Anschubfinanzierung 2016 für das Projekt:

Synthese von Digitoxigenine-amino-derivate und deren Test auf biologische Aktivität

Report

PART I

Synthesis of AMANTADIG and Digitoxigenine-amino-derivate

Part of that project is the synthesis after Sawlewicz et al., 1972 and Villhauer et al., 2003 of the

semisynthetic cardenolide analog 3β-[2-(1-amantadine)-1-on-ethylamine]-digitoxigenin

(AMANTADIG) that efficiently suppresses cell growth in human leukemia and urologic tumor

cell lines (Nolte et al., 2015).

Figure 1: Chemical structure of AMANTADIG (3β-[2-(1-amantadine)-1-one-

ethylamine]- digitoxigenin).

In addition to the synthesis of AMANTADIG, we try to synthesize further amino-digitoxigenin

derivates that contain different residues instead of 1-adamantine (Figure 2). The compounds

containing different hydrophobic, hydrophilic or small mimic residues might help

understanding the role of the free amino group and also the mechanism of action within the

bioactivity of the compounds.

Figure 2: Different amino-digitoxigenin derivates.

Methods

Chemical synthesis of AMANTADIG and further amino-digitoxigenin derivates

The chemical synthesis of 3β-[2-(1-amantadine)-1-one-ethylamine]- digitoxigenin

(AMANTADIG) is carried out mainly following Sawlewicz et al., 1972 (Reaction II – VI) and

Villhauer et al., 2003 (Reaction VII – VIII; Figure 7). The whole synthesis can be divided into

induvial reactions. Whereas the last reaction is important for connecting 1-adamantin and also

the different residues to the amino group of digitoxigenin.

O

H

O

OHH

NH2 H

CH3

CH3

CH3

CH3

O O

OH

OHH

H

H

Cl

O

Cl

N

O

H

O

OHH

NH H

CH3

CH3

O

Cl

HNH

2

H

H

OH

N

O

H

R

CH3

H

O

H

CH3

H

OH

N

after Sawlewicz et al., 1972

..

after Villhauer et al., 2003 after Villhauer et al., 2003

adding of differentgroups containing anamino fuction

Figure 3: chemical synthesis of AMANTADIG after Sawlewicz et al., 1972 and Villhauer et al.,

2003.

Reaction I

Hydrolysis of methanolic cardiac glycoside extract to digitoxigenin

O

OH

O

OH

methanolic extractof cardiac glycosides

H+

55 °C

Digitoxigenin

Figure 4: Reaction I – hydrolysis of cardiac glycoside extract to digitoxigenin

10 g of concentrated cardenolid extract is dissolved in 400 mL MeOH and stirred on an oil bath

until the reaction has reached 55 °C. 350 mL 1 M HCl is added and the reaction is further stirred

for 35 min and 55 °C. For lowering the temperature further 1 M HCl can be added. The reaction

is stopped by extracting with 3 x 200 mL chloroform or dichloromethane. The organic layer is

neutralized by 3 % NaHCO3 (1 x 50 mL) and further washed with 3 x 100 mL H2O and dried

over sodium sulfate. The reaction mixture is evaporated to dryness.

Purification of digitoxigenin

7 g of reaction mixture is dissolved in 100 mL dichloromethane is filtrated under vacuum

through around 60 g of silica gel (0.04-0,063 mm). The silica gel was washed with following

solvents of different polarities (Table 1).

Table 1: Polarity conditions used for column fractionation

polarity solvents amount/

filtration

number

of turns

fractions

I 100 % dichloromethane 100 mL 3 1 – 3

II 9:1 dichloromethane/ ethyl acetate 50 mL 15 4 – 18

III 7:3 dichloromethane/ ethyl acetate 50 mL 15 19 – 34

IV 1:1 dichloromethane/ ethyl acetate 50 mL 7 35 – 42

The 42 fractions are collected, evaporate to dryness, weighted to calculate the amount of each

fraction and analyzed by thin layer chromatography (TLC; silica gel: 0.063 – 0.2 mm). The

concentration for each sample spreading on TLC was set to 2 mg/mL in acetone. A mobile

phase of 100 % ethyl acetate is used and every TLC is run twice. Cardenolides are detected

