Chem. Res. Toxicol. 1994, 7, 815-822
816
Structure/ActivityRelationships Affecting the Ability of Monoanionic 3-Hydroxypyrid-4-onesto Mobilize Iron John J. Molenda, Mark M. Jones,* Kim M. Cecil, and Mark A. Basinger Department of Chemistry and Center in Molecular Toxicology, Vanderbilt University, Nashville, Tennessee 37235 Received December 7, 1993@
Current attempts to remove iron from individuals suffering from iron overload have encountered difficulty due to the toxicity of the administered chelating agent. In a search for iron chelators of potentially reduced toxicity, nine monoanionic compounds have been examined. To determine t h e chemical features which govern their ability to induce the excretion of iron, the compounds were administered to female Sprague-Dawley rats. All carboxylate derivatives were tested for biliary excretion following iv injection, as well as for urinary excretion following iv or PO injection. Sulfonate derivatives were tested for biliary and urinary excretion as well, but only one representative compound was tested PO. The biological activity of the new pyridinones was compared to t h a t of 1,2-dimethyl-3-hydroxypyrid-4-one,L1, which served as t h e standard. While none of the chelators was able to surpass L1 in both urinary and biliary iron excretion, all of the chelators at least equaled L1 in one of these two areas following iv administration. Two derivatives surpassed the standard in mobilizing iron into the bile, and all others were statistically equivalent. In terms of urinary excretion, two compounds were equivalent to L1 after iv administration, although none of the compounds equaled L1 when administered orally. The structure of (1,4-dihydro-3-hydroxy-2-methyl-4-oxo-l-pyridyl)methanecarboxylic acid was determined by X-ray diffraction, as this compound showed higher activity t h a n previously reported by other investigators. We speculate that these chelators utilize organ-specific, monoanionic transport systems in t h e liver and kidneys to mobilize iron and t h a t their toxicity may be substantially less than t h a t of their neutral analogs.
Introduction Although iron is an essential metal to almost all living organisms ( l ) ,its serious toxic effects when present in excess have prompted research into the mechanism of its toxicology. The toxicity of iron has been attributed largely to its participation in the formation and degradation of lipid hydroperoxides (2). Lipid peroxidation involves the insertion of 0 2 into lipids to form the lipid hydroperoxide, a spin forbidden reaction which must be initiated by the formation of hydroxyl radicals (HO') (3). Hydroxyl radicals are one of the most potent oxidizers of all oxygen species (4), and they are capable of reacting with nearly all known biological molecules, such as sugars and phospholipids (51, and serious damage has been observed extensively in DNA (6-8). These radicals are supplied directly by the Fenton reaction and by the breakdown of HzOz generated by the Haber-Weiss reaction, both of which require the presence of Fe3+that is not bound to ferritin or transferrin (5). It has also been suggested that Fez+ may have the ability to autoxidize in the presence of H+ and 0 2 , leading to further production of peroxides (3). In addition, iron can readily hasten the decomposition of preexisting lipid hydroperoxides into peroxy and alkoxy radicals (9). Unsaturated lipids within cell membranes are especially susceptible to peroxidation due to the weakening of the H bonds on carbons adjacent to double bonds (10). Niehaus has proposed that damage to cell membrane constituents also occurs by yet another, nonoxidative
* Address correspondence to this author at P.O. Box 1583, Station B, Vanderbilt University, Nashville, TN 37235;Phone: (615)-322-2987; FAX: (615)-322-4936. Abstract published in Advance ACS Abstracts, October 15, 1994.
