Surface Reactive Peptides and Polymers - American Chemical Society

Chapter 11

Downloaded by COLUMBIA UNIV on August 2, 2012 | http://pubs.acs.org Publication Date: November 26, 1991 | doi: 10.1021/bk-1991-0444.ch011

Phosphorylated and Nonphosphorylated Carboxylic Acids Influence of Group Substitutions and Comparison of Compounds to Phosphocitrate with Respect to Inhibition of Calcium Salt Crystallization J. D. Sallis, M. R. Brown, and Ν. M. Parker Department of Biochemistry, University of Tasmania, Hobart, Tasmania 7001, Australia Multinegatively charged molecules are recognized inhibitors of hydroxyapatite (HA) crystallization and phosphocitrate (PC) with its unique character is no exception. To gain insight into PC's inhibitory properties, a range of phosphorylated and non -phosphorylated carboxylic acids was studied. A useful synthetic strategy for some phosphorylated compounds was to couple 1,2-phenylene phosphochloridate to ester protected carboxylates followed by hydrogenation, base hydrolysis and chromato graphy. More elaborate strategies were devised for hydrocarbon chain lengthening, the incorporation of a sulfate, amino or carboxyl group to the parent compound. Their inhibitory influence on H A and/or CaOx crystallization was compared to PC. Data indicate that the group arrangement inherent in PC presents as the most ideal for preventing HA formation. Biological calcification with its several distinct crystalline salt forms can often be initiated from changing environmental circumstances leading to the pathological precipitation of the salt. In recognition of this aspect, considerable research effort has been applied to seek natural and synthetic compounds which might control or even regress unwanted calcification. The most successful compounds reported thus far have associated phosphate and/or carboxylate moieties. Interest, for example, in the role of pyrophosphate (P-OP) as a urinary inhibitor of stone formation led to extensive investigations of the potential of the bisphosphonate (P-C-P) class of compounds (1). The latter group are among the most powerful of the inhibitors of hydroxyapatite. Their usefulness clinically however poses some problems as secondary undesirable responses have been noted and the compounds are not degraded by enzymes (2). In respect to carboxylic acids alone, they too have elicited attention for their ability to inhibit although it is primarily the 0097-6156/91/0444-0149$06.00/0 © 1991 American Chemical Society In Surface Reactive Peptides and Polymers; Sikes, C., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.


150

SURFACE REACTIVE PEPTIDES AND POLYMERS

polycarboxylic acids which influence. Citric acid is recognized as a natural urinary inhibitor of calcium salt crystallization (3) but selected glycosaminoglycans appear more influential, displaying strong inhibitory action against calcium oxalate crystallization (4-6). Over the past decade, our studies have centered around the inhibitory potential of phosphocitrate (PC), a compound containing both a phosphate and carboxylate group (7). With a reported natural occurrence in animal mitochondria (8), this molecule also has been verified as a very powerful inhibitor of hydroxyapatite crystallization (9). Its inhibitory action for the most part seems to derive from its multinegative charge/size ratio which together with its stereochemical character allows it to bind to a crystalline lattice preventing growth and aggregation. Our previous studies (9) have highlighted some of the important chemical features which appear to confer inhibitory properties on a molecule. In the present studies, we have developed synthetic strategies for some additional compounds which have extended the range of compounds now examined, thus enabling consolidation and a reappraisal of the basic requirements. The structure-inhibitory activity relationships then of a range of phosphorylated and non-phosphorylated carboxylic compounds have been compared to the response evoked by PC. Consideration also has been given to the influence of group substitutions within some of the molecules. Methods Inhibitor studies: (a) hydroxyapatite (HA): The inhibitory activity of compounds toward apatite crystallization was determined by comparing the time at which the amorphous calcium phosphate (ACP) phase transformed into the H A crystalline state. The method was as previously described (9) and based on a report by Meyer and Eanes (10) in which a solution of calcium phosphate was induced to spontaneously precipitate at pH 7.4 and temperature 25 °C. Protons ejected by the transformation of ACP to HA were then neutralized and quantitated by base titration. The time (induction time = It) at which transformation starts was determined (see Fig. 1) so that the time difference between solutions with an inhibitor compound present to that seen in its absence could be expressed as Alt. (b) Calcium Oxalate: The rate of depletion of aqueous calcium from a metastable solution of a mixture of calcium chloride and sodium oxalate after seeding with mature calcium oxalate crystals was assessed as previously described

01). Test compounds: Unless otherwise indicated, compounds were obtained from commercial sources. In respect to those compounds for which a synthesis was required, outline strategies are given below. Ultimate purification was generally accomplished by ion exchange chromatography and characterization was pursued by a variety of spectroscopic and chemical techniques.

In Surface Reactive Peptides and Polymers; Sikes, C., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.


