The present invention relates to a process for the preparation of derivatives of Monosaccharides as novel Cell Adhesion Inhibitors. The compounds of this invention are useful for inhibition and prevention of cell adhesion and cell adhesion mediated pathologies including inflammatory and autoimmune diseases such as bronchial asthma, rheumatoid arthritis, type I diabetes, multiple
sclerosis and psoriasis.
This application is a divisional application out of the applecation No.

3108/Del/98 filed on 22/10/98
This invention also relates to pharmaceutical composition containing the
compounds of the present invention and the methods of treating bronchial asthma, rheumatoid arthritis, multiple sclerosis, type I diabetes, psoriasis, allograft rejection, and other inflammatory and/or autoimmune disorders, using the compounds.
Cell adhesion is a process by which cells associate with each other, migrate towards a specific target or localize within the extra-cellular matrix. These interactions are mediated by specialized molecules called cell adhesion molecules (CAM). CAMs have been demonstrated to participate in various cell-cell, cell-extracellular matrix, and platelet interactions. They influence the adhesion of leukocytes to the vascular endothelium, their transendothelial migration, retention at extravascular sites and activation of T cells and eosinophils. These processes are central to the pathogenesis of inflammatory and autoimmune diseases. Therefore, adhesion molecules are considered as potential targets to treat such disorders.
CAMs can be classified into three groups - integrins, selectins and the immunoglobulin superfamily. Out of these, integrins are key mediators in the adhesive interactions between hemopoietic cells and their microenvironment. They comprise of alpha-beta heterodimers and integrate signals from outside of the cells to inside and vice versa. Integrins can be classified on the basis of the beta subunits they contain. For example, beta-1 subfamily contains beta-1 subunit noncovalently linked to one of the 10 different alpha subunits.
The alpha-4 beta-1 integrin, also known as VLA4 (very late activation antigen 4),
is a member of beta 1 integrin family and consists of alpha-4 and beta-1 subunits. It interacts with two specific ligands - the vascular cell adhesion molecule (VCAM-1) and the the CS1 region of fibronectin. Adhesion mediated by
VUA4 is central to the process of transendothelial migration of leukocytes. Ligation of VLA4 is followed by gross rearrangement of the cytoskeleton leading
to flattening of cells along the blood vessel wall followed by expression of specific
molecules which digest the endothelial cell wall and diapedesis. Once in the extraluminal region, the interactions of VLA4 with extracellular fibronectin play a
crucial role in migration to the site of inflammation, T cell proliferation, expression of cytokines and inflammatory mediators. In addition, VLA4 ligation provides
costimulatory signal to the leukocytes resulting in enhanced immunoreactivity. Therefore, it is expected that VLA4 antagonists would ameliorate the immune
response through twofold actions - inhibition of T cell recruitment at the site of inflammation and inhibition of costimulatory activation of immune cells.
In support of this concept, inhibitors of VLA4 interactions have demonstrated
beneficial therapeutic effects in several animal models of inflammatory, and allergic diseases including sheep allergic asthma (Abraham et al. J. Clin. Invest.. 93, 776 (1994)), arthritis (Wahl et al, J. Clin. Invest. 94, 655 (1994)); experimental allergic encephomyelitis (Yednock et al, Nature (Lond). 356. 63 (1992) and Baron et al, J. Exp. Med.. 177. 57 (1993)); contact hypersensitivity (Chisolm et al, Eur J. Immunol.. 23,682 (1993)); type I diabetes (Yang et al, Proc. Natl. Acad. Sci. (USA), 90, 10494 (1993)) and inflammatory bowel disease (Podolsky et al, J. Clin. Invest.. 92. 372K1993).
Region of CS1 moiety of fibronectin involved in the interaction with VLA4 was
identified as the tripeptide Leu-Asp-Val. also known as LDV (Komoriya et al, J. Biol. Chem. 266. 15075(1991)). Taking a lead from this, several peptides
containing the LDV sequence were synthesised which have shown to inhibit the in vivo interaction of VLA4 to its ligands. (Ferguson et al, Proc. Natl. Acad.
Sci.,(USA), 88, 8072 (1991); Wahl et al, J. Clin. Invest.. 94. 655(1994); Nowlin et al. J. Biol. Chem.. 268(27). 20352(1993) and PCT publication WO91/4862).
Despite these advances, there remains a need for small and specific inhibitors of VLA4 dependent cell adhesion molecules. Ideally such inhibitors should be water
soluble with oral efficacy. Such compounds would provide useful agents for
treatment, prevention or suppression of various inflammatory pathologies mediated by VLA4 binding.
The main objective of the present invention is to provide a process for the synthesis of a new class of compounds that exhibit significant activity as VLA4
antagonists.
Thus the compounds of the present invention were screened for inhibitory activity in VLA4 mediated cell adhesion assay and the classical murine hypersensitivity
assay in mice. Several compounds exhibited significant inhibitory activity in both these tests. The salts of these compounds could be easily solubilised in water and used in treatment of chronic, cell adhesion mediated, allergic, autoimmune and inflammatory disorders such as bronchial asthma and rheumatoid arthritis. Some of the prior art describes development of peptide derivatives as cell adhesion antagonists for treatment of these diseases. However, because treatment of chronic diseases requires prolonged (mid term to long term) administration of drugs, development of small molecule, specific, orally available inhibitors of cell adhesion would be very beneficial.