mechanism, whereby Fez+autoxidizes to form the superoxide radical ( 0 2 ' - ) , which then acts as a nucleophile to deesterify fatty acid esters and phospholipids (11). This theory is substantiated by the work of Frimer, who has shown that 0 2 ' - is an extremely potent nucleophile in nonpolar environments and, thus, preferentially resides in cell membranes (10). Indeed, a large amount of 0 2 ' is manufactured by systems within cell membranes such as in the heart ( 5 ) and possibly within membranes themselves (13). Ultimately, these pathological reactions result in loss of cell membrane tension, a decreased ability to control the permeability of the membrane to ions, and the subsequent rupture of the membrane (11);it is therefore not surprising that quite a number of diverse disorders can be directly linked to iron intoxication. The accumulation of iron has been linked to the treatment of thalassemia, due to the frequent blood transfusions needed to replace faulty hemoglobin (12,131. Excess iron has also been implicated in the neurological disorder Parkinson's disease, where high concentrations of iron have been found in the basal ganglion (14-16). In addition, iron has been suspected as a contributor to lung cancer and emphysema in smokers (171, and several studies have shown a significant link between iron and various heart disorders such as ischemia-reperfusion injury (18, 19). While several iron chelators have displayed considerable potential as treatments for these disorders, reports of the toxicity of the chelators currently used have caused serious concern. Desferrioxamine (DFO), the premier iron chelator now in clinical use, has been reported to cause a wide range of toxic side effects, including potentially fatal pulmonary (20) and renal (21, 22)
0893-228x/94/2707-0815$04.50/00 1994 American Chemical Society
Molenda et al.
816 Chem. Res. Toxicol., Vol. 7, No. 6, 1994
toxicity, and the extensive work of Olivieri and other investigators have shown evidence of visual (23-271, auditory (23,28-301, and neurological toxicity (23). The ability of DFO to act as a n iron donor to infectious organisms such as Yersinia enterocolitica has also been observed (31). While compounds such as pyridoxal isonicotinoyl hydrazone (PINH) (32-34) and synthetic siderophores (35)have been investigated as replacements for DFO, the development of the 3-hydroxypyrid-4-ones as orally active iron mobilizing agents has been the most promising recent advance in this field (36-39). However, as with DFO, recent reports attest to the toxicity of the most extensively used of these compounds, 1,a-dimethyl3-hydroxypyrid-4-one (Ll) (40-421, for such reasons as the production of antinuclear antibodies (41,43)and bone marrow suppression (44). Still other 3-hydroxypyrid-4ones have been shown to inhibit the enzyme tyrosine hydroxylase (45). Concerns over toxicity have resulted in the decision to stop the clinical development of L1, a n action of some controversy (46,471. The toxicity of both the 3-hydroxypyrid-4-ones and DFO has indicated that the current approach to iron chelator design must be reevaluated. While numerous factors contribute to the ability of a compound to target specific organs and pass through membranes, the design parameters for iron chelators are known in part (41,42). Current approaches to oral iron chelator design have been unable to reconcile increasing the rate of chelator transport to cellular sites of storage without sacrificing organ specificity. Conventional design has focused too heavily on lipophilicity (48), stability constant (49-51), and neutrality of charge (42, 49, 50, 52-55). While neutral pyridinones are able to access iron quickly and may be administered orally, their lipophilicity enables them to penetrate numerous membranes where they exert their undesired toxic effects. In a recent work (56),we have focused on enhancing the organ specificity of the 3-hydroxypyrid-4-one iron chelators by the incorporation of a single negative charge to exploit monoanionic transport systems in the liver and the kidneys (57-60). We have obtained evidence that monoanionic 3-hydroxypyrid-4-ones can effectively mobilize iron deposits, and we proposed that incorporation of a single negative charge into the pyridinone structure could enable the monoanionic molecule to readily a n d selectively access intracellular iron (56). We hypothesize that these pyridinones may also be less toxic as their penetration of membranes would be restricted t o those organs which contain suitable anion transport systems. They may also function as superior iron chelators to both DFO, which is generally considered to be restricted to extracellular spaces (40, 61),and L1, whose lipophilicity enables it to readily penetrate membranes but not selectively (40). As a continuation of our work with this class of compounds, we have compared the results obtained from nine monoanionic pyridinone derivatives in order to determine the effect of different negatively charged functional groups, lipophilic character, molecular weight, and structure on their ability to enhance the excretion of iron in the unloaded rat.
