Patent Grant
9663548
This application is a national stage application under 35 U.S.C. §371 of PCT/IN2012/000617, filed Sep. 14, 2012, and published as WO 2013/038430 on Mar. 21, 2013, which claims priority to Indian Application No. 2655/DEL/2011, filed Sep. 14, 2011, which applications and publication are incorporated by reference as if reproduced herein and made a part hereof in their entirety, and the benefit of priority of each of which is claimed herein.
The present invention relates to 10-α/β-D-arabinofuranosyl-undecenes as anti-mycobacterial agents in vitro. The present invention also discloses a simple stereoselective synthesis of 10-undecenyl alpha and beta C-arabinofuranosides to target enzymes involved in the biosynthesis of cell wall of Mycobacterium and thus useful as inhibitors in the Mycobacterium tuberculosis drug development.
Tuberculosis (TB) caused by Mycobacterium tuberculosis remains a leading cause of mortality worldwide into 21st century. Tuberculosis is a respiratory transmitted disease affecting nearly 32% of the world's population, more than any other infectious disease. The mortality and spread of this disease has further been aggravated because of synergy of this disease with HIV. Approximately 50% of India's population is reported to be tuberculin test positive and one person dies from TB every minute.
Chemotherapy of tuberculosis started in early forties and since then a number of anti-tubercular agents have been discovered including para-aminosalicylic acid (PAS), isoniazid (INH), pyrazinamide (PZA), cycloserine, ethionamide, rifampicin (RMP), and ethambutol. Strategies have been devised to treat TB from time to time and current treatment involves a combination therapy that extends for months at a time, and the pharmacology of these treatment regimens can be complex. Moreover, a number of anti-TB drugs are found to be ineffective against the disease because of development of resistance strains.
The initial lack of understanding of drug action because of ignorance in the biochemistry of the Mycobacterium and the difficulty in manipulating M. tuberculosis had hindered efforts to define the mode of action of these agents. Based on the recent developments in evaluating the fine structure and biochemistry of Mycobacterium bacilli, it is observed that the cell wall of Mycobacterium plays a key role in growth and survival of Mycobacterium tuberculi (Mtb). The cell wall in M. tuberculosis being very complex and of very poor permeability, contributes significantly to the resistance against many therapeutic agents and for the long life in human lungs. [(a) Tam, P.-H.; Lowary, T. L., Curr. Opin. Chem. Biol. 2009, 13, 618-625; (b) P. J. Brennan, Tuberculosis, 2003, 83, 91-97; (c) M. Daffe and P. Draper, in Adv. Microb. Physiol. 1998, 131-203].
The major portion of the cell wall of Mtb is made up of the polysaccharides arabinogalactan (AG) and lipoarabinomannan (LAM). The AG and LAM polysaccharides are composed of arabinose and are synthesized inside the infected host cells. Arabinan component present in the polysaccharide contains approx 70 arabinofuranose residues. A key structural motif in this arabinan is the hexasaccharide, which is found at the non-reducing ends of both polymers. Mycobacterial viability is critically dependent upon its ability to produce both polysaccharides.
Since arabinose is foreign to the mammalian cells, the inhibition of the corresponding enzymes arabinosyltransferases (AraTs) play a critical role in mycobacterial cell wall biosynthesis and are considered as potential drug targets for the treatment of tuberculosis, especially multi-drug resistant forms of M. tuberculosis. Much of the research is now directed to the synthesis of natural products consisting of arabinofuranose rings as potential anti TB agents.
Article titled “Synthesis of octyl arabinofuranosides as substrates for mycobacterial arabinosyltransferases” by Jeongseok Han, Rajendrakumar Reddy et. al in Carbohydrate Research 338 (2003) 581-588, describes a panel of octyl oligosaccharides comprising of arabinofuranose rings. The process for glycosylation reactions involves coupling of octyl glycoside acceptors with the appropriate thioglycosides using N-iodosuccinimide and silver triflate activation. The synthesis disclosed provides substrates suitable for use in assays of mycobacterial arabinosyl transferases.
Article titled “Synthesis of deoxygenated all α(1→5)-linked arabinofuranose disaccharides as substrates and inhibitors of arabinosyltransferases of Mycobacterium tuberculosis” by Ashish K. Pathaka, Vibha Pathaka et al in Bioorganic & Medicinal Chemistry Volume 17, Issue 2, 15 Jan. 2009, Pages 872-881 discloses the synthesis and acceptor/inhibitory activity of Araf α(1→5) Araf disaccharides possessing deoxygenation at the reducing sugar of the disaccharide. Deoxygenation at either the C-2 or C-3 position of Araf was achieved via a free radical procedure using xanthate derivatives of the hydroxyl group (shown in figure below). The α(1→5)-linked disaccharides were produced by coupling n-octyl α-Araf 2-/3-deoxy, 2-fluoro glycosyl acceptors with an Araf thioglycosyl donor. The target disaccharides were tested in a cell free mycobacterial AraTs assay as well as an in vitro assay against MTB H37Ra and M. avium complex strains.
Article titled “Synthesis and Conformational Investigation of Methyl 4a-Carba-D-arabinofuranosides” by Christopher S. Callam and Todd L. Lowary J. Org. Chem. 2001, 66, 8961-8972 discloses the synthesis of carbasugar analogues of methyl α-D-arabinofuranoside and methyl β-D-arabinofuranoside (3 and 4) in identifying inhibitors of the arabinosyltransferases that are involved in the assembly of mycobacterial cell wall polysaccharides. Starting from D-mannose, the targets are obtained via a route in which the key steps are (i) a ring-closing metathesis and (ii) a subsequent stereoselective hydrogenation. The article further states that the route can also be applied to the preparation of other carbafuranoses through substitution of D-mannose with other pyranose sugars.
Article titled “Stereoselective synthesis of β-arabino glycosyl sulfones as potential inhibitors of mycobacterial Cell wall biosynthesis” by Benjamin Ayers, Hilary Long et. al in Carbohydrate Research Volume 344, Issue 6, 21 Apr. 2009, Pages 739-746, describes synthesis of a series of β-arabino glycosyl sulfones with varying alkyl chain lengths in a stereoselective fashion as putative mimics of decaprenolphosphoarabinose (DPA), and as potential inhibitors of mycobacterial cell wall biosynthesis.
Article titled “Synthesis of methyl 5-S-alkyl-5-thio-d-arabinofuranosides and evaluation of their anti-mycobacterial activity” by Aditya K. Sankia, Julie Boucaua et al. in Bioorganic & Medicinal Chemistry, Volume 16, Issue 10, 15 May 2008, Pages 5672-5682, discloses synthesis of methyl 5-S-alkyl-5-thio-d-arabinofuranoside analogues as potential inhibitors of mycobacterial antigen 85 complex. The antigen 85 (ag85) complex which is a family of mycolyl transferases is involved in the synthesis of trehalose-6,6′-dimycolate and the mycolated hexasaccharide motif found at the terminus of the arabinogalactan in mycobacterium. Two of the compounds, 5-S-octyl-5-thio-α-D-arabinofuranoside (8) and 5-S-octyl-5-thio-β-d-arabinofuranoside (11) are disclosed to exhibit potential antibacterial activity against Mycobacterium smegmatis ATCC 14468.
Further studies revealed that the enzyme arabinosyl transferases (AraT) involved in biosynthesis of AG and LAM utilize a single substrate i.e. β-decaprenyl-D-arabinofuranosyldiphosphate. This prompted a search for the mimics of the β-DPA as potential inhibitors for AraTs inter alia new anti-tubercular drug candidates.
C. A. Centrone and T. L. Lowary, J. Org. Chem., 2003, 68, 8115-8119; and in J. Org. Chem., 2002, 67, 8862-8870; have reported the synthesis of various alkylated-C-phosphoryl-D-arabino-β-furanosides A and B as stable mimics of β-DPA and showed that the inhibitory activity of these compounds changes proportionally with the length of the alkyl chain. Related sulfone analogues C are observed to be weakly to modestly active. [C. A. Centrone and T. L. Lowary, Bioorg. Med. Chem., 2004, 12, 5495-55038]
The present inventor in their earlier publication titled “Stereoselective Synthesis of β-C-Allyl- and β-C-Propargyl-D-arabinofuranosides” Synthesis, 2008, 1783-1787, have disclosed stereoselective synthesis of β-configured-C-allyl- and C-propargyl-D-arabinofuranosides (4,7-anhydro-1,2,3-deoxy-D-gluco-oct-1-enitols and -oct-1-ynitols) by employing allylation/propargylation of a dialdofuranose under aqueous Barbier reaction conditions and acid-catalyzed furan ring transposition of 5-O-mesyl-manno-oct-7-eno- or 5-O-mesyl-manno-oct-7-ynofuranoside derivatives.
With a view that C-glycosides have the potential to serve as carbohydrate analogues resistant to metabolic processes, consequently, this class of compounds is currently receiving much interest as a potential source of therapeutic agents for clinical use. Some of these C-glycosides are found to be better than the frontline drug ethambutol and equal to other drugs available in the market for the treatment of tuberculosis. Moreover, they are non-toxic up to 100 μg/ml towards human cell lines.
The current inventors therefore felt a need to provide a new series of α and β-C-arabinofuranosides having long alkyl chains in general and with 10-undecenyl alkyl chain in particular (considering the easy availability of requisite coupling partners) and a terminal olefin for further fictionalizations as β-DPA mimics for the treatment of tuberculosis. Also, it is the subject of the invention to provide a stereoselective process which is simple, efficient for the synthesis of 10-undecenyl alpha and beta-C-arabinofuranosides employing a novel tetrahydrofuran ring transposition reaction and use of Grignard reagents for the key C—C bond forming reaction.
Main objective of the present invention is to provide 10-α/β-d-arabinofuranosyl-undecenes as potential anti-mycobacterial agents.
Another object of the present invention is to provide process for the preparation of 10-α/β-d-arabinofuranosyl-undecenes of general formula 1.
Yet another objective of the present invention is to provide pharmaceutical composition comprising compound of general formula II optionally along with pharmaceutically acceptable additives.
Accordingly, present invention provides a compound of general Formula (II)
wherein, R″ represents 10-undecenyl;
R1 represents hydrogen or α-D-Arabinofuranosyl or β-D-Arabinofuranosyl of Formula (A″)
R in the general Formula (II) and in (A″) is selected independently from hydrogen, acetyl, benzyl, alkoxy, methane sulfonyl, unsubstituted or substituted carboxyl, unsubstituted or substituted phenyl as given below:
or unsubstituted or substituted alkyl, unsubstituted or substituted alkenyl, unsubstituted or substituted alkynyl, unsubstituted or substituted heterocycles.
In an embodiment; representative compounds of general formula (II) are:
wherein C1 is in β configuration wherein C1 is in α configuration;
R and R1 are as defined in claim 1.
In yet another embodiment, representative compounds are;
10-β-D-Arabinofuranosylundecene (1);
10-α-D-Arabinofuranosylundecene (2);
α-D-Arabinofuranosyl-(1→5)-10-β-D-arabinofuranosylundecene (3);
α-D-arabinofuranosyl-(1→5)-10-α-D-arabinofuranosylundecene (4);
2,3-Di-O-benzyl-10-α-D-Arabinofuranosylundecene (16);
α-D-Arabinofuranosyl-(1→5)-2,3-Di-O-benzyl-10-α-D-Arabinofuranosylundecene (17);
2,3-Di-O-phenyl-10-α-D-Arabinofuranosylundecene (18);
2,3-Di-O-(4-methyoxy phenyl)-10-α-D-Arabinofuranosylundecene (19);
2,3-Di-O-(1-naphthyl)-10-α-D-Arabinofuranosylundecene (20);
2,3-Di-O-(3-nitrophenyl)-10-α-D-Arabinofuranosylundecene (21);
2,3-Di-O-(4-nitro phenyl)-10-α-D-Arabinofuranosylundecene (22);
2,3-Di-O-(3-methyl phenyl)-10-α-D-Arabinofuranosylundecene (23);
2,3-Di-O-(4-methyl phenyl)-10-α-D-Arabinofuranosylundecene (24);
2,3-Di-O-(3-flurophenyl)-10-α-D-Arabinofuranosylundecene (25);
2,3-Di-O-methyl-10-α-D-Arabinofuranosylundecene (26);
2,3-Di-O-octyl-10-α-D-Arabinofuranosylundecene (27);
10-α-D-Arabinofuranosylundecane (28);
2,3-Di-O-benzyl-10-β-D-Arabinofuranosylundecene (29);
2,3-Di-O-phenyl-10-β-D-Arabinofuranosylundecene (30);
2,3-Di-O-(4-methyoxy phenyl)-10-β-D-Arabinofuranosylundecene (31);
2,3-Di-O-(1-naphthyl)-10-β-D-Arabinofuranosylundecene (32);
2,3-Di-O-(3-nitrophenyl)-10-β-D-Arabinofuranosylundecene (33);
2,3-Di-O-(4-nitro phenyl)-10-β-D-Arabinofuranosylundecene (34);
2,3-Di-O-(3-methyl phenyl)-10-β-D-Arabinofuranosylundecene (35);
2,3-Di-O-(4-methyl phenyl)-10-β-D-Arabinofuranosylundecene (36);
2,3-Di-O-(3-flurophenyl)-10-β-D-Arabinofuranosylundecene (37);
2,3-Di-O-methyl-10-β-D-Arabinofuranosylundecene (38);
2,3-Di-O-octyl-10-β-D-Arabinofuranosylundecene (39);
10-β-D-Arabinofuranosylundecane (40).