SALLIS ET AL.

Carboxylic Acids

5

15 TIME

25

35

(min)

Figure 1. Measurement of induction time.


152


Phosphocitrate (PC). Phosphorylation of triethylcitrate followed by hydrogénation, base hydrolysis and chromatographic purification was effected as previously described to yield PC (7,12). A similar strategy was used to derive phosphomalate (PM), phosphomethylglutarate (PMG) and phosphoisocitrate (PIQ. O

CH-COOH

O

I l HO—Ρ—O—C—COOH OH

1

HO—P—0~"Ç—COOH

CHjCOOH

OH

PC


ÇH^OOH

«

H

PM

OH

I

HO-P=0

C^COOH

I

HO—P—O—Ô—CH.

I OH

I

0 H—C—COOH H—C-COOH

PMG

1

CHXOOH

Tetracarballylate (TETCA: propane 1,2,2'3-tetracarboxylic acid). The intermediate, 2-cyano trimethyltricarballylate was initially prepared by dialkylation of methyl cyanoacetate with methyl bromoacetate in the presence of base (13). NBC-CH COOCH 2

CH COOCH

5

2

5

Na*MeO" N B C - - Ç - C O O C H , 2Br-CH COOCH 2

CH COOCH

5

2

3

1N NaOH

CH COONa

CH COOH

2

2

7

Na ooc—c—coo Na

S

C

^ V«' ,

Hooc-|-cooH COOH

CH COONa 2

TETCA

Subsequent hydrolysis yielded the desired product, 2,4-Dimethylene tetracarballylate (DMTETCA: pentane 1,3,3',5 tetracarboxylic acid). Reaction of methyl


11.

153

Carboxylic Acids

SALLISETAL.

3-bromopropionate with methyl cyanoacetate in the presence of base yielded the trimethyl ester of pentane 3' cyano 1,3,5 tricarboxylic acid. Hydrolysis of this product produced pentane 1,3,3',5 tetracarboxylic acid or as trivialized here, 2,4 dimethylene tetracarballylate. NsC-CH COOCH 2

CH CH COOCH 2

5

N

Bl

-CH CH COOCH 2

2

A

*

M

E

O

;

2

3

N=CJ>-COOCH, CH CH COOCH

3

2

2

3


NaOH

CH.CHXOONa I 2

CH-CH-COOH ι 2

2

2

Naooc—C—COONa

4

HOOC—C—COOH

I

I

CH CH COONa 2

2

7/H S0 ,4C

CH CH COOH

2

2

2

DMTETCA

Phospho-2-amino tricarballylate (PAT). Trimethyl 2-nitro tricarballylate was synthesized as described by Kaji and Zen (14). Catalytic hydrogénation of this product yielded the intermediate, trimethyl 2-amino tricarballylate. Phosphorylation of this product was achieved by 2-cyanoethyl phosphate in the presence of dicyclohexyl carbodiimide (DCCD) and pyridine. The final product was obtained by base hydrolysis in the presence of calcium. OH NC—CH — C H _ — P = 0

I

Ο

H

CH COOCH 2

DCCD/Pyrldine II I I • NC-CH —CH —o-P-N-C-COOCH

OH

2

2

OH

CH COOCH, 9

I

3

CH COOCH 2

5

3

Coupled Intermediate"

H N—C—COOCH, 9

I CH COOCH 2

NaOH 5

Trimethyl 2-amino tricarballylate

II I I HO—P-N—C—COOH OH

CH COOH 2

PAT


154


Sulfo-2-amino tricarballylate (SAT): We have previously reported a method for synthesis of this compound (15). Initially, 2-amino tricarballylate was prepared from diethyl 1,3-acetonedicarboxylate (16). Condensation of this reactant with pyridine-sulfur trioxide yielded SAT.

Ο

CH COOH 9

I

I

o=s=o II


ο

1

CH COOH 2

1

0 = S — Ν —C—COOH

+ H,N—C—COOH CH COOH

I

I

OH

2

Pyridine

H

11

2

CH COOH 2

SAT

2-aminotricarballylate

-sulfur trioxide

2-Methylidine aminophosphonate tricarballylate (MAPT: 2-methylidine aminophosphonate propane 1,2,3-tricarboxylic acid). Using as starting product 2-cyanotrimethyl tricarballylate, a series of reactions involving the isolation of an imidoaldehyde hydrochloride tin (IV) complex was employed in a similar manner to that described by Gancarz and Wieckzorke (17). Subsequent phosphorylation followed by hydrolysis led to the crude compound. NSC-CH C00CH 3

CH COOCH

2

2

N

2Br-CH COOCH 2

A

*

M

E

° ;

