BACKGROUND OF THE INVENTION Field of the Invention

The present invention relates to novel azo compound derivatives which exhibit anti-HIV activity, methods for synthesizing these azo compound derivatives, pharmaceutical compositions containing these azo compound derivatives and methods for treating HIV infections in mammals, especially humans, by administering these derivatives in a pharmacologically acceptable form. More particularly, the present invention is related to substantially pure azo compound derivatives of stilbenes and related compounds wherein the stilbene double bond is replaced with, for example, a methylene-, ethylene-, hetero atom-, amido-, ureido- or thioureido-linkage, as well as pharmacological uses and compositions thereof. Description of Related Art

Research has been conducted concerning a variety of compounds which potentially exhibit antiviral activity. For example, compounds which exhibit potential antiherpes activity include the dye Trypan blue (Alarcon et al, "Screening for New Compounds with Antiherpes Activity," Antiviral Res.. 4. (1984), pp. 231-243; and Thome et al , "Inactivation of Measles and Herpes Simplex Viruses by Tripan Blue," J. Gen. Virol. (1983), 64., PP. 1365-1368), as well as Indigocarnine and Paraorange (Westin et al, "Aromatic Sulfonic Acids as Inhibitors: Structure-Activity Study Using Rhino, Adeno 3, Herpes Simplex, and Influenza Viruses," J. of Med. Chem.. 1971, Vol. 14, No. 7, pp. 596-

600) . The dye Congo Red and derivatives thereof have been investigated for potential anti-AIDS activity (Mohan et al, "Potential Anti-AIDS Agents. Synthesis and Anti-viral Activity of Naphthalenesulfonic Acid Derivatives Against HIV-l and HIV-2," J. of Med. Chem.. 1991, 3_4, pp. 212-217), as have Suramin (De Clercq, Suramin in the Treatment of AIDS "Mechanism of Action," Antiviral Res.. 1, (1987), pp. 1-10) and Evans Blue (Scholes et al, "Flow Cytometric Method to Demonstrate Whether Anti-HIV-1 Agents Inhibit Virion Binding to T4* Cells," J. of Acquired Immune Deficiency Syndromes. (1989) , 2, pp. 10-15) . Also a number of azo dyes were demonstrated to exhibit protective properties in mice infected by equine encephalomyelitis virus (Hurst et al, Brit. J. Pharmacol., 2. p. 455 (1952)). Eric De Clercq in Anticancer Research, 2. p. 1023 (1987) and in the Int. J. Cancer, 37, p. 451 (1986) summarizes the perspectives for the chemotherapy of AIDS. These reviews mention two polyanionic dyes with proven anti-HIV activity, i.e. Evans Blue, an azo dye, which is available in 85% purity (Aldrich 1991) and aurintricarboxylic acid which has recently been shown to be a polymeric mixture, not a monomeric substance. Thus, some dye compounds have been shown to exhibit antiviral activity.

In 1883 Walter discovered and Geigy manufactured Sun Yellow CI 620, the first of a class of stilbene dyes which dye cotton (H.A. Lubs, The Chemistry of Synthetic Dyes and Pigments , page 119, Reinhold Publishing Co., 1955). A brief summary on this class of dyes is given in the "Colour Index. Third edition page 4365, volume 4, which states that, "The stilbene dyes are in most cases mixtures of dyes of indeterminate constitution which result from the condensation of 4-nitrotoluene-2-sulfonic acid (5-nitro-o- toluenesulfonic acid) in an aqueous caustic alkaline medium alone or with other aromatic compounds, usually arylamines. Azo and/or azoxy groups are probably the chromophores. The first products of the self condensation of 5-nitro-o- toluenesulfonic acid include 4,4'-dinitro-2,2*-

stilbenedisulfonic acid and 4,4 '-dinitrobibenzyl-2,2'- disulfonic acid which therefore form convenient starting points for the secondary condensations with arylamines. The largest and probably now the most important class of stilbene dyes consist of such secondary condensations in which the arylamine is an aminoazo compound. The constitutions and therefore the properties of the stilbene dyes vary with the proportions and concentrations of the reactants, and with the temperature and the duration of heating. Probably no two manufacturers use exactly the same conditions for any given dye."