Isopropylidene and benzylidene groups are the most commonly used protective groups in carbohydrate chemistry.These groups are introduced into a molecule under similar conditions; however, the location of the protection can be quite different. The reason for this difference is directly related to the stability of each protected molecule. Since protection normally occurs under conditions which allow reversibility, reaction proceeds until equilibrium is reached. The distribution of products at equilibrium is determined by their relative thermodynamic stabilities. In other words, these reactions are thermodynamically controlled. Benzylidene groups prefer to be part of six-membered ring acetals, while the ketals resulting from acetonation generally are 5-membered rings. The difference is attributed to the effect of the methyl and phenyl substituents on the stability of the particular ring systems. These blocking methods are described in the U.S. Pat. Nos. 2,715,121; 4,056,322; 4,735,934; 4,996,195; and 5,010,058 the disclosures of which are incorporated herein by reference. Other blocking methods are also described in J. Carbohydr. Chem., 4,227(1985); 3,331(1984); Methods in Carbohydr. Chem.1, 191 (1962); 1, 107 (1962); Can J. Chem., 62, 2728 (1984); 47, 1195, 1455 (1969) ; 48, 1754 (1970) all incorporated herein by reference. Literature reveals that in the case of D-glucose, which is blocked in its furanose ring structure, the 1,2- and 5,6-hydroxyl groups can be blocked using with isopropylidene or cyclohexylidene blocking group with the 3-position left
open to undergo derivatization. The therapeutic activity of hexoses and their derivatives are also disclosed in some of the above applications.
Intermediates mentioned in U.S.Pat. Nos. 4,996,195; 5,637,570; 5,367, 062; 5,360,794; 5,360,792; 5,298,494 and 5,010,058 were used as core nucleus and were prepared similarly as described in these patents.However, in the present application, it has been discovered, that the introduction of urea moiety at various positions of pentose and hexose monosaccharides introduces VLA4 antagonism
activity. It was also discovered that the tetrapetide sequence LDVP (leucyl-aspartyl- valyl- prolyl) or any other amino acid, dipeptide or tripeptide as present
in fibronectin is not necessary for the compounds to be active as inhibitors of VLA4.
Other objects and advantages of the invention will be set forth in the description which follows, and in part will be apparent from the description, or may be learned by the practice of the invention. The objects and the advantages of the invention may be realised and obtained by means of the mechanisms and combinations pointed out in the appended claims.
In order to achieve the above mentioned objects and in accordance with the purpose of the invention as embodied and broadly described herein, there is provided a process for the preparation of monosaccharide derivatives having the general formula III as shown in the accompanied drawings,
The term "Pharmaceutically acceptable salts" refer to derivatives of the disclosed compounds of Formula III are modified by making its acid or base salts. Examples of pharmaceutically acceptable salts include, but are not limited to, mineral or organic acidic residues such as carboxylic acids; and the like.
A process for the preparation of derivatives of monosaccharides as novel cell adhesion inhibitors of compounds of Formula III (as shown in the accompanied drawings) and it's pharmaceutically acceptable salts,
which comprises reacting 2,3;4,6-Di-O-isopropylidene a,L-xylo-2-hexulofurasonic acid of Formula XV with LD (Obzl) VP (Obzl) followed by hydrogenation with Pd/C at the temperature ranging from 20-80°C to give compounds of FormulaXXI
wherein in COLDVP represents tetrapeptide (Leucyl-aspartyl-valyl-prolyl) and selective hydrolysis with perchloric acid at the temperature ranging from -4°C - 20°C to give the compounds of the Formula III.
Soluble salts such as TRIS, sodium, potassium, ammonium etc. are prepared so as to solubilise the compound in aqueous medium for biological evaluations are also included in the present invention.
In the above synthesis, where specific acids, bases, solvents reducing agents etc are mentioned, it is to be understood that the other acids, bases, solvents, reducing etc. may be used. Similarly the reaction temperature and duration of the reaction may be adjusted according to the need.
An illustrative list of particular compounds according to the invention include:
Compound Chemical Name
No.
1. 2,3;4,6-Di-O-isopropylidene-1-carbonyl-L-Leucyl-,L-Aspartyl-L-Valyl-L-Proline-,L-
xylo 2-hexulofuranosonic acid
2. 2,3;4,6-Di-O-isopropylidene-1-carbonyl-,L-Aspartyl-L-Valyl-L-Proline-,L-xylo-2-
hexulofuranosonic acid
3. 2,3-O-isopropylidene-1-carbonyl-L-Leucyl-a,L-Aspartyl-L-Valyl-L-Proline-,L-xylo-2-
hexulofuranosonic acid
4. 2,3-O-isopropylidene-1-carbonyl-,L-Aspartyl-L-Valyl-L-Proline-,L-xylo-2-
hexulofuranosonic acid
5. 2,3-O-isopropylidene-1-carbonyl-L-Valyl-L-Proline-,L-xylo-2-hexulofuranosonicacid
The examples mentioned below demonstrate the general synthetic procedure as well as the specific preparation for the preferred compound. The examples are given to illustrate the details of the invention and should not be constrained to limit the scope of the present invention.