Materials and Methods Chemicals. Maltol (1)and ethyl maltol (Veltol Plus) (6)were obtained from the Specialty Chemical Group of Pfker Inc. (Doraville, GA), and kojic acid (8) (99+%) was obtained from Tokyo Kasei Chemical Co. (Portland, OR). The amines used i n
Scheme 1 0
2 t a
HOOC
e5 L O , H A
b. c
h O , "
6
b, f
HO&
I HO
b O , H 9
11
Hofl
HO
"A 13 a
o l / 12
(a)CH3NH2, HzO, 100 "C; (b) NHz(CH2)2SOsH,HzO, NaHC03,
100 "C; (c) dilute HC1; (d) glycine, HzO, N a C 0 3 , 100 "C; (e) 4-(aminomethyl)benzoic acid, HzO, NaHC03, 100 "C; (fl concentrated HC1; (g) 0 2 , PcUC 5%, NaOH, HzO, 10 "C; (h)CH3CH2NH2, 3:l H20/EtOH, 100 "C; (i) ( C H ~ ~ C H N HHzO, Z , 100 "C; 0') BzNH2, 3:l H20/EtOH, 100 "C.
the preparations were purchased from the Aldrich Chemical Co. (Milwaukee, WI). All reagents were used without further purification.
Animals. Animal studies were performed with female Sprague-Dawley rats (180-240 g) obtained from Sasco (Omaha, NB). Between collection periods, the rats were provided with food and water ad libitum,while during urinary or biliary collection periods, the rats were only provided with t a p water in their metabolic cages. The animals were kept in an AAALACapproved facility. Instrumentation. NMFt experiments were run on Bruker 300 and 400 MHz spectrometers, and combustion analyses were performed on a Carlo Erba Strumentazione elemental analyzer Model 1106, at Vanderbilt University or by Atlantic Microlabs, Norcross, GA. All mass spectra were obtained from the Department of Pharmacology, Vanderbilt University. Preparation of Monoanionic 3-Hydroxypyrid-4-ones. The preparation of the monoanionic 3-hydroxypyrid-4-ones shown in Scheme 1 followed t h e same general procedure first published by Kleipool and Wibaut (62) and later used by Kontoghiorghes (63) as a more facile route than that used previously (64). All reactions were performed under N2 in either a strictly aqueous medium or one containing EtOH as a minor component in order to preclude the possibility of obtaining Schiff
Iron Mobilization by 3-Hydroxypyrid-4-ones
Chem.Res. Toxicol.,Vol. 7,No. 6,1994 817
bases and to facilitate the Michael reaction. Despite this precaution, high yields were difficult to obtain due to decomposition products; moderate to low yields are not uncommon for these types of reactions (56, 63, 65, 66). All reaction mixtures were slowly acidified with 3:l HZO/HCl in a n ice bath until maximum yield was obtained. Careful acidification resulted in facile purification; the majority of the compounds were found t o be analytically pure after repeated washings with polar solvents such as HzO, MeOH, and acetone, while others were purified via recrystallization from the appropriate solvent. The extremely hygroscopic nature of some derivatives necessitated extensive drying a t elevated temperature to remove fractional water of hydration, and in the cases of 5 and 13,mass spectra were obtained to further establish that the target molecules were indeed obtained. The yields and characterization of the pyridinones is provided below; more detailed preparations may be obtained from the supplementary material, with the exception of compounds 2-4 and 9-11 which have been prepared previously (34, 56, 67-69).
