FIELD OF THE INVENTION
The present invention relates to harmonine analogues. Particularly, the present invention relates to harmonine analogues of formula (I) and a pharmaceutically acceptable salt thereof. More particularly, present invention relates to a process for preparation of harmonine and harmonine analogues of formula (I).
BACKGROUND AND PRIOR ART OF THE INVENTION
The Asian lady beetle or the harlequin ladybird, known as Harmonia axyridis is a ladybird beetle native to continental, temperate and subtropical parts of East and Central Asia. It has been used as bio pesticide against aphid and/or coccid pests in many parts of the world. But the last two decades has seen an issue of its invasiveness and potential threat to the native lady bird species. Its invasive success has been attributed to its enduring resistance against diverse pathogens and dominance over native European ladybirds, Coccinellaseptempunctata and Adaliabipunctata. When threatened or attacked, ladybirds exude droplets of haemolymph containing deterrent alkaloids through their leg joints. And one such alkaloid is Harmonine which is the possible reason for Asian lady bird’s exceptional survival in foreign environments.
Article titled “Harmonine, a defence compound from the harlequin ladybird, inhibits mycobacterial growth and demonstrates multi-stage antimalarial activity” by CR Rohrich et al published in Biol. Lett., 2012 ; 8(2); 308-311 reports that harmonine ((17R,9Z)-1,17-diaminooctadec-9-ene), present in H. axyridis haemolymph, displays broad-spectrum antimicrobial activity that includes human pathogens. Antibacterial activity is most pronounced against fast-growing mycobacteria and Mycobacterium tuberculosis, and the growth of both chloroquine-sensitive and -resistant Plasmodium falciparum strains is inhibited.
Article titled “Enantioselective total synthesis of harmonine, a defence alkaloid of ladybugs (Coleoptera: Coccinellidae)” by D Enders et al. published in European Journal of Organic Chemistry, 1991; 1991(6), 569 – 574 reports an efficient and highly enantioselective (ee>97%) total synthesis of the ladybug defence alkaloid (17R,9Z)-1,17-diaminooctadec-9-ene [(R,Z)-1, harmonine]. The key step is the generation of the stereogeniccenter via asymmetric addition of methyllithium to an aldehyde SAMP hydrazone. However, the main drawback of said process is having low yield of 5% only.
Article titled “Biosynthesis of Defensive Coccinellidae Alkaloids: Incorporation of Fatty Acids in Adaline, Coccinelline, and Harmonine” by E Haulotte et al. published in European Journal of Organic Chemistry, 2012, 2012 (10); pp 1907-1912 reports in vitro incorporation experiments of several labelled fatty acids in the ladybird alkaloids coccinelline (Coccinella 7-punctata), adaline (Adalia 2-punctata), and harmonine (Harmonia axyridis). The results clearly indicate that stearic acid is the precursor of coccinelline and harmonine, whereas myristic acid is at the origin of the carbon skeleton of adaline.
With increasing problems related to drug resistance development, against existing antibiotics and anti-mycobacterial agents, there is always a need for identifying and developing novel compounds towards various infections. Another therapeutic area that is in a continuous quest for novel agents for treatment is cancer. While harmonine has been
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disclosed to possess these activities and its processes of synthesis are known, however, there is no synthesis known in the literature to make harmonine and its related compounds in good quantities.
OBJECTIVE OF THE INVENTION
The main objective of the present invention is to provide analogues of harmonine of formula (I) or a pharmaceutically acceptable salt thereof and process for preparation thereof.
Another objective of present invention is to provide process for the preparation of harmonine and analogues of harmonine of formula (I) from tert-Butyl dec-9-enylcarbamate and (R)-tert-Butyl dec-9-en-2-ylcarbamate via cross metathesis reaction using Z-selective catalyst or Grubbs’ II generation catalyst.
Still another objective of present invention is to provide a process for the preparation of harmonine from N-Boc-alaninal.
Yet another objective of present invention is to provide a process for the preparation of harmonine fromsophorolipids.
SUMMARY OF THE INVENTION
Accordingly, present invention provides a compound of formula (I)
Formula (I)
wherein
m= 0, 1, 2, 3, 4, or 5;
n= 0, 1, 2, 3, 4, or 5;
R= NH2, OH, NHR1 or NHOH;
R’= NH2, OH, NHR1 or NHOH;
R1= Alkyl, acyl or alkoxycarbonyl;
or pharmaceutically acceptable salt thereof.
In an embodiment of the present invention, said compounds are selected from the group consisting of:
i. di-tert-Butyl (2R,17R,Z)-octadec-9-ene-2,17-diyldicarbamate (10),
ii. (Z)-di-tert-Butyl octadec-9-ene-1,18-diyldicarbamate (11),
4
iii. di-tert-Butyl (2R,17R,E)-octadec-9-ene-2,17-diyldicarbamate (12),
iv. (E)-di-tert-Butyl octadec-9-ene-1,18-diyldicarbamate (13),
v. (2R,17R,Z)-octadec-9-ene-2,17-diaminium bis(2,2,2-trifluoroacetate) (17),
vi. (Z)-octadec-9-ene-1,18-diaminium bis(2,2,2-trifluoroacetate) (18),
vii. (2R,17R,E)-octadec-9-ene-2,17-diaminium bis(2,2,2-trifluoroacetate) (19),
viii. (E)-octadec-9-ene-1,18-diaminium bis(2,2,2-trifluoroacetate) (20)
5
In another embodiment, present invention provides a process for the preparation of compound of formula (I) comprising the steps of:
i. adding a degassed solution of Z-selective Grubbs’ catalyst or Grubbs’ II generation catalyst to a solution of compound (R)-tert-Butyl dec-9-en-2-ylcarbamate or tert-Butyl dec-9-enylcarbamate in dichloromethane followed by heating the mixture at temperature in the range of 40-60 ºC for period in the range of 10 to 12 hours to afford the desired compounds of formula (I).
In yet another embodiment of the present invention, temperature is preferably in the range of 45-55°C.
In yet another embodiment of the present invention, pharmaceutical composition comprising compound of formula (I) as claimed in claim 1, or pharmaceutically acceptable salts in combination with at least one pharmaceutical excipient.
In yet another embodiment of the present invention, a process for the preparation of harmonine comprising the steps of:
i. adding a solution of compound C and compound D dissolved in solvent to a degassed solution of Z-selective catalyst in solvent followed by heating the mixture at temperature in the range of 40 to 45 °C for period in the range of 10-12 hrs to afford desired cross metathesis product (R,Z)-di-tert-Butyl octadec-9-ene-1,17-diyldicarbamate (7);
ii. adding trifluroacetic acid to a solution of (7) of step (i) in solvent followed by stirring the resulting reaction mixture at room temperature for the period ranging from 4 to 6hrsfollowed by removal of trifluoroacetic acid(TFA) and neutralization using saturated bicarbonate solution to afford cis-harmonine.
In yet another embodiment of the present invention, step (i) may also be carried out by Grubbs’ II generation catalyst instead of Z-selective catalyst to obtain trans-harmonine.
In yet another embodiment of the present invention, solvent used is dichloromethane.
In yet another embodiment of the present invention the present invention provides for a process for the preparation of harmonine, wherein said process comprising the steps of:
i. adding potassium tert. butoxide to a solution of (6-hydroxyhexyl)triphenylphosphonium bromide (3) in THF followed by addition of N-Bocalaninal(2)and stirring the reaction mixture at temperature ranging from 0 to 25 ºC for 5 to 6 hrs to obtain unsaturated alcohol 3a;
6
ii. adding Pd/C to a solution of unsaturated alcohol3a of step (i) in ethyl acetate followed by stirring the mixture at room temperature in the range of 20 to 30oC for 1 hour to afford (R)-tert-butyl 9-hydroxynonan-2-ylcarbamate (4);
iii. treating a solution of oxalyl chloride in DCM cooled at ̶ 78 °C with DMSO and then with alcohol 4 in DCM at temperature -20 °C for the period ranging from 3 to4 hrs;
iv. cooling the mixture of step (ii) to ̶ 78 °C and treating with Et3N followed by dilution with DCM to afford (R)-tert-Butyl 9-oxononan-2-ylcarbamate (A);
v. adding potassium tertiary butoxide to a compound 6 and cooling the mixture to 0°C followed by adding THF and stirred for 5 min;
vi. adding aldehyde A of step (iv) in THF to a reaction mixture of step (v) followed by stirring the reaction mixture for 2-3 hrs at room temperature to afford (R,Z)-di-tert-Butyl octadec-9-ene-1,17-diyldicarbamate (7);
vii. adding trifluroacetic acid to a solution of compound of step (vi) in DCM at 0°C followed by stirring reaction mixture for 4 hrs at room temperature to afford harmonine(1).