with anisaldehyde straining reagent. Pure fractions of digitoxigenin are combined and store

until further usage. Impure fractions are combined also and purified a second time by column

chromatography

Reaction II

Oxidation reaction – Digitoxigenin to Digitoxigenon

O O

OH

OH

O

OH

O

O

CrO3

Digitoxigenin Digitoxigenon

Figure 5: Oxidation of digitoxigenin to digitoxigenon with CrO3

1 g of digitoxigenin is dissolved in 112 mL acetone and is cooled to down to 0 °C by stirring

on ice. 1.4 mL of Kiliani solution is added drop by drop till a yellow – orange solution is

maintained. The reaction is stirred for 20 min on ice. After adding 20-25 mL of methanol to

remove surplus CrO3 the reaction is further stirred for 20 min at 0 °C. After adding 40 mL of

water the acetone is reduced under vacuum. The reaction is extracted with 4 x 30 mL chloroform

or dichloromethane and neutralized by 20 mL of 3 % Na2CO3 and washed with 3 x 30 mL

water. The organic layer is dried over sodium sulfate and evaporated to dryness. The reaction

product is analyzed by TLC. A mobile phase of 100 % ethyl acetate is used and every TLC is

run twice. Cardenolides are detected with anisaldehyde straining reagent.

Kiliani solution (Jones solution; Kiliani et al., 1913)

Adding of 2.3 mL H2SO4 to a solution of 2.6 g CrO3 in 7 mL of water

Reaction III

Reduction of Digitoxigenon to 3α-Digitoxigenin

OO

O

OH

OO

OH

OH

NaBH4

Digitoxigenon 3-Digitoxigenin

Figure 6: Reduction of digitoxigenon to 3α-digitoxigenin

1 g digitoxigenon are dissolved in 40 mL dioxane and 10 mL water. 365 mg NaBH4 in 37.5 mL

of 80 % dioxane (30 mL dioxane + 7.5 mL H2O) is added to the solution and stirred for 30 min

at 20 °C. The reaction is neutralized with 30 mL of 5 % acetic acid. It is quite important to reach

a pH value around 5. The reaction color is turning from yellow to clear and milky after adding

the acid. For stopping the reaction and avoiding the formation of additional reaction products

the fast reducing of dioxane under vacuum is also essential. The aqueous residue is extracted

with 5 x 30 mL Chloroform/ Ethanol (3:1) and the organic layer is washed 3 x 30 mL of water.

The organic layer is dried over sodium sulfate and evaporated to dryness. The reaction product

is analyzed by TLC. A mobile phase of ethyl acetate/ hexane (3:2) is used and every TLC is

run two times. Cardenolides are detected with anisaldehyde straining reagent. The Rf values

are decreasing from digitoxigenon, 3β-digitoxigenin to 3α-digitoxigenin.

Reaction IV

Tosylation of 3α-Digitoxigenin

Figure 7: Tosylation of 3α-digitoxigenin

500 mg of 3α-digitoxigenin is dissolved in 12.5 mL of dried pyridine. 465 mg Tosylchloride in

3 mL Pyridin is added and the reaction is stirred for 15 h at 20 °C. The pH is adjusted to 4 by

adding 140 - 180 mL 1 M HCl. By adding HCl, pyridine is getting protonated and is mainly

remaining in the water layer after extraction. The reaction is extracted 6 x 30 mL with

chloroform and neutralized with 3 % Na2CO3 (2 x 10 mL) and washed with water (3 x 30 mL).

The organic layer is dried over Na2SO4 and evaporated to dryness. The dark yellow reaction

product is analyzed by TLC. A mobile phase of ethyl acetate/ hexane (3:2) is used and every

TLC is run three times. Cardenolides are detected with anisaldehyde straining reagent. The

maintained reaction product is purified not further purified over silica gel.

