p-alkyloxybenzhydroxamic acids, effective inhibitors of the trypanosome glycerol-3-phosphate oxidase

Molecular and Biochemical Parasitology, 19 (1986) 231-240 Elsevier

231

MBP 00668

p - A l k y l o x y b e n z h y d r o x a m i c acids, effective inhibitors of the t r y p a n o s o m e glycerol-3-phosphate oxidase Robert W. Grady 1, E. Jay Bienen 2 and Allen B. Clarkson, Jr. 2 IDepartment of Pediatrics, Cornell University Medical College, 1300 York Avenue, New York, NY 10021, and ZDepartment of Medical and Molecular Parasitology, New York University School of Medicine, 550 First Avenue, New York, NY 10016, U.S.A.

(Received 14 November 1985; accepted 13 January 1986)

Energy production in bloodstream forms of African trypanosomes of the genus Trypanosoma involves two pathways unique to the parasite and which can be blocked by a combination of salicylhydroxamic acid (SHAM) and glycerol. Although this leads to rapid parasite destruction both in vitro and in vivo, the toxicity of SHAM precludes practical use of SHAM/glycerol as a therapeutic regimen. Based on our hypothesis that SHAM operates by interfering with ubiquinone, we attempted to develop this approach by synthesizing and screening a series of hydroxamic acids which more closely resemble ubiquinone: the p-n-alkyloxybenzhydroxamic acids. We also examined a variety of mono-, di- and trisubstituted benzhydroxamic acids together with a selected group of secondary heterocyclic hydroxamic acids. We found an increase in activity of the p-n-alkyloxy compounds with increasing chain length up to 12 carbon atoms with longer chains offering little advantage. The most active compound, p-n-tetradecyloxybenzhydroxamic acid, had an apparent Ki of 0.43 laM indicating a specific activity 70 times greater than SHAM. Although this represents a vast improvement, the low water solubility of these compounds reduces their bioavailability to the point where they are not practical substitutes for SHAM. Consequently, improvement in the SHAM/glycerol approach to chemotherapy appears to lie with improving solubility by altering the,lipophilicity of the alkyl side chain. Key words: Trypanosoma brucei; Glycerol-3-phosphate oxidase; Hydroxamic acid; Ubiquinone; Chemotherapy; Electron transport

Introduction The bloodstream forms of African trypanos o m e s utilize a u n i q u e g l y c e r o l - 3 - p h o s p h a t e oxid a s e s y s t e m ( G P O ) to s h u t t l e e l e c t r o n s d e r i v e d from glycolysis to m o l e c u l a r oxygen [1]. T h e G P O can b e c o m p l e t e l y i n h i b i t e d b y h y d r o x a m i c acids such as s a l i c y l h y d r o x a m i c acid ( S H A M ) [2,3], w h e r e u p o n t h e p a r a s i t e s shift f r o m a e r o b i c to ana e r o b i c glycolysis, p r o d u c i n g p y r u v a t e a n d glycAbbreviations: AB, assay buffer; BB, breaking buffer; BSA, bovine serum albumin - Cohn fraction V, fatty acid free; DE52, Whatman microgranular diethylaminoethyl cellulose resin; DMSO, dimethylsulfoxide; EDTA, tetrasodium ethylenediaminetetraacetate; FBS, fetal bovine serum; GPO, glycerol3-phosphate oxidase system; Hepes, N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid; PMSF, phenylmethylsulfonyl fluoride; PS, phosphate-saline buffer; PSG, PS with glucose; SHAM, salicylhydroxamic acid; TLCK, N-e~-p-tosyl-L-lysine chloromethyl ketone-HCl.

erol in e q u i m o l a r a m o u n t s [4]. This a l t e r n a t i v e glycolytic p a t h w a y is s u s c e p t i b l e to i n h i b i t i o n b y glycerol via m a s s a c t i o n [5]. T h u s , c o m b i n i n g S H A M and glycerol blocks energy production and l e a d s to d e s t r u c t i o n o f t h e p a r a s i t e s b o t h in vitro a n d in vivo [5-11]. C u r e s of p a r a s i t i z e d hosts req u i r e t h a t sufficient levels of b o t h a g e n t s b e prese n t s i m u l t a n e o u s l y in all i n f e c t e d tissues as neit h e r a g e n t is active a l o n e [5,10]. W h i l e g l y c e r o l p r e s e n t s few p r o b l e m s , t h e d o s e of S H A M req u i r e d to a c h i e v e a 100% cure r a t e is e x c e e d i n g l y high. In t h e case o f T r y p a n o s o m a brucei bruceii n f e c t e d rats, the c u r a t i v e d o s e c a u s e d 4 4 % m o r tality d u e to a c u t e d r u g toxicity [10]. C l e a r l y t h e r e is a n e e d to d e v e l o p a less toxic a n d m o r e effective a l t e r n a t i v e to S H A M . T o w a r d s t h a t e n d we p r e v i o u s l y s c r e e n e d a w i d e variety o f h y d r o x a m i c acids a g a i n s t a cell-free extract o f T.b. brucei [12]. This assay was c h o s e n as

