Abstract:
The present invention is directed to the field of organic chemistry in general and specifically to the preparation of hydrophobic derivatives of cyclic ADP ribose. One form of the present invention is the composition of one or more hydrophobic derivatives of cyclic ADP ribose. In another form of the present invention, a method for preparing a hydrophobic composition is described. Compositions of the present invention are useful for the study of in vivo calcium metabolism.

Description:
This application claims priority from U.S. Provisional Patent Application Ser. No. 60/299,556, filed Jun. 20, 2001. 
    
    
     FIELD OF THE INVENTION 
     The present invention is directed to the field of organic chemistry in general and specifically to the preparation of hydrophobic derivatives of cyclic ADP ribose. 
     BACKGROUND OF THE INVENTION 
     Intracellular calcium plays key roles in stimulation-secretion coupling in pancreatic islet β-cells. The elevation of cellular cytosolic calcium concentration ([Ca 2+ ] c ) is mediated through two pathways: Ca 2+  release from intracellular calcium stores and Ca 2+  influx from extracellular medium. The mechanisms underlying internal calcium release in β-cells remain incompletely understood, and the relative contribution of intracellular Ca 2+  release to the overall [Ca 2+ ] c  increase and subsequent insulin secretion needs to be determined. 
     Ca 2+  release from intracellular stores is an important signaling mechanism for a variety of cellular processes and is generally controlled by two systems, the IP 3  and cADPR systems (FIG.  1 ). IP 3  acts directly on the IP 3  receptor (IP3R) localized in the endoplasmic reticulum (ER). IP3R forms the Ca 2+  releasing channel and regulates the efflux of Ca 2+  from the ER to the cytosol. Cyclic ADP ribose increases the opening probability of other intracellular Ca 2+  releasing channel formed by the ryanodine receptor (RyR) in the ER. 
     Ca 2+  influx through voltage gated Ca 2+  channels is a well-characterized phenomenon in β-cells, and it is thought to play an important role in maintaining Ca 2+  homeostasis, especially during glucose stimulation. However, contributions from internal calcium release cannot be ignored. Ca 2+  influx from extracellular sources and Ca 2+  release from the intracellular pool in human β-cells has been examined, and showed that 42-75% of the increase in intracellular Ca 2+  by glucose stimulation was due to the release of Ca 2+  from the intracellular stores. Both IP 3  and cADPR signaling systems have been reported in insulin secreting β-cells, but controversies remain regarding which system is more important for maintaining proper insulin secretion responses. 
     To examine IP 3  or cADPR induced Ca 2+  release in β-cells, it is necessary to deliver these second messengers inside cells and assay their effects on cellular calcium homeostasis and insulin secretion. Methods relying on triggering cell surface receptors to produce endogenous IP 3  or cADPR inevitably activating other signaling pathways, making it impossible to separate the effects caused by IP 3  or cADPR from those caused by other signaling branches. To deliver exogenous IP 3  or cADPR inside cells, one need to overcome the difficulty of getting them across cell membranes. Both IP 3  and cADPR are charged and hydrophilic molecules at physiological pH, thus are membrane impermeant. Previous techniques of getting these two molecules across hydrophobic cell membranes include microinjection, patch clamping, electroporation or detergent assisted permeabilization. All these methods are invasive and suffer from major drawbacks such as disrupting intact cell membranes, letting cytosolic factors leak out of cells, and compromising long term viability of cells. In addition, techniques such as microinjection or patch clamping can only be applied to single cells, making it practically impossible to study more physiological preparations such as islets. 
     SUMMARY OF THE INVENTION 
     One form of the present invention is a hydrophobic compound of the general formula:                           
     where R 1 , R 2 , R 3  and R 4  are each independently hydrogen or linear or branched alkyl groups having from 1 to 12 carbon atoms. R 5  and R 6  are each an alkyl group, metallic cation, a photo-labile caging group, or an acyloxymethylgroup or a homologue thereof. W is CH 2 , CF 2 , or CHF. X is N or CH. Y is N or CH. Z is chosen from the group including H, Br, NH 2 , OCH 3 , CH 3  and N 3 . 