In yet another the embodiment of the present invention, said process comprising the steps of:
a) subjecting sophorolipids to acid hydrolysis in presence of suitable hydrolyzing agent selected from sulfuric acid or methanol at temperature ranging from 60 to 65°C for the period ranging from 16 to 20h to afford ester methyl (S,Z)-17-hydroxyoctadec-9-enoate(14);
b) treating a solution of lithium aluminium hydride in THF with ester methyl (S,Z)-17-hydroxyoctadec-9-enoate(14) in THF followed by stirring the reaction mixture for 8-10 h at room temperature to afford (R,Z)-octadec-9-ene-1,17-diol (14a);
c) adding triphenylphosphine to a solution of diol 14a of step (b) in THF followed by adding a cooled mixture of diisopropylazodicarboxylate and diphenoxyphosphorylazide in THF and stirring the resulting mixture at room temperature for 16-17 hrs to obtain (R,Z)-1,17-diazidooctadec-9-ene (15).
d) Subjecting a solution of diazide 15 of step (c) in ethanol to hydrogenation for 1 hour at temperature ranging from 25 to 30 ºC in presence of Lindlar catalyst to obtain harmonine.
BRIEF DESCRIPTION OF THE DRAWINGS
Scheme 1-3 represents steps for the preparation of compound of formula I.
Scheme 1 represents synthesis of harmonine from tert-Butyl dec-9-enylcarbamate and (R)-tert-Butyl dec-9-en-2-ylcarbamate via cross metathesis reaction using Z-selective catalyst and/or Grubbs’ II generation catalyst.
Scheme 2 represents process for the preparation of harmonine from N-Bocalaninal.
Scheme 3represents process for the preparation of harmonine from sophorolipids.
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Figure 1represents (A) The UV- Visible spectra, (B) XRD pattern, (C) and (D) the TEM images of Harmonine capped AuNPs. The inset in (C) shows the particle size distribution plot and the inset in (D) shows the higher magnification TEM image where the (111) planes of the gold lattice are clearly visible.
DETAILED DESCRIPTION OF THE INVENTION
The present invention provide compounds of formula (I) or a pharmaceutically acceptable salt thereof;
Wherein,
m= 0, 1, 2, 3, 4, or 5;
n= 0, 1, 2, 3, 4, or 5;
R= NH2, OH, NHR1 or NHOH;
R’= NH2, OH, NHR1 or NHOH;
R1= Alkyl, acyl or alkoxycarbonyl.
The present invention also provides a process for the preparation of harmonine from N-Boc-alaninaland a process for the preparation of harmonine fromsophorolipids.
The compound of formula (I)or a pharmaceutically acceptable salt thereof are selected from the group consisting of:
i. di-tert-Butyl (2R,17R,Z)-octadec-9-ene-2,17-diyldicarbamate (10),
ii. (Z)-di-tert-Butyl octadec-9-ene-1,18-diyldicarbamate (11),
iii. di-tert-Butyl ((2R,17R,E)-octadec-9-ene-2,17-diyl)dicarbamate (12),
iv. (E)-di-tert-Butyl octadec-9-ene-1,18-diyldicarbamate (13),
v. (2R,17R,Z)-octadec-9-ene-2,17-diaminium bis(2,2,2-trifluoroacetate) (17),
vi. (Z)-octadec-9-ene-1,18-diaminium bis(2,2,2-trifluoroacetate) (18),
vii. (2R,17R,E)-octadec-9-ene-2,17-diaminium bis(2,2,2-trifluoroacetate) (19),
viii. (E)-octadec-9-ene-1,18-diaminium bis(2,2,2-trifluoroacetate) (20).
The present invention providesa process for the preparation of harmonine from tert-Butyl dec-9-enylcarbamate and (R)-tert-Butyl dec-9-en-2-ylcarbamate via cross metathesis reaction using Z-selective catalystor Grubbs’ II generation catalystand the said process comprising the steps of:
i. adding Z-selective Grubbs catalyst or Grubbs II generation catalyst to a degassed solution of compound (R)-tert-Butyl dec-9-en-2-ylcarbamate (C) or tert-Butyl dec-9-enylcarbamate(D) in dry DCM followed by heating the mixture at 40-60 ºC for 12 hours to
8
afford the desired compounds of formula (I).
Reactant
Catalyst
Solvent
Yield
Product
C +C
Z-selective Grubbs catalyst
10 mol% DCM
84%
C +D
Z-selective Grubbs catalyst
10 mol% DCM
80%
C +C
Grubbs II generation catalyst
10 mol% DCM
67%
C +D
Grubbs II generation catalyst
10 mol% DCM
81%
More preferably, the temperature is in the range of 45-55ºC.
The compound is (R)-tert-Butyl dec-9-en-2-ylcarbamate (C) and the Z-selective Grubbs’ catalyst is used the compound of formula (I) is di-tert-Butyl (2R,17R,Z)-octadec-9-ene-2,17-diyldicarbamate(10).
The compound is tert-Butyl dec-9-enylcarbamate (D) and the Z-selective Grubbs’ catalyst is used the compound of formula (I) is (Z)-di-tert-Butyl octadec-9-ene-1,18-diyldicarbamate (11).
The compound is (R)-tert-Butyl dec-9-en-2-ylcarbamate (C) and the Grubbs’ II generation catalyst is used the compound of formula (I) is di-tert-butyl ((2R,17S,E)-octadec-9-ene-2,17-diyl)dicarbamate(12).
9
The compound is tert-Butyl dec-9-enylcarbamate (D) and the Grubbs’ II generation catalyst is used the compound of formula (I) is (E)-di-tert-Butyl octadec-9-ene-1,18-diyldicarbamate (13).
The pharmaceutical compositions of the invention can be prepared by combining a compound of formula I or a stereoisomer, or ester or pharmaceutically acceptable salt thereof with an appropriate pharmaceutically acceptable carrier, diluent or excipient, and may be formulated into preparations in solid, semi-solid, liquid or gaseous forms, such as tablets, capsules, powders, granules, ointments, solutions, injections, gels and microspheres.
The present invention relates to administering 'an effective amount' of the 'composition of invention ' to the subject suffering from said disease.
Accordingly, compound of formula (I) or a pharmaceutically acceptable salt thereof and pharmaceutical compositions containing them may be administered using any amount, any form of pharmaceutical composition via any route of administration effective for treating the disease. Typical routes of administering such pharmaceutical compositions include, without limitation, oral, topical, transdermal, inhalation, parenteral, sublingual, buccal, rectal, vaginal, and intranasal.
Pharmaceutical compositions of the invention are formulated so as to allow the active ingredients contained therein to be bioavailable upon administration of the composition to a patient. Compositions that will be administered to a subject or patient may take the form of one or more dosage units. The dosage forms can also be prepared as sustained, controlled, modified and immediate dosage forms.
The compounds of formula (I), or a stereoisomer, or ester or pharmaceutically acceptable salt thereof are used as anti-malarial agents.
The present invention provides a method for treating malarial infection in a subject, wherein the method comprises administering to the said subject an effective amount of a compounds of formula (I) or pharmaceutically acceptable salt thereof.
The compounds of the present invention possess anti-bacterial activity against a wide spectrum of Gram-positive and Gram-negative bacteria, aerobic and anaerobic organisms such as Staphylococcus, Lactobacillus, Streptococcus, Escherichia, Enterobacter, Pseudomonas, Proteus, Citrobacter, Baccillus, Clostridium, Salmonella, and other organisms. Also, the compounds of the present invention possess antibacterial activity against bacterial species resistant to conventional [beta]- lactams, such as MRSA. Further, the compounds of the instant invention are effective as antiplasmodium agent for the treatment of malaria. The compound of formula (I)or a pharmaceutically acceptable salt thereof disclosed herein is present in the composition in an amount which is effective to treat the disease or the condition caused by the bacterial strains mentioned above.