232 a primary screen in order to focus on the GPO itself, thereby obviating effects due to membrane transport and tissue distribution within the host. We demonstrated that activity against the GPO requires the hydroxamate moiety to be on an aromatic ring and that the activity of this nucleus can be enhanced by lipophilic substituents. Of particular interest, increasing the length of a p-nalkyloxy substituent on benzhydroxamic acid resuited in a more active compound. Together with the fact that hydroxamic acids are capable of undergoing oxidation [13], these results support the hypothesis that the inhibitory effect of the hydroxamates is due to their ability to displace reduced ubiquinone, the putative electron carrier, from the dehydrogenase to the terminal oxidase of the GPO [12] and of a similar system in plants [14]. Recently, additional and more direct evidence has been found for the role of ubiquinone in the GPO [15]. Materials and Methods

Chemicals. DL-Glycerol-3-phosphate, Tris, N-et-ptosyl-L-lysine chloromethyl ketone-HC1 (TLCK), catalase (2 x recrystallized, 30000 Sigma units (mg protein) -1) bovine serum albumin (BSA; Cohn fraction V), fetal bovine serum (FBS), D-a-tocopherol acetate, putrescine-2HC1, N-2hydroxyethylpiperazine-N'-2-ethanesulfonic acid (Hepes), glycerol, dimethylsulfoxide (DMSO), phenylmethylsulfonyl fluoride (PMSF), antipain2HC1, leupeptin, aprotinin, dithiothreitol, 13-mercaptoethanol and tetrasodium ethylenediaminetetraacetate (EDTA) were obtained from Sigma Chemical Co. (St. Louis, MO). DEAE-cellulose (DE-52) was obtained from Whatman, Inc. (Clifton, NJ). RPMI-1640 was from GIBCO (Grand Island, NY). Silicon carbide (No. 400 B, carborundum) was from the Norton Co. (Akron, OH) and was pretreated by washing three successive times alternately in 6 N HC1 and 6 N NaOH with six water washes between each step after which the abrasive was finally washed in reagent grade acetone and air dried. In general, the hydroxamic acids were synthesized from the corresponding methyl esters by the method of Scott and Wood [16] as modified by Gale et al. [17]. The p-n-alkyloxybenzhydrox-

amic acids were prepared according to Hase et al. [18]. The amides were prepared via acylation of methyl p-aminobenzoate and subsequent conversion to the corresponding hydroxamic acid via the general procedure referred to above. A number of compounds were obtained from other sources: 3,4-dihydroxybenzhydroxamic acid and 3,4,5-trihydroxybenzhydroxamic acid from B. van 't Riet (Virginia Commonwealth University, Richmond, VA); m-n-alkyloxybenzhydroxamic acids from J.C. Powers (Pace University, New York, NY); 1-hydroxy-2(1H)-quinolinone, 1,4dihydroxy-2(1H)-quinolinone and 3-bromo-l,4dihydroxy-2(1H)-quinolinone from R.T. Coutts (University of Alberta, Edmonton, Canada); and 2-hydroxy-lH-isoindol- 1,3(2H)-dione (N-hydroxyphthalimide), 1-hydroxy- 1H-benzimidazol-2(3H)one, 1,3-dihydroxy-lH-benzimidazol-2(3H)-one, 1hydroxy-3-methylquinazolin-2,4(1H,3H)-dione and dimethyl 1,2,3,4-tetrahydro-3-hydroxy-4-oxoquinazolin-3,3-diyldiacetate from J.F. Ryley (ICI, Pharmaceuticals Division, Macclesfield, U.K.). Coumatin, 4-hydroxycoumatin and 2-mercapto-4(3H)quinazolinone were obtained from the Aldrich Chemical Co. (Milwaukee, WI).