Furthermore, the alkaline condensation of 4- nitrotulene-2-sulfonic acid may be carried in the presence of reducing agents such as glucose or oxidizing agents such as NaOCL. The agents may be added during or after the condensation (H.R. Schweizer, Kunstliche Orσanische Farbstoffe und ihre Zwischenprodukte. page 406, Springer Verlag, 1964). Reaction conditions, and references for the stilbene dyes have been tabulated (Colour Index, third edition, pages 4365-4373) . As stated by Zollinger, Color Chemistry, page 101, VCH 1987, except for the primary reaction product, 4,4 •-dinitrostilbene-2,2'-disulfonic acid, their structures are not known exactly. Even the simple stilbene dye, Hessian Yellow, i.e., the coupling product of one mole of diazotized 4,4'-diaminostilbene- 2,2'-disulfonic acid and 2 moles of salicylic acid possesses a dye content of only 50% when analyzed. (Chem. Abstr. 3_5, 6796) . The observation that a commercial sample of Direct Orange 15 exhibits anti-HIV properties i.e. EC ₅₀ of 2μg/ml suggested separation of the biologically active dye components via column chromatography, HPLC or centrifugal partition chromatography. This approach has been unsuccessful. Thus, it is difficult to obtain such dye compounds in pure form.

In view of the above, it is desirable to obtain azo compound derivatives which may potentially exhibit

biological activity such as antiviral activity, wherein the derivatives are in substantially pure form.

SUMMARY OF THE INVENTION It is therefore an object of the present invention to provide substantially pure azo stilbene and related compounds with potent anti-HIV activity.

It is a further object of the present invention to provide methods for synthesizing pure azo stilbenes and related compounds with potent anti-HIV activity.

It is another object of the present invention to provide a method of treating HIV infections by administering substantially pure azo stilbenes and related dyes.

It is yet a further object of the present invention to treat HIV infected humans with azo stilbene and related compounds.

It is still a further object of the present invention to provide pharmaceutical compositions containing azo stilbenes and related compounds.

The foregoing objects and other are accomplished in accordance with the present invention by providing substantially pure azo compound derivatives of the following formula:

wherein R is SOjH-E, C0 ₂ H* E or PO(OH) ₂ *E, N0 ₂ or halogen;

R ¹ , R ² , R ³ , R*, R ⁵ and R ⁶ are each independently H, NH _2/ NHCH _j , N(CH _j ) ₂ , OH, CH ₃ 0, S0 ₃ H-E, N0 ₂ , halogen, C0 ₂ H* E and PO(OH) ₂ -E;

X is -CHCH-, -CH ₂ CH ₂ -, -CONH-, -NHCONH-, -NHCSNH-, -S-, -SO-, -S0 ₂ -, -NH-, -CH ₂ - or cyclopropyl; and ring "A" is an optional benzene ring fused to the phenyl ring to provide a naphthyl ring system or an optional pyridine ring fused to the phenyl ring to provide a quinoline ring system; wherein E represents sodium, potassium, ammonium, magnesium, (HOC ₂ H ₃ N, (CH ₃ ) ₃ N, CH ₃ N(C ₂ H ₄ OH) ₂ , or N-methylglucamine.

The present invention further relates to pharmaceutical compositions containing the above-noted compounds of Formula 1 and appropriate pharmaceutically acceptable excipients. In another embodiment of the present invention, a method is provided for treating viral infections of a host by administering to the host an antiviral effective amount of a pharmaceutical composition containing the above-noted azo compound derivatives of Formula 1.

Further scope of the applicability of the present invention will become apparent from the detailed description and drawings provided below. However, it should be understood that the detailed description and

specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any method and materials similar or equivalent to those described therein can be used in the practice or testing of the present invention, the preferred methods and materials are now described. All publications mentioned hereunder are hereby incorporated herein by reference.

BRIEF DESCRIPTION OF THE DRAWINGS The invention is further illustrated in the accompanying drawings wherein:

Figures 1 and 2 are graphs showing .in vitro testing results for anti-HIV activity for Examples 1 and 13. DETAILED DESCRIPTION OF THE INVENTION The present invention relates to novel azo compound derivatives of the above-noted Formula 1. In order to form the novel azo compound derivatives of the present invention, azo compounds of structure 1 are prepared by reacting the requisite amine 2, with nitrous acid to give the corresponding arene diazonium ion 3.. Coupling of 3. at either acid or basic pH with the appropriate aromatic or heteroaromatic amine, hydroxy or methoxy compound 4. yields compounds of structure _L as shown in the reaction scheme below.