Decorating biologically active molecules on a nanoparticle scaffold is an attractive strategy to improve their bio-availability and enhanced cellular uptake. Considering the structural features present in harmonine, the inventorshave envisaged that the harmonines could be easily decorated on gold nanoparticles. The UV-Vis spectrum of the harmonine coated nanoparticle dispersion, refer Fig 1 shows an absorption peak around 578 nm,
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which is typical for Au nanoparticles. The pure metallic phase of the nanoparticles was also confirmed by powder X - ray diffraction pattern corresponding to the fcc lattice system.
The process for the preparation of harmoninefrom tert-Butyl dec-9-enylcarbamate and (R)-tert-Butyl dec-9-en-2-ylcarbamate via cross metathesis reaction using Z-selective catalyst or Grubbs’ II generation catalyst as given in scheme 1, wherein said process comprising the steps of:
i. adding a solution of compound C and compound D dissolved in dichloromethane to a degassed solution of Z-selective catalyst in dichloromethane followed by heating the mixture at 45 °C for 10-12 hrs to afford desired cross metathesis product (R,Z)-di-tert-Butyl octadec-9-ene-1,17-diyldicarbamate (7);
ii. adding trifluroacetic acid to a solution of (7) of step (i) in DCM followed by stirring the resulting reaction mixture at room temperature for 4 hrs to afford trifluoroacetic acid salt of harmonine ;
iii. neutralizingthesalt of step (ii) with saturated sodium bicarbonate solution until the solution becomes basic followed by work-up to afford cis harmonine;
iv. Wherein, the step (i) may also be carried out by Grubbs’ II generation catalyst instead of Z-selective catalyst to obtain trans-harmonine.
The invention providesa novel process for the preparation of harmonine from N-Bocalaninal as given in scheme 2 comprising the steps of:
i. Adding solid potassium tert. butoxide to a solution of compound 3 in THF and stirring followed by addition of N-Bocalaninal 2 in THF and stirring the reaction mixture at temperature ranging from0 ºC to 25 ºCfor 5-6 hrs to obtain unsaturated alcohol 3a;
ii. adding Pd/C to a solution of unsaturated alcohol 3a of step (i) in ethyl acetate followed by stirring the mixture at room temperature for 1 hour to afford (R)-tert-butyl 9-hydroxynonan-2-ylcarbamate 4;
iii. treating a solution of oxalyl chloride in DCM cooled at ̶ 78 °C with DMSO and then with alcohol 4 in DCM at temperature -20 °C for 3-4 hrs to obtain a mixture;
iv. cooling the mixture of step (iii) to ̶ 78 °C and treating with Et3N followed by dilution with DCM to afford (R)-tert-Butyl 9-oxononan-2-ylcarbamate (A).
v. adding potassium tertiary butoxide to a compound 6 and cooling the mixture to 0°C followed by adding THF and stirred for 20 min;
vi. adding aldehyde A in THF to a reaction mixture of step (v) followed by stirring the reaction mixture for 2-3 hrs at room temperature in the range of 20 to 30oC to afford (R,Z)-di-tert-Butyl octadec-9-ene-1,17-diyldicarbamate;
vii. adding trifluroacetic acid to a solution of compound of step (vi) in DCM at 0°C followed by stirring the reaction mixture for 4 hrs at room temperature in the range of 20 to 30oC to afford trifluoroacetic acid salt of harmonine.
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viii. Neutralizing the salt of step (vii) with saturated sodium bicarbonate solution until the solution becomes
basic followed by work-up to afford harmonine 1.
The invention providesa process for the preparation of harmonine from sophorolipids as given in scheme 3and the
said process comprising the steps of:
i. subjecting sophorolipids to acid hydrolysis in presence of suitable hydrolyzing agent at temperature
ranging from 60 to 65°C for the period ranging from 16 to 20h to afford methyl (S,Z)-17-
hydroxyoctadec-9-enoate(14);
ii. treating a solution of lithium aluminium hydride in THF with methyl (S,Z)-17-hydroxyoctadec-9-
enoate14 in THF followed by stirring the reaction mixture for 8-10 h at room temperature to afford
(S,Z)-octadec-9-ene-1,17-diol(14a);
iii. adding triphenylphosphine to a solution of (S,Z)-octadec-9-ene-1,17-diol14a of step (ii) in THF followed
by adding a cooled mixture of diisopropylazodicarboxylate and diphenylphosphorylazide in THF and
stirring the resulting mixture at room temperature for 16-17 hrs to obtain (R,Z)-1,17-diazidooctadec-9-
ene (15).
iv. adding Lindlar catalyst to a degassed solution of diazide 15 of step (iii) in ethanol followed by subjecting
the reaction mixture to o hydrogenation for 1 hour at temperature ranging from 25 ºC to 30 ºC to obtain
harmonine.
The hydrolyzing agent is selected from sulfuric acid/methanol.
EXAMPLES
Following examples are given by way of illustration and therefore should not be construed to limit the scope of
the invention.
EXAMPLE 1
Preparation of (R)-tert-butyl 9-hydroxynonan-2-ylcarbamate (4)
(a) To an ice cooled solution of compound 3 (6.0 g, 13.81 mmol) in dry THF (60 mL) was added solid potassium
tertiary butoxide (1.28 g, 11.42 mmol) portion wise and stirred at 0 ºC for 20 min, which was followed by
addition of N-Bocalaninal2(1.80 g, 10.39 mmol) in dry THF drop wise and stirred at same temperature for 1
hour. After the reaction mixture was allowed to stir for another 4 hours and then quenched with saturated aqueous
NH4Cl (40 mL). The resulting solution was extracted with EtOAc (3 × 30 mL) and the combined organic layer
was washed with brine (40 mL), dried over anhydrous Na2SO4, and concentrated in vacuo to give crude residue.
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Silica gel flash column chromatography (using 40% EtOAc : petroleum ether) of the residue obtained above
afforded tert-butyl (R,E)-(9-hydroxynon-3-en-2-yl)carbamate 3a (1.80 g, 68%) as colorless oil: 1H NMR(400
MHz, CDCl3) 5.39 - 5.36 (m, 1 H), 5.24 - 5.19 (m, 1 H), 4.49 (br s, 2 H), 3.61 (dt, J = 2.1, 6.5 Hz, 2 H), 2.12 -
2.10 (m, 3 H), 1.48 - 1.55 (m, 2 H), 1.50 - 1.45 (m, 4 H), 1.44 - 1.41 (m, 9 H), 1.15 (t, 6.4 Hz, 3 H); 13C NMR
(100 MHz, CDCl3) 155.1, 132.2, 131.0, 79.3, 62.9, 44.0, 32.6, 29.2, 28.5, 28.5, 28.4, 27.4, 25.3, 22.2; IR
υmax(film): cm-1 3333, 1682, 1515, 1246, 1046, 1028; HRMS (ESI): m/zcalculated for C14H27NO3 [M+Na]+
280.1883 found 280.1884.
(b) To a solution of unsaturated alcohol tert-butyl (R,E)-(9-hydroxynon-3-en-2-yl)carbamate 3a(1.80 g, 10.39 mmol)
in EtOAc (25 mL) was added 5% Pd/C(20 mg) and the mixture was stirred under hydrogen balloon pressure at
room temperature for 1hour.The catalyst was filtered off and the filtrate obtained was concentratedinvacuoto
afford 4(1.70 g,99%) as colorless oil:[]D
25 = + 0.67 (c = 1.2, CHCl3); 1H NMR (400 MHz, CDCl3) 4.34 (br s, 1
H), 3.62 (t, J = 6.6 Hz, 2 H), 1.67 - 1.52 (m, 6 H), 1.52 - 1.47 (m, 9 H), 1.40 - 1.29 (m, 8 H), 1.07 (d, J = 6.4 Hz, 3
H); 13C NMR(100 MHz,CDCl3) 155.4, 78.9, 62.9, 46.5, 37.3, 32.7, 29.4, 29.3, 28.4 (2C), 28.3, 25.9, 25.6, 21.3;
IR υmax(film): cm-1 3339, 2974, 2929, 2856, 1682, 1521, 1453, 1390, 1365, 1247, 1092; HRMS
(ESI):m/zcalculated for C14H29NO3[M+Na]+282.2040 found 282.2039.