     Another form of the invention is a method for preparing a hydrophobic composition comprising the following steps:                           
     where RO and R′O comprise independently in each location carboxylate groups further comprising from 2 to 20 carbon atoms. 
    
    
     BRIEF DESCRIPTION OF THE FIGURE 
     The above and further advantages of the invention may be better understood by referring to the following detailed description in conjunction with the accompanying drawings in which corresponding numerals in the different FIGURES refer to the corresponding parts in which: 
     FIG. 1 depicts aspects of calcium metabolism in accordance with the present invention; 
     FIG. 2 depicts a pathway in accordance with the present invention; 
     FIG. 3 depicts an 8-amino cADPR (left) and a hydrophobic derivative of cyclic ADP ribose (right) in accordance with the present invention; 
     FIG. 4 depicts a pathway of the cellular delivery of phosphate- or phosphanate-containing compounds in accordance with the present invention; 
     FIG. 5 depicts methanodiphosphonate alanogues of pyrophosphates and neutral esters in accordance with the present invention; 
     FIG. 6 depicts a synthetic scheme for preparing a photocaged and hydrophobic derivative of cyclic ADP ribose in accordance with the present invention; 
     FIG. 7 depicts another synthetic scheme for preparing hydrophobic derivative of cyclic ADP ribose in accordance with the present invention; and 
     FIG. 8 depicts the synthesis of an ester of a methane-diphosphonate derivative of cyclic ADP ribose. 
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     While the making and using of various embodiments of the present invention are discussed herein in terms of organic chemistry, it should be appreciated that the present invention provides many inventive concepts that may be embodied in a wide variety of specific contexts. The specific embodiments discussed herein are merely illustrative of ways to make and use the invention are not meant to limit the scope of the present invention in any way. 
     Terms used herein have meanings as commonly understood by a person of ordinary skill in the areas relevant to the present invention. Terms such as “a,” “an,” and “the” are not intended to refer to only a singular entity, but include the general class of which a specific example may be used for illustration. The terminology herein is used to describe specific embodiments of the invention, but their usage does not limit the invention, except as outlined in the claims. 
     Novel techniques to prepare hydrophobic derivatives of phosphate-containing molecules including cADPR have been developed. These hydrophobic derivatives are expected to diffuse across cell membranes of fully intact cells and regenerate their parent molecules by cellular esterase hydrolysis. Photo-chemical uncaging techniques may also be used to activate these molecules with desired temporal and spatial precision. A “caged” molecule is masked by a photo-labile protecting group, and is thus biologically inactive. Photolysis with a flash of UV light (“uncaging”) removes the photo-labile protecting group to restore the biological activity of the molecule abruptly. Caged and hydrophobic derivatives of cADPR serve as powerful pharmacological tools for the to study of their roles in cellular Ca 2+  signaling, and allow the assay of their effects on insulin secretion in intact cell populations. 
     A natural metabolite, cADPR, has been purified from sea urchin egg homogenates and was found to have Ca 2+ -mobilizing activities. Cyclic ADP ribose showed distinct Ca 2+  releasing properties from ones caused by IP 3 . Pharmacological studies suggested that cADPR mediates Ca 2+  release through ryanadine receptor (RyR), one of the two major intracellular Ca 2+  releasing channels (the other is IP3R). Cyclic ADP ribose has been shown to be able to release Ca 2+  from intracellular stores in a number of mammalian cells. 
     Cyclic ADP ribose is formed in one step from NAD + , a common reduction-oxidation cofactor. The reaction is catalyzed by an enzyme, ADP ribosyl cyclase, that was first purified, sequenced, and cloned from the ovotestis of the marine mollusk Aplysia. The enzyme activity was also found to be present in many, if not most, mammalian tissues. Considerable homology (69%) of the amino acid sequence between Aplysia ADP-ribosyl cyclase and human lymphocyte surface antigen CD38 has been observed. Subsequent studies from a number of laboratories showed that CD38 from human, mouse, and rat possess ADP-ribosyl cyclase activity, synthesizing cADPR from NAD + . CD38 has also been found to exist in many animal tissues, and in both plasma membrane and microsomal membrane fractions. 