OO

OH

OH

OO

OH

TsO

TsCl

Pyridin

3-Digitoxigenin 3-Tosyl-digitoxigenin

Reaction V

Azidation of 3α-Tosyl-digitoxigenin to 3β-acido-3-desoxy-digitoxigenin

OO

OH

TsO

OO

OH

N

N+

N

3-Tosyl-digitoxigenin 3-Azido-3-desoxy-digitoxigenin

NaN3

Figure 8: Azidation of 3α-Tosyl-digitoxigenin to 3β-acido-3-desoxy-digitoxigenin

500 mg of 3α-Tosyl-digitoxigenin is dissolved in 43 mL dimethylformamid and 700 mg of

NaN3 is added. The reaction is heated to 75 °C and stirred for 3 h under the exclusion of water.

After 16 h at 20 °C the NaN3 is filtrated twice through cotton and 3 volumes of EtOAc is added

to the flow through. The organic layer is extracted with 8 times with 20 mL of water. DMF

remains in water layer and the organic layer is dried over sodium sulfate and evaporated. The

reaction product is analyzed by TLC. A mobile phase of ethyl acetate/ hexane (3:2) is used and

every TLC is run two times and strained with Kedde reagent and anisaldehyd reagent. The Rf

value and color of 3α-Tosyl-digitoxigenin and 3β-acido-3-desoxy-digitoxigenin is similar on

TLC.

Reaction VI

Catalytic reduction of 3β-acido-3-desoxy-digitoxigenin to 3-amino-digitoxigenin

Alternative reaction after Patent WO2013000286 A1

OO

OH

N

N+

N

OO

OH

NH2

3-Azido-3-desoxy-digitoxigenin

TPP, THF

3-amino-digitoxigenin

Figure 9: Catalytic reduction of 3β-acido-3-desoxy-digitoxigenin to 3-amino-

digitoxigenin

300 mg 3β-acido-3-desoxy-digitoxigenin (0.75 mmol, EQ 1) and 236 mg TPP (tris phenyl

phosphine, 0.9 mmol, EQ 1.2) is dissolved in 5 mL of tetrahydrofuran. 1 mL of water is added

and the reaction is kept under reflux at 70 °C overnight. The remaining residue is dissolved in

80 mL dichloromethane. The organic layer (CH2Cl2 I, containing mainly the intermediate ring

product) is extracted 4 x 100 mL with a mixture of 390 mL of H2O and 10 mL 2 M HCl. The

acid aqueous phases are extracted with 8 x 30 mL dichloromethane and the organic phase

(CH2Cl2 II) is dried over sodium sulfate and evaporated. The residue contains only 0.015 g

neutral reaction products, like digitoxigenine. The aqueous phases are combined and adjusted

to pH 8-9 by NH3-solution (13 – 15 mL 3 % NH3 solution) and is extracted 8 times with 30 mL

of dichloromethane. The organic layer is washed with 2 x 50 mL water (containing little volume

of 3 % NH3-solution pH 8-9), dried of sodium sulfate and evaporated to dryness. The resulting

residue remained as yellow oil. The reaction product is analyzed by TLC. A mobile phase of

MeOH/ EtOAc/ Triethylamine (4.99/4.99/0.02) is used and every TLC is run two times and

strained with Kedde reagent and anisaldehyd reagent.

Reaction VII

Adding of Chloroacetyl chloride to 3β-amino-digitoxigenin

OO

OH

NH2

OO

OH

N

H

O

Cl

O

ClCl

3-amino-digitoxigenin Chloracetyl-NH2-digitoxigenin

Figure 10: Adding of Chloroacetyl chloride to 3β-NH2-digitoxigenin

A solution of 50 mg 3β-NH2-digitoxigenin (0.13 mmol) in 300 µl tetrahydrofuran and is added

dropwise over 30 min to stirred mixture of 10 µL chloroacetyl chloride (0.13 mmol) and 72 mg

K2CO3 (0.52 mmol) in tetrahydrofuran (150 µL) at room temperature. The reaction mixture is

then stirred for 18 hours at room temperature. The reaction mixture is then filtrated through

cotton to remove the K2CO3 and diluted to maximal 2 mL with THF. The reaction is directly

used for reaction VIII. MeOH/ EtOAc/ Triethylamine (4.99/4.99/0.02) is used and every TLC

is run two times and strained with Kedde reagent and anisaldehyd reagent.