suture. The second incision was closed, and the tubing was then passed through a dorsal access point close to the shoulder by insertion of a needle large enough to allow the tubing to go through. The first incision made was sutured closed, and a Velcro backpack was placed around the animal to hold the 10 x 75 polystyrene collecting tube. Biliary Excretion. All pyridinones were tested for biliary iron excretion based on the model used by Pippard e t al. (70) and Bergeron et al. (55). The rats were cannulated as described, and their bile was collected for a control period of 30 min. The animals were reanaesthetized after the control collecting period in order to give the iv injection. Injections were given via tail veins, and if necessary, the tail was passed under warm water to make the veins more visible. Bile was then collected for a 2-h period, after which time the animals were sacrificed. Urinary Excretion. All compounds were tested for the urinary excretion of iron following iv injection. All carboxylates were tested for urinary excretion PO, but since sulfonates are not readily absorbed into the gastrointestinal tract, only one (A)2-(1,4-Dihydro-3-hydroxy-2-methyl-4-oxo-l-pyridyl)-representative sulfonate was examined. The rats served as their ethane-l-sulfonicAcid (3). Yield 26.9% (white solid); mp own controls, with the controls receiving 1mL of Millipore HzO 2300 "C; 'H NMR (DzO) 6 8.1 (d, l H ) , 7.1 (d, lH), 4.74 (t, 2H), PO or iv, and the following day, each r a t was administered the 3.46 (t, 2H), 2.62 (s, 3H). Anal. Calcd for C S H I ~ N O ~ S - H ~C, O: appropriate amount of chelate solution. Urine was collected for 38.24; H, 5.22; N, 5.58. Found: C, 38.22; H, 5.24; N, 5.43. both control and treated excretion for 15 h in metabolic cages (B) (1,4-Dihydro-3-hydroxy-2-methyl-4-oxo-l-pyridyl~which separated the urine from the feces. Collection was methanecarboxylic Acid (4). Yield 43.2% (white solid); mp performed overnight due to the nocturnal nature of the animals, 271-272 "C; 'H NMR (D2O) 6 7.58 (d, lH), 6.5 (d, l H ) , 4.64 (5, with administration performed a t -6 PM. 2H), 2.3 (9, 3H). Anal. Calcd for CsHgN04: C, 51.97; H, 4.98; Iron Analysis. Both bile and urine analyses were performed N, 7.57. Found: C, 51.91; H, 4.95; N, 7.24. directly or with dilution on a Perkin-Elmer 4000 atomic absorp(C)1-(4-Carboxybenzyl)-1,4-dihydro-3-hydroxy-2-meth-tion spectrometer with a Perkin-Elmer 400 graphite furnace. yl-4-oxopyridine(5). Yield 32.8% (white solid); mp '300; 'H The analyzer was run using standard conditions and deuterium NMR (DzO) 6 7.77 (d, 3H), 7.10 (d, 2H), 6.55 (d, lH), 5.3 (s,2H), background correction. 2.23 (s, 3H); SLSIMS calcd for 260.09228 (MH+),found 260.09265 X-rayCrystallography. All measurements were performed (A = 0.3 mmu). Anal. Calcd for C14H13N04: C, 63.78; H, 5.12; on a Rigaku AFC6S diffractometer a t Vanderbilt University N, 5.31. Found: C, 64.64; H, 5.11; N, 5.55. with graphite monochromated Cu Ka radiation. The structure (D)2-(1,4-Dihydro-2-ethyl-3-hydroxy-4-oxo-l-pyridyl)-of 4 was solved by direct methods using SHELXS-86. All nonethane-l-sulfonicAcid (7). Yield 15.9% (white solid); mp hydrogen atoms were refined anisotropically, and all hydrogens > 300; 'H NMR (D2O) 6 8.1 (d, l H ) , 7.1 (d, lH), 4.7 (t, 2H), 3.45 were placed in positions of idealized geometry. (t, 2H), 3.05 (9, 2H), 1.25 (t, 3H). Anal. Calcd for CgH13N05S: C, 43.72; H, 5.30; N, 5.67. Found: C, 43.58; H, 5.47; N, 5.54.
Results and Discussion
(E)1,4-Dihydro-l-ethyl-3-hydroxy-4-oxopyridne-6-carboxylic Acid (12). Yield 8% (golden brown solid); mp 224(A) Design. After examining two model monoanionic 225 "C [lit. (62) mp 168.5-170 "Cl; 'H NMR (DzO) 6 7.60 (s, pyridinones which both displayed activity (one sulfonate lH), 6.54 (s, lH), 4.12 (9, 2H), 1.38 (t, 3H). Anal. Calcd for and one carboxylate derivative) (561, we desired to CSH9NO4:C, 52.44; H, 4.96; N, 7.65. Found: C, 52.09, H, 5.08;