     There have been a number of discrepancies regarding the signaling role of cyclic ADP ribose. The mechanism of how cADPR activates ryanadine receptors is not fully understood at the moment, but it appears that cADPR requires the presence of other proteins such as calmodulin to exhibit its biological activity. The lack of Ca 2+  responses to cADPR using permeabilized cells, microinjection or patch clamping technique may be due to diluting cytosolic factors required for the action of cADPR. Moreover, since the extracellular calcium concentration is more than 10 5  fold higher than [Ca 2+ ] c , it is difficult to keep cellular calcium under low levels during these invasive manipulations. In contrast, hydrophobic derivatives of cADPR or IP 3  can be applied to fully intact cells, thus allowing us to test their effects on Ca 2+  release, glucose stimulated insulin secretion (GSIS), and other downstream biochemical events reliably. 
     Prodrug Design and Intracellular Delivery of Phosphate-containing Molecules. 
     Because phosphates are ionized and hydrophilic species at physiological pH, phosphate-containing molecules usually do not cross hydrophobic lipid membranes. The concept of prodrug design from pharmaceutical industry has been used to design hydrophobic derivatives of IP 3  and other inositol polyphosphates. Prodrug design comprises an area of drug research that is concerned with the optimization of drug delivery. A prodrug is a pharmacologically inactive derivative of a drug that requires spontaneous or enzymatic transformation within the body in order to regenerate its active parent drug molecule. 
     Analogues of Cyclic ADP Ribose and Their Hydrophobic Derivatives 
     To deliver cADPR inside cells, the negative charges on the pyrophosphate must be covered. However, neutral esters of pyrophosphates are highly unstable in aqueous solutions, spontaneously breaking down into two phosphates (FIG.  5 ). Replacing the center oxygen atom with a methylene group forms a methanediphosphonate. The neutral esters of methanediphosphonate are stable because the center P-C bond is not susceptible to hydrolysis. 
     The synthetic scheme of compound 1 (as shown in FIG. 2) is outlined in FIG.  6 . Briefly, the starting material dibutyryl adenosine is coupled with the methanediphosphonate methyl ester. The resulting intermediate is coupled to another ribose derivative. Formation of the macrocycle is catalyzed TMS triflate using Hilbert-Johnson reaction to form the N1-glycosidic bond (step b in FIG.  6 ). After removing methyl groups with lithium cyanide (step c), the resulting methanediphosphonate is sequentially protected with one equivalent of NPE group (step d) and PM group (step e) to generate the target molecule 1. An alternative synthetic pathway for another hydrophobic derivative is shown in FIG.  7 . 
     Example of Synthesis of an Ester of a Methane-diphosphonate Derivative of Cyclic ADP Ribose 
     The synthesis of a methane-diphosphanate derivative of cyclic ADP ribose is shown in FIG.  8 . Initially, 2′, 3′-dibutyryl-5′-O-tosyl adenosine (Compound 9) is prepared from adenosine in 4 steps following general procedures apparent to those of ordinary skill in the art. The structure was analyzed by  1 H NMR (i.e., CDCl 3 ; chemical shifts in ppm) and showed results of 0.95 (6H, m, CH 3 ), 1.6 (4H, m, CH 2 ), 2.25 (4H, m, CH 2 ), 2.4 (3H, s, CH 3 ), 4.39 (3H, m, H4′ &amp; H5′), 5.56-6.14 (3H, H3′, H2′ &amp; H1′), 7.26 (2H, d, ArH, J=8.4 Hz), 7.75 (2H, d, ArH, J=8.4 Hz), 7.93 (1H, s, H2), 8.28 (1H, s, H8). 
     The synthetic intermediate 1,2,3-tri-O-acetyl-5-O-tosyl ribofuranose is prepared from D-ribose using the literature procedure.  1 H NMR results (CDCl 3 ; in ppm) are as follows: 2.04-2.11 (9H, m, COCH 3 ), 2.45 (3H, s, Ar—CH 3 ), 4.05-4.2 (3H, m, H4, H5), 5.02-5.4 (1H, m, H3), 5.31 (1H, m, H2), 6.09 (s, H1β) &amp; 6.25 (d, J=7Hz, H1α, 1H combined), 7.36 (2H, t, ArH, J=6.3 Hz), 7.79 (2H, t, ArH, J=6.3 Hz). 