EXAMPLE 2
Preparation of(R)-tert-Butyl 9-oxononan-2-ylcarbamate (A)
A stirred solution of oxalyl chloride (2.30 mL, 28.22 mmol) in 8.0 mL of CH2Cl2, under nitrogen, was cooled to ̶
78 °C, treated drop wise over 15 min with DMSO (3.0 mL,28.22 mmol ) and stirred at ̶ 78 °C for 20 min. Then
the reaction mixture was treated over 7 min with alcohol 4 (3.80 g, 21.71 mmol) dissolved in 10mL of CH2Cl2and
was allowed to warm to ̶ 20 °C during 2.5 hours. It was further kept at this temperature for 1.5 hours, re-cooled
to ̶ 78 °C and treatedwith Et3N (15 mL) during 5 min.This mixture was warmed to ambient temperature during 30
min, diluted with CH2Cl2, washed with water and brine, dried over anhydrous Na2SO4, and concentratedinvacuo
to give 3.40 g (90%) ofA.The aldehyde Awas always freshly prepared and immediately forwarded for next step
due to its unstable nature:1H NMR (400 MHz,CDCl3) 9.71 (t, J = 6.4 Hz, 1 H), 4.38 (br s, 1 H), 3.58 (br s, 1
H), 2.42 - 2.38 (m, 1 H), 2.33 - 2.13 (m, 1 H), 1.60 - 1.54 (m, 2 H), 1.40 (br s, 9 H), 1.28 - 1.21 (m, 8 H), 1.06 (d,
J = 6.4 Hz, 3 H); IR υmax(film): cm-1 3354, 2930, 1700, 1520, 1456, 1365, 1248,.
EXAMPLE 3
Preparation of (R)-tert-Butyl dec-9-en-2-ylcarbamate (C)
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To an ice cooled solution of methyltriphenylphosphonium bromide (7.22 g, 20.23 mmol) was added potassium
tertiary butoxide (1.98 g, 17.68 mmol) portion wise, upon which the solution turns to bright yellow. The resulting
reaction mixture was stirred at same temperature for 20 min and then, a solution of freshly prepared A(1.30 g,
5.05 mmol) in THF was added drop wise over a period of 15 min. The same temperature was maintained for
another 30 min before warming to room temperature and stirring it for further 4 hours. The reaction mixture was
quenched with saturated aqueous NH4Cl (20mL) and extracted with EtOAc (3 × 5mL). The combined organic
layer was washed with brine (20mL), dried over anhydrous Na2SO4, and concentrated. Silica gel flash column
chromatography (5% EtOAc : petroleum ether) of the residue obtained afforded the olefin C (0.90 g, 68%) as
colorless oil:[]D
25 = +0.12 (c = 1.02, CHCl3); 1H NMR (500 MHz,CDCl3)  5.83 - 5.78 (m, 1 H), 5.01 - 4.92 (m,
2 H), 4.31 (br s, 1 H), 3.62 (br s, 1 H), 2.06 - 2.02 (m, 2 H), 1.44 (s, 9 H), 1.37 - 1.29 (m, 10 H), 1.10 (d, J = 6.4
Hz, 3 H); 13C NMR (125 MHz,CDCl3) 155.4, 139.2, 114.2, 78.9, 46.5, 37.4, 33.8, 29.4, 29.1, 28.8, 28.4 (3C),
26.0, 21.3; IR υmax(film): cm-1 3345, 2927, 1687, 1505, 1389, 1364, 1245, 1086; HRMS (ESI): m/z calculated for
C15H29O2N [M+Na]+ 278.2091, found 278.2092.
EXAMPLE 4
Preparation oftert-Butyl dec-9-enylcarbamate (D)
A stirred solution of the undecylinicacid(10.0 g, 54.30 mmol), diphenylphosphorylazide (15 mL, 70.65 mmol)
and triethylamine (18 mL, 135.70 mmol) in 1:1 tert-butyl alcohol (100 mL), toluene (100 mL) was refluxed for
18 hours. The reaction mixture was cooled, and the solvent was removed under reduced pressure to obtain crude
residue, which was re-dissolved in EtOAc (20 mL) and washed successively with water (50 mL), brine (50 mL),
dried over anhydrous Na2SO4, and then concentrated in vacuo to give crude residue. Silica gel flash column
chromatography of the residue using 5% EtOAc: petroleum ether, afforded N-Boc amine D(5.80 g, 41% ) as a
colorless oil: 1H NMR (400 MHz, CDCl3)  5.81 - 5.74 (m,1 H), 4.99 - 4.89 (m, 2 H), 4.59 (br s, 1 H), 3.07 (m, 2
H), 2.03 - 1.98 (m, 2 H), 1.41 (br s, 10 H), 1.34 - 1.31 (m, 2 H), 1.26 (m, 9 H); 13C NMR (100 MHz, CDCl3)
155.9, 139.0, 114.1, 78.8, 40.5, 33.7, 30.0, 29.3, 29.1, 28.9, 28.8 (3C), 28.3, 26.7; IR υmax(film): cm-1 3354, 2925,
1690, 1640, 1507; HRMS (ESI): m/z calculated for C15H29O2N [M+Na]+ 278.2091, found 278.2090.
EXAMPLE 5
14
Preparation oftert-butyl 9-hydroxynonylcarbamate (5):
(a) To a solution of alkene D(1.50 g, 5.88 mmol) in an 4:1 dioxane: water mixture (30 mL) were added a
2.5%solution ofOsO4 in tBuOH (3.0 mL , 0.294 mmol). After a period of 10 min, sodium meta-periodate (5.0 g,
23.52 mmol) was added and the resultingwhite mixture was stirred at room temperature for 5 hours,followed by
addition of saturated aqueous NaHSO3and further stirred for another 30 min. The dioxane was removed under
reduced pressure, brine was added and the aqueous layer was extracted with EtOAc (3 × 15mL).The combined
organic layer was dried over anhydrous Na2SO4, and concentratedinvacuo. Silica gel flash column
chromatography of the residue using 25% EtOAc: petroleum ether, afforded aldehydetert-butyl (9-
oxononyl)carbamate 5a (1.29 g, 85%) as a colorless oil: 1H NMR (400 MHz, CDCl3)  9.70-9.68 (m,1 H), 4.64
(br s, 1 H), 3.03 - 3.02 (m, 2 H), 2.33 - 2.38 (m, 2 H), 1.54 - 1.56 (m, 2 H), 1.37 (br s, 10 H), 1.24 (br s, 9 H); 13C
NMR (100 MHz, CDCl3)  202.9, 156.0, 78.9, 43.9, 40.6, 30.0, 29.3, 29.1(2C), 28.4 (3C), 26.7, 22.0; IR
υmax(film): cm-1 3362, 2855, 1694, 1516, 1390, 1247, 1167; HRMS (ESI): m/z calculated for C14H27O3N [M+Na]+
280.1883,found 280.1883.
(b) To a solution of above aldehyde5a(1.20 g. 4.66 mmol) in EtOH (10 mL), was added NaBH4 (0.21 g, 5.67 mmol)
portion wise at ̶ 10 oC. After stirring for 5 min at the same temperature, the reaction was warmed to room
temperature and continued stirring for 6 hours. Then reaction was quenched with saturated aqueousNH4Cl (10
mL) and extracted with EtOAc (3 × 10 mL). The combined organic layer was washed with brine (15mL), dried
over anhydrous Na2SO4,filtered and concentrated in vacuo. Silica gel flash column chromatography using 40%
EtOAc : petroleum ether afforded alcohol 5 (1.10 g, 90%) as a colorless oil:
1H NMR (400 MHz, CDCl3)  4.67 (br s, 1 H), 3.55 (t, J = 8 Hz, 2 H), 3.04 - 3.03 (m, 2 H), 2.49 - 2.47 (m, 1 H),
1.53 - 1.46 (m, 2 H), 1.38 (s, 12 H), 1.24 - 1.18 (m, 9 H); 13C NMR (100 MHz,CDCl3)  155.9, 78.8, 62.6, 40.4,
32.6, 29.9, 29.3, 29.2, 29.0, 28.3(3C), 26.6, 25.6; IR υmax(film): cm-1 3353, 1687, 1524, 1455, 1390; HRMS (ESI):
m/z calculated for C14H29O3N [M+Na]+ 282.2040, found 282.2042.