NSC-LcQOCK

5

CH COOCH 2

1. Diethyl phosphite 3

2. Aqueous HCI CI Sn.CIH.HN=CH- - Ç — C O O C H 4

3

;H COOCH 2

3

3

3. H S(g) 2

Ο

NH,CH,COOH 2

II π

HO-P-C—C—COOH OH

CH COOH 2

MAPT


11. SALLis ET AL.

155

Carboxylic Acids

Results and Discussion


Table I describes differences in induction time for series A, carboxylated, nonphosphorylated compounds and series B, phosphorylated, non-carboxylated compounds. In general, the carboxylated compounds are very poor inhibitors, only those compounds with three or more carboxyl groups having any influence at all. Citrate requires at least a concentration in excess of ΙΟΟμΜ to exhibit any inhibitory properties. TETCA on the other hand with four carboxyls is much stronger. It was interesting to note that extension of the side chains of TETCA did reduce the inhibitory power of the parent compound. Delocalization of the net negative charge over the larger extended molecule, thus in effect providing a smaller charge to size ratio could be one explanation. Table I. Influence of Some Carboxylic Acids and Some Phosphorylated, Noncarboxylated Compounds on the Transformation of Amorphous Calcium Phosphate to Hydroxyapatite.

Series A Formate Oxalate, Succinate, Malate Tricarballylate Citrate Tetracarballyate 2,4 Dimethylene Tetracarballylate Series Β Pyrophosphate Adenosine 5'-diphosphate Adenosine 5-triphosphate Imidobisphosphonate Hydroxy ethane bisphosphonate Tripolyphosphate

Inhibitor Cone. ADP), the configuration of groups can overide such an advantage. HEBP, the most active compound in the series probably gained its increase in potency over pyrophosphate through the attachment of the OH group at CI. Figure 2 reveals however that much more inhibitory power is displayed by compounds possessing both phosphate and carboxylate moieties in comparison



156


[INHIBITOR]

(μΜ)

Figure 2. A comparison of the inhibitory influence of some phosphorylated, carboxylic acids on the induction time required for the transformation of amorphous calcium phosphate to hydroxyapatite.



11.

SALLis ET AL.

Carboxylic Acids

157

to compounds with only one of the moieties. The number and positioning of these negatively charged groups then assumes major importance. Phosphonoformate (PF) and phosphomalate for example, are stronger inhibitors than their non-phosphorylated counterparts. Phosphomalate and phosphomethyl glutarate both display moderate inhibitory activity, suggesting that the inclusion of two carboxylates instead of one is more beneficial. Clearly however, phosphocitrate with three carboxylates is much more impressive. Of interest in this series, phosphoisocitrate is not quite as powerful as phosphocitrate suggesting that a C3 position of the PO4 is important. Modification of the basic phosphocitrate molecule by the inclusion of other groups does significantly alter inhibitory potential as shown in Fig. 3. An amino grouping appeared to lessen activity, probably as a result of the positive charge of this moiety. Although PAT revealed itself to be a very powerful inhibitor, other characteristics inherent in the molecule do not make it suitable as a potential drug. Under acid conditions it is very labile and additionally, the compound is also subject to enzyme deactivation. Replacement of the phosphate moiety in PAT with a sulfo-group (SAT) renders the compound less inhibitory. This effect could be explained by the fact that when SO4 is incorporated into the molecule, there is a loss of a chelating group compared to phosphate. In addition, the moiety change also confers other interesting properties on the molecule. We have previously reported, that SAT is enzyme resistant and possesses good absorptive properties (18). MAPT, the other N-compound tested, displayed only relatively weak activity. Whilst most of the research has focused on controlling hydroxyapatite formation, some limited observations have also been made regarding the influence of the more powerful compounds on calcium oxalate crystallization. As can be seen in Fig. 4 phosphocitrate is not as effective an inhibitor as HEBP suggesting that underlying differences in configuration are important for binding to the particular type of crystal lattice formed. Of interest, TETCA at ΙΟΟμΜ proved almost as potent as phosphocitrate in this system. Overall, the data emerging from these studies allow for some generalizations and conclusions to be drawn concerning the nature of groups required for inhibition and the importance of their relationship to one another. Inhibitors of calcium salt crystallizations are generally thought to act primarily by occupying defect sites on various faces of the crystals (19). Through negative charge for example, compounds bind tightly to hydroxyapatite. Although charge is important, size also influences as it is clear that commonly a larger molecule can exert a weaker association because of its respective spatial group orientation. TETCA for example with four negative groups tightly arranged around the central C possesses stronger inhibitory powers than the extension product, DMTETCA. Comparatively however, in respect to hydroxyapatite inhibition, TETCA is not as strong as phosphocitrate which probably reflects the fact that with phosphate present, five chelation sites are available as compared to four with TETCA. Also of course, the properties of hydroxyl (e.g. P-OH, C-OH, S-OH) or carboxyl groups would


158



50r

[INHIBITOR] ( U M )

Figure 3. Comparative influence of group substituents on the time required to transform amorphous calcium phosphate into hydroxyapatite. HEBP

0

20

40 60 [INHIBITOR] ( μ Μ )

80

100

Figure 4. Dose-response relationship of some selected hydroxyapatite inhibitors to restrict calcium oxalate crystallization.