Reaction VIII

Adding of 1-Adamantylamine (1-Aminoadamantane) or further residues (after

recommendation of Saulo)

Substrate I – 4-chloraniline

OO

OH

N

H

O

Cl

OO

OH

N

H

O

N

Cl

Chloracetyl-NH2-digitoxigenin4-chloraniline

4-chloraniline

Figure 11: Adding of 4-chloraniline to obtain 3β-amino-chloraniline digitoxigenin

A 30 mg solution of chloroacetyl-amino-digitoxigenin (0.07 mmol, 1 equiv) in 3 ml THF is

added to a mixture of 17 mg 4-chloraniline (0.13, 2 equiv) and 12 mg KI (0.07 mmol, 1 equiv).

Maximal 10 drops of water are added to dissolve the potassium iodide. The resulting reaction

is stirred 4 hours at 25 °C. The reaction was diluted with 50 mL H2O and extracted with 4 x 20

mL dichloromethane. The organic layer is washed with 1 x 10mL 3 % NaHCO3 and with 2 x

10 mL water, dried over Na2SO4 and concentrated under vacuum. The reaction product need to

be purified by silica gel. MeOH/ EtOAc (3:7) is used for TLC analysis and the TLC is strained

with Kedde reagent and anisaldehyd reagent.

Reaction VIII

Adding of 1-Adamantylamine (1-Aminoadamantane)

OO

OH

N

H

O

Cl

OO

OH

N

H

O

N

H

H

H

NH2

H

H

H

Chloracetyl-NH2-digitoxigeninAMANDTADIG

N

NCH

3

OO

OH

N

O

H

methylpiperazine -amin-digitoxigenin

N

OO

OH

N

O

H

4-phenylpiperidine-amin-digitoxigenin

Figure 12: Adding of (1) - 1-Adamantylamine to obtain 3β-[2-(1-amantadine)-1-one-

ethylamine]- digitoxigenin (AMANTADIG) (exact mass 564 m/z); adding of

1-methylpiperazine to obtain methylpiperazine-amin-digitoxigenin (exact mass

513m/z), adding of 4-phenylpiperidine to obtain 4-phenylpiperidine-amin-

digitoxigenin (exact mass 574 m/z).

A 0.5 M solution of chloroacetyl-amino-digitoxigenin (1 equiv) in THF is added dropwise over

30 min into an ice-water-cooled 0.3 M mixture of 1-adamantylamine (2-3 equiv) and K2CO3 (3

equiv) in THF. The resulting reaction is stirred on ice-water for 2 hours and after 12 hours the

volume was increased to 2 mL THF and to each sample 100 µL trieethylamine was added as a

base. Furthermore the temperature was increased for 1 hour and then set back to 25 °C.

Afterwards the reaction was kept shaking under observation for 1-3 days at room temperature.

The reaction is then filtrated to remove the K2CO3, concentrated and the residue is dissolved in

dichloromethane. The organic layer is washed with 5 % NaHCO3. The aqueous layer is

extracted twice with dichloromethane and the combined organic layers are washed with water,

dried over Na2SO4 and concentrated under vacuum. The reaction product need to be purified

by silica gel. MeOH/ EtOAc (3:7) is used for TLC and the TLC is strained with Kedde reagent

and anisaldehyd reagent.

2.4 Chromatography analysis

UPLC/MS

All UPLC/MS-analyses were conducted using an ACQUITY Ultra Performance LC™ system

(Waters, Milford, MA, USA) linked simultaneously to both a PDA 2996 photo diode array

detector (Waters, Milford, MA, USA) and a ACQUITY TQ Detector (Waters MS

Technologies, Manchester, UK), equipped with a Z-spray electrospray ionization (ESI) source

operating in positive mode. MassLynx™ software (version 4.1, Waters, Milford, MA, USA)

was used to control the instruments, as well as for data acquisition and processing. Sample

solutions (3 µL; 0.5 mg/mL) were injected into a reversed phase column (BEHC18, 1.7 μm,

1×50 mm, Waters, Milford, MA), which was maintained at 40°C. The mobile phase consisted

of solvent A (H2O/0.1 HCOOH) and solvent B (acetonitrile/0.1 HCOOH) at a flow rate of

300 μL/min: T=0 min, 5% B; T=10 min, 95% B; T=11 min, 5% B; T=13 min, 5% B. The

effluent was introduced into a PDA detector (scanning range 210–400 nm, resolution 1.2 nm)

and subsequently into an electrospray source (source block temperature 120°C, desolvation

temperature 350°C, capillary voltage 3.5 kV, cone voltage 30 V) and nitrogen as the

desolvation gas (600 L/h).Mass chromatograms were recorded in the positive and negative

ionization mode. Compounds were identified by either known standard solutions (1 mg/mL) or

by specific masses.