In yet another embodiment of the present invention, said compounds are useful as anti mycobacterial agent in vitro.
In yet another embodiment, present invention provides a process for the preparation of compounds of formula (II) as claimed in claim 1 comprising the steps of:
In yet another embodiment of the present invention, Grignard reagent used is selected from the group consisting of 9-decenyl-magnesium bromide or 10-undecenyl magnesium bromide.
In yet another embodiment of the present invention, the copper catalyst is selected from the group consisting of copper powder, Cu(II) halides, copper cyanide, copper triflate preferably copper cyanide.
In yet another embodiment of the present invention, the solvent used is selected from group consisting of diethyl ether, acetonitrile, THF or DMF.
In yet another embodiment of the present invention, the process for alkylation or arylation at C2 and C3 position of α-acetals (5) and (11) or disaccharides (3) and (4) comprising the steps of:
In yet another embodiment, present invention provides a pharmaceutical composition comprising the antimycobacterial compounds of Formula (II) in association with at least one pharmaceutically acceptable excipients.
In yet another embodiment, present invention provides method for treating tuberculosis in a subject, comprising administering an effective amount of the compounds of Formula (II) in association with pharmaceutical excipients.
In yet another embodiment, present invention provides use of the compounds of Formula (II) for preparation of medicament useful for treatment of tuberculosis.
In yet another embodiment, present invention provides a process for the preparation of compound of Formula 1 and the said process comprising the steps of:
In yet another embodiment, present invention provides a process for the preparation of compound of Formula 2 and the said process comprising the steps of:
In yet another embodiment, present invention provides a process for the preparation of compound of Formula 3 and 4 and the said process comprising the steps of:
The present invention relates to 10-α/β-D-Arabinofuranosylundecene (10-undecenyl α/β-C-arabinofuranosides) and to the process of synthesis thereof for inhibition of M. bovis BCG growth thus revealing them as potential candidates in the Mycobacterium tuberculosis drug development. All the known arabinofuranosyl transferases utilize a single substrate i.e. β-decaprenyl-D-arabino furanosyldiphosphate (I)
In this regard, the present invention provides novel C-glycosides which entail methylene substitution for the anomeric oxygen offering a great deal of stability without substantial conformational amendment.
Present invention relates to 10-α/β-D-Arabinofuranosylundecene with anomeric carbon atom at C(1) of the general Formula (II) for inhibition of M. bovis BCG growth
Wherein
R″ represents 10-undecenyl;
R1 represents, hydrogen or α-D-Arabinofuranosyl or β-D-Arabinofuranosyl of Formula
R in the general Formula (II) and in (A″) is selected independently from hydrogen, acetyl, benzyl, alkoxy, methane sulfonyl, unsubstituted or substituted carboxyl, unsubstituted or substituted phenyl as given below:
or unsubstituted or substituted alkyl, or unsubstituted or substituted alkenyl, or unsubstituted or substituted alkynyl, unsubstituted or substituted heterocycles.
The anomeric carbon at C(1) in compound of the general Formula (II) is represented as beta anomer of formula (II-A) and alpha anomer of formula (II-A′) as below:
Wherein R1 and R in (II-A) and (II-A′) are as described above.
Present invention discloses the compounds 1-4 to target enzymes involved in the biosynthesis of cell wall of Mycobacterium and thus useful as inhibitors against Mycobacterium tuberculosis.
Representative compounds of General formula (II) are:
10-β-D-Arabinofuranosylundecene
α-D-Arabinofuranosyl-(1 → 5)-10-β-D- arabinofuranosylundecene
1-α-D-Arabinofuranosyl-undec-10-ene
α-D-arabinofuranosyl-(1 → 5)-10-α-D- arabinofuranosylundecene
The compound of general formula (II) of the current invention encompasses the salts, enantiomers thereof.
General Process for the Preparation of Compound of Formula 1 and Formula 2
Present invention provides a simple, efficient process for the stereoselective synthesis of 10-α/β-D-Arabinofuranosylundecene with anomeric carbon atom at C(1), of the general Formula (2 or 1) comprises reacting an aldehyde (7) or an epoxide (12) with a grignard reagent under suitable reaction conditions to obtain the alcohol (9) or (8) followed by mesylation and acid mediated ring transposition to obtain acetals of formula (11) or (5). This is followed by hydrolysis and reduction to obtain the desired product (1) or (2). The relative stereochemistry obtained in the current process was observed to possess the 5R (6) and 5S (10) configuration.
1. General Process for the Preparation of Compound of Formula 2
The present invention provides a process for the preparation of α-C-arabinofuranosides (compound of Formula 2) comprising the steps of:
The process is given below in Scheme 1
2. General Process for the Preparation of Compound of Formula 1
Present invention provides a process for the preparation of β-C-arabinofuranosides (compound of Formula 1) comprising:
The process is given below in Scheme 2
General Process for the Preparation of Compound of Formula 3 and 4
Present invention provides a process for the preparation of the compounds of formula (3) and (4) which are the analogues of motif C of AG complex involving selective glycosylation at the C(5)-O— of α/β-C-glycosides (1) and (2) which, includes:
The process is given below in Scheme 3
General Process for the Alkylation or Arylation at C2 And C3 Position of A-Acetals (5) and (11) or Disaccharides (3) and (4)
Present invention provides alkylation or arylation at C2 and C3 position of acetal 5 and 11 and disaccharides 3 and 4 which exhibit potent inhibitory effect against Mycobacterium Bovis BCG.
The process of alkylation includes:
Accordingly, the alkylation or arylation at C2 and C3 position of acetals 5 and 11 includes reacting α-C-Undec-11-enyl-D-Arabinofuranoside with the corresponding alkyl halide of the formula R—X in presence of copper catalyst and Cs2CO3 in a solvent to obtain diaryl compounds. The copper catalyst selected from copper powder, Cu(II) halide, copper cyanide, copper triflate, and the like.
The alkylation is effected in presence of sodium hydride as base in presence of polar aprotic solvents selected from diethyl ether, THF, DMF, DMSO and the like; preferably DMF and in presence of catalyst selected from Bu4NBr.
The alkylation or arylation is given in Scheme 4 to 6 below
Anti-Mycobacterial Activity
Present invention provides the anti-mycobacterial activity of the synthesized compounds 1-4. Accordingly, the Mycobacterium bovis BCG strain is used for the purpose and the inhibition studies are carried out on the whole cell based HTS assay employing rifampicin and ethambutol as controls. All the C-glycosides (except 3) displayed significant anti-mycobacterial effect at concentrations of 1 μg/mL (Table 1). The dose dependent inhibition of M. bovis BCG by compounds 1-4 [each at concentrations of 0 (control), 0.05, 0.1, 0.2, 0.3, 0.5 and 1 μg/mL] were carried out. As given in the table 1, compound 2 showed the best activity amongst the four tested with the IC50 (0.19 μg/ml), MIC (1.6 μg/ml,
Surprisingly, the β-glycoside 1 (originally designed β-DPA mimic) is less active than 2 (
Further, to evaluate the cytotoxicity of these compounds, the effect of compounds 1-4 on the metabolic function of the Thp-1, A431 and HL-60 human monocytic cells were examined using a standard MTT assay. No substantial growth inhibition of these cell lines is found up to 100 μg/ml concentration of these compounds (
From the data presented in Table 1, it is evident that the simple C-arabinofuranosides are more potent inhibitors than the corresponding disaccharides and their inhibitory activity is superior than the other C-analogues documented earlier. The superior inhibition of mono-C-arabinofuranosides over the disaccharides is quite remarkable and also important. Besra and co-workers have examined various O- and S-arabinofuranosides, the alkyl O-disaccharides and trisaccharides as acceptors for the arabinofuranosyl transferases and also as inhibitors of glycan synthesis. It has been revealed that the C8-alkyl disaccharides are good substrates for the arabinofuranosyl transferases. The monosaccharides however turned out to be the poor substrates of the arabinofuranosyl transferases. The O-disaccharides showed inhibition with moderate MICs.
Moreover, the disaccharides with a α-anomeric configuration at the reducing end have better acceptor abilities inter alia the better inhibition when compared with the corresponding β-disaccharides. A similar trend is noticed with the α- and β-C-disaccharides 4 and 3 suggests that they might be acting as substrates for the arabinofuranosyl transferases. However, the superior inhibition of the monosaccharide 1 and 2 over corresponding disaccharides indicate that the mode of inhibition of 1 and 2 might be different from simply acting as acceptors.
The 2,3-di-methyl-α-C-undec-11-enyl-D-arabinofuranoside is shown to exhibit 91% inhibition at 20 ug/ml and all rest dialkylated or arylated derivative shown 0 to 45% inhibition at 20 ug/ml more potential inhibiting activity against Mycobacterium bovis BCG than the corresponding Motif C disaccharides.
The invention relates to a pharmaceutical composition comprising of the active ingredient of Formula (II), as defined above either alone or as its salts along with pharmaceutically acceptable excipients.
The pharmaceutical composition according to the invention can be in the form of a solid, for example, powders, granules, tablets, capsules or can be present in the liquid form such as solutions, emulsions, suspensions etc. or as an injectable composition.
The invention discloses the use of the potent compounds of formula II against mycobacterium bacilli M. bovis BCG.
The invention also provides methods for the treatment of the disorder discussed above. The α/β10-undecenyl C-arabinosides of Formula (II) and pharmaceutical compositions containing them may, according to the invention, be administered using any amount, any form of pharmaceutical composition and any route of administration effective for the treatment. After formulation with an appropriate pharmaceutically acceptable carrier in a desired dosage, as known by those of skill in the art, the pharmaceutical compositions of this invention can be administered by any means that delivers the active pharmaceutical ingredient (s) to the site of the body whereby it can exert a therapeutic effect on the patient.
Following examples are given by way of illustration therefore should not be construed to limit the scope of the invention.
General Methods: Air and/or moisture sensitive reactions were carried out in anhydrous solvents under an atmosphere of argon in oven-dried glassware. All anhydrous solvents were distilled prior to use: THF and diethyl ether from Na and benzophenone; MeOH, CH2Cl2 from CaH2. Commercial reagents were used without purification. Column chromatography was carried out by using Spectrochem silica gel (60-120, 230-400 mesh). Optical rotations were determined on a Jasco DIP-370 digital polarimeter. Specific optical rotations []D are given in 10−1 degcm2g−1. 1H and 13C NMR spectroscopy measurements were carried out on Bruker AC 200 MHz, or Bruker DRX 400 MHz or Bruker DRX 500 MHz spectrometers, and TMS was used as internal standard. 1H and 13C NMR chemical shifts are reported in ppm downfield from tetramethylsilane and coupling constants (J) are reported in hertz (Hz). The following abbreviations are used to designate signal multiplicity: s=singlet, d=doublet, t=triplet, q=quartet, m=multiplet, br=broad. Mass spectroscopy (ESI, API-QStar Pulsar) was carried out on a Finnigan MAT-1020 spectrometer. M. bovis BCG (ATCC35745) was obtained from AstraZeneca, India. The monocyte Thp-1 cell line was obtained from National Centre for Cell Science (NCCS), Pune, India.