Buffers. PS buffer (100 mM sodium phosphate, 73 mM NaC1, pH 8.0) was as described by Lanham [19]. PSG is PS with 1.5% glucose added. The standard buffer used for cell homogenization or breaking buffer (BB) consisted of 100 mM Ttis, 50 mM KC1, 5 mM EDTA and 5 mM putrescine adjusted to pH 8.0. Assay buffer (AB) consisted of 20 mM glycerol-3-phosphate, 5 mM EDTA, 5 mM putrescine, 1% BSA, 73 mM NaC1 and 100 mM sodium phosphate, pH 8.0. Complete medium was RPMI-1640 containing 10% FBS and 10 mM Hepes (pH 7.4). Growth, and harvesting of parasites. Frozen stocks of T. b. brucei (EATRO 110) were prepared and used as described previously [8]. Blood was collected from female Sprague-Dawley rats (Taconic Farms, Germantown, NY) 3 days after inoculation with 106 trypanosomes and the parasites were isolated with a DE-52 column by the method of Lanham [19] and washed three times with PSG by centrifugation at 1000 × g for 15 min at 4°C.

233

Preparation of cell-free homogenates. The cells were washed three more times in PS and the final pellet added to an equal amount (v/w) of silicon carbide and then ground in a mortar at 4°C [20,21]. After dilution with a volume of cold BB equal to 10 times the original volume of the pellet, the carbide was removed by centrifugation at 150 x g for 10 min at 4°C. Unbroken cells and cell nuclei were removed by two additional centrifugations at 1000 x g for 30 min. The homogenates were dispensed into 1.2-ml aliquots and stored at in liquid nitrogen until needed. There was no significant loss of activity after storage for 6 months.

GPO activity assay. Activity (oxygen uptake) was monitored at 37°C in the chamber of a polarographic oxygen electrode (Yellow Springs Instrument Co., Yellow Springs, OH). The general procedures and precautions were as previously described [12]. AB, 3.0 ml, and a maximum of 50 Ixl of inhibitor solution were added to the chamber. The solvents used had no effect on activity. After temperature and oxygen equilibration, 100 Ixl of cell homogenate was added and the rate of oxygen consumption at 10 rain taken as the G P O activity. A consistent time point was important as there is always a decrease in the rate of oxygen consumption caused both by loss of enzyme activity due to degradation (seen with no inhibitor present) and by the approach to equilibrium of the enzyme-inhibitor complex. At 10 min the slope was linear in the case of most inhibitors.

all measurements were made at one substrate concentration. Some inhibitors did not yield a straight Dixon plot, thus a meaningful K i could not be obtained. Accordingly we used the concentration which inhibited 90% of the G P O activity (90%1) as an alternative measure of relative activity. Although based on only those points surrounding that at 90% inhibition, this value is relevant since pharmacologically useful inhibition is that which is virtually complete. In some cases the 90%1 was estimated by extrapolation of the curve from points at less than 90% inhibition. Plots of 90%I and Ki vs. carbon chain length (Fig. 1) were nearly identical.

Determination of inhibitor constants using whole cells. The procedures used with whole cells were similar to those described above with cell-free homogenate. Whole trypanosomes isolated from infected blood replaced the homogenate and complete medium replaced AB. Approximately 2 x 107 cells were added to the chamber of the oxygen electrode for each assay.

Determination of the osmotic fragility of erythrocytes in the presence of inhibitor. Osmotic fragility of rat erythrocytes was determined by incubation in 10 mM Tris containing 1% glucose and 1.0, 0.8, 0.6, 0.5, 0.4, 0.3, 0.2 or 0.0% NaC1. One series contained no inhibitor, a second 100 ixM pn-dodecyloxybenzhydroxamic acid, and a third

25~-

t 250

Calculation of apparent inhibitor constants (Ki). G P O activity in the presence of inhibitor was expressed as a percentage of the uninhibited rate. To insure the validity of this comparative method, all measurements in a given experiment were done with the same cell homogenate. At the beginning, at 2-h intervals and at the end of each set of assays, the uninhibited rate was measured to detect and correct for any change in cell homogenate activity. Apparent K i was determined by Dixon plots [22] using linear regression analysis. These are apparent values because inhibition by SHAM, the prototype inhibitor, is non-competitive [12] and

150

::L

F ~00

o

C

5o

0

2

4

6 Alkyq

8 Chain

I0

12

14

|6

f0 t8

Length

Fig. 1. K~ and 90%• of p-n-alkyloxybenzhydroxamic acids as functions of the length of the alkyl side chain. (A) mean Ki; (m) mean 90%1.