Results and Disscusion

Chemical synthesis of AMANTADIG and further amino-digitoxigenin derivates

Reaction I

Hydrolysis of methanolic cardiac glycoside extract to digitoxigenin

10 g of an acetone extract of a mixture of cardenolides was used for hydrolysis reaction. The

extract was obtained as it was described in the report Munkert 2014. The 10 g of cardiac

glycoside resulted in 7 g of a mixture mainly consisting of digitoxigenine. The 7 g of reaction

mixture was purified as it was described in reaction I (methods) to obtain the pure digitoxigenin.

Fractions 21 – 26 contained digitoxigenin and the combined fractions resulted in 1.3 g of

digitoxigenin that was analyzed by TLC (Figure 13).

Figure 13: Reaction I – Hydrolysis of cardenolide mixture to digitgoxgenin. A) TLC

analysis, 1) reaction mixture 2) digitoxigenin standard solution; B) design of

reaction; C) purified digitoxigenin of combined fractions F 21 – 26.

Reaction II

Oxidation reaction – Digitoxigenin to Digitoxigenon

The oxidation reaction of digitoxigenin is performed as it was described in the section methods

– reaction II. In total 5 g of digitoxigenin was oxidized to 4.2 g digitoxigenon. This is a

conversion rate of 84 %. The reaction product was analyzed by TLC and UPLC/MS and directly

used for the following reduction reaction (Figure 14).

Figure 14: Reaction II – Oxidation of digitoxigenin to digitoxigenon. A) Reaction design;

B) TLC analysis of reaction (1) digitoxigenin standard, (2) digitoxigenon

standard, (3) reaction mixture, (4) extracted reaction containing digitoxigenon;

C) Chromatogram of reaction product – digitoxigenon: Rt = 5.17; D) MS spectra

in positive and negative ion mode of digitoxigenone with exact mass 372 m/Z.

Reaction III

Reduction of Digitoxigenon to 3α-Digitoxigenin

The reduction of 2.35 g digitoxigenon was achieved by obtaining in total 2.32 g of mainely 3α-

digitoxigenin and only traces of 3β-digitoxigenin (Figure 14 B). This was a total conversion

rate of 98 %. The reaction mixture was analyzed on TLC with mobile phase of EtOAc (Figure

14 A) and EtOAc: Hexane (3:2; Figure 14 B). Using the first mobile phase digitoxigenon could

be separated from the possible reaction products 3β-digitoxigenin and 3α-digitoxigenin whereas

using the last-mentioned solvent combination 3β-digitoxigenin and 3α-digitoxigenin could be

separated by decreased Rf value.

Figure 15: Reaction III – Reduction of digitoxigenon.to 3α-digitoxingenin A) TLC analysis

of reaction in EtOAc (1) digitoxigenon standard, (2) reaction mixture, (3) 3α-

digitoxingenin stamdard; B) TLC analysis of reaction (1) digitoxigenon

standard, (2) 3β-digitoxigenin standard, (3) reaction mixture, (4) 3α-

digitoxingenin standard; C) Chromatogram of reaction product – 3α-

digitoxingenin: Rt = 5.11; D) MS spectra in positive and negative ion mode of

digitoxigenone with exact mass 374 m/z; E) change of reaction color after pH

shift to 5.

Increasing the amount of starting material, a main focus has to be set on reducing the dioxan

almost completely and also setting the pH value to 5 by using acetic acid so there is no

possibility of forming a 17α-digitoxingenin by conversion of the lactone ring in basic pH and

in dioxan. Furthermore the changing of pH towards 5 could be observed as the color of the

reaction mixture is turning from yellow into clear and finally the reaction products are

precipitating (Figure 14 E).