The azo compound derivatives of the present invention may be employed in pharmaceutical compositions and used for treating viral infections, such as Hepatitis B, HTLV-1, HTLV-II, HTLV-IV, HTLV-V, HIV-1, HIV-2, HIV-3 and HIV-4, herpes simplex I, herpes simplex II, herpes zoster, Epstein-Barr virus, cytomegalo viruses and influenza.

The azo compound derivatives employed in the present invention may be made into pharmaceutical compositions by combination with appropriate pharmaceutically acceptable carriers or diluents, and may be formulated into preparations in solid, semi-solid, liquid or gaseous forms such as tablets, capsules, powders, granules ointments.

solutions, suppositories, injections, inhalants, and aerosols in the usual ways for their respective route of administration. The following methods and excipients are merely exemplary and are in no way limiting.

In pharmaceutical dosage forms, the azo compound derivatives employed in the present invention may be used in the form of their pharmaceutically acceptable salts, and also may be used alone or in appropriate association, as well as in combination with other pharmaceutically active compounds.

In the case of oral preparations, the azo compound derivatives may be used alone or in combination with appropriate additives to make tablets, powders, granules or capsules, e.g. with conventional additives such as lactose, mannitol, corn starch or potato starch; with binders such as crystalline cellulose, cellulose derivatives, acacia, corn starch or gelatins; with disintegrators such as corn starch, potato starch or sodium carboxymethylcellulose; with lubricants such as talc or magnesium stearate; and if desired, with diluents, buffering agents, moistening agents, preservatives and flavoring agents.

Furthermore, the azo compound derivatives employed in the present invention may be made into suppositories by mixing with a variety of bases such as emulsifying bases or water-soluble bases.

The azo compound derivatives employed in the present invention may be formulated into preparations for injections by dissolving, suspending or emulsifying them in an aqueous or non-aqueous solvent, such as vegetable oil, synthetic aliphatic acid glycerides, esters of higher aliphatic acids or propylene glycol; and if desired, with conventional additives such as solubilizers, isotonic agents, suspending agents, emulsifying agents, stabilizers and preservatives.

In the cases of inhalations or aerosol preparations, the azo compound derivatives employed in the invention in the form of a liquid or minute powder may be filled up in

an aerosol container with gas or liquid spraying agents, and if desired, together with conventional adjuvants such as humidifying agents. They may also be formulated as pharmaceuticals for non-pressured preparations such as in a nebulizer or an atomizer.

The amount of the azo compound derivatives employed in the present invention to be used varies according to the degree of the infection encountered, and the stages of the disease. A suitable dosage is that which will result in concentration of the azo dye derivative in blood and/or tissues harboring virus which are known to inhibit the virus, e.g. about 1 x 10 ^* * M to 1 x 10 ^"6 M, more preferably 1 x 10' ⁵ M to 1 x 10 ^"6 M. The preferred dosage is that amount sufficient to beneficially affect a host and possibly render the host asymptomatic to the particular viral infection. The dose may vary when the compounds are used prophylactically.

Unit dosage forms for oral administration such as syrups, elixirs, and suspensions wherein each dosage unit, e.g., teaspoonful, tablespoonful, contains a predetermined amount of the azo compound derivatives employed in the present invention can be by a pharmaceutically acceptable carrier, such as Sterile Water for Injection, USP, or by normal saline.

The azo compound derivatives employed in the present invention can be administered rectally via a suppository. The suppository can include vehicles such as cocoa butter, carbowaxes and polyethylene glycols, which melt at body temperature, yet are solidified at room temperature.

The azo compound derivatives employed in the present invention can be utilized in aerosol formulation to be administered via inhalation. The azo compound derivatives employed in the present invention can be formulated into pressurized acceptable propellants such as dichlorodifluororaethane, propane, nitrogen and the like.

The term "unit dosage form" as used herein refers to physically discrete units suitable as unitary dosages for

human and animal subjects, each unit containing a predetermined quantity of the azo compound derivatives calculated in an amount sufficient to produce the desired effect in association with a pharmaceutically acceptable, diluent, carrier or vehicle. The specifications for the novel unit dosage forms of the present invention depend on the particular compound employed and the effect to be achieved, and the pharmacodynamics associated with each compound in the host.