To a solution of aldehyde 7 (13.8 g, 68 mmol) in diethyl ether (150 mL) at −10° C. was added a solution of 10-undecenylmagnesium bromide [prepared from Mg (3.3 g, 136 mmol) and 11-bromo-undec-1-ene (27 mL, 123 mmol) in diethyl ether (150 mL) drop wise over 15 min]. The reaction mixture was warmed and allowed to stir at room temperature for 2 h, then it was treated with saturated NH4Cl solution (30 mL) and stirred at room temperature for 30 min. The organic phase was separated and the aqueous layer was extracted 3 times with 90 mL of ethyl acetate. The combined organic phase was washed with water, brine, dried over Na2SO4 and concentrated under reduced pressure. The crude product was purified by column chromatography (15:85 EtOAc:Hexane) to afford alcohol 9 (17.3 g, 71% yield) as colorless oil.
Rf=0.7 (25:75 EtOAc/pet ether); [α] D: +39.9 (c 1 CHCl3); 1H NMR (200 MHz, CDCl3): δ1.24-1.28 (br m, 17H), 1.45 (s, 3H), 1.51-1.60 (m, 2H), 1.96-2.06 (m, 2H), 2.88 (br s, 1H —OH), 3.31 (s, 3H), 3.75 (dd, J=3.7, 5.2 Hz, 1H), 3.99 (br dt, J=4.0, 6.0 Hz, 1H), 4.54 (d, J=6.0 Hz, 1H), 4.69 (dd, J=3.6, 6.0 Hz, 1H), 4.89 (ddt, J=1.2, 2.3, 10.1 Hz, 1H), 4.97 (ddt, J=1.6, 2.2, 17.0 Hz, 1H), 4.92 (s, 1H), 5.78 (ddt, J=6.7, 10.1, 17.1 Hz, 1H) ppm; 13C NMR (50 MHz, CDCl3): δ24.5 (q), 25.3 (t), 25.9 (q), 28.9 (t), 29.1. (t), 29.4 (t), 29.5 (t, 3C), 33.1 (t), 33.8 (t), 54.5 (q), 69.9 (d), 80.5 (d), 81.7 (d), 85.4 (d), 106.7 (d), 112.6 (s), 114.1 (t), 139.2 (d) ppm; ESI-MS: Anal. (C20H36O5) 379.30 ([M+Na]+, 100%), 395.39 ([M+K]+, 3%).
At 0° C., to a solution of alcohol 9 (4.5 g, 12.62 mmol) in anhydrous dichloromethane (60 mL) triethyl amine (5 mL, 38 mmol), mesyl chloride (1.2 mL, 15 mmol) was added slowly and the contents were stirred at the same temperature for 3 h. The reaction mixture was treated with ice water and partitioned between DCM and water. Organic layer was washed with aq. NaHCO3 solution, brine, dried over Na2SO4 and concentrated under reduced pressure. The crude product was purified by column chromatography (15:85 EtOAc:Hexane) to afford light yellow oil 10 (5.3 g, 96% yield).
Rf=0.7 (25:75 EtOAc:Hexane); [α]D: +36.9 (c 1, CHCl3); 1H NMR (200 MHz, CDCl3): δ1.20-1.35 (br m, 17H), 1.44 (s, 3H), 1.63-1.81 (m, 2H), 1.96-2.06 (m, 2H), 3.10 (s, 3H), 3.28 (s, 3H), 3.98 (dd, J=3.4, 9.0 Hz, 1H), 4.55 (d, J=5.9 Hz, 1H), 4.64 (dd, J=3.5, 5.9 Hz, 1H), 4.85 (br dt, J=3.3, 9.0 Hz, 1H), 4.86-4.95 (m, 1H), 4.96 (ddt, J=1.6, 2.2, 17.2 Hz, 1H), 4.89 (s, 1H), 5.79 (ddt, J=6.7, 10.1, 17.2 Hz, 1H) ppm; 13C NMR (50 MHz, CDCl3): δ24.4 (t), 24.8 (q), 26.0 (q), 28.8 (t), 29.0 (t), 29.2 (t), 29.2 (t), 29.4 (t), 29.4 (t), 31.0 (t), 33.7 (t), 38.5 (q), 54.5 (q), 79.4 (d), 81.0 (d), 83.3 (d), 85.0 (d), 106.9 (d), 112.8 (s), 114.0 (t), 139.2 (d) ppm; ESI-MS: Anal. (C21H38O7) 457.41 ([M+Na]+, 15%), 379.44 (38%), 301.25 (38%), 304.30 (100%).
To a solution of mesylate 10 (4.3 g, 9.9 mmol) in anhydrous methanol (110 mL) was added p-TSA (340 mg, 1.98 mmol) and allowed to reflux at 80° C. for 72 h. After complete consumption of 10 as indicated by TLC, the reaction mixture was cooled and treated with solid NaHCO3 and stirred for 1 h. The contents were filtered through celite and concentrated under reduced pressure. The crude product was purified by column chromatography (30:70 EtOAc/Pet ether) to procure 11 (2.90 gm 88%). as a pale yellow color oil.
Rf=0.4 (50:50 EtOAc/Pet ether); [α]D: +20.0 (c 1, CHCl3); 1H NMR (200 MHz, CDCl3): δ1.20-1.40 (br m, 14H), 1.50-1.60 (m, 2H), 1.94-2.04 (m, 2H), 3.41 (s, 3H), 3.45 (s, 3H), 3.67 (br s, 1H —OH), 3.61-3.80 (br m, 2H), 3.83 (t, J=5.0 Hz, 1H), 3.97 (br s, 1H —OH), 4.14 (br t, J=4.4 Hz, 1H), 4.35 (d, J=4.8 Hz, 1H), 4.86 (ddt, J=1.2, 2.2, 10.1 Hz, 1H), 4.95 (ddt, J=1.5, 2.2, 17.1 Hz, 1H), 5.76 (ddt, J=6.7, 10.1, 17.1 Hz, 1H) ppm; 13C NMR (50 MHz, CDCl3): δ25.6 (t), 28.8 (t), 29.0 (t), 29.4 (t), 29.5 (t, 2C), 29.6 (t), 33.2 (t), 33.7 (t), 55.0 (q), 56.4 (q), 78.4 (d), 81.0 (d), 82.0 (d), 83.6 (d), 105.4 (d), 114.0 (t), 139.1 (d) ppm; ESI-MS: Anal. (C18H34O5) 353.26 ([M+Na]+, 100%), 301.27 (4%).
The dimethyl acetal 11 (350 mg, 1.06 mmol) was dissolved in 7 mL ice cold 70% aq. trifluoroacetic acid (TFA) and stirred at rt for 2 h. After complete consumption of the acetal 11 as indicated by TLC the reaction was concentrated under reduced pressure and the resulting crude (301 mg) was dissolved in isopropanol and treated with a solution of NaBH4 (120 mg, 3.18 mmol) in water (1.5 mL) and stirred at rt for 4 h. The reaction mixture was brought to acidic pH by adding 1N hydrochloric acid and extracted with diethyl ether (15×3 mL). The combined organic layer were washed with brine, dried over Na2SO4 and concentrated under reduced pressure. The crude product was purified by column chromatography (10:90 CH3OH/CH2Cl2) to afford compound 2 (233 mg, 77% yield).
Rf=0.2 (10:90 CH3OH/CH2Cl2) [α]D: +29.77 (c 0.8, CHCl3); 1H NMR (400 MHz, CDCl3): δ1.28-1.40 (br m, 14H), 1.56-1.63 (m, 2H), 2.02-2.06 (m, 2H), 3.61 (dd, J=5.3, 11.8 Hz, 2H), 3.69 (dd, J=3.5, 11.8 Hz, 1H), 3.71-3.74 (br m, 2H), 3.76 (dt, J=3.5, 5.3 Hz, 2H), 3.93 (t, J=5.5 Hz, 1H), 4.91 (ddt, J=1.1, 2.2, 10.2 Hz, 1H), 4.98 (br ddt, J=1.6, 2, 17.1 Hz, 1H), 5.80 (ddt, J=6.7, 10.2, 17.1 Hz., 1H) ppm; 13C NMR (100 MHz, CDCl3): δ26.7 (t), 31.0 (t), 30.2 (t), 30.6 (t), 30.7 (t, 2C), 30.8 (t), 34.7 (t), 34.9 (t), 63.4 (t), 79.1 (d), 82.7 (d), 84.1 (d), 84.5 (d), 114.7 (t), 140.1 (d) ppm; ESI-MS: Anal. (C16H30O4) 309.22 ([M+Na]+, 100%), 301.28 (22%).
To an ice cooled solution of diol 11 (30 mg, 91 μmol) in pyridine (0.3 mL) acetic anhydride (0.2 mL) was added and reaction mixture was stirred for 2 h. The contents were poured in water and extracted with ethyl acetate. Combined organic layer was washed with sat. CuSO4 solution, water followed by brine. The organic layer was dried over Na2SO4, concentrated under reduced pressure and the crude was purified by column chromatography (30:70 EtOAc/Pet ether) to afford 11 as a yellowish gum (36 mg, 97%).
Rf=0.3 (30:70 EtOAc/Pet ether). [α]D: +32.1 (c 1.3, CHCl3); IR (CHCl3) v: 3019, 2929, 2856, 1743, 1371, 1216, 1048, 757, 668; 1H NMR (400 MHz, CDCl3): δ1.20-1.30 (br m, 12H), 1.34-1.37 (m, 2H), 1.55-1.64 (m, 2H), 1.99-2.05 (m, 2H), 2.06 (s, 6H), 3.42 (s, 6H), 3.99 (dt, J=4.0, 6.0 Hz, 1H), 4.02 (dd, J=4.0, 6.0 Hz, 1H), 4.42 (d, J=6.1 Hz, 1H), 4.99 (dd, J=3.0, 4.0 Hz, 1H), 5.29 (dd, J=3.8, 2.8 Hz, 1H), 4.90 (ddt, J=1.2, 2.1, 10.2 Hz, 1H), 4.98 (br ddt, J=1.7, 2.0, 17.2 Hz, 1H), 5.79 (ddt, J=6.7, 10.2, 17.1 Hz, 1H); 13C NMR (100 MHz, CDCl3): δ20.9 (q, 2C), 25.4 (t), 28.9 (t), 29.1 (t), 29.4 (t, 2C), 29.5 (t), 29.7 (t), 32.4 (t), 33.8 (t), 53.9 (q), 55.3 (q), 78.6 (d), 81.2 (d, 2C), 83.2 (d), 103.4 (d), 114.1 (t), 139.2 (d), 169.8 (s), 170.1 (s); ESI-MS: Anal. (C22H38O7) 437.38 ([M+K]+, 100%), 455.35 (40.5%), 301.20 (38.5%), 485.40 (11.1%).
To a suspension of magnesium turnings (2.55 g, 104 mmol) in anhydrous diethyl ether (50 mL) was added 10-bromo-dec-1-ene (7.66 mL, 52 mmol) and the contents were heated to reflux for 2 h. The reaction mixture was diluted with diethyl ether (50 mL) and was transferred slowly to a stirring solution of anhydrous cuprous cyanide (7.52 g, 83 mmol) in diethyl ether (50 mL) at 0° C. then the contents stirred for additional 30 min at the same temperature. To this cooled dark brown colloidal suspension of the cuprate, was added a solution of the oxirane 12 (7.56 g, 35 mmol) in diethyl ether (50 mL) and stirred for 1 h at 0° C. and for 5 h at rt. Then the reaction mixture was quenched by adding cold water and extracted with ethyl acetate. Combined organic layer was washed with water, brine, dried over Na2SO4 and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (15:85 EtOAc:Hexane) to afford the compound 8 (9.7 g, 78% yield) as a low melting solid.
Rf=0.7 (25:75 EtOAc:Hexane). [α] D: +59.7 (c 1.0 CHCl3); 1H NMR (200 MHz, CDCl3): δ1.23-1.30 (br m, 17H), 1.46 (s, 3H), 1.50-1.68 (m, 2H), 1.96-2.06 (m, 2H), 2.52 (d, J=5.7 Hz, 1H), 3.30 (s, 3H), 3.74 (dd, J=3.6, 7.3 Hz, 1H), 3.81-3.93 (m, 1H), 4.53 (d, J=6.0 Hz, 1H), 4.79 (dd, J=3.7, 6.0 Hz, 1H), 4.89 (br ddt, J=1.2, 2.2, 10.2 Hz, 1H), 4.90 (s, 1H), 4.98 (br ddt, J=1.6, 3.6, 16.0 Hz, 1H), 5.78 (ddt, J=6.7, 10.2, 17.2 Hz, 1H) ppm; 13C NMR (50 MHz, CDCl3): δ24.6 (q), 25.5 (t), 25.9 (q), 28.9 (t), 29.1 (t), 29.4 (t), 29.5 (t), 29.5 (t), 29.7 (t), 33.7 (t), 34.5 (t), 54.5 (q), 70.3 (d), 80.1 (d), 81.8 (d), 84.8 (d), 106.9 (d), 112.5 (s), 114.0 (t), 139.1 (d) ppm; ESI-MS: Anal. (C20H36O5) 304.28 (100%), 379.37 ([M+Na]+ 3%), 360.43 (4%).