examine various analogs of these compounds to obtain a (F)l,4-Dihydro-l-isopropyl-3-hydroxy-4-oxopyridine-6-general idea of the properties responsible for their in vivo activity. The existence of several pyran core structures carboxylic Acid (13). Yield 4.3% (yellowish white plates); mp provided an ideal way to examine pyridinone derivatives 211-212 "C [lit. (62)mp 196-197 "C]; lH NMR (CD30D) 6 7.94 possessing sulfonate groups. The effect of lipophilicity (s, l H ) , 6.94 (s, lH), 5.10 (m, lH), 1.41 (d, 6H). (SLSI, mlz) 198.1 (loo), 156.1 (33), 138.0 (32). Anal. Calcd for CgHllN04: on these pyridinones can be seen in compounds 3,7,and C, 54.82; H, 5.62; N, 7.10. Found: C, 54.55; H, 5.76; N, 7.05. 9,with 7 being the most lipophilic, while 9 with the (G)1,4-Dihydro-l-benzyl-3-hydroxy-4-oxopyridine-6-addition of the hydroxyl group is the most hydrophilic. carboxylic Acid (14). Yield 24% (brown solid); mp 221-222 Both 3 and 7 are analogs of 1,2-dimethyl-3-hydroxypyrid"C; 1H NMR (D20)6 7.65 (s, l H ) , 7.30 (m, 5H), 6.6 (s, lH), 5.35 4-one (2) and its even more effective analog, 1,2-diethyl(s, 2H). Anal. Calcd for C13HllN04: C, 63.66; H, 4.52; N, 5.71. 3-hydroxypyrid-4-one (421,respectively. Here, we specuFound: C, 63.85; H, 4.64; N, 5.55. lated that it might be possible to achieve a similar Preparation of Chelator Solutions for Administration. enhancement in activity by a n increase in lipophilicity, All compounds were prepared for administration by neutralizawhile at the same time avoiding the substantial increase tion with a roughly equal molar amount of sodium bicarbonate, in toxicity (71)through incorporation of the sulfonate which also facilitated the water solubility of the compounds. IV group. Structural factors remain generally constant injection solutions consisted of 0.2 mmol of chelatorkg in 1mL between the molecules, though in 9, the methylhydroxy of Millipore HzO, while PO solutions were comprised of 1 mmol/ kg of chelator also in 1 mL of Millipore HzO. The volume of functionality is located para to t h e hydroxyl group rather these solutions depended on animal weight, with the typical than adjacent to it. Regarding molecular weight, the injection for the 200-g r a t equaling 1 mL. The purity of all compounds were nearly identical. chelators was verified by 'H NMR and combustion analysis. Having obtained the parent comenic acid molecule 10 Cannulation Procedure. The bile ducts of the rats were from 8,we were in the fortuitous position to synthesize cannulated using PE-10 tubing, the procedure being performed a series of molecules with carboxylate groups as well. To while the rats were anesthetized with ether. Two small this end, a series of four 3-hydroxypyrid-4-onesof varying incisions just below the xiphoid process were made in order to lipophilicity were prepared using 10 as t h e core structure gain access to the bile duct. Following a small incision into the to generate the N-methyl (ll),N-ethyl(12),N-isopropyl bile duct, the tubing was inserted and then tied firmly with N, 7.48.
Molenda et al.
818 Chem. Res. Toxicol., Vol. 7, No. 6, 1994
Table 3. Urinary Iron Excretion following po Administrationa
Table 1. Chelating Agent-InducedBiliary Iron Excretionapb compd
N
control iron excretion (ng of Feh)
treatment iron excretionc (ng of Fe/h)
2 3 4 5 7 9 11 12 13 14
5 3 3 3 3 5 5 3 3 3
407 f 73 595 f 172 162 f 35 251 f 194 722 f 200 350 f 68 343 f 44 579 f 125 733 f 333 390 f 214
4810 f 2130 2608 f 239 4245 f 1390 13678 f 2137d 3491 f 948 10305 f 349od 5870 f 895 3387 f 1056 3637 f 257 24.46 f 446
Chelators were administered, and bile was collected as described in the experimental section. Data on compounds 9 and 11 are from our previous study (56). Following administration of the chelating agent, biliary excretion of iron appeared to increase substantially within a 2-hperiod. From the dark coloration of the bile, most chelated iron was excreted after -30 min. All treated groups were shown to be at least Statistically equivalent to 2. Significantly different from 2 (p I0.05).
Table 2. Urinary Iron Excretion following iv Administrationa compd
N
control iron excretion (ng of Fen5 h)
treatment iron excretion (ng of Fe/15 h)
2 3 4 5 7 9 11 12 13 14
6 4 4 4 4 3 3 4 4 4
490 f 170 339 f 141 333 f 127 286 f 100 240 f 96 504 f 220 410 f 77 349 f 226 410 f 42 294 f 83
8370 f 3110 2310 f 498 2288 f 577 1373 f 374 1580 f 631 2196 f 662 6932 f BOOb 3657 f 2200b 1634 f 120 3387 f 1318
"Chelators were administered, and urine was collected as described in the experimental section. Data on compounds 9 and 11 are from our previous study (56). Not statistically different from 2 (p z 0.05).