The pharmaceutically acceptable excipients, for example, vehicles, adjuvants, carriers or diluents are readily available to the public.

Any necessary adjustments in dose can be readily made to meet the severity of the infection and adjusted accordingly by the skilled practitioner.

EXAMPLES Materials and Methods for Synthesizing Azo Compounds

Melting points were determined with a ofler hot stage microscope and are corrected. All temperatures are in degrees centigrade. NMR spectra were determined with either a Bruker WM-300 or Varian XL-400 spectrometer, chemical shifts are in ppm relative to TMS (0.00). Mass spectra were recorded on a Varian-Mat 112S or a VG ZAB spectrometer. Preparative centrifugal TLC was done on a Harrison Research Model 7924T (Chromatotron) . The azo compounds of structure 1 are prepared by reacting the requisite amine 2. with nitrous acid to give the corresponding arene diazonium ion 3_.

Coupling of 2 at either acid or basic pH with the appropriate aromatic or heteroaromatic amine, hydroxy, or ethoxy compound 4. yields compounds of structure 1. Generally, the conditions for coupling compound 3_ with compound 4. include chilling the freshly prepared diazonium salt solution or suspension to 10-15"C and then with rapid stirring adding it rapidly to the appropriate aromatic or heteroaromatic amine, hydroxy or methoxy compound 4.. The mixture is then stirred for a period of about two hours at ice bath temperature and then for about three hours at room temperature. The resulting solid is then filtered off, washed with ice water and repeatedly purified.

Reaction conditions for these transformations can be found in text books such as Fieser's Experiments in Organic Chemistry, third edition, Heath and Co., 1957, Vogel's Textbook of Practical Organic Chemistry, third edition, J. Wiley, 1966, Barton's Comprehensive Organic Chemistry. Volume 2, page 154-157, Perga on Press, 1979 and the like. Catalytic reduction of 4,4'-diaminostilbene-2,2'-disulfonic acid will furnish the requisite intermediate 2 for compounds 1, wherein X is -CH ₂ CH ₂ - using standard procedures. References for this transformation are compiled in Barton's Comprehensive Organic Chemistry volume 1, page 166, Pergamon Press, 1979.

Sodium, potassium and ammonium salts of 1. are water soluble, but the calcium, barium and lead analogs are much less so and often insoluble. Salts with organic cations such as the S-benzylthiouronium ion have frequently defined melting points and may be used to characterize the acids whose melting points are usually ill defined. Further details regarding the sale formation may be found in Houben-Weyl's Methoden der Organischen Chemie. Vol 9, chapter 16, Thie e Verlag, 1955.

Pure compounds of structure 1 are obtained by subjecting the crude reaction mixture to centrifugal partition chromatography which upon repeated cycles will furnish spectrally pure dye. The following examples are provided for illustration purposes only without departing from the spirit or scope of the invention. Example 1

A general procedure for diazotization and coupling which may be adapted and used to synthesize the azo dye compounds of the present invention is disclosed in Organic Synthesis Col. Vol. 2, pp. 145-150. The compound of Example 1 was synthesized as described below.

A. Diazotization of 4, '-diaminostilbene-2,2 '-disulfonic acid

To a stirred suspension of 4,4 '-diaminostilbene-2,2 ^• - disulfonic acid (1.852 g, 5.0 mmole) in water (15 mL) concentrated hydrochloric acid (1 mL, 11.5 mmole) was added dropwise. The mixture was stirred, warmed to 50"C and then cooled to 10*C. An additional portion of concentrated hydrochloric acid (1.05 L, 12.0 mmole) was added followed by rapid addition of sodium nitrite (0.704 g, 10.2 mmole) solution in water (2 L) . A yellow mixture was stirred at 10-15'C for 30 min., to give a suspension of diazonium salt.