At 0° C., to a solution of alcohol 8 (8.8 g, 25 mmol) in anhydrous dichloromethane (150 mL) was added triethyl amine (8 mL, 61 mmol) and mesyl chloride (2.1 mL, 27 mmol) and stirred at same temperature for 3 h. Excess of mesyl chloride was quenched by adding ice. Usual work up followed by purification by column chromatography (15:85 EtOAc:Hexane) gave 6 (10.2 g, 95% yield) as light yellow oil.
Rf=0.7 (25:75 EtOAc:Hexane); [α] D: +12.8 (c 1, CHCl3); 1H NMR (200 MHz, CDCl3): δ1.23-1.28 (br m, 17H), 1.45 (s, 3H), 1.70-1.93 (m, 2H), 1.96-2.00 (m, 2H), 3.06 (s, 3H), 3.30 (s, 3H), 3.98 (dd, J=3.4, 8.4 Hz, 1H), 4.55 (d, J=5.9 Hz, 1H), 4.69 (dd, J=3.5, 5.8 Hz, 1H), 4.86 (s, 1H), 4.87-4.93 (br m, 2H), 4.96 (br ddt, J=1.3, 2.0, 17.0 Hz, 1H), 5.78 (ddt, J=6.7, 10.2, 17.2 Hz, 1H) ppm; 13C NMR (50 MHz, CDCl3): δ23.6 (t), 24.9 (q), 26.0 (q), 28.9 (t), 29.1 (t), 29.3 (t), 29.4 (t, 2C), 29.6 (t), 32.2 (t), 33.8 (t), 38.3 (q), 54.7 (q), 78.8 (d), 79.0 (d), 80.2 (d), 84.8 (d), 107.0 (d), 112.8 (s), 114.0 (t), 139.2 (d) ppm; ESI-MS: (C21H38O7) 457.34 ([M+Na]+, 11%), 116.16 (100%), 301.34 (6%).
Following the procedure used for compound 11, the acid catalyzed rearrangement of the mesylate (4.0 g, 9.2 mmol) crude was purified by column chromatography (25:75 EtOAc/pet ether) to procure 5 (2.52 g, 83% yield) as light yellow color oil.
Rf=0.5 (50:50 EtOAc/pet ether); [α]D: +20.8 (c 1, CHCl3); 1H NMR (200 MHz, CDCl3): 1.24-1.37 (br m, 14H), 1.59-1.65 (m, 2H), 1.97-2.06 (m, 2H), 3.3 (br d, J=11.1 Hz, 1H), 3.48 (s, 3H), 3.54 (s, 3H), 3.74 (dd, J=2.6, 11.0 Hz, 1H), 3.82 (dd, J=1.7, 2.9 Hz, 1H), 3.92 (dt, J=2.7, 6.8 Hz, 1H) 4.25 (brs, 1H), 4.36 (d, J=3, 1H), 4.89 (ddt, J=1.3, 2.3, 10.1 Hz, 1H), 4.96 (ddt, J=1.5, 2.2, 17.1 Hz, 1H), 5.79 (ddt, J=6.7, 10.1, 17.2 Hz, 1H) ppm; 13C NMR (50 MHz, CDCl3): δ26.1 (t), 28.2 (t), 28.9 (t), 29.1 (t), 29.5 (t, 3C), 29.7 (t), 33.7 (t), 56.4 (q), 57.8 (q), 77.1 (d), 77.9 (d), 82.1 (d), 85.7 (d), 105.2 (d), 114.1 (t), 139.2 (d) ppm; ESI-MS: Anal. (C18H34O5) 301.27 ([M+1]+, 100%), 353.35 ([M+Na]+, 100%), 369.35 ([M+K]+, 6%), 339.33 (30%), 301.28 (35%).
The dimethyl acetal 5 (500 mg, 1.5 mmol) was subjected for hydrolysis in 50% aq. trifluoro acetic acid (10 mL) followed by reduction with NaBH4 (171 mg, 4.5 mmol) according to the procedure used for 2, and the crude was purified by column chromatography (10:90 CH3OH/CH2Cl2) to acquire 1 (313 mg, 72% yield).
Rf=0.3 (10:90 CH3OH/CH2Cl2); [α]D: +25.9 (c 0.6, CHCl3). 1H NMR (400 MHz, CDCl3): δ1.31-1.44 (br s, 14H), 1.61-1.66 (m, 2H), 2.01-2.07 (m, 2H), 3.63 (dd, J=4.8, 11.5 Hz, 1H), 3.68 (dd, J=3.9, 11.5 Hz, 1H), 3.73 (ddd, J=2.5, 3.7, 4.6 Hz, 1H), 3.78 (br dd, J=0.9, 3.0 Hz, 1H), 3.91 (dt, J=3.1, 6.9 Hz, 1H), 3.96 (br dd, J=1.0, 2.4 Hz, 1H), 4.91 (ddt, J=1.2, 2.2, 10.1 Hz 1H), 4.98 (ddt, J=1.5, 2.2, 17.1 Hz 1H), 5.81 (ddt, J=6.8, 10.2, 17.1 Hz, 1H) ppm; 13C NMR (100 MHz, CDCl3): δ27.4 (t), 29.8 (t), 30.2 (t), 30.3 (t), 30.7 (t), 30.8 (t, 2C), 31.1 (t), 35.0 (t), 63.7 (t), 78.9 (d), 80.7 (d), 81.2 (d), 87.4 (d), 114.8 (t), 140.3 (d) ppm; ESI-MS: Anal. (C16H30O4) 2 87.40 ([M+1]+ 2%), 309.29 ([M+Na]+, 100%), 325.37 ([M+1<]+, 2%), 301.21 (11%).
Following the procedure for synthesis of 11Ac, the diol 5 (25 mg, 76 μmol) was converted to the corresponding diacetate (30 mg, 95%).
Rf=0.4 (30:70 EtOAc/Pet ether). [α]D: +13.3 (c 2, CHCl3); IR (CHCl3) : 3019, 2928, 2855, 1743, 1372, 1215, 1088, 1047, 755, 668; 1H NMR (400 MHz, CDCl3): δ1.20-1.30 (br m, 14H), 1.32-1.38 (m, 2H), 1.99-2.04 (m, 2H), 2.07 (s, 3H), 2.09 (s, 3H), 3.39 (s, 3H), 3.42 (s, 3H), 3.84 (dd, J=3.7, 6.4 Hz 1H), 3.94 (br dt, J=3.6, 6.0 Hz, 1H), 4.38 (d, J=6.4 Hz 1H), 4.90 (ddt, J=1.1, 2.0, 10.0 Hz, 1H), 4.96 (ddt, J=1.5, 2.6, 17.1 Hz, 1H), 5.13 (br d, J=3.5 Hz, 2H), 5.79 (ddt, J=6.7, 10.2, 17.1 Hz, 1H); 13C NMR (50 MHz, CDCl3): δ20.7 (q), 20.9 (q), 25.9 (t), 28.3 (t), 28.9 (t), 29.1 (t), 29.39 (t), 29.4 (t, 2C), 29.6 (t), 33.8 (t), 53.8 (d), 55.1 (d), 77.3 (d), 78.9 (d), 81.0 (d), 82.2 (d), 103.6 (d), 114.1 (t), 139.2 (d), 169.4 (s), 169.6 (s); ESI-MS: Anal. (C22H38O7) 437.32 ([M+Na]+, 100%), 453.30 ([M+K]+, 27.7%), 301.2 (38.5%), 432.36 (16.6%).
At 0° C., to a solution of orthoester (330 mg, 700 μmol) in anhydrous dichloromethane (5 mL) was added dibutyl phosphate (550 μl, 2.77 mmol) and stirred for 2 h at room temperature. The reaction mixture was treated with triethyl amine (2 mL) and concentrated under reduced pressure. The resulting crude product was purified on flash silica gel to get the phosphate 13 (344 mg, 73% yield) which was immediately subjected to next reaction.
At −33° C., in a solution of triol 1 (100 mg, 350 μmol) and phosphate 13 (344 mg, 523 μmol) in anhydrous dichloromethane was added TMSOTf (0.2 mL, 1.05 mmol) and stirred for 1 h at same temperature, to this triethylamine (3 mL) was added allowed to come at room temperature. The reaction mixture was concentrated under reduced pressure and purified by column chromatography (50:50 EtOAc/Pet ether) to procure the disaccharide (150 mg, 59% yield) as a colorless gum.
Rf=0.3 (50:50 EtOAc/Pet ether). [α]D: +1.7 (c 1, CHCl3); 1H NMR (400 MHz, CDCl3): δ1.24-1.31 (br s, 14H), 1.65-1.70 (m, 2H), 1.99-2.07 (m, 2H), 3.82 (br t, J=2.5 Hz, 1H), 3.87 (dd, J=2.9, 11.0 Hz, 1H), 3.88-3.89 (br m, 1H), 3.90 (dt, J=2.7, 6.8 Hz, 1H), 3.97 (dd, J=2.1, 11.0 Hz, 1H), 4.26 (d, J=2.2 Hz, 1H), 4.59 (ddd, J=3.3, 4.8, 5.3 Hz, 1H), 4.67 (dd, J=4.8, 11.9 Hz, 1H), 4.84 (dd, J=3.2, 11.9 Hz, 1H), 4.91 (br ddt, J=1.2, 2.0, 10.1 Hz, 1H), 4.98 (br ddt, J=1.7, 2.2, 17.0 Hz, 1H), 5.38 (s, 1H), 5.50 (d, J=1.2 Hz, 1H), 5.60 (dd, J=1.0, 5.2 Hz, 1H), 5.80 (ddt, J=6.7, 10.2, 17.0 Hz, 1H), 7.28 (t, J=7.9 Hz 2H), 7.38 (t, J=7.9 Hz, 2H), 7.44 (t, J=7.9 Hz, 2H), 7.49 (t, J=1.3, 7.5 Hz, 1H), 7.56-7.59 (m, 2H), 7.98 (dd, J=1.3, 7.9 Hz, 2H), 8.02 (dd, J=1.3, 7.9 Hz, 2H), 8.13 (dd, J=1.3, 7.9 Hz, 2H) ppm; 13C NMR (100 MHz, CDCl3): δ26.2 (t), 28.1 (t), 28.9 (t), 29.4 (t), 29.5 (t), 29.8 (t), 29.7 (t), 29.8 (t), 33.8 (t), 63.6 (t), 67.4 (t), 77.6 (d), 78.5 (d), 79.7 (d), 81.4 (d), 82.0 (d), 82.2 (d), 84.3 (d), 106.4 (d), 114.1 (t), 128.3 (d, 2C), 128.4 (d, 2C), 128.5 (d, 2C), 128.7 (s), 128.8 (s), 129.6 (s), 129.7 (d, 2C), 129.9 (d, 2C), 130.1 (d, 2C), 133.1 (d), 133.6 (d), 133.63 (d), 139.2 (d), 165.5 (s), 165.9 (s), 166.2 (s) ppm; MALDI-TOF: Anal (C42H50O11) 445.21 (100%), 533.32 (48%), 753.25 ([M+Na]+, 38%), 769.20 ([M+K]+, 10%).
The tribenzoate 14 (50 mg, 68 μmol) was taken in methanol (5 mL) and a catalytic Na was added. After 2 h stirring at room temperature, the reaction mixture was concentrated and purified by column chromatography (10:90 CH2Cl2/CH3OH) to procure the free disaccharide (27 mg, 95% yield).