Various solvents such as acetone, methanol, pyridine, ether, tetrahydrofuran, Hexanes, dichloromethane were dried using various drying reagents following the procedure as described in the literature. Wet solvents gave poor yields of the products and intermediates. IR spectra were recorded as nujol mulls or a thin neat film on a Perkin Elmer Paragon instrument. NMR (H, C) were recorded on a varian XL-300 MHz instrument using tetramethylsilane as an internal standard. CIMS were obtained on a Finnigan MAT-4510 mass spectrometer eqquipped with an INCOS data system. Generally a direct exposure probe was used and methane was used as a reagent gas ( 0.33 mm Hg, 120 ^C source temp.)
EXAMPLE 1
Preparation of 2,3-O-isopropylidene-1-carbonyl-L-Leucyl-,L-Aspartyl-L-Valyl-LProline- ,L-xylo-2-hexulofuranosonic acid (Compound No. 3)
Step-7:2,3;4,6-Di-O-isopropylidene-1-carbonyl-L-Leucyl-,L-Aspartyl-L-Valyl-L-Proline- ,L-xylo-2-hexulofuranosonic acid
To a suspension of 1-(3-dimethylaminepropyl)-3-ethylcarbodimide (1.15 gm) and 1-hydroxybenzotriazolehydrate (0.76 gm) in DMF (10 ml) was added triethyl amine (1.5 gm) at room temp, and the reaction mixture was stirred for about 30 min., To this was added the acid 2,3-0-isopropylidene-1-O-dodecyl-5-deoxy-5-N-{[4-(2-hydroxy-2-oxoethyl)phenyl]aminocarbonylamino}-,D-lyxofuranoside (1.46 gm) and Leu-Asp(obzl)Val-Pro(obzl) HCI and stirred for about 24 hrs.The reaction mixture thus obtained was poured into water, extracted with methylene
chloride, washed the methylene chloride layer with saturated sodium bicarbonate, water and brine. Dried the organic layer over anhydrous Na2SO4
and solvent was removed under reduced pressure to get white foamy solid which was used for the next step without any purification.
To a solution of above benzyl ester (3.6 gm) in ethylacetate (70 ml) was added Pd/C (2.0 gm) at room temperature and subjected to hydrogenation using parr shaker for about 5 hrs. Filtered the catalyst , evaporated ethylacetate under
vacuum to obtain a white solid in 91% yield. The product was recrystallized from ethylacetate hexane.
The compounds prepared similarly were as follows :
2,3;4,6-Di-O-isopropylidene-1-carbonyl-,L-Aspartyl-L-Valyl-L-Proline-,L-xylo-2-hexulofuranosonic acid (Compound No. 1)
2,3;4,6-Di-O-isopropylidene-1-carbonyl-L-Valyl-L-Proline-,L-xylo-2-hexulofuranosonic acid (Compound No. 2)
Step-2 : 2,3-O-isopropylidene-1 -carbonyl-L-Leucyl-,L-Aspartyl-L-Valyl-L-Proline-,L-xylo-2-hexulofuranosonic acid
To a solution of the compound (0.5 gm) obtained in step 1 in THF (10 ml) was added a 30 % solution of perchloric acid (0.5 ml) dropwise at 0-5°C and reaction mixture was stirred at this temp, for about 4 hrs. The reaction was quenched with a saturated solution of potassium carbonate, the solid salt formed was filtered out and discarded. The solvent from the filtrate was evaporated under
vacuum completely and the residue was dissolved in ethyl acetate washed with water and brine. The organic layer was dried over anhydrous Na2SO4 and
solvent was removed to get a low melting solid in 90% yield.
The compounds prepared similarly were as follows 2,3-O-isopropylidene-carbonyl-,L-Aspartyl-L-Valyl-L-Proline-,L-xylo-2-hexulofuranosonic acid (Compound No. 4)
2,3-O-isopropylidene-1-carbonyl-L-Valyl-L-Proline-,L-xylo-2-hexulofuranosonic acid (Compound No. 5)

We Claim:
1. A process for the preparation of derivatives of monosaccharides as novel
cell adhesion inhibitors of compounds of Formula III (as shown in the
accompanied drawings) and it's pharmaceutically acceptable salts,
which comprises reacting 2,3;4,6-Di-O-isopropylidene a,L-xylo-2-hexulofurasonic acid of Formula XV with LD (Obzl) VP (Obzl) followed by hydrogenation with palladium on carbon at the temperature ranging from 20-80°C to give compounds of Formula XXI wherein in COLDVP is defined as tetrapeptide (Leucyl-aspartyl-valyl-prolyl) and selective hydrolysis with perchloric acid at the temperature ranging from -4°C- 20°C to give the compounds of the Formula III.
2. The process for the preparation of derivatives of monosaccharides of
compound of Formula III, substantially as herein described and illustrated
by the examples herein.