B. Coupling of diazonium salt with 8-hydroxyquinoline-5- sulfonic acid

To 8-hydroxyquinoline-5-sulfonic acid (2.481 g, 10.2 mmole) in water (35 L) an aqueous solution of sodium hydroxide (0.400 g., 10.0 mmole in 1.5 mL) followed by anhydrous sodium carbonate (1.750 g, 16.5 mmole) were added. The solution was cooled to 10*C and vigorously stirred. The diazonium salt suspension then was rapidly run into the prepared yellow solution. The color of the reaction mixture quickly changed to brown and then to burgundy, and the dye began to precipitate. The mixture was stirred in an ice bath for 2 hours and an additional 5 hours at room temperature. A thick paste was filtered off under reduced pressure, washed with small amount of water followed by 95% ethanol and dried, giving a greenish-black solid (4.280 g) in 92% yield. This material was redissolved in water (about 200 mL) with heating to 70- 80'C, stirred and then salted off using hydrated sodium acetate (approx. 40 g) . After cooling the solid was filtered off under reduced pressure, thoroughly washed with copious amounts of ethanol and dried, giving the dye in yield of 83% (3.550 g) . The product was once again redissolved in water, freeze dried and then dried under reduced pressure at 110*C for 24 h to give a dye of Example

1, i.e. 2,2'-stilbenedisulfonic acid, 4, '-bis[ (8-hydroxy- 5-sulfo-7-quinolinyl)azo]-, tetrasodium salt. Its substituents, their position of attachment, biological activity, and type of salt are shown in Table 1 under Example 1.

The melting point of Example 1 is over 300'C. TLC

(silica gel, n-BuOH-Py-NH _t OH-H ₂ 0 = 3.5:2:3:1.5) R.0.45. 1H

NMR (DMSO-d ₆ ) δ (ppm) : 9«13 (dd, J=8.7; 1.5 Hz, 2H, H-a) ;

8.94 (dd, J=4.2; 1.5 Hz, 2H, H-c); 8.38 (s, 2H, H-h); 8.37

(d, J-2.1 Hz, 2H, H-e) ; 8.31 (ε, 2H, H-d) ; 8.04 (dd, J=8.7;

2.1 Hz, 2H, H-f) ; 7.88 (d, J=9.0 Hz, 2H, H-g) ; 7.71 (dd,

J=8.7; 4.2 Hz, 2H, H-b) ; and 3.33 HOD. UV (H ₂₀ ) *„_ (nm) :

210, 322, 544 (log β 4.94, 4.62, 4.93). Elemental analysis: C__H ₁₈ N ₆ 0 _li S _, Na ₁ ' 5H ₂ 0 calc. found

%C 37.65 37.61 %H 2.76 2.78 %N 8.23 8.18

%S 12.56 12.44

IR (KBr) v. _ιx (cm ¹ ): 3442, 1635, 1627, 1588, 1577, 1497,

1315, 1194, 1117, 1075, 1049, 1025, 954, 799, 689, 680, 623 and 588.

Example 1

Examples 2-12

Following the procedures of Example 1 and coupling diazonium salt 2 with the appropriate amine-, hydroxy-, or methoxyquinoline of structure 4. the compounds of Examples 2-12 as shown in Table 1 are obtained. Examples 13-25

Following the procedures of Example 1 and coupling diazonium salt 2 with the appropriate amine-, hydroxy-, or methoxybenzene of structure 4. the compounds of Examples 13- 25 as shown in Table 2 are obtained. The structure of Example 13, i.e. 2,2 '-stilbenedisulfonic acid, 4,4'-bis[(2- carboxy-3,6-dihydroxyphenyl)azo]-, tetrasodium salt, is indicated below.

Examples 26-42

Following the procedures of Example 1 and coupling diazonium salt 2 with the appropriate amine-, hydroxy- or methoxynaphthalene of structure 4. the compounds of Examples 26-42 as shown in Table 3 are obtained. The structure of Example 26 is indicated below.

TABLE 1

Ex. Formula 3 Formula 4. Ring System: Quinoline

$ Postion of diazonium group

* Position

** Position of Coupling

*** 50% effective concentration for jln vitro anti-HIV activity

SUBSTITUTESHEET

TABLE 1 (Cont'd)

Ex. Form. 1

Pos. of Coupling** Salt EC ₅₀ M ***

1 7

2 5

3 7

4 7

5 7

6 7

7 7

8 7

9 7

10 7

11 5

12 5

$ Postion of diazonium group

* Position

** Position of Coupling

*** 50% effective concentration for in vitro anti-HIV activity

SUBSTITUTESHEET

TABLE 2

$ Position of diazonium group

* Position

** Position of Coupling

*** 50% effective concentration for jLn vitro anti-HIV activity

SUBSTITUTE SHEET

TABLE 2 (Cont ' d)