(131, and the N-benzyl (14) derivatives. As we were interested in the effect of varying the position of the carboxyl group, the synthesis of compounds 6 and 14 enabled the observation of the differences in placement of the negatively charged group on the pyridinone ring or on the N-substituted benzene ring. Additionally, the activity and sites of iron removal of the high molecular weight compounds relative to the smaller pyridinones could also be examined. Conveniently, the carboxylic acid moiety, which is converted to the sodium salt for administration, allowed for the testing of the pyridinones with benzyl groups as their neutral analogs were previously observed to be insoluble in water (63). One may also compare two structurally similar monoanionic variations of L1, one with the carboxyl group on the pyridinone ring, 11, and the other with it attached to the methyl group itself as in the glycine (aminoacetic acid) derivative 4. The activity of 4 and 6, together with derivatives 1114, provided us with a general idea of the ability of the carboxylate compounds to mobilize iron relative to the sulfonates. (B) Biological Evaluation. Initial results from the biliary data (Table l ) , iv urinary data (Table 2), and the PO data (Table 3) suggest that monoanionic 3-hydroxypyrid-4ones with either a carboxylate or sulfonate moiety significantly enhance the excretion of iron. The carboxylate functional group results in chelators of more versatile effectiveness, enhancing iron excretion when given orally or iv, while the sulfonates, on the other hand, enhance
compd
N
control iron excretion (ng of Fd15 h)
treatment iron excretionb (ng of Fd15 h)
2 4 5 9 11 12 13 14
4 4 4 3 3 3 4 4
401 f 110 983 f 152 736 f 204 295 f 113 497 f 52 555 f 39 1397 f 535 675 f 160
17326 f 6404b 1828 f 102 1554 f 925' 350 f 12e 6737 f 370 6847 i 3056 2344 f 88 986 f 54oC
" Chelators were administered, and urine was collected as described in the experimental section. Data on compounds 9 and 11 are from our previous study (56). Significantly more effective than any of the other compounds (p 5 0.05). Not significantly different from control values (p z 0.05). urinary and biliary excretion only when given iv. Among the sulfonates, the biliary excretion data showed that all derivatives were statistically equivalent to L1, although 9, the most hydrophilic compound, was at least twice as effective as the more lipophilic derivatives 3 and 7 and statistically more effective than L1. Differences in lipophilicity and structure had virtually no effect on the ability of these compounds to mobilize iron into the urine when given iv; however, and relative to L1, they were a t best half as effective. Since sulfonates are generally not well absorbed into the gastrointestinal tract, only 9 was tested to affirm its inability to remove iron when given PO* Substantial biliary excretion of iron was observed for all of the carboxylate derivatives, and all were a t least statistically equivalent to L1. Among the comenic acid series, the compounds with aliphatic chains 11-13 possessed roughly equal activity among themselves and compared with L1. Surprisingly, 14 showed the weakest activity of the series (roughly half), almost equaling its iv urinary excretion ability. Placement of the carboxylate group on the benzyl ring as in 6 enhanced its iron mobilizing ability by 5-fold, nearly a 2-fold increase over L1. However, little difference in activity was observed when the carboxylate group was shifted off of the pyridinone ring and onto the side chain between 11 and 4.