EXAMPLE 6
Preparation oftert-Butyl 9-bromononylcarbamate (B):
To a solution of5(1.00 g, 3.89 mmol) in dry CH2Cl2 (20mL), was added triphenyl phosphine (1.52 g, 5.83 mmol)
at 0 oC. After stirring for 5 min at same temperature, carbon tetrabromide (2.57 g, 7.78 mmol) was added portion
wise and the reaction mixtre was allowed to stir for 3 more hours at 0 oC. The reaction mixture was concentrated
15
in vacuo and the crude residue obtained was purified by silica gel flash column chromatography using 10%
EtOAc : petroleum ether to affordcompoundB (0.98 g, 79%) as white solid:
1H NMR (400 MHz,CDCl3)  4.54 (br s, 1 H), 3.39 (t, J = 6.8 Hz, 2 H), 3.08 (q, J = 6.3 Hz, 2 H), 1.87 - 1.79 (m,
2 H), 1.38 (br s, 12 H), 1.28 - 1.24 (m, 9 H); 13C NMR (100 MHz, CDCl3)  156.0, 79.0, 40.6, 34.0, 32.8, 30.0,
29.3, 29.2, 28.7, 28.4(3C), 28.1, 26.7; IR υmax(film): cm-1 3375, 2928, 1685, 1507, 1467; HRMS (ESI): m/z
calculated for C14H28O2N [M+Na]+ 344.1196, found 344.1201.
EXAMPLE 7
Preparation of (R,Z)-di-tert-Butyl octadec-9-ene-1,17-diyldicarbamate (7)
(a) To a solution ofB(1.20 g, 3.72 mmol) in dry acetonitrile (25 mL), was added triphenyl phosphine (1.20 g, 4.47
mmol) and heated under reflux for 36 hours. The resulting reaction mixture was concentrated in vacuo to give
crude phosphonium salt, which was subjected to repeated washings with hexanes to obtain 6 as white solid. This
salt obtained was used further without any extensive characterization. The same reaction was also performed
under microwave conditions with similar results, in which the reaction mixture in 5mL MeCN was irradiated in
microwave instrument (Monowave 300, Anton paar) for 1 hour at 140 oC.
(b) Compound6 (0.50 g, 0.85 mmol)was taken in a clean dry round bottom flask and solid potassium tertiary
butoxide (0.10 g, 0.85 mmol) was added to it. The reaction mixture was cooled to 0 oC and freshly distilled dry
THF was added, which instantaneously gave deep red/orange color indicating the formation of ylide. The reaction
mixture was stirred at same temperature for 5 min and then aldehyde A (0.13 g, 0.51 mmol), dissolved in dry
THF, was added slowly and the reaction mixture was allowed to warm to room temperature. The reaction mixture
was stirred further for 2 hours before quenching it with saturated aqueous NH4Cl solution and extracting with
EtOAc (3 x 3 mL). The combined organic layer was washed with brine (10mL), dried over anhydrous
Na2SO4,filtered and concentrated in vacuo. Silica gel flash column chromatography of the residue obtained, using
10% EtOAc: petroleum ether, afforded compound 7 (0.112 g, 46%) as a colorless oil:
[]D
26 = + 0.82 (c = 0.38, CHCl3); 1H NMR (400 MHz, CDCl3)  5.32 (t, J = 4.8 Hz, 2 H), 4.57 (br s, 1 H), 4.35
(br s, 1 H), 3.60 (br s, 1 H), 3.09 - 3.07 (m, 2 H), 1.99 - 1.96 (m, 4 H), 1.46 - 1.38 (m, 22 H), 1.27 (br s, 18 H),
1.08 (d, J = 6.9 Hz, 3 H); 13C NMR (100 MHz, CDCl3)  155.9, 155.3, 129.8, 129.8, 78.9, 78.8, 46.4, 40.6, 37.3,
30. 0, 29.7, 29.6, 29.4, 29.2, 29.2, 29.1, 28.4(6C), 28.3, 27.1(2C), 26.8, 25.9, 21.2; IR υmax(film): cm-1 3683, 3449,
2980, 2930, 1707, 1505; HRMS (ESI): m/z calculated for C28H54O4N2 [M+Na]+ 505.3976, found 505.3977.
EXAMPLE 8
Preparation of (R,Z)-octadec-9-ene-1,17-diamine (1) (Harmonine):
16
To a solution of7(0.10 g, 0.23 mmol) in CH2Cl2(5.0 mL), was added trifluroacetic acid (1.0 mL) at 0 oC and
stirred for 4 hours at room temperature. The resulting reaction mixture was concentrated in vacuo and neutralized
with saturated aqueous sodium bicarbonate until the solution becomes basic (pH = 9-10). The aqueous layer was
extracted with dichloromethane (3 x 3mL) and the combined organic layer was washed with brine, dried over
anhydrous Na2SO4and concentrated in vacuo to afford harmonine1
(41 mg, 71%) as a green oil: []D
26 = ̶ 1.75 (c = 0.6, CHCl3); 1H NMR (200 MHz, CDCl3)  5.35 (t, J = 4.5 Hz, 2
H), 2.88-2.85 (m, 1 H), 2.68 - 2.65 (m, 2 H), 2.02 - 2.00 (m, 4 H), 1.40 - 1.30 (m, 26 H), 1.05 (d, J = 6.4 Hz, 3 H);
13C NMR (100 MHz, CDCl3) 129.9, 129.8, 46.9, 42.1, 39.9, 33.5, 29.7, 29.7, 29.6, 29.5, 29.4, 29.2, 29.2,
27.1(2C), 26.8, 26.4, 23.7; HRMS (ESI): m/z calculated for C18H38N2 [M+H]+ 283.3108, found 283.3101.
EXAMPLE 9
Preparation of (R,Z)-di-tert-Butyl octadec-9-ene-1,17-diyldicarbamate (7): (Using Z-selective catalyst)
(a) To a degassed solution of Z-selective catalyst (Aldrich product code: 771082) (10 mol%, 12.4 mg, 0.1 mmol) in
dry CH2Cl2 (4.0 mL), equipped with a reflux condenser and syringe pump, was added a solution of compounds C
(50 mg, 0.196 mmol) and D(50 mg, 0.196 mmol) dissolved in degassed CH2Cl2 (3.0 mL), over a period of 2 hours
at a rate of 2.0 mL per hour at 45 ºC. The heating was continued at 45 ºC for another 10 hours, before
concentrating the reaction mixture under reduced pressure to obtain a crude mixture, which was separated by
silica gel column chromatography using 10% EtOAc : petroleum ether to afford the desired cross metathesis
product(R,Z)-di-tert-Butyl octadec-9-ene-1,17-diyldicarbamate 7 (70 mg, 74%) as major isomer (>90% desired
cis isomer). The spectral data obtained is identical to that obtained via Wittig reaction.
17
(b) To a solution of 7(0.10 g, 0.207 mmol) in CH2Cl2 (4.0 mL), was added trifluroacetic acid (0.8 mL) and resulting
reaction mixture was stirred for 4 hours at room temperature.Then reaction mixture was concentrated in vacuo
and neutralized with saturated aqueous sodium bicarbonate until the solution becomes basic (pH = 9-10). The
aqueous layer was extracted with dichloromethane (3 x 5 mL) and the combined organic layer was washed with
brine, dried over anhydrous Na2SO4 to afford the natural product harmonine1 (42 mg, 71%) as a liquid. The
spectral data obtained is identical to the harmonine obtained via Wittig reaction.