Ex. Form. 1

Pos. of Cplng** Salt Ec ₅₀ M***

÷ Position of diazonium group

* Position

** Position of Coupling

*** 50% effective concentration for in vitro anti-HIV activity

SUBSTITUTE SHEET

X Postion of diazonium group * Position

SUBSTITUTE SHEHT

TABLE 3 (Cont'd)

Ex. Form. 1

Pos. of

Cplng. Salt

Na

Na

Na

Na

Na

Na

Na

Na

Na

Na

Na

Na

Na

Na

Na

Na

Ca NH.,

X Postion of diazonium group * Position

TABLE 3 (cont'd)

^* 50% effective concentration for in vitro anti-HIV activity

In Vitro Anti-HIV Testing

The procedure used in the National Cancer Institute's test for agents active against Human Immunodeficiency Virus (HIV) (Weislow, O.W. et al, "New soluble-formazan assay for HIV-1 cytopathic effects: application to high-flux screening of synthetic and natural products for AIDS- antiviral activity," J. Natl. Cancer Inst.. £1:577-586, 1989) is designed to detect agents acting at any stage of the virus reproductive cycle. The assay basically involves the killing of T4 lymphocytes by HIV. Small amounts of HIV are added to cells, and a complete cycle of virus reproduction is necessary to obtain the required cell killing. Agents that interact with virions, cells, or virus gene-products to interfere with viral activities will protect cells from cytolysis. The system is automated in several features to accommodate large numbers of candidate agents and is generally designed to detect anti-HIV activity. However, compounds that degenerate or are rapidly metabolized in the culture conditions may not show activity in this screen. All tests are compared with at least one positive (e.g., AZT-treated) control done at the same time under identical conditions. The Procedure:

1. Candidate agent is dissolved in dimethyl sulfoxide (unless otherwise instructed) then diluted 1:100 in cell culture medium before preparing serial half-log. ₀ dilutions. T4 lymphocytes (CEM cell line) are added and after a brief interval HIV-1 is added, resulting in a 1:200 final dilution of the compound. Uninfected cells with the compound serve as a toxicity control, and infected and uninfected cells without the compound serve as basic controls.

2. Cultures are incubated at 37 ^* in a 5% carbon dioxide atmosphere for 6 days.

3. The tetrazoliu salt, XTT, is added to all wells, and cultures are incubated to allow for azan color development by viable cells.

4. Individual wells are analyzed εpectrophotometrically to quantitate formazan production, and in addition are viewed microscopically for detection of viable cells and confirmation of protective activity.

5. Drug-treated virus-infected cells are compared with drug-treated noninfected cells and with other appropriate controls (untreated infected and untreated noninfected cells, drug-containing wells without cells, etc.) on the same plate.

6. Data are reviewed in comparison with other tests done at the same time and a determination about activity is made.

Figures 1 and 2 display a plot of the log ₁₀ of the concentrations (as μg/mL or molar) of the Examples 1 and 13 compounds, respectively, against the measured test values expressed as a percentage of the uninfected, untreated control values. The solid line connecting the diamond symbols depicts the percentage of surviving HIV-infected cells treated with your sample (at the indicated concentration) relative to uninfected, untreated controls. This line expresses the in vitro anti-HIV activity of the Example. The dashed line connecting the triangular syrnbols depicts the percentage of surviving uninfected cells treated with the Example relative to the same uninfected, untreated controls. This line expresses the in vitro growth inhibitory properties of the Example. The viral cytopathic effect in this particular experiment is indicated by a dotted reference line. This line shows the extent of destruction of cells by the virus in the absence of treatment and is used as a quality control parameter. Survival values of this parameter less then 50% are considered acceptable in the current protocol. The percent of protection has been calculated from the data and is presented on the right side of the graph.

Approximate values for 50% effective concentration (EC ₅₀ ) have been calculated for each test and are provided for most Examples in Tables 1-3.

The invention being thus described, it will be obvious that the same may be varied in many ways. Such variations are not to be regarded as a departure from the spirit and scope of the invention, and all such modifications as would be obvious to one skilled in the art are intended to be included within the scope of the following claims.