Abstract:
The present invention relates to novel compounds selected from R 1  --COO(CH 2 ) n  --R 2  and ##STR1## wherein R 1  is selected from heterocyclic aromatic and hydrophobic naturally occuring and modified amino acids in the D- and in the L-form, and from peptides, wherein n is an integer of from 1 to 5 and R 2  is selected from unsubstituted and substituted phenyl rings, and from heterocyclic rings, as well as to pharmaceutical compositions for preventing sickling of erythrocytes during sickle cell crisis and for desickling of sickle cells of patients afflicted by sickle cell disease, the active ingredient of which is a compound as defined above.

Description:
FIELD OF THE INVENTION 
     The present invention relates to novel compounds and to pharmaceutical agents which are of value in the treatment of sickle cell disease. The novel pharmaceutical compositions are adapted to prevent sickling of erythrocytes and even to reverse sickling of erythrocytes from persons afflicted by the disease. 
     BACKGROUND OF THE INVENTION 
     Sickle cell disease is caused by a haemoglobin mutant called haemoglobin S, hereinafter HbS. The mutation of HbS comprises the replacement of a polar residue, glutamic acid, by a hyrophobic one, valine, in the 6th position of the β-chains, and this renders the HbS capable of polymerization in the desoxy form: see Pauling et al., Science 110, 543 (1949); Ingram:Nature, (London) 178, 792 (1956). In the deoxygenated state the HbS molecules aggregate in the form of elongated microtubular structures which distort the shape of the red cell to a sickle shape. The sickled cells tend to block the blood capillaries and ultimately give rise to the squelae of sickle cell disease. 
     There are known compounds which affect polymerization of HbS, and there must be made a clear distinction between those which are antisickling agents and those which are antigelling agents. 
     The antisickling agents are those which are able to pass through the cell membrane of the erythrocytes and prevent or reverse sickling; the latter are those which are adapted to prevent polymerization of deoxygenated HbS, but which do not pass the cell membrane in sufficient quantities and thus are not adapted to prevent or reverse sickling when contacted with red blood cells of a patient suffering from sickle cell disease. 
     In Table II there is presented a summary of the activity of known compounds, tried for use as antisickling agents, the comparison being on the basis of effective concentrations in vitro. The last item of the Table relates to compounds of the present invention. 
     Antigelling agents are summarized in Table III. The antigelling agents are not able to prevent sickling nor are they suited to reverse sickling when incubated with erythrocytes. 
     Sickling cell disease has been studied extensively, but in spite of this there does not exist a universally acceptable therapeutic agent for the treatment of this disease. During recent years attempts have been made to provide such agents. Some of these are based on the use of three types of compounds: 
     a. Agents which bind covalently with the haemoglobin molecule; 
     b. Agents which bind non-covalently to this molecule; 
     c. Agents affecting the cell membrane. 
     Some of the known antisickling agents have a rather high degree of toxicity, one of these being, for example, potassium cyanate. 
     According to the present invention there are provided novel active agents for use in the prevention and treatment of sickle cell crises which are both effective and non-toxic. 
     SUMMARY OF THE INVENTION 
     The present invention relates to novel compounds for use as active ingredients, along with conventional pharmaceutical excipients, in pharmaceutical compositions for the prevention and/or for the treatment of sickle cell crises. The invention also relates to the pharmaceutical compositions themselves and the method of treatment of sickle cell disease patients using such compounds and composition. The novel compounds inhibit sickling of erythrocytes from sickle cell disease patients, when such cells are incubated together with the novel agents. The antisickling effect is a very pronounced one and the novel agents are effective at low concentrations. 