EXAMPLE 10
Preparation of (R,E)-di-tert-Butyl octadec-9-ene-1,17-diyldicarbamate (8): (using Grubbs’ II catalyst)
To a degassed solution of Grubbs’ II generation catalyst (8 mol%, 20 mg, 0.024 mmol) in dry CH2Cl2 (5.0 mL),
equipped with a reflux condenser and syringe pump, was added a solution of compounds C (77 mg, 0.30 mmol)
and D (77 mg, 0.30 mmol) dissolved in degassed CH2Cl2 (3.0 mL), over a period of 2 hours at a rate of 2 mL per
hour at 45 ºC. The heating was continued for another 10 hours before concentrating the reaction mixture under
reduced pressure to obtain crude mixture, which was separated by silica gel column chromatography using 10%
EtOAc : petroleum ether to afford the desired cross metathesis product (R,E)-di-tert-butyl octadec-9-ene-1,17-
diyldicarbamate 8 (103 mg, 70%)as major compound (>90%):
1H NMR (400 MHz, CDCl3)  5.34 (m, 2 H), 4.59 (br s, 1 H), 4.37 (br s, 1 H), 3.59 (br s, 1 H), 3.08 - 3.06 (m, 2
H), 1.93 - 1.92 (m, 4 H), 1.41 - 1.34 (m, 22 H), 1.27 (br s, 18 H), 1.07 (d, J = 6.9 Hz, 3 H); 13C NMR (100 MHz,
CDCl3) 155.8, 155.4, 130.3, 130.3, 78.9, 78.8, 46.4, 40.5, 37.2, 32.4, 32.4, 30.0, 29.6, 29.5, 29.4, 29.3, 29.2,
29.0, 28.3(6C), 27.1, 26.7, 26.0, 21.2; IR υmax(film): cm-1 3683, 3020, 2928, 2855, 1687, 1525; HRMS (ESI): m/z
calculated for C28H54O4N2 [M+Na]+ 505.3976, found 505.3976.
EXAMPLE 11
Preparation of (R,Z)-octadec-9-ene-1,17-diamine (1) (Harmonine):
18
To a solution of8(90 mg, 0.186 mmol) in CH2Cl2 (5.0 mL), was added trifluroacetic acid (1.0 mL) and resulting
reaction mixture was stirred for 4 hours at room temperature. The reaction mixture was then concentrated in
vacuo and neutralized with saturated aqueous sodium bicarbonate until the solution becomes basic (pH = 9-10).
The aqueous layer was extracted with dichloromethane (3 x 5 mL) and the combined organic layer was washed
with brine, dried over anhydrous Na2SO4 to afford transharmonine9
(35 mg, 66%, light yellow oil).1H NMR (400 MHz, CDCl3)  5.37 (br s, 2 H), 2.86 - 2.85 (m, 1 H), 2.67 (t, J =
6.9 Hz, 2 H), 2.01 - 1.96 (m, 4 H), 1.49 (br s, 4 H), 1.43-1.41 (m, 2 H) 1.28 (br s, 20 H), 1.03 (d, J = 6.4 Hz, 3 H);
13C NMR (100 MHz, CDCl3) 130.2, 130.2, 46.9, 42.1, 40.1, 33.7, 32.5 (2C), 29.6, 29.5, 29.4, 29.3, 29.2, 29.2,
29.1, 26.8, 26.4, 23.9; IR υmax(film): cm-1 3672, 1665, 1582, 1463, 1216; HRMS (ESI): m/z calculated for
C18H38N2 [M+H]+ 283.3099, found 283.3108.
EXAMPLE 12
Preparation of di-tert-Butyl (2R,17R,Z)-octadec-9-ene-2,17-diyldicarbamate (10)
To a degassed solution of D(55 mg, 0.215 mmol) in dry CH2Cl2 (4.0 mL) in a 10 mL round bottomed flask fixed
with a reflux condenser was added Z-selective Grubbs’ catalyst (Aldrich product code: 771082) (10 mol%, 13
mg, 0.021 mmol) and was heated to 55 ºC for 12 hours. The reaction mixture was concentrated under reduced
pressure and the crude mixture was separated by silicagel column chromatography using 15% EtOAc : petroleum
ether to afford compound 10 (44 mg, 84%) as colorless liquid:
1H NMR (200 MHz, CDCl3) 5.34 (t, J = 4.7 Hz, 2 H), 4.34 (br s, 2 H), 3.62 (br s, 2 H), 2.02-1.99 (m, 4 H),
1.44 (br s, 20 H), 1.28 - 1.26 (m, 18 H), 1.10 (d, J = 8 Hz, 6 H); 13C NMR (50 MHz, CDCl3)  155.4 (2C), 129.8
(2C), 78.9 (2C), 46.6 (2C), 37.4 (2C), 29.7 (2C), 29.4 (2C), 29.2 (2C), 28.5 (6C), 27.2 (2C), 26.0 (2C), 21.3 (2C);
IR υmax(film): cm-1 3028, 2932, 2856, 1710, 1503, 1352, 1356, 1210.
EXAMPLE 13
Preparationof (Z)-di-tert-Butyl octadec-9-ene-1,18-diyldicarbamate (11)
19
To a degassed solution of C(70 mg,0.274 mmol) in dry CH2Cl2(4.0 mL) in a 10 mL round bottomed flask fixed
with a reflux condenser was added Z-selective catalyst (Aldrich product code: 771082) (10 mol%, 17 mg, 0.027
mmol) and heated to 45 ºC for 12 hours. The reaction mixture was concentrated under reduced pressure and the
crude mixture was separated by silicagel column chromatography using 15% EtOAc : petroleum ether to afford
compound 11(53 mg, 80%) as a colorless oil:
1H NMR (200 MHz, CDCl3) 5.34 (t, J = 4.6 Hz, 2 H), 4.53 (br s, 2 H), 3.09-3.11 ( m, 4 H), 2.02 - 1.98 (m, 4
H), 1.44 (br s, 24 H), 1.28 (br s, 18 H); 13C NMR (50 MHz, CDCl3)  155.9 (2C), 129.8 (2C), 79.0 (2C), 40.6
(2C), 30.1 (2C), 29.7 (2C), 29.4 (2C), 29.3 (2C), 29.2 (2C), 28.4 (6C), 27.2 (2C), 26.8 (2C); IR υmax(film): cm-1
3445, 3020, 2856, 1702, 1508, 1442, 1216; HRMS (ESI): m/z calculated for C28H54O4N2 [M+Na]+505.3976,
found 505.3979.
EXAMPLE 14
Preparation ofdi-tert-Butyl ((2R,17R,E)-octadec-9-ene-2,17-diyl)dicarbamate (12):
To a degassed solution of D(30 mg, 0.117 mmol) in dry CH2Cl2(5.0 mL) in a 10 mL round bottomed flask fixed
with a reflux condenser was added Grubbs’ II generation catalyst (5 mol%, 5.0 mg, 0.0058mmol) and heated to
55 ºC for 12 hours. The reaction mixture was concentrated under reduced pressure and the crude mixture was
separated by silicagel column chromatography using 15% EtOAc : petroleum ether to afford compound 12(19
mg, 67%) as colorless liquid:
1H NMR (200 MHz, CDCl3) 5.39 (m, 2 H), 4.33 (br s, 2 H), 3.64 (br s, 2 H), 1.98 - 1.97 (m, 4 H), 1.46 (br s,
20 H), 1.34 - 1.28 (m, 18 H), 1.12 (d, J = 8 Hz, 6 H); 13C NMR (50 MHz, CDCl3)  155.4 (2C), 130.4 (2C), 79.0
(2C), 46.6 (2C), 37.4 (2C), 32.6 (2C), 29.7 (2C), 29.5 (2C), 29.2 (2C), 28.5 (6C), 26.1 (2C), 21.3 (2C); IR
υmax(film): cm-1 3020, 2932, 2856, 1705, 1503, 1367, 1216; HRMS (ESI): m/z calculated for C28H54O4N2
[M+Na]+ 505.3976, found 505.3978.