234 10 ~M p-n-dodecyloxybenzhydroxamic acid. Ethanol (final concentration 10%) was used to dissolve the inhibitor and was added to the controis. After incubation for 1 h at room temperature the suspensions were centrifuged at 1800 × g for 10 min to sediment unbroken cells. Lysis was quantitated by measuring the hemoglobin released (A415). The absorbance after incubation in 0.0% NaC1 was defined as 100% lysis and that after incubation in 1.0% NaC1 as 0% lysis.

Determination of the reversibility of inhibition in vitro. Inhibitors were removed from a suspension of trypanosomes by tenfold dilution with cold (0°C) inhibitor-free medium followed by centrifugation at 1900 × g for 15 min (4°C); and resuspension in PSG containing 1% BSA. A second set of samples was treated the same but the inhibitors were present throughout. Control cells never exposed to either inhibitor (but centrifuged and resuspended so as to control for cell loss due to handling) were defined as 0% inhibited; lack of oxygen consumption was defined as 100% inhibition.

Killing of trypanosomes in vitro. Incubations were made in a 24-well tissue culture plate with each well containing 1-5 × 107 purified trypanosomes in 1 ml of 10 mM glycerol, 120 mM NaC1, 1 mM CaC12, 1% glucose and 25 mM Hepes at pH 8.0. GPO inhibitors were added over a range extending from roughly the Ki to 100 times this value. Appropriate controls were included to account for the effects of glycerol and the inhibitor alone as well as for the solvents used to dissolve the inhibitors. Killing was determined with the aid of an inverted microscope. Thin wet mount preparations were made periodically for more detailed examination. Results

Optimization of the GPO assay. Measurement of the GPO is complicated by time- and temperature-dependent loss of activity upon thawing the homogenate. While we compensate for this by relating rates to the base rates measured during the course of the day and choosing a standard time point for determining activity after addition of the

cell-free homogenate, increased activity and improved stability would be very helpful. Accordingly, we tested the following additives. E D T A (5 mM) was evaluated since we showed previously that autolysis of the parasites after exposure to GPO inhibitors is faster in the presence of calcium [23]. The effects of dithiothreitol and [3mercaptoethanol (1 mM) were investigated since it has been suggested that thiols are important components of the GPO [24] and maintaining them in a reduced state may be advantageous. Putrescine (0.0, 0.5 and 5.0 mM) was tested because it had been observed to enhance the activity of the GPO in both intact cells and cell-free homogenates (B.F. Giffin et al., personal communication). Two types of protease inhibitors were evaluated: (1) TLCK (1 mM) and (2) a cocktail of protease inhibitors - PMSF (1 mM), leupeptin (25 ~zg ml-1), antipain (25 ~g ml 1) and aprotinin (1.7 U ml-l). Tocopherol (1 p~M) was tested since it is an excellent scavenger of free radicals and there have been reports that hydrogen peroxide, a source of free radicals, is produced by the GPO [25]. For the same reason catalase (1250 U ml l) was tested. In addition various concentrations of BSA (1-5%) were evaluated and the optimum pH was determined. In that E D T A (5 mM) and putrescine (5 mM) produced slightly higher mean specific activities, these were included in the standard GPO assay even though the observed differences were not significant (p > 0.05). BSA concentrations in excess of 1% were of no added benefit. The other additives tested were without effect. A broad peak of GPO activity occurred from pH 7.9 to 8.2; a standard of 8.0 was chosen for all homogenization and assay processes. The method of homogenization was important. The mean specific activity of cell homogenates prepared with a French pressure cell [12] was 67.6 nmol 0 2 consumed min -1 (mg protein) -1 while that of cell homogenates prepared by grinding was 105.6, the difference being highly significant (p < 0.006). As one variation we ground trypanosomes in BB rather than PS. Another variation was to substitute 0.25 M sucrose, 10 mM KCI, 10 mM Tris, 5 mM EDTA, 5 mM putrescine (pH 8.0) for grinding and suspension of the broken cells. The standard grinding method