Rf=0.2 (10:90 CH2Cl2/CH3OH); [α]D: +61.7 (c 1, CH3OH); 1H NMR (400 MHz, CDCl3): δ1.29-136 (br m, 14H), 1.61-1.66 (m, 2H), 2.02-2.07 (m, 2H), 3.59 (dd, J=4, 9.7 Hz, 1H), 3.63 (dd, J=5.4, 11.9 Hz, 1H), 3.73 (dd, J=3.4, 11.9 Hz, 1H), 3.79-3.85 (m, 5H), 3.89 (dt, J=3.4, 7.0 Hz, 1H), 3.96-3.97 (br m, 2H) 3.98 (d, J=1.1 Hz, 1H), 4.90 (ddt, J=1.2, 2.1, 10.1 Hz, 1H), 4.93 (d, J=1.2 Hz, 1H), 4.97 (ddt, J=1.7, 2.1, 17.0 Hz, 1H) 5.81 (ddt, J=6.7, 10.2, 17.1 Hz, 1H) ppm; 13C NMR (100 MHz, CDCl3): δ27.3 (t), 29.7 (t), 30.2 (t), 30.3 (t), 30.7 (t), 30.8 (t), 30.9 (t), 31.0 (t), 35.0 (t), 63.2 (t), 69.1 (t), 78.8 (d), 79.2 (d), 80.9 (d), 83.2 (d), 83.3 (d), 85.6 (d), 86.1 (d), 109.7 (d), 114.8 (t), 140.3 (d) ppm; MALDI-TOF: Anal (C21H38O8) 441.21 ([M+Na]+, 100%), 457.21 ([M+K]+, 39%).
The glycosidation of the compound 2 (115 mg, 401 μmol) was carried out as outlined in the preparation of 14 employing freshly prepared phosphonate (394 mg, 602 μmol), the crude was purified by column chromatography (50:50 EtOAc/Pet ether) to get the tribenzoyl disaccharide
Rf=0.2 (50:50 EtOAc/Pet ether); [α]D: +7.6 (c 0.5, CHCl3); 1H NMR (400 MHz, CDCl3): δ1.22-1.38 (br m, 14H), 1.58-1.61 (m, 2H), 2.0-2.04 (m, 2H,), 3.2 (br s, 2H —OH), 3.77 (dd, J=4.0, 10.8 Hz, 1H), 3.84 (dd, J=6.3, 12.5 Hz, 1H), 3.86 (t, J=5.2 Hz, 1H), 3.95 (dd, J=3.8, 10.9 Hz, 1H), 3.98 (dt, J=4.0, 5.7 Hz, 1H), 4.24 (t, J=5.2 Hz, 1H), 4.61 (dt, J=3.5, 4.8 Hz, 1H), 4.65 (dd, J=4.8, 11.8 Hz, 1H), 4.81 (dd, J=3.3, 11.8 Hz, 1H), 4.92 (ddt, J=1.2, 2.0, 10.2 Hz, 1H), 4.98 (ddt, J=1.7, 2.0, 17.1 Hz, 1H), 5.38 (s, 1H), 5.53 (d, J=1.4, 1H), 5.59 (dd, J=1.4, 5.1 Hz, 1H), 5.80 (ddt, J=6.7, 10.2, 17.1 Hz, 1H), 7.28 (t, J=7.8 Hz, 2H), 7.37 (t, J=7.8 Hz, 2H), 7.43 (t, J=7.8 Hz, 2H), 7.49 (t, J=1.2, 7.2 Hz, 1H), 7.54-7.58 (m, 2H), 7.97 (dd, J=1.2, 8.1 Hz, 2H), 8.02 (dd, J=1.2, 8.1 Hz, 2H), 8.13 (dd, J=1.2, 8.2 Hz, 2H) ppm; 13C NMR (100 MHz, CDCl3): δ25.6 (t), 28.9 (t), 29.1 (t), 29.4 (t), 29.5 (t, 2C), 29.6 (t), 33.3 (t), 33.8 (t), 63.6 (t), 67.6 (t), 77.6 (d), 79.5, (d), 80.9 (d), 81.0 (d), 82.0 (d), 82.3 (d), 83.5 (d), 106.2 (d), 114.1 (t), 128.3 (d, 2C), 128.5 (d, 4C), 128.7 (s), 128.8 (s), 129.5 (s), 129.7 (d, 2C), 129.9 (d, 2C), 130.0 (d, 2C), 133.1 (d, 2C), 133.5 (d), 133.6 (d), 139.2 (d), 165.7 (s), 165.8 (s), 166.3 (s) ppm; MALDI-TOF: Anal (C42H50O11) 445.05 (100%), 518.38 (54%), 533.16 (46%), 753.11 ([M+Na]+, 47%), 769.03 ([M+K]+, 18%). (161 mg, 55% yield) as a colourless gum.
To a solution of tribenzoate 15 (40 mg, 54 μmol) in methanol (5 mL) catalytic amount of Na was added and stirred for 2 h. The reaction mixture was concentrated and purified by column, chromatography (10:90 CH2Cl2/CH3OH) to obtain 4 (22 mg, 96% yield).
Rf=0.2 (10:90 CH2Cl2/CH3OH); [α]D: +31.1 (c 0.4, CH3OH); 1H NMR (400 MHz, CDCl3): δ1.29-137 (br m, 14H), 1.56-1.63 (m, 2H), 2.02-2.07 (m, 2H), 3.59 (dd, J=3.6, 10.7 Hz, 1H), 3.62 (d, J=5.2 Hz, 1H), 3.65 (d, J=5.4 Hz, 1H), 3.71-3.74 (m, 2H), 3.75 (t, J=3.3 Hz 1H), 3.82 (dd, J=5.3, 11 Hz, 1H), 3.83 (dd, J=3.0, 6.0 Hz, 1H), 3.87 (dt, J=3.8, 5.3 Hz, 1H), 3.97 (dd, J=3.3, 5.7 Hz, 1H), 3.98-4.02 (br m, 2H), 4.92 (ddt, J=1.1, 2.0, 10.1 Hz, 1H), 4.93 (d, J=1.3 Hz, 1H), 4.97 (ddt, J=1.6, 2.0, 17.2 Hz, 1H), 5.80 (ddt, J=6.7, 10.2, 17.0 Hz, 1H) ppm; 13C NMR (100 MHz, CDCl3): δ26.7 (t), 30.1 (t), 30.2 (t), 30.6 (t), 30.7 (t, 2C), 30.8 (t), 34.6 (t), 34.9 (t), 63.1 (t), 68.7 (t), 78.9 (d), 79.6 (d), 82.8 (d), 82.9 (d), 83.2 (d), 84.3 (d), 85.9 (d), 109.6 (d), 114.7 (t), 140.2 (d) ppm; MALDI-TOF: Anal (C21H38O8) 441.10 ([M+Na]+, 100%), 533.15 (71%), 551.15 (54%), 628.48 (55%).
Following the procedure for synthesis of 11Ac, the triol 1 (42 mg, 146 μmol) was converted to the corresponding triacetate (58 mg, 97%).
Rf=0.7 (25:75 EtOAc/Petether); [α]D: +6.4 (c 3.4, CHCl3). 1H NMR (400 MHz, CDCl3): δ1.24 (bs, 14H), 1.48-1.63 (m, 2H), 1.95-2.04 (m, 2H), 2.06 (s, 6H), 2.09 (s, 3H), 3.98-402 (m, 2H), 4.12 (dd, J=6.5, 11.5 Hz, 1H), 4.33 (dd, J=4.7, 11.5 Hz, 1H), 4.86-4.91 (m, 1H), 4.95 (ddt, J=1.7, 2.2, 17.2 Hz, 1H), 5.16 (dd, J=0.83, 3.5 Hz, 1H), 5.77 (ddt, J=6.8, 10.1, 17.2 Hz, 1H) ppm; 13C NMR (100 MHz, CDCl3): δ 20.68 (q), 20.74 (q), 20.79 (q), 25.92 (t), 28.24 (t), 28.83 (t), 29.03 (t), 29.32 (t), 29.36 (t), 29.38 (t), 29.49 (t), 33.72 (t), 63.90 (t), 76.99 (d), 79.01 (d), 80.92 (d), 80.99 (d), 114.05 (t), 139.13 (d), 169.64 (s), 169.71 (s), 170.71 (s); ESI-MS: Anal. (C22H36O7) 413.14 ([M+1]+, 4%), 435.19 ([M+Na]+, 100%), 451.15 ([M+K]+, 3%).
Following the procedure for synthesis of 11Ac, the triol 2 (35 mg, 122 μmol) was converted to the corresponding triacetate (47 mg, 95%).
Rf=0.6 (20:80 EtOAc/Petether) [α]D: +16.50 (c 0.761, CHCl3). 1H NMR (400 MHz, CDCl3): δ 1.26 (bs, 14H), 1.34-1.38 (m, 2H), 1.59-1.63 (m, 2H), 2.08 (s, 6H), 2.09 (s, 3H), 4.0 (ddd, J=3.8, 7.0, 13.7 Hz 1H), 4.14 (ddd, J=3.9, 5.0, 8.7 Hz 1H), 4.22 (dd, J=6.0, 11.6 Hz 1H), 4.26 (dd, J=5, 11.6 Hz 1H), 4.91 (ddt, J=1.2, 2.4, 10.3 Hz 1H), 4.97 (ddt, J=1.1, 2.1, 17.1 Hz 1H), 5.06 (dd, J=2.4, 3.7 Hz 1H), 5.80 (ddt, J=6.7, 10.2, 17.3. Hz, 1H) ppm; 13C NMR (100 MHz, CDCl3): δ 20.80 (q), 20.82 (q), 20.90 (q), 25.46 (t), 28.92 (t), 29.10 (t), 29.39 (t), 29.44 (t), 29.46 (t, 2C), 32.43 (t), 33.78 (t), 63.46 (t), 78.92 (d), 80.21 (d), 80.95 (d), 83.15 (d), 114.10 (t), 139.21 (d), 170.02 (s), 170.05 (s), 170.72 (s); ESI-MS: Anal. (C22H36O7) 413.14 ([M+1]+, ˜1%), 435.31 ([M+Na]+, 100%).
1H NMR (400 MHz, CDCl3): δ 1.20-1.40 (br m, 14H), 1.56-1.64 (m, 2H), 1.99-2.08 (m, 2H), 2.16 (br s, 1H, —OH), 3.68-3.70 (m, 2H), 3.82 (dd, J=0.7, 2.6 Hz, 1H), 3.96-4.04 (br m, 2H), 4.09 (dd, J=4.3, 9.2 Hz, 1H), 4.53 (s, 2H), 4.55 (s, 2H), 4.88-5.06 (br m, 2H), 5.85 (ddt, J=6.6, 10.3, 17.03 Hz, 1H), 7.26-7.40 (br m, 10H); 13C NMR (100 MHz, CDCl3): 25.6 (t), 28.8 (t), 29.0 (t), 29.4 (t, 3C), 32.9 (t), 33.7 (t, 2C), 62.6 (t), 71.5 (t), 71.9 (t), 82.6 (d), 82.7 (d), 84.4 (d), 87.2 (d), 114.0 (t), 127.6 (d), 127.6 (d, 2C), 127.7 (d), 128.3 (t, 2C), 128.3 (t, 2C), 137.5 (s), 137.6 (s), 139.0 (s); ESI-MS: Anal. (C30H42O4) 505.65 ([M+K]+, 100%), 489.68 (40.5%).
1H NMR (400 MHz, CDCl3): δ1.20-1.40 (br m, 14H), 1.55-1.64 (m, 2H), 2.00-2.05 (m, 2H), 2.87 (s, 1H), 2.95 (s, 1H), 3.56 (dd, J=3.8, 10.3 Hz, 1H), 3.70-3.80 (m, 2H), 3.83 (dd, J=6.2, 10 Hz, 1H), 3.86 (dd, J=1.8, 3.6 Hz, 1H), 3.95 (br s, 1H), 3.97 (br m, 1H), 3.99 (br s, 1H), 4.02 (br s, 1H), 4.12 (ddd, J=3.8, 5.9, 9.4 Hz, 1H), 4.40-4.57 (m, 4H), 5.0 (s, 1H), 4.89-5.02 (br m, 2H), 5.80 (ddt, J=6.6, 10.3, 16.9 Hz, 1H), 7.26-7.38 (br m, 10H); 13C NMR (100 MHz, CDCl3): 28.9 (t), 29.1 (t), 29.3 (t), 29.5 (t), 29.7 (t, 3C), 31.9 (t), 33.8 (t), 62.0 (t), 62.6 (t), 71.7 (t), 71.8 (t), 77.2 (d), 78.0 (d), 78.9 (d), 81.1 (d), 83.0 (d), 85.4 (d), 87.1 (d), 87.3 (d), 107.6 (d), 114.1 (t), 127.7 (d), 128.0 (d, 2C), 128.5 (d), 129.7 (d), 137.5 (s), 139.2 (s), 7.27-7.38 (br m, 10H) ppm; ESI-MS: Anal. (C35H50O8): 621.78 [M+Na]+
[α]D26: +1.6 (c 1.1, CHCl3); 1H NMR (400 MHz, CDCl3): δ 1.20-1.40 (br m, 16H), 1.99-2.10 (m, 2H), 2.17 (br s, 1H, —OH), 3.80-3.91 (m, 2H), 4.23 (ddd, J=1.8, 5.4, 11.0 Hz, 1H), 4.30 (ddd, J=4.1, 7.5, 12.1 Hz, 1H), 4.65 (br m, 1H), 4.85 (d, J=3.2 Hz, 1H), 4.93 (ddd, J=1.2, 3.5, 10.2 Hz, 1H), 4.93 (ddd, J=1.6, 3.5, 16.5 Hz, 1H), 5.85 (ddt, J=6.6, 10.2, 16.5 Hz, 1H), 6.65-7.05 (m, 6H), 7.22-7.33 (m, 4H); 13C NMR (100 MHz, CDCl3): 25.7 (t), 28.9 (t), 29.1 (t), 29.4 (t), 29.5 (t, 3C), 32.2 (t), 33.8 (t), 62.3 (t), 82.4 (d), 82.8 (d), 82.3 (d), 85.6 (d), 114.1 (t), 115.7 (d, 2C), 115.8 (d, 2C), 121.7 (d, 2C), 129.7 (d, 4C), 139.2 (d), 157.0 (s), 157.1 (s) ppm; ESI-MS: Anal. (C28H38O4) 461.59 [M+Na]+.