     The synthetic intermediate P1, P2-diethyl methanediphosphonate bis(tetra-n-butyl ammonium) salt is prepared according to a previously reported method from the corresponding tetraethyl ester. The  1 H NMR results (CDCl 3 , ppm) include: 0.84 (24H, t, CH 3 ), 1.05 (6H, m, CH 2 ), 1.31 (16H, m, CH 2 ), 1.52 (16H, m, CH 2 ), 1.91 (2H, m, P—CH 2 —P), 3.25 (16H, m, CH 2 ), 3.87 (4H, m, CH 2 );  31 P NMR results (CDCl 3 ): 15.99 (s). 
     Compound 10 or P1-5-O-(1, 2, 3-triacetyl)ribosyl P2-5′-O-(2′,3′-dibutyryl)adenosyl P1,P2-diethyl methylenediphosphonate is prepared as discussed below (see FIG.  8 ). In brief, 2′, 3′-dibutyryl-5′-O-tosyl adenosine (at least about 0.925 g, 1.65 mmol) and P1, P2-diethyl methanediphosphonate bis(tetra-n-butyl ammonium) salt (at least about 1.135 g, 1.59 mmol) are heated in DMF (1 mL) for 18 hours at 80-90 degrees Centigrade under argon. Next, 1,2,3-tri-O-acetyl-5-O-tosyl ribofuranose (0.817g, 1.9 mmol) was added to the reaction mixture and the mixture was heated for another 20 hours. After removing the solvent under vacuum, the residue was purified on a silica gel column (e.g., CH 2 Cl 2 /MeOH) to yield the unsymmetrical tetraester Compound 10 (the yield may be at least around 0.323 g or a 22% yield). Results of  1 H NMR(CDCl 3 , ppm) show the following: 0.8-09(6H, CH 3 ), 1.32 (6H, m, CH 3 ), 1.56 (4H, CH 2 ), 2.00 (9H, m, COCH 3 ), 2.22 (4H, COCH 2 ), 2.5 (2H, m, P—CH 2 —P), 4.1 (4H, m, OCH 2 ), 4.38 (6H, m), 5.35 (3H, m), 5.6-5.9 (3H, m), 6.1 (1H, s), 6.2 (1H,d), 6.62 (2H, m), 8.45 (2H, m, H2 &amp; H8);  31 P NMR (CDCl 3 , ppm) 20.4-21.8 (m). Mass spectroscopy analysis was performed, where the mass (Electrospray, positive) that was calculated for C 34 H 51 N 5 O 18 P 2  was 880.27 ([M+H] + ) and found to be 880.56. 
     The neutral ester of a methane-diphosphonate derivative of cyclic ADP ribose or Compound 11 is prepared as follows (see FIG.  8 ). First, BSTFA (6 equivalents) was added to a solution of the Compound 10 (at least about 20 mg or 2.27×10 −5  mol) in 5 mL CH 3 CN. TMSOTf (2 equivalents) was added subsequently and the mixture was stirred at room temperature for 6 hours. Another two equivalents of TMSOTf were then added. The reaction was quenched about two hours later by 1 mL saturated NaHCO 3  and extracted with dichloromethane. The organic layer was dried and purified on a silica gel column (e.g., CH 2 Cl 2 /MeOH) to give Compound 11 as analyzed by mass spectroscopy where the Electrospray, positive calculation for C 32 H 47 N 5 O 16 P 2  was 820.25 ([M+H] + ) and found to be 820.22. 
     Although this invention has been described in reference to illustrative embodiments, the description is not intended to be construed in a limiting sense. Various modifications and combinations of the illustrative embodiments, as well as other embodiments of the invention, will be apparent to persons skilled in the art upon reference to the description. It is therefore intended that the appended claims encompass any such modifications or embodiments.