235 TABLE I Inhibition of the GPO by various alkyloxybenzhydroxamic acids and related compounds Compound

*p-Methoxybenzhydroxamic acid a p-Ethoxybenzhydroxamic acid p-n-Propyloxybenzhydroxamic acid *p-n-Butyloxybenzhydroxamic acid p-n-Pentyloxybenzhydroxamic acid *p-n-Hexyloxybenzhydroxamic acid p-n-Heptyloxybenzhydroxamic acid p-n-Octyloxybenzhydroxamic acid p-n-Decyloxybenzhydroxamic acid p-n-Dodecyloxybenzhydroxamic acid p-n-Tetradecyloxybenzhydroxamic acid p-n-Hexadecyloxybenzhydroxamic acid p-Benzyloxybenzhydroxamic acid m-n-Butyloxybenzhydroxamic acid m-n-Octyloxybenzhydroxamic acid m-n-Dodecyloxybenzhydroxamic acid

Solvent a

A,C A,C A,C A,C A,C A,C B,C B,C B,C B,C B B A A A A m-n-Octanoylaminobenzhydroxamic acid A p-n-Octanoylaminobenzhydroxamic acid B p-n-Dodecanoylaminobenzhydroxamic acid C

nb

K i txMc

(range)

90%• IxM

(range)

4 2 2 7 2 5 2 3 2 3 3 2 1 3 3 2 2 1 2

18.6 13.9 8.3 13.9 19.3 9.4 5.8 7.0 3.6 1.1 0.43 1.0 e 71.0 18.2 9.4 50.0 e 103.0

(14.0-26.0) (13.0-14.8) (6.0-10.5) (4.0-20.0) (16.0-22.6) (4.0-12.5) (5.5-6.1) (4.8-8.8) (3.5-3.6) (0.7-1.1) (0.34-0.57) (0.9-1.1)

191 120 70 136 201 77 55 59 36 10 2.8 19 200 640 163 79 483 >1000 910

(150-250) (112-120) (45-96) (56-188) (198-203) (42-105) (53-57) (42-68) (35-37) (2-12) (2.7-2.8) (17-21)

(66.6-77.0) (15.5-19.9) (8.9-9.9) (48.8-51.2) (73-133)

(617-654) (142-183) (67-91) (420-543) (660-1159)

a The following solvents were used to dissolve the inhibitors: (A), 50% ethanol; (B), 95% ethanol; (C), DMSO. Where two solvents are indicated, both were used on different occasions. None of the solvents inhibited the GPO. b This is the number of independent times the compound was assayed.

c The values are apparent K~ values as explained in Methods. d The compounds marked with an asterisk were reported earlier [12] and were included in this study for comparative purposes. Because we modified our assay, the K~ values may be slightly different from those previously reported. e A meaningful K~ could not be determined due to the curvature of the Dixon plot.

was slightly superior to these and was more convenient. While inclusion of putrescine in the BB did not produce a statistically significant enhancement of specific activity, it did nearly double the total yield of activity. This was apparently due to the production of a more compact pellet of nuclei and unbroken cells after centrifugation from BB when putrescine was present. Consequently, 5 mM putrescine was added to BB in the standard procedure. The same protease inhibition schemes described for the AB were tested in the BB and were without effect on specific activity or loss of activity upon storage on ice and were therefore not included in the BB.

Structure-activity of GPO inhibitors. Increasing the length of the alkyl chain of p-n-alkyloxybenzhydroxamates and related compounds resulted in an increase in activity (Table I) confirming our earlier preliminary results [12]. n-Dodecyl, p-ntetradecyl and n-hexadecyl derivatives are at least 30 times more active than SHAM with the n-te-

tradecyl derivative being 70 times more active. The effect of increasing the length of the p_n_ alkyloxy substituent was not linear (Fig. 1). A peak of activity, a minimum Ki, was reached at three carbon atoms, the n-butyl and n-pentyl derivatives being less active. Increasing the length of the carbon chain beyond five again led to a decrease in the Ki to a floor beginning at a chain length of twelve. Our previous work indicated that a lipophilic substituent (especially a halogen) in the meta position on the benzhydroxamate nucleus resulted in a somewhat lower K i than one in the para position. Therefore a series of m-n-alkyloxybenzhydroxamates (n-butyl, n-octyl and n-dodecyl) was synthesized for comparison with the corresponding para-substituted compounds. All proved to be less inhibitory than the para analogues with the difference increasing with increasing carbon chain length (Table I). An undesirable property of the long chain p-nalkyloxybenzhydroxamates is limited aqueous