Reaction IV

Tosylation of 3α-Digitoxigenin

The tosylation of 3α-digitoxigenin followed the protocol as it was described above. In total 2.1

g of 3α-digitoxigenin was used in the reaction to obtain finally 2.75 g of 3α-tosyl-digitoxigenin.

This is in total a recovery of 93 %. However, besides the tosylation product an additional

product was obtained. In addition to the remaining substrate and the 3β-digitoxigenin, this side

product decreased the conversion rate down to 50 %. Over the 15 hours of reaction time the

reaction mixture turned from yellow to orange (Figure 15). Due to their property as a leaving

group tosylates are used as intermediates in the preparative organic chemistry. By converting

alcohols in tosylates poor leaving group HO- is converted into a good leaving group, whereby

substitution reactions are made possible at this position of the carbon skeleton. The tosyl acts

electron withdrawing, so the underlying tosyl chloride is a strong acid and the tosylate is a good

leaving group. The anion (= tosylate) of tosyl chloride occurs as a leaving group. The

preparation of tosyl esters and amides are conducted in the presence of a base, which absorbs

hydrogen chloride. The selection of the base is often crucial to the efficiency of tosylation. Here

pyridine was used as an appropriate base. Adding HCl at the end to achieve a pH of 4 to the

reaction, leaded to a further protonation of the pyridine and therefore pyridine remained in the

water layer, during the extraction with chloroform. The extracted reaction mixture was analyzed

on TLC with mobile phase of EtOAc: Hexane (3:2; Figure 15 B) and also by UPLC/MS (Figure

15 C).

Figure 16: Reaction IV – Tosylation of 3α-digitoxingenin A) TLC analysis of reaction in

EtOAc/Hexane (3:2) (1) 3α-digitoxingenin standard, (2) reaction mixture, (3)

tosyl standard; B) color change during reaction from light yellow to orange; (C)

Chromatogram of reaction mixture – 3α/3β-digitoxingenin: Rt = 5.11; unknown

product: Rt = 6.42; 3α-tosyl digitoxigenin Rt = 7.26;D) MS spectra in positive

and negative ion mode of 3α-tosyl digitoxigenin exact mass 528 m/z; E) MS

spectra in positive and negative ion mode of unknown product.

The unknown compound is not elucidate so far. However it is only appearing in the tosylation

reaction and can be eliminated within the next reaction.

Reaction V

Azidation of 3α-Tosyl-digitoxigenin to 3β-acido-3-desoxy-digitoxigenin

The formation of 3β-acido-3-desoxy-digitoxigenin followed as it is described above. 2.4 g of

3α-tosyl-digitoxigenin was converted to 1.7 g 3β-acido-3-desoxy-digitoxigenin. This is

corresponding to a recovery rate of 94 %. However the conversion rate towards pure 3β-acido-

3-desoxy-digitoxigenin is around 55 %. For avoiding the DMF the reaction mixture was diluted

with 3 times volume of EtOAc and washed extensively with water. The organic layer is

afterwards free of DMF. The 3β-acido-3-desoxy-digitoxigenin product as well as the reaction

mixture was analyzed by on TLC with mobile phase of EtOAc: Hexane (3:2; Figure 16 B) and

also by UPLC/MS (Figure 16 C). 3β-acido-3-desoxy-digitoxigenin could only be clearly

identified by UPLC/MS as substrate and product are too similar within their Rf values.

Figure 17: Reaction V – Azidation of 3α-tosyl digitoxigenin A) TLC analysis of reaction in

EtOAc/Hexane (3:2) (1) 3β-digitoxingenin standard, (2) 3α-tosyl digitoxigenin,

(3) reaction mixture, (4) 3α-digitoxigenin B) reaction design; (C) Chromatogram

of reaction mixture – 3α/3β-digitoxingenin: Rt = 5.11; unknown product: Rt =

6.43; 3β-acido-3-desoxy-digitoxigenin Rt = 7.12; D) MS spectra in positive and

negative ion mode of 3β-acido-3-desoxy-digitoxigenin exact mass 399 m/z.