Large differences were observed in iv-induced urinary excretion among the carboxylates, depending on functional group variations. Among the comenic acid derivatives 11- 13, excretion decreased substantially with increasing lipophilicity and molecular weight, with the methyl derivative 11showing similar activity to L1. The ethyl derivative 12 showed significant enhancement as well, but not a t the same level as 11. While it would follow that a n increase in molecular weight may account for the decrease in urinary excretion (as high molecular weight compounds are metabolized in the liver), compound 14 with the benzyl functionality showed equal activity to 12, and this may be explained by the unique polarity afforded by the benzyl group. Shifting the position of the carboxyl group to a less sterically hindered position on the benzyl group itself in compound 6 actually reduced excretion by a factor of 2-fold relative to 14. A similar decrease in activity was seen in moving the carboxylate group off the pyridinone ring as seen between 4 and 11. Regarding po-induced urinary excretion by the carboxylates, molecular weight appeared to play the most obvious role in the chelators' effectiveness. Compounds
Iron Mobilization by 3-Hydroxypyrid-4-ones
Chem. Res. Toxicol., Vol. 7, No. 6, 1994 819 Table 4. Summary of Crystal Data and Intensity Collection
01
formula formula weight color of crystal crystal system
(A)
CsHgN04 183.16
golden monoclinic
8.384(1) 10.477(2) 9.540(2) c (A) 90 a (deg) 103 /3 (deg) 90 vel( 1 817.0(2) 1.117 D (g/cm3) 288 F(OO0) 7.41 p (cm-l) absorption correction none space group P2JC 2 4 0.65 x 0.60x 0.20 crystal dimensions (mm) temperature 20 f 1 “C Cu Ka radiation w-scan data collection mode scan width 1.78 0.30tan 0 scan speed (deg/min) 8.0 background counts stationary counts; peaW bkgd counting time = 2:l 28 limits (deg) 60 5 28 5 1200 toatl reflections collected 1397 1302 no. of unique intensities no. of intensities with F =- 3.00(F) 1036 0.055,0.079 R, R w a
b (A)
(dT)
+
characteristics were previously reported, it was unclear as to what product was obtained. Similar questions arose from the differences in melting points obtained for the ethyl and isopropyl derivatives 12 and 13 and were again compounded by the lack of previous structural characterization. In light of this ambiguity, we decided to verify our structural predictions of 4 and accurately relate them to the observed activity via X-ray analysis, as shown in O4 the ORTEP diagram (Figure 1). The crystal data and Figure 1. ORTEP diagram of (1,4-dihydro-3-hydroxy-2-methyl- intensity collection information are shown in Table 4. 4-0XO-l-pfldyl)methanecarboxylicacid (4). The X-ray structure of 4 clearly reveals the insertion of nitrogen into the pyran ring to give the substituted 11 and 12 showed roughly equivalent behavior, both pyridinone. While several structures have been deterincreasing the excretion of iron by roughly half that of mined for neutral pyridinones chelated to iron (73-75) L1. The high molecular weight compounds 5, 13,and 14 all showed relatively similar low urinary iron excreand other tripositive metals (76-81), little work has been tion. Minor differences in excretion were observed in the done on free pyridinones themselves, with the exception placement of the carboxyl group between compounds 5 of L1 (82, 83) and our previous structures of 9 and 11 and 14 , although a significant drop was observed in (56). From the equivalent C - 0 bond distances of the going from 11 to 4. chelating oxygens and nearly equivalent (3a) bond distances of the atoms within the ring, we concluded that 9 (C)X-ray Characterization. While performing the syntheses of neutral derivatives in previous work (661, and 11 exist essentially as aromatic species. Surprisingly, such aromatic character was strongly diminished we noted that Schiff base formation can be preferentially in the structure of 4. The bond distances between atoms induced if the solvent medium is nonaqueous, and this prompted us to definitively establish the structure of the within the ring as well as between the C-0 bonds of the monoanionic pyridinones that we had synthesized in chelating oxygens point toward a “partially aromatic” aqueous media (56). While considerably higher melting species, resembling both L1 and the ligands of its iron(II1) complex (65). The C-0 distances (1.308 and 1.365 A) points (-40 “ C )and in some cases proton shifts in NMR (more up field) can provide reasonably accurate strucshown in Table 5 are between what one would expect for tural verification, X-ray analysis supplies the only definieither a single- or double-bonded oxygen (-1.23-1.43 A), tive data regarding the differences between isomers. and the ring atom distances are similar but by no means Since our goal has been to prove that monoanionic equivalent, ranging from 1.347 to 1.406 A. The data transport systems can be utilized to mobilize iron, it was point to the possible correlation between substituent imperative to establish positively that we were obtaining effects and degree of aromaticity, specifically on the the desired target molecules. As most of the monoanionic location of the substituent rather than the particular substituent. In the cases of 9 and 11, both molecules pyridinones tested thus far exhibited significant activity, the reported absence of activity of compound 4 (72) possess a substituent para to the hydroxyl group, though interestingly, the hydroxymethyl group of 9 is electron appeared inconsistent with our current animal data on releasing while the carboxylate group of 11 is electron similar monoanionic pyridinones. Since neither the method of preparation of this compound nor its physical withdrawing. As 4 contains a methyl group at position
w
Molenda et al.