EXAMPLE 15
Preparation of (E)-di-tert-Butyl (octadec-9-ene-1,18-diyl)dicarbamate (13):
20
To a degassed solution of C (30 mg, 0.117mmol) in dry CH2Cl2 (5.0 mL) in a 10 mL round bottomed flask fixed
with a reflux condenser was added Grubbs’ II generation catalyst (5 mol%, 5.0 mg, 0.0058 mmol) and heated to
55 ºC for 12 hours. The reaction mixture was concentrated under reduced pressure and the crude mixture was
separated by silicagel column chromatography using 15% EtOAc : petroleum ether to afford product13 (23 mg,
81%) as white solid:
1H NMR (200 MHz, CDCl3) 5.37 - 5.34 (m, 2 H), 4.53 (br s, 2 H), 3.12 - 3.07 (m, 4 H), 2.01 - 1.95 (m, 4 H),
1.44 (br s, 24 H), 1.28 - 1.26 (m, 18 H); 13C NMR (50MHz, CDCl3)  = 156.4 (2C), 130.4(2C), 79.1 (2C), 40.7
(2C), 32.6 (2C), 30.1 (2C), 29.7 (2C), 29.6 (2C), 29.5 (2C), 29.1(2C), 28.5 (6C), 26.9 (2C); IR υmax(film): cm-
13445, 3018, 2856, 1708, 1508, 1456, 1393,1169; HRMS (ESI): m/z calculated for C28H54O4N2 [M+Na]+
505.3976, found 505.3979.
EXAMPLE 16
Preparation of (R,Z)-1,17-diazidooctadec-9-ene (15):
A stirred solution of lithium aluminium hydride (10 mg, 0.25 mmol) in 5.0 mL of dry THF, under argon, was
cooled to 0 °C and treated drop wise over 15 min with a solution of ester 14, (63 mg, 0.20 mmol) in dry THF.The
resulting reaction mixture was warmed to room temperature during 30 min, stirred for another 8 hours, and then
re-cooled to 0 oC and quenched simultaneously with saturated aqueous sodium sulphate and EtOAc until all of the
turbidity is lost. The reaction mixture was then filtered through a pad of celite, washed with water and brine, dried
over Na2SO4, and concentrated in vacuo to obtain diol 14a which was used as such for azide displacement
reaction without any further purification:
1H NMR (400 MHz, CDCl3)  5.35 - 5.33 (m, 2 H), 3.77 - 3.76 (m, 1 H), 3.59 (t, J = 6.6 Hz, 2 H), 2.64 (br s, 2
H), 2.04 - 1.97 (m, 4 H), 1.56 - 1.51 (m, 2 H), 1.30 (br s, 20 H), 1.17 (d, J = 6.1 Hz, 3 H).
A solution of crude diol 14a (0.20 mmol) in anhydrous THF (3.0 mL) was cooledto 0 oC, and triphenylphosphine
(3.35 g, 12.80 mmol) was added atonce. After 5 min of stirring,add this solution to previously cooled solution of
21
DIAD (0.157 g, 0.80 mmol) and DPPA (0.166 g, 0.13 mL, 0.6 mmol) in dry THF (4 mL), at 0 oC. Then the
icebath was removed, and the resulting mixture was stirred at room temperature for 16 hours. Volatiles were
evaporated in vacuo, and the residue was dissolved in EtOAc and subsequently washed with water and brine
solution. Silica gel flash column chromatography using 3% EtOAc: petroleum ether afforded the diazide 15 (50
mg, 74 % for two steps) as colorless oil:
[]D
26 =̶ 18.23 (c = 1.06, CHCl3); 1H NMR (400 MHz, CDCl3) 5.37 - 5.34 (m, 2 H), 3.43 - 3.41 (m, 1 H), 3.26
(t, J = 7.0 Hz, 2 H), 2.03 - 2.02 (m, 4 H), 1.62 - 1.59 (m, 2 H), 1.43 - 1.28 (m, 20 H), 1.25 (d, J = 6.6 Hz, 3 H); 13C
NMR (100 MHz, CDCl3)  129.9, 129.8, 58.0, 51.5, 36.2, 29.7, 29.6, 29.5, 29.5, 29.4, 29.3, 29.1, 28.8, 27.2,
27.1, 26.7, 26.1, 19.5; IR υmax(film): cm-1 3019, 2930, 2856, 2100, 1729, 1465, 1216.
EXAMPLE 17
Preparation of Harmonine (1)from diazide (15):
To a degassed solution of diazide15 (43 mg, 0.128 mmol) in ethanol was added commercially
availableLindlarcatalyst(10mg) and subjected to hydrogenation under balloon pressure for 1 hour. The reaction
mixture was filtered through the celite pad, washed with ethanol/EtOAc and the filtrate was then concentrated in
vacuo to obtain harmonine1(33 mg, 90%):
[]D
26.9 = ̶ 3.44 (c = 1.62, C6H6); 1H NMR (400 MHz, CDCl3)  5.33 (t, J = 4.8 Hz, 2 H), 2.87 - 2.83 (m, 1 H),
2.67 (t, J = 6.9 Hz, 2 H), 2.01 - 1.94 (m, 4 H), 1.44 - 1.40 (m, 2 H), 1.29 (br s, 24 H), 1.05 (d, J = 6.4 Hz, 3 H);
13C NMR (100 MHz, CDCl3) 129.9, 129.8, 46.9, 42.2, 40.1, 33.8, 29.7, 29.6, 29.6, 29.5, 29.4, 29.2, 29.2,
27.1(2C), 26.8, 26.4, 23.7; HRMS (ESI): m/z calculated for C18H38N2[M+H]+ 283.3108, found 283.3109.
EXAMPLE 18
Preparation of HarmonineSalt: (16)
To a solution of compound7 in CH2Cl2 (5.0 mL for 0.10g of compound), was added trifluroacetic acid (TFA) (1.0
mL) at 0 ºC and stirred for 4 hours at room temperature. The resulting reaction mixture was concentrated in vacuo
and the resulting trifluoroaceticacid salt16 was washed with hexanes and then dried completely to obtain
corresponding salt of harmonine.
22
EXAMPLE 19
PREPARATION OF TRIFLUOROACETIC ACID SALTS OF HARMONINE ANALOGUES
Compounds 10, 11, 12, 13were converted to respective trifluoracetic acid salts using 1:1 TFA: DCM, resulting with structures 17, 18, 19, 20 respectively using the following general procedure.
To a solution of compound in CH2Cl2(5.0 mL for 0.10g of compound), was added trifluroacetic acid (1.0 mL) at 0 oC and stirred for 4 hours at room temperature. The resulting reaction mixture was concentrated in vacuo and the resulting trifluoroaceticacid salt was washed with hexanes and then dried completely to obtain corresponding salts of the starting material.
EXAMPLE20 PREPARATION OF GOLD-NANOPARTICLES OF HARMONINE:
In a 50 mL Round bottom flask, 0.025 gm of DDAB(didodecyldimethylammoniumbromide) was added to 2.5 mL of toluene (dry). To that 1.5 mg of AuCl3 (2 x 10-3M) was added under vigorous stirring conditions leading to the formation of an orange coloured solution. An aqueous solution of NaBH4 (40 - 50 μL, 9.4 M) was added drop wise to this orange coloured solution. The stirring was allowed to proceed for 1 - 2 h to ensure complete reduction. The reduction of Au+3 ions to AuNPs containing Au0 was confirmed by the appearance of wine red colour.
To the wine red solution, harmonine (14 mg) was added in such a way that the metal to ligand molar ratio was 1:10. After 2 hours, 7.5 mL of absolute EtOH was added to this solution to separate the unreacted ligand, DDAB and other side products from the harmonine coated nanoparticles. The residual EtOH was removed under reduced pressure. The black colour precipitate was redispersed again in 3 mL of toluene (dry). The so obtained nanoparticles were subjected to further characterization like UV - Vis, XRD, and TEM.
EXAMPLE 21
ANTI-MALARIAL SCREENING OF HARMONINE AND ITS ANALOGUES:
1. Plasmodium culture
Plasmodium falciparum (3D7; Malaria Research and Reference Reagent Resource Center ID NO: MRA-102) is cultured in the laboratory as per standard protocols[Moll et.al, 2008]. Briefly, P. falciparum is cultured using 2% hematocrit washed RBCs, separated from freshly collected human blood, in RPMI media containing glutamine, sodium bicarbonate and antibiotics. Parasites are routinely synchronized using 5% sorbitol treatment.