11. SALLis ET AL.

159

Carboxylic Acids

^,C-(P0 H ) 3

O

CH.COOH

H O — P - O — Cil— CI— C O O H C-COOH I I OH

O

CH-o

II

ï

2

«

HO-P—O-C —C—(ΡΟ,Η,)

CHXOOH

OH

CH. V îO

\J!

O

HO-P-O—Ç-O—(P0 H )


HO—Ρ—Ο—Ρ—O—C—COOH

I

CHjCOOH 3

I

ί III

2

I IV

0

ÇH -(PO H ) 2

s

2

HO—P—O—C—COOH

1 OH

I CH —(P0 H ) 2

3

2

Figure 5. Structures of some compounds predicted to exert a strong inhibitory influence on calcium salt crystallization.

be subject to change under the influence of other groups. Substitution of groups at C3 appears to play a key role in the molecule's inhibitory properties. PC, TETCA, MAPT and 2,4 DMTETCA all have a central C atom from which chelating groups radiate tetrahedrally. The removal of one chelating arm (e.g. P M vs PC) or the substitution of one chelating functionality with that of a lower chelating ability or charge, leads to a significant depletion of inhibitory activity. In terms of contribution to inhibitory potential, the following order appears to emerge: O-PO3H2 > - C H 2 - P O 3 H 2 > - N H - P O 3 H 2 > - O - S O 3 H (predicted) > -NH-SO3H > -COOH > -OH. On the basis of the information available then, it might be predicted that the following compounds shown in Fig. 5 would also show similar or even more powerful inhibitory action. To date, these compounds have not been synthesized and of course if they were to become useful compounds with a clinical application they would need to possess the characteristics of being non-toxic and of being eventually cleared from systems. Alternatively, a search for natural compounds with a similar arrangement of chemical groups as suggested might provide a greater understanding on how biological mineralization can be controllable.


160



Literature Cited 1. Fleisch, H. Kidney Int. 1978, 13, 361-71. 2. Sallis, J. D. In Urolithiasis and related clinical research; Schwille, P. O.; Smith, L. H.; Robertson, W. G.; Vahlensieck, W. Eds.; Plenum: New York, 1985; pp 803-9. 3. Bisaz, S.; Felix, R.; Neuman, W. F.; Fleisch, H. Min. Electr. Metab. 1978, 1, 74-83. 4. Robertson, W. G.; Peacock, M.; Nordin, B. E. C. Clin. Chim. Acta. 1973, 43, 31-7. 5. Ryall, R. L.; Harnett, R. M.; Marshall, V. R. Clin. Chim. Acta. 1981, 112, 349-56. 6. Kok, D. J.; Papapoulos, S. E.; Blomen, L. J. M . J.; Bijvoet, O. L. M . Kidney Int. 1988, 34, 346-50. 7. Williams, G.; Sallis, J. D. Anal. Biochem. 1980, 102, 365-73. 8. Williams, G.; Sallis, J. D. In Urolithiasis, Clinical and Basic Research. Smith, L. H.; Robertson, W. G.; Finlayson, B. Eds.; Plenum: New York, 1981; pp 569-77. 9. Williams, G.; Sallis, J. D. Calc. Tiss. Int. 1982, 34, 169-77. 10. Meyer, J. L.; Eanes, E. D. Calc. Tiss. Res. 1978, 25, 59-68. 11. Meyer, J. L.; Smith, L. H. Invest. Urol. 1975, 13, 31-5. 12. Tew, W. P.; Mahle, C.; Benavides, J.; Howard, J. E.; Lehninger, A. L. Biochemistry 1980, 19, 1983-8. 13. Cope, A. C.; Holmes, H. L.; House, H. O. Organic Reactions 1957, 9, 107331. 14. Kaji, E.; Zen, S. Bull. Chem. Soc. Jap. 1973, 46, 337-8. 15. Brown, M . R.; Sallis, J. D. Anal. Biochem. 1983, 132, 115-23. 16. Dornow, Α.; Rombusch, K. Chem. Ber. 1955, 88, 1334-41. 17. Gancarz, M.; Wieckzorke, C. Synthesis 1977, 9, 625. 18. Brown, M . R.; Sallis, J. D. In Urolithiasis and Related Clinical Research; Schwille, P.O.; Smith, L.H.; Robertson, W.G.; Vahlensieck, W., Eds.; Plenum: New York, 1985; pp 891-4. RECEIVED August 27, 1990


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