820 Chem. Res. Toxicol., Vol. 7, No. 6, 1994 Table 5. Intramolecular Distances (A) and Angles (deg) Involving the Non-HydrogenAtoms for (3,4-Dihydro-3-hydroxy-2-methyl-4-oxopyridyl)1methanecarboxylicAcid atoms
distance
atoms
distance
0(1)-C(3) 0(2)-C(4) 0(3)-C(8) 0(4)-C(8) N(l)-C(l) N(l)-C(5) N(l)-C(7)
1.308 (3) 1.365 (4) 1.224 (4) 1.266 (3) 1.350 (4) 1.367 (4) 1.470 (4)
C(l)-C(2) C(2)-C(3) C(3)-C(4) C(4)-C(5) C(5)-C(6) C(7)-C(8)
1.347 (4) 1.406 (4) 1.396 (4) 1.386 (4) 1.487 (5) 1.523 (4)
atoms
angle
atoms
angle
C(l)-N(l)-C(5) C(l)-N(l)-C(7) C(5)-N(l)-C(7) N(l)-C(l)-C(2) C(I)-C(2)-C(3) 0(1)-C(3)-C(2) 0(1)-C(3)-C(4) C(2)-C(3)-C(4) 0(2)-C(4)-C(3)
120.7 (3) 117.9 (3) 121.2 (3) 121.8 (3) 120.5 (3) 124.0 (3) 119.4 (3) 116.7 (3) 120.4 (3)
0(2)-C(4)-C(5) c(3)-C(4)-c(5) N(l)-C(5)-C(4) N(l)-C(5)-C(6) C(4)-C(5)-C(6) N(l)-C(7)-C(8) 0(3)-C(8)-0(4) 0(3)-C(8)-C(7) 0(4)-C(8)-C(7)
117.8 (3) 121.8 (3) 118.5 (3) 119.9 (3) 121.6 (3) 111.6 (2) 125.1 (3) 119.5(3) 115.4 (3)
2 and only a hydrogen para to the hydroxyl group, this may point to the importance of structural factors in the ultimate conformation of the free ligand over the physical organic properties of the substituents.
Conclusions The urinary and biliary excretion data for these monoanionic 3-hydroxypyrid-4-ones significantly extend the data collected previously for two model compounds of this type (56),contributing further evidence that the assumption that these compounds use monoanionic transport systems may be exploited to enhance the excretion of toxic iron deposits. While no apparent pathological effects were observed in the animals following a single dosage either PO or iv, further studies are needed to examine the toxicity of these pyridinones in greater detail. Since several of these compounds are a t least as active or better than L1 in influencing the biliary or urinary iron excretion when administered iv, this class of chelators may not only be as effective but substantially less toxic due to increased organ specificity. The animal data on these compounds have also shown that structural factors, lipophilicity, and molecular weight are all factors which influence the path by which iron is excreted. Continued manipulation of these chemical features may result in a molecule that will be able to enhance both the biliary and urinary excretion of iron. As iron has been linked to numerous pathological conditions due to its complex toxicology and the iron chelators presently in use have displayed numerous adverse effects, the exploitation of monoanionic chelating agents may facilitate the search for effective, less toxic Fe3+ chelators.
Acknowledgment. We wish to acknowledge with thanks the support received for this study from the Center in Molecular Toxicology a t Vanderbilt University via NIEHS Grant 0268 and from the National Institute of Environmental Health Sciences via Grant 02638. We are also grateful for assistance from Dr. Glen R. Gale with the statistical analysis, as well as from Drs. Ernest Walker and Don Cannon for their contributions in the iron analyses. Supplementary Material Available: Detailed preparations for the monoanionic 3-hydroxypyrid-4-ones and Tables S1 and S2 for the positional and Uij parameters, respectively, for
compound 4 (5 pages). Ordering information is given on any current masthead page.
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