2. Setting up the anti-malarial screen
200 μls of the diluted parasite culture (2% parasitemia and 2% hematocrit; at late ring stage), is added to each well in a 96 well plate pre-seeded with the compound of interest at the required concentration. Usually, compounds being screened for the first time are tested at 10 μM concentration. The master stocks and dilutions of the compounds are prepared in cell culture grade DMSO. NOTE: DMSO only treatment control is done and
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the results are used for normalizing the data obtained from compound treatment. All compounds are plated in triplicates. Each 96 well plate also includes negative control (compound untreated culture) and positive control (standard anti-malarial treated culture). Standard anti-malarials used are chloroquine, artemisinine and atovaquone, each at 1 μM concentration. After plating, the culture is incubated in standard growth condition for 60 hours, after which the cultures are processed for testing the effect of compounds on parasite growth as given below.
3. Estimating parasite growth by SybrGreen staining
We have standardized a SybrGreen dye based staining protocol for estimating parasite growth. SybrGreen dye binds to DNA and only the DNA bound dye gives a specific fluorescence emission at 520 nM, when excited at 498 nM. Since only the parasites have DNA (as RBCs are devoid of nucleus), the SybrGreen signal is a direct indictor of parasite density in respective samples. We use a plate reader based read out assay for quantifying relative parasite growth (or conversely % inhibition of growth) in untreated, standard anti-malarial treated and test molecules treated P. falciparum cultures. 25 μl of 10X staining solution (0.5% Triton X100 & 10X SybrGreen dye in PBSA) is added to each sample in 96 well plate, mixed and incubated for 30 min before taking readings.
Data obtained is then tabulated to compare parasite growth between untreated, standard anti-malarial treated and compound treated parasite cultures. Compounds showing greater than 80% reduction in parasitemia are selected from this primary screening for carrying out dose response analysis using the respective test molecules from 10μM to 1nM. Data from this experiment is used for the calculation of IC50 values for the respective test molecules.
Data from the preliminary screen (@ 10μM only) is shown in Figure-1 and data from the dose response experiment is shown in Figure-2. The IC-50 values for the selected molecules are tabulated in following Table-1.
Compound
MIC IC50
16
4.94 μM/ 4.13/ 4.54
17
2.25 μM/ 2.14/ 2.30
ADVANTAGES OF INVENTION
 Short sequence of synthesis
 Improved yield
 The compounds shows anti-malarial activity.

WE CLAIM
1. A compound of formula (I)
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Formula (I)
wherein
m= 0, 1, 2, 3, 4, or 5;
n= 0, 1, 2, 3, 4, or 5;
R= NH2, OH, NHR1 or NHOH;
R’= NH2, OH, NHR1 or NHOH;
R1= Alkyl, acyl or alkoxycarbonyl;
or pharmaceutically acceptable salt thereof.
2. The compound as claimed in claim 1, wherein said compounds are selected from the group consisting of :
i. di-tert-Butyl (2R,17R,Z)-octadec-9-ene-2,17-diyldicarbamate (10),
ii. (Z)-di-tert-Butyl octadec-9-ene-1,18-diyldicarbamate (11),
iii. di-tert-Butyl (2R,17R,E)-octadec-9-ene-2,17-diyldicarbamate (12),
iv. (E)-di-tert-Butyl octadec-9-ene-1,18-diyldicarbamate (13),
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v. (2R,17R,Z)-octadec-9-ene-2,17-diaminium bis(2,2,2-trifluoroacetate) (17),
vi. (Z)-octadec-9-ene-1,18-diaminium bis(2,2,2-trifluoroacetate) (18),
vii. (2R,17R,E)-octadec-9-ene-2,17-diaminium bis(2,2,2-trifluoroacetate) (19),
viii. (E)-octadec-9-ene-1,18-diaminium bis(2,2,2-trifluoroacetate) (20)
3. A process for the preparation of compound of formula (I) comprising the steps of:
i. adding a degassed solution of Z-selective Grubbs’ catalyst or Grubbs II generation catalyst to a solution of compound (R)-tert-Butyl dec-9-en-2-ylcarbamate or tert-Butyl dec-9-enylcarbamate in dichloromethane followed by heating the mixture at temperature in the range of 40-60 ºC preferably in the range of 45-55°Cfor period in the range of 10 to 12 hours to afford the desired compounds of formula (I).
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4. The pharmaceutical composition comprisingcompound of formula (I) as claimed in claim 1, or pharmaceutically acceptable salts in combination with at least one pharmaceutical excipient.
5. A process for the preparation of harmonine comprising the steps of:
i. adding a solution of compound C and compound D dissolved in solvent to a degassed solution of Z-selective catalyst in solvent followed by heating the mixture at temperature in the range of 40 to 45 °C for period in the range of 10-12 hrs to afford desired cross metathesis product (R,Z)-di-tert-Butyl octadec-9-ene-1,17-diyldicarbamate (7);
ii. adding trifluroacetic acid to a solution of (7) of step (i) in solvent followed by stirring the resulting reaction mixture at room temperature in the range of 20 to 30oC for the period ranging from 4 to 6hrsfollowed by removal of trifluoroacetic acid(TFA) and neutralization using saturated bicarbonate solution to afford cis-harmonine.
6. The process for the preparation of harmonine claimed in claim 5, wherein said step (i) may also be carried out by Grubbs’ II generation catalyst instead of Z-selective catalyst to obtain trans-harmonine.
7. The process for the preparation of harmonine claimed in claim5, whereinsolvent is dichloromethane.
8. A process for the preparation of harmonine, wherein said process comprising the steps of:
i. adding potassium tert. butoxide to a solution of (6-hydroxyhexyl)triphenylphosphonium bromide (3)in THF followed by addition of N-Bocalaninal(2)and stirring the reaction mixture at temperature ranging from 0 to 25 ºC for 5 to 6 hrs to obtain unsaturated alcohol 3a;
ii. adding Pd/C to a solution of unsaturated alcohol3a of step (i) in ethyl acetate followed by stirring the mixture at room temperature in the range of 20 to 30oC for 1 hour to afford (R)-tert-butyl 9-hydroxynonan-2-ylcarbamate (4);
iii. treating a solution of oxalyl chloride in DCM cooled at ̶ 78 °C with DMSO and then with alcohol 4 in DCM at temperature -20 °C for the period ranging from 3 to4 hrs;
iv. cooling the mixture of step (iii) to ̶ 78 °C and treating with Et3N followed by dilution with DCM to afford (R)-tert-Butyl 9-oxononan-2-ylcarbamate (A);
v. adding potassium tertiary butoxide to a compound 6 and cooling the mixture to 0°C followed by adding THF and stirred for 5 min;
vi. adding aldehyde A of step (iii) in THF to a reaction mixture of step (v) followed by stirring the reaction mixture for 2-3 hrs at room temperature to afford (R,Z)-di-tert-Butyl octadec-9-ene-1,17-diyldicarbamate (7);
vii. adding trifluroacetic acid to a solution of compound of step (vi) in DCM at 0°C followed by stirring the reaction mixture for 4 hrs at room temperature to afford harmonine(1).
9. The process for the preparation of harmonine claimed in claim 8, whereinunsaturated alcohol used is tert-butyl (R,E)-(9-hydroxynon-3-en-2-yl)carbamate.
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10. A process for the preparation of harmonine comprising the steps of:
a) subjecting sophorolipids to acid hydrolysis in presence of suitable hydrolyzing agent selected from sulfuric acid or methanol at temperature ranging from 60 to 65°C for the period ranging from 16 to 20h to afford estermethyl (S,Z)-17-hydroxyoctadec-9-enoate(14);
b) treating a solution of lithium aluminium hydride in THF with ester methyl (S,Z)-17-hydroxyoctadec-9-enoate14 in THF followed by stirring the reaction mixture for 8-10 h at room temperature to afford (R,Z)-octadec-9-ene-1,17-diol (14a) ;
c) adding triphenylphosphine to a solution of diol 14a of step (b) in THF followed by adding a cooled mixture of diisopropylazodicarboxylate and diphenoxyphosphorylazide in THF and stirring the resulting mixture at room temperature for 16-17 hrs to obtain (R,Z)-1,17-diazidooctadec-9-ene (15).
d) subjecting a solution of diazide 15 of step (c) in ethanol to hydrogenation for 1 hour at temperature ranging from 25 to 30 ºCin presence of Lindlar catalyst to obtain harmonine.