236 TABLE II Inhibition of the GPO by various primary aromatic hydroxamic acids Compound (A) Monosubstituted *Benzhydroxamic acid a *Salicylhydroxamic acid (SHAM) o-Fluorobenzhydroxamic acid p-Hydroxybenzhydroxamic acid N,N-Dimethyl-p-aminobenzhydroxamic acid p-Benzylbenzhydroxamic acid (B) Disubstituted *2,3-Dihydroxybenzhydroxamic acid *2,5-Dihydroxybenzhydroxamic acid 3,4-Dihydroxybenzhydroxamic acid 5-Chloro-2-hydroxybenzhydroxamic acid *5-Bromo-2-hydroxybenzhydroxamic acid 3-Methyl-2-hydroxybenzhydroxamic acid 5-Methyl-2-hydroxybenzhydroxamic acid 3,5-Dibromobenzhydroxamic acid 2-Naphthohydroxamic acid Nicotinobenzhydroxamic acid

Solvent a

nb

K, ~M c

A,C A A C A

3 2 1 3 3

147 29 300 1043 13.2

A

1

e

450

C C C

1

15 40 700

C B B B C C

(C) Trisubstituted 3,4,5-Trihydroxybenzhydroxamic acid B 3,5-Diisopropyl-2-hydroxybenzhydroxamic A acid 3-Hydroxy-2-naphthohydroxamic acid B,C

(range) (145-150) (28--30) (800-1170) (10.7-16.5)

90%• IxM

(range)

900 282 >1000 >2000 95

(800-1000) (275-290)

1 2 1

600

125 315 >2000 399 250 240 520 >1000 175 1000

1 1

132 e

900 300

3

c

70

1

1 1 1 3 1

60 40 77

(20-65)

e

(80-105)

(170-300)

(110-240)

(50-130)

See Table I footnotes. TABLE III Inhibition of the GPO by various secondary heterocyclic hydroxamic acids and related compounds Compound

Solvent a

2-Hydroxy-lH-isoindol-l,3(2H)-dione (N- B hydroxyphthalimide) 1-Hydroxy-lH-benzimidazol-2(3H)-one C 1,3-Dihydroxy- 1H-benzimidazol-2(3H)-one C *1-Hydroxy-2(1H)-quinolinone d C *1,4-Dihydroxy-2(1H)-quinolinone C *3-Bromo-1,4-dihydroxy-2(1 H)-quinolinone C 2-Mercapto-4(3H)-quinazolinone C 1-Hydroxy-3-methylquinazolinC 2,4(1H,3H)-dione Dimethyl 1,2,3,4-tetrahydro-3-hydroxyC 4-oxoquinazolin-3,3-diyldiacetate Coumarin C 4-Hydroxycoumarin C

nb

K i p~Mc

(range)

1 1 1 4 2 2 1 1

90%• ixM

(range)

>2000

1650 172 27 83 1500

1

>2000

1 1

>2000 2000

(145-210) (22-32) (75-90)

>2000 >2000 1100 715 805 >2000 >2000

(1000-1300) (680-750) (780-830)

See Table I footnotes. s o l u b i l i t y . T h i s is r e f l e c t e d by t h e fact t h a t inc r e a s i n g t h e c o n c e n t r a t i o n o f t h e 12, 14 a n d 16 carbon derivatives allowed a maximum inhibition o f a b o u t 8 3 % a l t h o u g h t h e D i x o n p l o t s w e r e lin-

e a r u p to this p o i n t ( d a t a n o t s h o w n ) . U n d o u b t edly, lack of solubility was a contributing factor in l i m i t i n g t h e d e c r e a s e in Ki. S i n c e l o w s o l u b i l i t y w o u l d also b e e x p e c t e d to l i m i t in v i v o a v a i l a b i l -