[α]D: +29.7.1 (c 2.3, CHCl3); 1H NMR (400 MHz, CDCl3): δ 1.20-1.40 (br m, 14H), 1.55-1.78 (m, 2H), 1.97-2.07 (m, 2H), 2.16 (s, 1H), 3.75 (S, 6H), 3.76-3.85 (m, 2H), 4.18 (ddd, J=2.2, 5.3, 8.74 Hz, 1H), 4.26 (ddd, J=3.9, 5.3, 8.74 Hz, 1H), 4.52 (dd, J=1.3, 2.2 Hz, 1H), 4.71 (br dd, J=1.3, 3.3 Hz, 1H), 4.91 (ddd, J=1.2, 2.6, 10.2 Hz, 1H), 4.98 (ddd, J=1.4, 2.6, 17.0 Hz, 1H), 5.80 (ddt, J=6.7, 10.2, 17.0 Hz, 1H), 6.78-6.83 (br m, 8H); 13C NMR (100 MHz, CDCl3): 25.8 (t), 29.0 (t), 29.2 (t), 29.4 (t), 29.5 (t, 3C), 32.4 (t), 33.8 (t), 55.7 (q, 2C), 62.4 (t), 82.8 (d), 83.2 (d), 83.3 (d), 86.5 (d), 114.1 (t), 114.8 (d, 4C), 117.0 (d, 2C), 117.2 (d, 2C), 139.26 (d), 151.1 (s), 151.2 (s), 154.6 (s), 158.0 (s) ppm; ESI-MS: Anal. (C30H42O6) 521.64 ([M+Na]+
1H NMR (400 MHz, CDCl3): δ1.20-1.40 (br m, 14H), 1.53-186 (m, 2H), 1.98-2.07 (m, 2H), 2.15 (br s, 1H, —OH), 3.96 (d, J=4.7 Hz, 2H), 4.44 (ddd, J=1.6, 5.3, 7.9 Hz, 1H), 4.52 (ddd, J=2.9, 4.6, 7.9 Hz, 1H), 4.92 (ddd, J=1.2, 3.5, 10.2 Hz, 1H), 4.98 (ddd, J=1.6, 3.6, 17.0 Hz, 1H), 5.00 (br s, 1H), 5.23 (d, J=3.0 Hz, 1H), 5.8 (ddt, J=6.6, 10.2, 17.0 Hz, 1H), 6.72 (d, J=7.6 Hz, 1H), 6.86 (d, J=7.6 Hz, 1H), 7.25 (t, J=8.3 Hz, 1H), 7.26 (t, J=7.8 Hz, 1H), 7.42-7.58 (m, 6H), 7.75-7.85 (m, 2H), 8.28 (dd, J=3.0, 5.8 Hz, 1H), 8.32 (dd, J=3.0, 5.8 Hz, 1H); 13C NMR (100 MHz, CDCl3): δ 25.7 (t), 28.9 (t), 29.1 (t), 29.4 (t), 29.5 (t, 3C), 32.3 (t), 33.8 (t), 62.4 (t), 82.7 (d), 83.1 (d), 83.4 (d), 85.6 (d), 106.1 ( ), 106.3 ( ), 114.1 (t), 121.2 (d, 2C), 121.7 (d), 121.8 (d), 125.5 (d), 125.6 (d), 125.7 (d), 125.7 (d), 125.8 (d), 125.8 (d), 126.6 (d), 126.7 (d), 127.6 (d, 2C), 134.7 (d, 2C), 139.2 (d), 152.6 (s), 152.7 (s) ppm; ESI-MS: Anal. (C36H42O4) 561.32 [M+Na]+.
[α]D26: +8.5 (c 2.3, CHCl3); 1H NMR (400 MHz, CDCl3): δ 1.20-1.48 (br m, 14H), 1.71-1.87 (m, 2H), 1.98-2.10 (m, 2H), 2.14 (br s, 1H, —OH), 3.78-3.90 (br m, 2H), 3.96 (dd, J=4.2, 11.7 Hz, 1H), 4.26 (ddd, J=3.1, 5.3, 8.7 Hz, 1H), 4.31 (dd, J=4.4, 8.7 Hz, 1H), 4.75 (dd, J=1.9, 2.8 Hz, 1H), 4.87-5.07 (br m, 3H), 5.85 (ddt, J=6.6, 10.2, 17.0 Hz, 1H), 7.23-7.36 (br m, 2H), 7.45 (t, J=8.2 Hz, 1H), 7.46 (t, J=8.2 Hz, 1H), 7.75 (t, J=2.2 Hz, 1H), 7.82-7.90 (br m, 3H); 13C NMR (100 MHz, CDCl3): 25.6 (t), 28.9 (t), 29.1 (t), 29.3 (t), 29.4 (t, 3C), 32.4 (t), 33.8 (t), 61.7 (t), 81.9 (d), 82.6 (d), 82.8 (d), 86.6 (d), 109.8 (d), 110.1 (d), 114.1 (t), 116.9 (d, 2C), 122.2 (d), 122.6 (d), 130.4 (d, 2C), 139.22 (d), 149.2 (s, 2C), 157.5 (s, 2C) ppm; ESI-MS: Anal. (C28H36N2O8) 551.32 [M+Na]+
[α]D26: +35.8 (c 2.6, CHCl3); 1H NMR (400 MHz, CDCl3): δ 1.20-1.48 (br m, 14H), 1.57-1.75 (m, 2H), 1.97-2.07 (m, 2H), 2.16 (s, 1H, —OH), 3.75-3.90 (br m, 2H), 3.94 (dd, J=4.4, 11.7 Hz, 1H), 4.21 (br dd, J=2.5, 4.9 Hz, 1H), 4.28 (dd, J=4.4, 8.5 Hz, 1H), 4.75 (br s, 1H), 4.85-5.10 (br m, 3H), 5.79 (ddt, J=6.7, 10.2, 17.0 Hz, 1H), 6.95 (d, J=9.2 Hz, 2H), 7.05 (d, J=9.2 Hz, 2H), 8.15 (d, J=1.2 Hz, 2H), 8.19 (d, J=1.2 Hz, 2H); 13C NMR (100 MHz, CDCl3): 25.6 (t), 28.9 (t), 29.1 (t), 29.3 (t), 29.4 (t, 3C), 32.3 (t), 33.7 (t), 61.6 (t), 82.0 (d), 82.6 (d), 82.8 (d), 86.5 (d), 114.1 (t), 115.4 (d, 4C), 126.1 (d, 4C), 139.1 (d), 142.3 (s, 2C), 161.7 (s, 2C), 161.8 (s, 2C) ppm; ESI-MS: Anal. (C28H36N2O8) 551.33 [M+Na]+
[α]D26: +11.6 (c 1.2, CHCl3); 1H NMR (400 MHz, CDCl3): δ 1.20-1.48 (br m, 14H), 1.59-1.79 (m, 2H), 1.99-2.16 (m, 2H), 2.28 (s, 6H), 3.78-3.88 (br m, 2H), 4.16-4.25 (br m, 2H), 4.29 (dd, J=4.4, 8.5 Hz, 1H), 4.62 (br s, 1H), 4.80 (d, J=2.9 Hz, 1H), 4.87-5.07 (br m, 2H), 5.81 (ddt, J=6.7, 10.1, 17.0 Hz, 1H), 6.68 (d, J=2.2 Hz, 2H), 6.72 (d, J=7.0 Hz, 2H), 7.70 (d, J=6.8 Hz, 2H), 7.14 (t, J=7.6 Hz, 2H); 13C NMR (100 MHz, CDCl3): 21.4 (q, 2C), 25.8 (t), 28.9 (t), 29.1 (t), 29.4 (t), 29.5 (t, 3C), 32.2 (t), 33.8 (t), 61.3 (t), 82.4 (d), 83.0 (d), 83.4 (d), 85.5 (d), 112.5 (d), 112.6 (d), 114.1 (t), 116.6 (d), 116.8 (d), 122.5 (d), 129.4 (d), 139.2 (d), 139.8 (d, 2C), 157.0 (s), 157.2 (s) ppm; ESI-MS: Anal. (C30H42O4) 489.29 [M+Na]+
[α]D26: +22.44 (c 3.8, CHCl3); 1H NMR (400 MHz, CDCl3): δ 1.20-1.48 (br m, 14H), 1.59-1.84 (m, 2H), 1.98-2.10 (m, 2H), 2.27 (s, 6H), 3.78-3.88 (br m, 2H), 4.16-4.24 (br m, 2H), 4.27 (dd, J=4.0, 8.6 Hz, 1H), 4.59 (br s, 1H), 4.77 (d, J=3 Hz, 1H), 4.86-5.06 (br m, 2H), 5.81 (ddt, J=6.7, 10.1, 17.0 Hz, 1H), 6.77 (d, J=6.7 Hz, 2H), 6.81 (d, J=6.7 Hz, 2H), 7.05 (d, J=8.4 Hz, 4H); 13C NMR (100 MHz, CDCl3): 20.4 (q, 2C), 25.8 (t), 28.9 (t), 29.1 (t), 29.4 (t), 29.5 (t, 3C), 32.2 (t), 33.8 (t), 62.3 (t), 82.5 (d), 82.9 (d), 83.2 (d), 85.6 (d), 114.1 (t), 115.5 (d, 2C), 115.7 (d, 2C), 130.1 (d, 4C), 130.9 (s, 2C), 139.2 (d), 154.8 (s), 155.0 (s) ppm; ESI-MS: Anal. (C30H42O4) 489.31 [M+Na]+
[α]D26: +2.4 (c 8.4, CHCl3); 1H NMR (400 MHz, CDCl3): δ 1.20-1.45 (br m, 14H), 1.59-1.82 (m, 2H), 1.97-2.11 (m, 2H), 2.24 (s, 1H), 3.77 (br dd, J=5.5, 11.7 Hz, 1H), 3.89 (br dd, J=4.6, 11.9 Hz, 1H), 4.19 (dd, J=2.3, 5.3 Hz, 1H), 4.27 (dd, J=4.4, 8.7 Hz, 1H), 4.62 (dd, J=1.3, 2.2 Hz, 1H), 4.83 (d, J=3.0 Hz, 1H), 4.88-5.06 (br m, 2H), 5.81 (ddt, J=6.7, 10.1, 17.0 Hz, 1H), 6.60-6.75 (br m, 6H), 7.17 (d, J=7.9 Hz, 2H), 7.24 (t, J=7.6 Hz, 2H); 13C NMR (100 MHz, CDCl3): 25.8 (t), 28.9 (t), 29.1 (t), 29.3 (t), 29.4 (t, 3C), 32.2 (t), 33.8 (t), 61.9 (t), 82.4 (d), 82.5 (d), 83.0 (d), 86.0 (d), 103.4 (d, J=2.5 Hz, 1C), 103.9 (d, J=2.5 Hz, 1C), 103.6 (d, J=25 Hz, 1C), 103.7 (d, J=25 Hz, 1C), 108.7 (d, J=21.2 Hz, 2C), 111.0 (d, J=2.8 Hz, 1C), 111.2 (d, J=2.8 Hz, 1C), 130.5 (d, J=9.9 Hz, 1C), 139.2 (s), 158.2 (d, J=10.7 Hz, 1C), 158.3 (d, J=10.7 Hz, 1C), 163.0 (d, J=246.6 Hz, 1C), ppm; ESI-MS: Anal. (C28H36F2O4) 497.26 [M+Na]+
[α]D≈: +33.6 (c 2.6, CHCl3); 1H NMR (400 MHz, CDCl3): δ 1.20-1.40 (br m, 14H), 1.53-1.70 (m, 2H), 1.97-2.07 (m, 2H), 2.16 (br s, 1H, —OH), 3.37 (s, 3H), 3.38 (s, 3H), 3.47-3.55 (br m, 1H),), 3.66-3.75 (br m, 3H), 3.91 (br dd, J=3.4, 8.3 Hz, 1H), 3.99 (dd, J=4.0, 8.2 Hz, 1H), 4.85-5.06 (br m, 2H), 5.85 (ddt, J=6.7, 10.0, 17.0 Hz, 1H); 13C NMR (100 MHz, CDCl3): 25.8 (t), 28.9 (t), 29.1 (t), 29.5 (t, 4C), 33.0 (t), 33.8 (t), 57.3 (q), 57.7 (q), 62.9 (t) 82.4 (t), 82.8 (t), 86.3 (d), 89.3 (d), 114.1 (t), 139.2 (d) ppm; ESI-MS: Anal. (C18H34O4) 314.46 [M+Na]+.