     The novel compounds according to the present invention comprise an aromatic compound, such as aromatic amino acid, linked via an ester linkage to an aromatic or heterocyclic alcohol or linked via a peptide bond to an aromatic or heterocyclyc amine. Suitable aromatic amino acids are, for example, D- or L-phenylalanine, tryptophan or tyrosine. A typical effective ester linkage is, for example a carboxy-ester bond. Typical heterocyclic groups when used in the alcohol include thienyl, morpholine, pyridine and pyrrolidone groups. 
     Amongst typical compounds according to the present invention there may be mentioned L-Phe-OBz.HCl, L-Tyr-OBz.HCl, L-Try-OBz.HCl, L-Pro-OBz.HCl, L-Phe-OBz-(pNO 2 ).HBr, L-Pro-OBz(pNO 2 ).HBr, L-His-OBz(pNO 2 )-pTs, 1-Ala-L-Phe-OBz.HBr, L-Gly-L-Phe-OBz.HBr, L-Phe-Benzyl amide, L-Val-OBz, L-Leu-OBz, L-Phe-L-Ala-OBz.HBr, L-Try-Lys-OBz.HBr, D-Phe-OBz.HCl, D-Tyr-OBz.HCl, D-Try-OBz.HCl, L-Phe-(2-thienyl)ethyl ester, L-Phe-(2-morpholine)ethyl ester, L-Phe-(2-pyridine)ethyl ester, L-Phe-(2-pyrrolidone)ethyl ester, L-Phe-(2-phenyl)ethyl ester, L-Phe-(3-phenyl)propyl ester, and L-Phe-(4-phenyl)butyl ester. Compounds according to the present invention are adapted to prevent and to reverse sickling of red cells at a concentration as low as about 0.5 to 3 mM when incubated with red blood cells of sickle disease patients. The novel compounds have a very low toxicity. They were tested in mice as regards toxicity. They were found to be non-mutagenic as determined by the Ames method (Ames et al, Mutation Res. 31, 347 (1975)). 
     DESCRIPTION OF THE PREFERRED EMBODIMENT 
     The novel compounds according to the present invention comprise in combination an aromatic amino acid linked to an aromatic or heterocyclic alcohol or amine through a carboxylic ester bond or a peptide bond. The novel compound can be designated as benzyl esters of aromatic amino acids and they can be synthesized by reacting such amino acid with phosgene at room temperature in dioxane to form the corresponding N-carboxy anhydride hydrochloride, which latter is reacted with the aromatic alcohol, such as for example C 6  H 5  CH 2  OH to form the desired product in the form of its hydrochloride. 
     In the present specification and claims, the following abbreviations are used: Alanine (Ala), arginine (Arg), asparagine (Asn), aspartic acid (Asp), cysteine (Cys), glycine (Gly), glutamic acid (Gln), glycine (Gly), histidine (His), isoleucine (Ilu), leucine (Leu), lysine (Lys), methionine (Met), phenylalanine (Phe), proline (Pro), sernine (Ser), threonine (Thr), tryptophan (Trp), tyrosine (Tyr), valine (Val), hydrochloric acid (HCl), benzyl ester (-OBz), and p-toluene sulphonate (pTs). 
     It is clear that the above method is by illustration only and that various other routes of preparation can be resorted to. 
     The novel compounds defined above are effective agents for the intended purposes. Further improved compounds which have a lower degree of hydrolysis can be prepared as follows: ##STR2## where R is an amino acid residue. 
     As amino acid there can be used any amino acid listed, or any modification thereof, or any other acid which enhances the product with hydrolysis stability. 
     Instead of D-Phe, any other aromatic amino acids, or its functional derivative, can be used. The benzene ring can be substituted by conventional substituents such as p-nitro. It is also possible to link the aromatic amino acid to a benzylamine group through a peptide bond. 
     The compounds set out in Table IV were found to be effective agents for the inhibition of erythrocyte sickling and for the restoration of sickled cells. 
    