237 ity and thus effectiveness as a drug, we sought to modify this parameter. Accordingly, we synthesized three acylaminobenzhydroxamic acids, the amide linkage being utilized because of its tendency to increase aqueous solubility in other situations and because it offers more possibilities than the ether for further derivatization of the side chain. These compounds proved to be significantly less active than the corresponding ethers (Table I) while their solubility was not significantly improved (data not shown). In addition to the p-n-alkyloxybenzhydroxamic acids, we evaluated a variety of other primary aromatic hydroxamates (Table II). Among these, two further leads were uncovered. The Ki of pdimethylaminobenzhydroxamic acid was 13.2 IxM, nearly half that of p-methoxybenzhydroxamate. One might expect extension of the alkyl chain(s) to lead to increased inhibitory activity as in the case of the alkyl ethers reported above while the amino nitrogen should enhance water solubility. The other lead stems from the two naphthohydroxamic acids studied. 2-Naphtho- and 3-hydroxy-2-naphthohydroxamic acids had 90%• values of 175 and 70 ixM, respectively, significantly lower than that of SHAM (282 ixM). These compounds bear a close resemblance to the aromatic nucleus of ubiquinone. They are also structurally similar to the 2(1H)-quinolinones which we previously showed to be good inhibitors [12]. That the closely related quinazolinones, benzimidazolones and isoindolones studied were inactive (Table III) indicates that aromaticity and a lipophilic substituent are not the only criteria required in order to achieve effective inhibition of the GPO. A suitable redox potential and the ability to form charge transfer complexes also seem to be very important. Thus, cinnamohydroxamic acid was inactive despite conjugation of the hydroxamate moiety with the aromatic ring while p-dimethylaminobenzhydroxamic acid was nearly twice as active as the corresponding methoxy derivative. None of the other benzhydroxamic acids examined showed significant promise. These include the 3-methyl, 5-methyl, 5-chloro and 5bromo derivatives of SHAM. These results were unexpected given the enhanced activity of mchlorobenzhydroxamic acid relative to that of the parent compound, benzhydroxamic acid. p-Hy-

droxybenzhydroxamic acid also behaved anomalously. While more water soluble than the related n-alkyloxy compounds, it was 30 times less active than SHAM and 50 times less active than the pmethoxy derivative.

Potential of p-n-dodecyloxybenzhydroxamic acid as a trypanocidal drug. Since this compound was 30 times more active than SHAM and somewhat more soluble than the n-tetradecyl and n-hexadecyl analogues, we further evaluated its potential. We measured the Ki using whole cells instead of cell-free homogenate to determine if the cell membrane would act as a barrier to permeability. The whole cell K i was 0.6 p~M and, by extrapolation, the 90%• was 5.8 ~M. These values are approximately half those observed with a cellfree homogenate, thus the activity of this compound appears to be enhanced rather than diminished in the presence of the outer membrane of the trypanosome. Next we considered that p-n-alkyloxybenzhydroxamic acids might inhibit the GPO by interfering with the inner mitochondrial membrane in a nonspecific, detergent-like manner. An aromatic nucleus with a hydrophilic group on one side and a long alkyl chain on the other fits the general structure of several well-known membrane-active, nonionic detergents which readily inactivate this oxidase complex ([15], A. Fairlamb, Ph.D. Thesis, University of Edinburgh, 1973). We used osmotic fragility of rat erythrocytes to detect detergent activity. The plots of percent lysis versus NaCI concentration were identical (data not shown) with a sharp increase in lysis between 0.5 and 0.4% NaC1. Since the concentrations of inhibitor tested were well above those which blocked the GPO, the inhibitor is not acting as a detergent. The linear Dixon plots achieved with the p-n-alkyloxy compounds provide additional evidence that they are not acting as detergents since a detergent would be expected to have a threshold of activity rather than the smooth increase in inhibition which we observed. We then determined whether inhibition of the GPO was reversible as would be expected if it were competing with reduced ubiquinone, the putative natural substrate. Addition of a DMSO