[α]D26: +22.2 (c 2.1, CHCl3); 1H NMR (400 MHz, CDCl3): δ 0.9 (t, J=6.6, Hz), 1.20-1.40 (br m, 34H), 1.48-1.65 (m, 6H), 1.97-2.08 (br m, 2H), 3.39-3.52 (br m, 4H), 3.59 (dd, J=2.6, 3.2 Hz, 1H), 3.69 (dd, J=6.3, 11.0 Hz, 1H), 3.73 (dd, J=2.4, 4.0 Hz, 1H), 3.90 (ddd, J=3.6, 5.6, 8.5 Hz, 1H), 3.99 (ddd, J=4.0, 5.0, 9.0 Hz, 1H), 4.65 (br m, 1H), 4.86-5.05 (br m, 2H), 5.80 (ddt, J=6.7, 10.0, 16.9 Hz, 1H); 13C NMR (100 MHz, CDCl3): 14.1 (q, 2C), 22.6 (t, 2C), 25.9 (t), 26.1 (t, 2C), 28.9 (t), 29.1 (t), 29.3 (t, 2C), 29.4 (t, 2C), 29.5 (t, 4C), 29.8 (t), 31.8 (t, 3C), 331 (t), 33.81 (t), 63.1 (t), 69.9 (t), 70.2 (t), 82.6 (d), 82.9 (d), 85.0 (d), 87.8 (d), 114.1 (t), 139.2 (d) ppm; ESI-MS: Anal. (C32H62O4) 533.56 [M+Na]+.
1H NMR (400 MHz, CDCl3): δ δ 0.9 (t, J=6.6, Hz), 1.20-1.40 (br m, 14H), 1.56-1.64 (m, 2H), 1.99-2.08 (m, 2H), 2.16 (br s, 1H, —OH), 3.68-3.70 (m, 2H), 3.82 (dd, J=0.7, 2.6 Hz, 1H), 3.96-4.04 (br m, 2H), 4.09 (dd, J=4.3, 9.2 Hz, 1H), 4.53 (s, 2H), 4.55 (s, 2H); 13C NMR (100 MHz, CDCl3): 14.3 (q), 25.6 (t), 28.8 (t), 29.0 (t), 29.4 (t, 3C), 32.9 (t), 33.7 (t, 2C), 62.6 (t), 71.5 (t), 71.9 (t), 82.6 (d), 82.7 (d), 84.4 (d), 87.2 (d) ppm; ESI-MS: Anal. (C16H32O4) 311.46 ([M+Na]+.
1H NMR (400 MHz, CDCl3): δ 1.21-1.44 (br m, 14H), 1.56-1.64 (m, 2H), 1.99-2.08 (m, 2H), 2.16 (br s, 1H, —OH), 3.69-3.71 (m, 2H), 3.82 (m, 1H), 4.00-4.14 (br m, 2H), 4.18 (m, 1H), 4.55 (s, 2H), 4.57 (s, 2H), 4.93-5.09 (br m, 2H), 5.88 (ddt, J=6.7, 10.2, 16.9 Hz, 1H), 7.30-7.42 (br m, 10H); 13C NMR (100 MHz, CDCl3): 25.7 (t), 28.9 (t), 29.0 (t), 29.5 (t, 3C), 33.0 (t), 33.8 (t, 2C), 63.0 (t), 71.4 (t), 71.8 (t), 82.3 (d), 82.6 (d), 84.4 (d), 87.0 (d), 114.1 (t), 127.5 (d), 127.6 (d, 2C), 127.7 (d), 128.3 (t, 2C), 128.3 (t, 2C), 137.5 (s), 137.6 (s), 139.0 (s); ESI-MS: Anal. (C30H42O4) 505.67 ([M+K]+
1H NMR (400 MHz, CDCl3): δ 1.21-1.41 (br m, 16H), 1.99-2.10 (m, 2H), 2.16 (br s, 1H, —OH), 3.80-3.90 (m, 2H), 4.20-4.25 (m, 1H), 4.28-4.36 (m, 1H), 4.68-4.90 (br m, 1H), 4.87-4.89 (m, 1H), 4.94 (ddd, J=1.2, 3.5, 10.2 Hz, 1H), 4.95 (ddd, J=1.6, 3.5, 16.5 Hz, 1H), 5.86 (ddt, J=6.6, 10.2, 16.5 Hz, 1H), 6.66-7.07 (m, 6H), 7.24-7.35 (m, 4H); 13C NMR (100 MHz, CDCl3): 25.7 (t), 28.9 (t), 29.1 (t), 29.4 (t), 29.5 (t, 3C), 32.2 (t), 33.8 (t), 62.6 (t), 81.1 (d), 80.8 (d), 82.2 (d), 88.4 (d), 114.1 (t), 115.7 (d, 2C), 115.8 (d, 2C), 121.7 (d, 2C), 129.7 (d, 4C), 139.2 (d), 157.0 (s), 157.1 (s) ppm; ESI-MS: Anal. (C28H38O4) 461.66 [M+Na]+.
[α]D: +29.7.1 (c 2.3, CHCl3); 1H NMR (400 MHz, CDCl3): δ 1.20-1.40 (br m, 14H), 1.55-1.81 (m, 2H), 1.97-2.07 (m, 2H), 2.17 (s, 1H), 3.76 (S, 6H), 3.77-3.86 (m, 2H), 4.19-4.25 (m, 1H), 4.22-4.28 (m, 1H), 4.62-4.65 (m, 1H), 4.85-4.88 (m, 1H), 4.93 (ddd, J=1.2, 2.6, 10.2 Hz, 1H), 5.00 (ddd, J=1.4, 2.6, 17.0 Hz, 1H), 5.85 (ddt, J=6.7, 10.2, 17.0 Hz, 1H), 6.81-6.86 (br m, 8H); 13C NMR (100 MHz, CDCl3): 25.80 (t), 28.95 (t), 29.15 (t), 29.41 (t), 29.52 (t, 3C), 32.37 (t), 33.83 (t), 55.7 (q, 2C), 62.7 (t), 82.5 (d), 81.2 (d), 82.3 (d), 89.3 (d), 114.13 (t), 113.5 (d, 4C), 117.0 (d, 2C), 117.21 (d, 2C), 139:26 (d), 151.05 (s), 151.22 (s), 154.57 (s), 158.01 (s) ppm; ESI-MS: Anal. (C30H42O6) 521.77 [M+Na]+.
1H NMR (400 MHz, CDCl3): δ1.21-1.42 (br m, 14H), 1.54-187 (m, 2H), 1.98-2.07 (m, 2H), 2.17 (br s, 1H, −OH), 3.93-3.98 (m, 2H), 4.42-4.45 (m, 1H), 4.58-5.12 (m, 1H), 5.13-5.16 (m, 1H), 5.22 (ddd, J=1.6, 3.6, 17.0 Hz, 1H), 5.30 (br s, 1H), 5.55-5.73 (m, 1H), 5.8 (ddt, J=6.6, 10.2, 17.0 Hz, 1H), 6.73 (d, J=7.6 Hz, 1H), 6.89 (d, J=7.6 Hz, 1H), 7.26 (t, J=8.3 Hz, 1H), 7.28 (t, J=7.8 Hz, 1H), 7.45-7.62 (m, 6H), 7.78-7.88 (m, 2H), 8.32 (dd, J=3.0, 5.8 Hz, 1H), 8.36 (dd, J=3.0, 5.8 Hz, 1H); 13C NMR (100 MHz, CDCl3): δ 25.8 (t), 29.0 (t), 29.2 (t), 29.6 (t), 29.6 (t, 3C), 32.4 (t), 33.9 (t), 62.7 (t), 82.4 (d), 81.0 (d), 82.6 (d), 88.4 (d), 106.1 (d), 106.3 (d), 113.5 (t), 121.3 (d, 2C), 121.8 (d), 121.9 (d), 125.6 (d), 125.7 (d), 125.8 (d), 125.9 (d), 125.8 (d), 125.8 (d), 126.6 (d), 126.7 (d), 127.6 (d, 2C), 134.7 (d, 2C), 139.2 (d), 152.6 (s), 152.7 (s) ppm; ESI-MS: Anal. (C36H42O4) 561.33 [M+Na]+.
