1956). dium acetate hydrate (4 g) were refluxed in EtOH (300 ml) for 2 (a) British Patent 874,519 issued to Badische Anilin & Sodahr. On cooling a small amount of inorganic material separated Fabrik A. G. (Aug 10, 1961); (b) U.S. Patent 3,153,095 issued and was filtered off. The filtrate was evaporated in vacuo to give to Commercial Solvents Corp. (Oct 13, 1964); (c) E. Jucker an oil which on treatment with CHC13 gave further inorganic and A. Lindenmann, Helu. Chim. Acta, 45,2316 (1962).
Synthesis and Biological Evaluation of Xanthine Oxidase Inhibitors. Pyrazolo[3,4d]pyrimidines and Pyrazolo[3,4-b]pyridinest Ih C h u j and Brian Maurice Lynch* Department
of
Chemistry, Saint Francis Xauier Uniuersity, Antigonish, Nova Scotia, BOH I C O , Canada. Receiued May 20, 97.
1-, 3-, and 5-substituted pyrazolo[3,4-d]pyrimidinesand pyrazolo[3,4-b]pyridines related to allopurinol were synthesized and evaluated as xanthine oxidase inhibitors. Among these compounds, 4-hydroxypyrazolo[3,4-b]pyridine5-carboxylic acids 12 were found to possess potency in the same order of allopurinol. The influence of the substitutions on the enzyme inhibitory effect and the bulk tolerance of the enzyme-inhibitor complex are discussed.
4-Hydroxypyrazolo[3,4-d]pyrimidine (allopurinol, la) has been shown to be an effective inhibitor of xanthine oxidase and thus a clinically useful agent in gout treatt Research supported by National Research Council of Canada Regional Development Grant RD-29 and by the Saint Francis Xavier University Council for Research. 1 Postdoctorate Fellow, 1972-1974.
ment; it is also an effective adjuvant in antitumor chemotherapy.1 The objective of the work now reported is to develop more effective enzyme inhibitors using the parent skeleton (pyrazolo[3,4-d]pyrimidine)of allopurinol as the basis species, both by exploring the effects of substituent insertion on the activity of a ~ ~ o p u r i nand o ~ also by exploring various deaza species related t o this molecule. Previ-