238 solution of this compound to PSG in an amount sufficient to yield a 20 IxM solution resulted in a cloudy mixture which was undoubtedly saturated with respect to the inhibitor. The respiratory rate of trypanosomes suspended in this mixture was reduced by 83% compared to that of parasites suspended in PSG. After incubation at 37°C for 10 min, washing out the inhibitor and compensation for loss of cells due to the manipulations involved, no residual inhibition of oxygen consumption was observed. For comparison, 100 ~xM SHAM inhibited oxygen consumption by 71% with 1% inhibition being observed following washing. Thus, neither S H A M nor p-n-dodecyloxybenzhydroxamic acid appears to act irreversibly v i s a vis the GPO. We next evaluated the ability of p-n-dodecyloxybenzhydroxamic acid to kill parasites in vitro when combined with glycerol. The minimum concentration of S H A M that killed 100% of the parasites (complete lysis by microscopic examination after incubation for 1 h in the presence of 10 mM glycerol) was 1000 ~M. The equivalent pn-dodecyloxybenzhydroxamic acid concentration was between 50 and 100 txM or 10-20 times less. This value is in reasonably close agreement with the 30-fold lower K i. This is particularly interesting as one might have expected the dodecyl derivative to have little trypanocidal activity when combined with glycerol due to its inability to completely inhibit the G P O because of limited solubility. Since p-n-dodecyloxybenzhydroxamic acid combined with glycerol was considerably more active than S H A M in killing trypanosomes in vitro, we tested the combination in vivo. Four mice with parasitemias of approximately 3 × 108 organisms ml i were treated, p-n-Dodecyloxybenzhydroxamic acid was dissolved in DMSO and delivered intraperitoneally in a total volume of 0.1 ml. Glycerol was administered per os as a 30% solution in water. The mice were treated according to the following protocol: at t=0 min, 5 g glycerol kg-1; at t=60 and 120 min, 2.5 g glycerol kg-1; at t=0 min, 800 mg kg -1 p-n-dodecyloxybenzhydroxamic acid. There was no effect on the parasitemia. The mice were sacrificed and the pn-dodecyloxybenzhydroxamic acid was found to have precipitated in the peritoneum, thus the

limited solubility of this compound prevented any in vivo activity. In retrospect, this is not surprising since, as noted earlier, the maximum in vitro inhibition with a saturated solution was only 83%. The above protocol was repeated with p-ntetradecyloxybenzhydroxamic acid with exactly the same result. Discussion

The data support our previous suggestion [12,26] that the action of aromatic hydroxamates as G P O inhibitors is best explained by the hypothesis that they interfere with ubiquinone/ubiquinol-mediated electron transport, more specifically that they bind to the ubiquinol receptor on the oxidase component of the GPO. This hypothesis is consistent with the reversibility of action we report here and the uncompetitive nature of the inhibition by S H A M which we observed when glycerol-3-phosphate was the independently controlled substrate and the velocity of the GPO was measured by the rate of oxygen consumption [12]. It is also consistent with the structure/activity analysis discussed below. Fig. 2 is a theoretical representation of the binding site of ubiquinone/ubiquinol on the ter-

1 o

0

\

N O'H

~

ORIENVAVIOS

~'~o ~

Fig. 2. Possible orientations of p-n-propyloxybenzhydroxamic acid in the receptor site for ubiquinol. Ubiquinol is shown in the upper drawing, with p-n-propyloxybenzhydroxamicin the lower two drawings.

239

minal oxidase which can account for the observed relative activities of the various p-n-alkyloxybenzhydroxamic acids. The short chain analogues could assume either orientation 'A' or 'B'. 'A' would be due to the attraction of the short alkyloxy chain to the site where the methoxy groups of ubiquinol bind. As the length of the alkyloxy substituent is increased, steric hindrance would lessen the tendency to bind in this way and instead favor orientation 'B' in which the alkyloxy group binds at the site normally occupied by the long lipophilic isoprenoid chain of ubiquinol. Flexibility within the active site would permit a lateral shift of the aromatic nucleus so as to assume a minimum energy conformation. For compounds in orientation 'B', increasing the alkyl chain length would better mimic the isoprenoid side chain, thus increasing activity of the inhibitor as we observed. However, this increase would be limited by the decreasing aqueous solubility associated with longer chains. We ascribe the high activity of the naphthohydroxamic acids (K i values of 13 and 5 txM) to their resemblance to the aromatic nucleus of ubiquinone. These naphthohydroxamic acids could assume either orientation within the active site of the oxidase because of their small size and planarity.

For p-n-dodecyloxybenzhydroxamic acid (30 times more active than SHAM) the parasite cell membrane is not a barrier, the mechanism of inhibition is not by detergent action, the inhibition is reversible and, when combined with glycerol, it is 10-20 times more active than SHAM in killing trypanosomes in vitro. However, activity in vivo is limited by poor aqueous solubility. In summary, we have demonstrated that a vast improvement in the activity of GPO inhibitors is possible by a rational modification of the basic structure. The results have given us better insight into the mode of action of GPO inhibition and have confirmed the utility of screening compounds against the GPO enzyme system as a means of identifying compounds with therapeutic potential. Improvement of in vivo activity awaits the development of compounds with improved bioavailability, p-Dimethylaminobenzhydroxamic acid offers one approach as does incorporation of solubilizing groups on the alkyl chain of the p-nalkyloxybenzhydroxamic acids.

Acknowledgement This work was supported by NIH grant AI17899.

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