    
     The following Examples illustrate the present invention, and these are to be construed in a non-limitative manner. 
     EXAMPLE 1 
     Determination of Antigelling Property 
     The antigelling property of the said new class of compounds was determined by a modified method of Bookchin et al. (1967); (see Bookchin et al, J. Biol. Chem., 242, 248-255, 1967). To 10 μl of a solution of the said new class of compounds in a micro test tube, at about 37° C., 150 μl of 35% of purified HbS were added and vortexed. 15 microliters of a 10% sodium dithionite in 0.05 M phosphate buffer, pH 7.2 were added. The mixture was thoroughly mixed and flushed with nitrogen and quickly stoppered. The micro test tube was incubated at 37° C. for 30 minutes and then transferred to 0° C. for 10 minutes and then returned to 37° C. for 30 minutes. A compound was defined as having antigelling properties when the HbS inhibition mixture remained in the fluid state for a period of more than 20 minutes at 37° C. In the control experiment the inhibitor was omitted and this resulted in gelation within 2 minutes of incubation at 37° C. 
     The novel agents were found to have an antigelling property. D- and L-Phe-OBz inhibited gelation of HbS at 6 moles to one mole HbS while L-Try-OBz inhibited gelation at 4 moles to one mole of HbS. 
     L-Lys-L-Phe-L-Phe which had previously been found to have antigelling activity was used as reference standard. It inhibited gelation at 15 moles L-Lys-L-Phe-L-Phe to one mole of HbS. 
     EXAMPLE 2 
     Inhibition of Sickling 
     A venous blood sample was obtained from the anticubital vein of a sickle cell disease patient and washed several times with phosphate buffer saline, pH 7.2, consisting of 145.4 mM NaCl, 4.0 mM KCl, 1.65 mM Na 2  HPO 4 , 0.16 mM KH 2  PO 4 , 0.01 mM MgCl 2  and 11.1 mM glucose. The buffy coats were removed by aspiration. To a 300 μl portion of a 20 percent cell suspension, a solution of a compound of the invention was added to give a final concentration of 0.5, 1.5 and 3 mM. The mixture was well stoppered, incubated at 37° C. and swirled at about 100 cycles per minute in a controlled environment incubator. The standard control consisted of a 300 μl of a 20% cell suspension in the buffer in which the novel compound was dissolved. After an incubation period, 20 μl of the treated cell suspension was deoxygenated with 20 μl of 2.0% sodium metabisulphite or with nitrogen gas and examined with a Zeiss microscope. 
     The shape of the cells, as seen under a Zeiss microscope, are the same as oxygenated untreated cells. The control cells, deoxygenated and untreated with the novel compounds are completely sickled. 
     EXAMPLE 3 
     Reversal of Sickling 
     A sickle cell suspension prepared by the method described in Example 2 was completely deoxygenated by mixing with an equal volume of 2.0% sodium metabisulphite and incubation at 37° C. for 10 minutes. The cells were centrifuged and resuspended in phosphate buffer saline, (see Example 1). A drop of the sickled cells were examined under a Zeiss microscope to ascertain complete sickling. 
     A solution of a compound of the invention was added to give a final concentration of 3.0 mM. The mixture was flushed with nitrogen gas, incubated at 37° C. and swirled at about 100 cycles per minute in a controlled environment incubator. The control sample consisted of a mixture of 1.0 ml of buffer and 1.0 ml of sickled cells. 
     A 5 μl aliquot was removed from both the treated cells and a control sample and examined with the Zeiss microscope. 
     The desickling process was observed within 10 minutes of incubation and completed within 30 minutes of incubation of the sickled cells with the novel class of compounds. The shapes of the erythrocytes as seen under a Zeiss microscope are the same as oxygenated cells. 
     EXAMPLE 4 
     Transport of the novel compounds into erythrocytes 
     As sickle cell disease is caused by a defect of the erythrocytes, in order to study transport of a therapeutic agent for the treatment of the disease into erythrocytes, erythrocytes and no other cells must be used for the transport studies. 
     Washed erythrocytes were prepared by the method described in Example 2 and used for transport studies. The novel class of compounds were radioactively labelled, using for example, [ 14  C]-L-Phe. 
     The results indicate that the novel compounds, as exemplified by L-Phe-OBz are rapidly transported into erythrocytes by a carrier mediated process as shown by a simple Michaelis plot with a Michaelis constant for transport of 0.175 mM. Time course experiment shows that L-Phe-OBz is rapidly transported into erythrocytes, see Table I. 
     