1H NMR (400 MHz, CDCl3): δ 1.19-1.50 (br m, 14H), 1.69-1.85 (m, 2H), 1.97-2.07 (m, 2H), 2.17 (br s, 1H, —OH), 3.74-3.92 (br m, 2H), 3.94-3.97 (m, 1H), 4.24-4.27 (m, 1H), 4.36-4.39 (m, 1H), 5.01-5.07 (m, 1H), 4.87-5.07 (br m, 3H), 5.86 (ddt, J=6.6, 10.2, 17.0 Hz, 1H), 7.24-7.37 (br m, 2H), 7.46 (t, J=8.2 Hz, 1H), 7.47 (t, J=8.2 Hz, 1H), 7.76 (t, J=2.2 Hz, 1H), 7.83-7.91 (br m, 3H); 13C NMR (100 MHz, CDCl3): 25.7 (t), 28.8 (t), 29.1 (t), 29.2 (t), 29.3 (t, 3C), 32.5 (t), 33.7 (t), 61.9 (t), 81.6 (d), 80.5 (d), 81.8 (d), 89.5 (d), 109.9 (d), 110.2 (d), 114.2 (t), 116.9 (d, 2C), 122.2 (d), 122.6 (d), 130.4 (d, 2C), 139.22 (d), 149.2 (s, 2C), 157.5 (s, 2C) ppm; ESI-MS: Anal. (C28H36N2O8) 551.67 [M+Na]+
1H NMR (400 MHz, CDCl3): δ 1.18-1.46 (br m, 14H), 1.48-1.66 (m, 2H), 1.96-2.04 (m, 2H), 2.17 (s, 1H, —OH), 3.74-3.91 (br m, 2H), 3.94-396 (m, 1H), 4.21-4.26 (m, 1H), 4.28-4.30 (m, 1H), 4.75 (br s, 1H), 4.85-5.10 (br m, 3H), 5.81 (ddt, J=6.6, 10.3, 17.1 Hz, 1H), 6.98 (d, J=9.2 Hz, 2H), 7.08 (d, J=9.2 Hz, 2H), 8.18 (d, J=1.2 Hz, 2H), 8.21 (d, J=1.2 Hz, 2H); 13C NMR (100 MHz, CDCl3): 25.7 (t), 29.0 (t), 29.2 (t), 29.4 (t), 29.5 (t, 3C), 32.2 (t), 33.6 (t), 62.0 (t), 80.7 (d), 81.8 (d), 81.9 (d), 89.1 (d), 115.5 (t), 115.6 (d, 4C), 126.2 (d, 4C), 139.3 (d), 142.2 (s, 2C), 161.6 (s, 2C), 161.9 (s, 2C) ppm; Anal. (C28H36N2O8) 551.44 [M+Na]+
1H NMR (400 MHz, CDCl3): δ 1.18-1.46 (br m, 14H), 1.57-1.77 (m, 2H), 1.97-2.14 (m, 2H), 2.30 (s, 6H), 3.80-3.87 (br m, 2H), 4.13-4.22 (br m, 2H), 4.27-4.29 (m, 1H), 4.62 (br s, 1H), 4.78-4.82 (m, 1H), 4.87-5.07 (br m, 2H), 5.80-5.83 (m, 1H), 6.67 (m, 2H), 6.73 (m, 2H), 7.70 (m, 2H), 7.74 (m, 2H); 13C NMR (100 MHz, CDCl3): 21.3 (q, 2C), 25.9 (t), 28.8 (t), 29.2 (t), 29.6 (t), 29.8 (t, 3C), 32.3 (t), 33.9 (t), 62.7 (t), 82.2 (d), 81.0 (d), 82.5 (d), 88.1 (d), 112.6 (d), 112.7 (d), 114.2 (t), 116.6 (d), 116.8 (d), 122.5 (d), 129.4 (d), 139.4 (d), 139.8 (d, 2C), 157.0 (s), 157.2 (s) ppm; ESI-MS: Anal. (C30H42O4) 489.31 [M+Na]+
1H NMR (400 MHz, CDCl3): δ 1.23-1.53 (br m, 14H), 1.63-1.89 (m, 2H), 210-2.20 (m, 2H), 2.29 (s, 6H), 3.75-3.86 (br m, 2H), 4.16-4.24 (br m, 2H), 4.27 (m, 1H), 4.59 (br s, 1H), 4.77 (m, 1H), 4.88-5.09 (br m, 2H), 5.83 (ddt, J=6.6, 10.2, 16.9 Hz, 1H), 6.79 (d, J=6.8 Hz, 2H), 6.85 (d, J=6.8 Hz, 2H), 7.05 (d, J=6.9 Hz, 4H); 13C NMR (100 MHz, CDCl3): 20.6 (q, 2C), 25.9 (t), 29.0 (t), 29.3 (t), 29.6 (t), 29.7 (t, 3C), 32.4 (t), 33.9 (t), 62.37 (t), 80.9 (d), 82.4 (d), 82.3 (d), 88.6 (d), 114.2 (t), 115.8 (d, 2C), 116.0 (d, 2C), 130.4 (d, 4C), 131.2 (s, 2C), 139.4 (d), 155.2 (s), 155.0 (s) ppm; ESI-MS: Anal. (C30H42O4) 489.39 [M+Na]+
1H NMR (400 MHz, CDCl3): δ 1.21-1.46 (br m, 14H), 1.61-1.83 (m, 2H), 1.997-2.13 (m, 2H), 2.17 (s, 1H), 3.80-3.85 (m, 1H), 3.87-3.89 (m, 1H), 4.17-4.20 (m, 1H), 4.22-4.26 (m, 1H), 4.58-4.63 (m, 1H), 4.80-4.85 (m, 1H), 4.88-5.06 (br m, 2H), 5.81 (ddt, J=6.7, 10.1, 17.0 Hz, 1H), 6.61-6.76 (br m, 6H), 7.18 (d, J=7.9 Hz, 2H), 7.25 (t, J=7.6 Hz, 2H); 13C NMR (100 MHz, CDCl3): 24.9 (t), 29.0 (t), 29.3 (t), 29.4 (t), 29.6 (t, 3C), 32.6 (t), 33.9 (t), 62.4 (t), 80.6 (d), 82.0 (d), 82.3 (d), 89.0 (d), 103.6 (d, J=2.5 Hz, 1C), 104.1 (d, J=2.5 Hz, 1C), 103.8 (d, J=25 Hz, 1C), 103.9 (d, J=25 Hz, 1C), 108.9 (d, J=21.2 Hz, 2C), 111.2 (d, J=2.8 Hz, 1C), 111.4 (d, J=2.8 Hz, 1C), 114.2 (d), 130.7 (d, J=9.9 Hz, 1C), 139.3 (s), 158.4 (d, J=10.7 Hz, 1C), 158.5 (d, J=10.7 Hz, 1C), 163.2 (d, J=246.6 Hz, 1C), ppm; ESI-MS: Anal. (C28H36F2O4) 497.29 [M+Na]+
1H NMR (400 MHz, CDCl3): δ 1.21-1.43 (br m, 14H), 1.55-1.71 (m, 2H), 1.20-2.10 (m, 2H), 2.17 (br s, 1H, —OH), 3.39 (s, 3H), 3.40 (s, 3H), 3.47-3.55 (br m, 1H), 3.72-3.89 (br m, 3H), 3.94-3.98 (m, 1H), 4.12-4.14 (m, 1H), 4.86-5.10 (br m, 2H), 5.86 (ddt, J=6.7, 10.0, 17.0 Hz, 1H); 13C NMR (100 MHz, CDCl3): 25.7 (t), 29.0 (t), 29.3 (t), 29.6 (t, 4C), 33.2 (t), 33.9 (t), 57.4 (q), 57.8 (q), 63.3 (t), 80.8 (t), 82.2 (t), 85.4 (d), 92.3 (d), 114.2 (t), 139.4 (d) ppm; ESI-MS: Anal. (C18H34O4) 314.53 [M+Na]+.
1H NMR (400 MHz, CDCl3): δ 1.0 (t, J=6.7, Hz), 1.21-1.48 (br m, 34H), 1.50-1.67 (m, 6H), 2.00-2.11 (br m, 2H), 3.40-3.53 (br m, 4H), 3.58-3.60 (m, 1H), 3.67-3.69 (m, 1H), 3.70-3.73 (m, 1H), 3.89-3.92 (m, 1H), 3.97-3.99 (m, 1H), 4.63-4.65 (br m, 1H), 4.86-5.05 (br m, 2H), 5.80 (ddt, J=6.7, 10.0, 16.9 Hz, 1H); 13C NMR (100 MHz, CDCl3): 14.2 (q, 2C), 22.7 (t, 2C), 26.0 (t), 26.3 (t, 2C), 29.0 (t), 29.2 (t), 29.3 (t, 2C), 29.5 (t, 2C), 29.6 (t, 4C), 29.9 (t), 32.0 (t, 3C), 33.2 (t), 33.8 (t), 63.3 (t), 70.0 (t), 70.3 (t), 80.8 (d), 82.2 (d), 85.4 (d), 92.3 (d), 114.2 (t), 139.4 (d) ppm; ESI-MS: Anal. (C32H62O4) 533.66 [M+Na]+.
1H NMR (400 MHz, CDCl3): δ 1.0 (t, J=6.6 3H),
1.32-1.46 (br s, 14H), 1.63-1.67 (m, 2H), 2.03-2.08 (m, 2H), 3.64 (dd, J=4.9, 11.0 Hz, 1H), 3.69 (dd, J=4.0, 11.0 Hz, 1H), 3.74 (ddd, J=2.9, 4.0, 4.5 Hz, 1H), 3.79 (br dd, J=1.0, 3.3 Hz, 1H), 3.93 (dt, J=3.3, 7.0 Hz, 1H), 3.98 (br dd, J=1.0, 2.5 Hz, 1H), 4.93 (ddt, J=1.4, 2.5, 10.0 Hz 1H). ppm; 13C NMR (100 MHz, CDCl3): 14.4 (q), 27.5 (t), 29.9 (t), 30.1 (t), 30.4 (t), 30.6 (t), 30.7 (t, 2C), 31.3 (t), 35.3 (t), 63.2 (t), 78.7 (d), 80.6 (d), 81.3 (d), 87.5 (d) ppm; ESI-MS: Anal. (C16H30O4) 309.29 [M+Na]+.
Growth Conditions for Bacterial Strains and Cell Lines
M. Bovis BCG Culture:
Sub-culturing of the Strain was routinely done in Dubos albumin agar slants or plates. Liquid inoculum of the organism was added in Dubos tween albumin broth medium and incubated at 37° C. and 150 rpm shaking conditions. One percent of 1.0 O.D at 620 nm of the culture was used as standard inoculum size for all the experiments, yielding a final inoculum of approximately 105 CFU/ml. viable cell counts were measured by following an earlier described method.1 Thp-1, MCF-7 and HL-60 cell line: Cells obtained were at passage numbers 90-93. Cells were cultured at 37° C. with 5% CO2 and 90% humidity in T-75 tissue culture flasks (Corning 430641). Cells were maintained in the culture medium used Minimum Essential Medium (MEM) without phenol red with 2.5 mM L-glutamine (Sigma, US) and supplemented with 10% fetal bovine serum.
Effect of Inhibitors on Growth of the Bacilli
The inhibitory effect of the compounds was monitored by following an earlier described method. Briefly, 247.5 μl of 1% of 1 O.D at 620 nm M. Bovis BCG culture was taken in Dubos medium without ADS enrichment in microplate wells. Already having 2.5 μl of inhibitor in DMSO, the plate was then covered with a sterile sealer and incubated at 37° C. for 8 days. Then the growth of the bacilli was measured by reading the absorbance at 620 nm as well as by determining CFU/ml of the culture at different time intervals. The lowest concentration of drugs yielding a differential absorbance (620 nm) of approximately zero was defined as MIC. The well representing positive controls have only DMSO (vehicle) and the negative controls were having rifampicin and ethambutol at their respective IC50 values. (
Cytotoxicity Assessment of the Inhibitors
To test the in vitro viability/cytotoxicity of the compounds, THP-1, A431 and HL-60 human cell lines were selected. The effect of the compounds were examined on metabolic function of the cells using a standard MTT assay 3 a widely adopted method of measuring cellular proliferation. The MTT assay consists of a yellow tetrazolium 3-(4,5-dimethylthiazolyl)-2,5-diphenyltetrazolium bromide) dye that is reduced by mitochondrial dehydrogenase enzymes to form purple formazan which gets precipitated within viable cells. The concentration of formazan formed is proportional to the number of viable cells. These crystals of reduced MTT dye i.e. formazan, absorbs light at 490 nm. The absorbance of light at 490 nm should be proportional to the viable cell count. For this, 100 μl of the culture containing 10,000 cells/ml was added to each of the 96 wells of the tissue culture plate containing 2.5 μl of inhibitors. Then, the cells were incubated in a CO2 incubator supplied with 5% CO2, 95% humidity at 37° C. At the end of the incubation period, 10 μl MTT (5 mg/ml) was added and incubated at 37° C. for 1 h. Then, add 200 μl of 100% isopropyl alcohol in all the wells and keep it at rt for 4 h. The reading was taking at 490 nm by using a plate reader (Model SPECTRA max PLUS384 from Molecular Devices, USA). In positive control there was no inhibitor added in the wells and in negative controls only medium was used instead of culture. Here, the values obtained from positive and negative controls are considered to have 100% and 0% equivalent growth of the cells respectively. (
A general protocol for the stereoselective synthesis of α- and β-10-undecenyl C-arabinofuranosides has been developed and the corresponding Motif C disaccharide analogs of cell wall of Mtb are synthesized. The anti-mycobacterial activity of these compounds was evaluated against the M. Bovis BCG. One of the C-arabinofuranosides (compound of formula 2) was found to be having the MIC similar to that of frontline anti-tubercular drug ethambutol. [Mikusova, K., Slayden, R. A., Besra, G. S. & Brennan, P. J. Antimicrob. Agents Chemother. 1995, 39, 2484-2489].
This disclosure reveals the promising anti-mycobacterial activity of 10-undecenyl C-/
-arabinofuranosides and of corresponding motif C (of cell wall AG complex of M.Tb) analogues as potential leads for the further development. Protocols for the stereoselective synthesis of both α- and β-C-arabinofuranosides have been disclosed. Selective glycosylation of 5-OH of a free C-arabinofuranoside employing glyclosyl phosphate as a donor has been developed. The α-C-glycoside (IC50=0.2
g/ml) was found to be better inhibitor of Mycobacterium than the corresponding β-glycoside (IC50=1.1
g/ml). One of the 2,3-di-O-benzyl derivative has turned to be the best candidate amongst the various compounds screened. To highlight, the
-C-glycosides are found to be inhibiting as good as that of ethambutol and are non-cytotoxic to Thp-1 cells at 100
g/ml concentration. This promising anti-mycobacterial activity and the appealing non-cytotoxicity of these C-glycoside makes them as potential candidates to further investigate.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2655/DEL/2011 | Sep 2011 | IN | national |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/IN2012/000617 | 9/14/2012 | WO | 00 | 5/28/2015 |
| Publishing Document | Publishing Date | Country | Kind |
|---|---|---|---|
| WO2013/038430 | 3/21/2013 | WO | A |
| Number | Name | Date | Kind |
|---|---|---|---|
| 20080015344 | Fraser-Reid et al. | Jan 2008 | A1 |
| Entry |
|---|
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| Number | Date | Country | |
|---|---|---|---|
| 20150259372 A1 | Sep 2015 | US |