                       TABLE I______________________________________L-Phe INTO ERYTHROCYTES14 C]      PERCENT OFTIME       INCORPORATION______________________________________ 0          010         3020         7030         9045         10060         90______________________________________ 
    
     
                       TABLE II______________________________________Antisickling Agents. Effective Concentration In Vitro      EffectiveAntisickling      Concen-Agent      tration  Reference______________________________________DL--Glyceral-      20 mM    Nigen, A.M. and Manning J.M.dehyde              Proc. Natl. Acad. Sci. (USA)               74, 367, 1977Various Aldehydes      5 mM     Zaugg, R.H.; Walder, J.A.; andand Ketones         Klotz, I.M., J. Biol. Chem.               252, 8548, 1977.Alkyl Urea and      100 mM   Elbaum, D.; Roth E.F. Jr.;Urea                Neumann, G.; Gaffe, E.; Bookchin,               R.M.; and Nagel, R.L., Blood,               48, 273, 1976.2(Benzoyl amino)      50 mM    Adhikary, P.K; Haynes, J.K.;pyridinium ben-     Patthey, H.L.; and Rhodes, R.S.zoate               Experientia, 34, 804, 1978.Dibromo aspirin      5 mM     Walder, J.A.; Zaugg, R.H.;               Iwaoka, R.S.; Watkin, W.G.;               and Klotz, I.W., Proc. Acad.               Sci. (USA) 74, 5499, 1977.3,4-dihydro-2,2-      10 mM    Ekong, D.E.U.; Okogun, J.I.;dimethyl 2H-1       Enyenihi, V.U.; Belgh-Nairm, V;Benzopyridirium-6-  Nakanishi, K; and Natt, C.;butyric acid        Nature (London) 258, 743, 1975.Pyridoxal  20 mM    Kark, J.A.; Kale, M.P.; Tara-               ssoff, P.G.; Woods, M.; Lessin,               L.S. and Hicks, C.U., J. Chim.               Invest., 62, 888, 1978.Cystamine  220 mM   Hassan, W.; Benzard Y Rosa J,               Proc. Natl.Acad. Sci.(USA),               73, 3288, 1976.Nitrogen Mustard      10 mM    Roth, E. Jr.; Nagel, L.R.;               and Bookchin, R.M., Biochem.               Biophys, Res. Comm., 48, 612,               1972.Potassium Cyanate      100 mM   Cerami, A.; and Manning, J.M.,               Proc. Natl. Acad. Sci., 68,               1180,1971.Dimethyl Adipi-      10 mM    Lubin, B.L.; Pena, V.; Mentzer,midate              W.C.; Bynum, E.; Parker, L.,               Proc. Natl. Acad. (USA) 72,               43, 1975.Benzyl esters of      0.5-     This invention.some Amino 3.0 mMAcids______________________________________ 
    
     
                                           TABLE III__________________________________________________________________________Antigelling AgentsAntigelling Agent        Examples    Reference__________________________________________________________________________Tri- and tetrapeptides        L--Phe--L--Phe--Arg                    Votano, J.R.; Gorecki, M.;                    Rich, A, Science, 196, 1216,                    1977.Aromatic compounds        Benzyl alcohol                    Ross, P.D. and Subramanian, S.                    Biochem. Biophys. Res. Comm. 77,                    1217, 1977.Aromatic amino acids        L--Phe      Noguchi, C.T. and Schechter,        L--Tyr      A.N., Biochem, Biophys. Res.                    Res. Comm. 74, 637,1977.Aromatic amino acids        L--Phe      Noguchi, C.T. and Schechter,and peptides             A.N., Biochemistry, 17,                    5455, 1977.Oligopeptides that mimic        Val--His--Leu--Thr--                    Kubota, S. and Yank, L.T.segments of the amino acid        Pro--Proc. Natl. Acad. Sci. (USA)sequence of HbS          74, 5431, 1977.__________________________________________________________________________ 
    
     
                       TABLE IV______________________________________Compounds Active as Antisickling Agents           Percentage of Normal Cells______________________________________1.  L--Phe--OBz.HCl   902.  *L--Tyr--OBz.HCl  703.  *L--Try--OBz.HCl  954.  L--Pro--OBz.HCl   605.  L--Phe--OMe        06.  L--Phe--OBz (pNO.sub.2).HBr                 907.  L--Pro--OBz (pNO.sub.2).HBr                 808.  L--His--OBz (pNO.sub.2) pTS                 709.  L--Ala--L--Phe--OBz.HBr                 9010. L--Gly--L--Phe--OBz.HBr                 9011. L--Phe--Benzyl amide                 7012. L--Val--OBz       5013. L--Leu--OBz       5514. L--Phe--L--Ala--OBz.HBr                 7015. L--Try--Lys--OBz.HBr                 7016. D--Phe--OBz.HCl   9017. L--Tyr--O Octadecyl.HCl                 1018. L--Phe             0______________________________________ *0.5 mM of compound was used for the incubation of cells. 
    
     Erythrocytes were incubated with 3 mM of the compound, and tested for 30 minutes. They were deoxygenated with sodium metabisulphite and examined under a Zeiss microscope. 
     Compounds No. 5, 17 and 18 are included for comparison and are not part of the invention.