Reaction VI

Catalytic reduction of 3β-acido-3-desoxy-digitoxigenin to 3-amino-digitoxigenin

The catalytic reduction was performed on the patent WO2013000286 and is based on the

Staudinger Reaction (Staudinger et al., 1919), however the extraction of the amin followed the

description of Salewicz et al., 1972. The mechanism starts with a nucleophilic attack of

triphenylphosphine at the azid. The resulted phosphazid is than cyclized to a four ring structure

that reacts by the elimination of molecular nitrogen towards a phosphazen. As water is present

in the reaction, this is leading to the conversion to an amin while the triphenylphosphine oxide

is removed (Figure 18).

R´

RN

N+

N

P

Ph

Ph Ph

R´

RN

N

N

PPh3

R´

R NN

NP

Ph3

R´

R´ NPPh

3

R´

RNH

2

+

H2O

O=PPh3

Figure 18: Mechanism of the Staudinger Reaction (1919).

In total 1,47 g of the reaction mixture, containing mainly 3β-acido-3-desoxy-digitoxigenin of

reaction V was used. This resulted in around 830 mg of pure 3β-amino-digitoxigenin,

corresponding to a 60 % conversion rate (Figure 18). However one should consider that the

reaction mixture was in total only containing around 1 g of pure 3β-acido-3-desoxy-

digitoxigenin. The first extraction of the reaction mixture with acid water leads a remaining

organic layer (I) containing mainly the intermediate product (Figure 18 A, E). The extraction

of the acid water layer with dichloromethane resulted in an organic layer (II) containing

digitoxigenin. (Figure 18 A). Whereas the extraction of the basic water layer finally yielded

into the pure 3β-amino-digitoxigenin (Figure 18 A, D). Extracting the reaction mixture by

different pH values leads to a purification of the final product. The obtained 3β-amine-

digitoxigenin remained as a yellow oil. The organic layers were analyzed by TLC with a mobile

phase of EtOAc: MeOH containing trimethylamine as base (1:1) and also by UPLC/MS (Figure

18).

Figure 19: Reaction VI – catalytic reduction of 3β-acido-3-desoxy-digitoxigenin to 3β-

amine-digitoxigenine A) TLC analysis of reaction in EtOAc/MeOH (1:1) with

triethylamine (1) 3β-acido-3-desoxy-digitoxigenin, (2) organic layer III, (3)

organic layer III of second reduction of organic layer I , (4) organic layer II B)

reaction design; finally obtained 3β-amine-digitoxigenin (C) Chromatogram of

organic layer III - 3β-amine-digitoxigenine, Rt = 2.89; D) MS spectra in positive

and negative ion mode of 3β-amine-digitoxigenin exact mass 373 m/z; E)

Chromatogram of organic layer I – intermediate ring product, Rt = 5.51, exact

mass 661 m/z.

The organic layer II weighted around 70 mg in total. As it contained mainly digitoxigenin, this

could be recovered and used again in reaction II. However a major loss of the reaction was the

high amount of intermediate product that could only be converted barely to further 3β-amine-

digitoxigenin (100 mg) by adding water to the in THF and acetone dissolved intermediate

product. Maybe the conversion rate will be increasing by starting with a larger volume of water

in the reaction and also by elucidating the additional product in reaction VI, so an optimization

can already be started at that point.

Reaction VII

Adding of Chloroacetyl chloride to 3β-amino-digitoxigenin

For reaction VII maximal 50 mg was used, as it was not extracted and directly used for reaction

VIII. However the conversion rate of the substrate could be determined by around 90 %. In case

the reaction product was used in the reaction VIII described after Villhauer et al., 2003 the

reaction mixture was filtrated, diluted with dried CH2Cl2 and evaporated (Figure 20).

Figure 20: Reaction VII – Adding of chloroacetylchloride to 3β-amine-digitoxigenine A)

TLC analysis of reaction in EtOAc/MeOH (1:1) with triethylamine (1) 3β-

amine-digitoxigenine, (2) reaction mixture I, (3) reaction mixture II B) reaction

design; evaporating of the filtrated and diluted reaction mixture; C)

Chromatogram of reaction mixture - 3β-amine-digitoxigenine, Rt = 3.07; 3β-

chlor-amine-digitoxigenine Rt = 5.66 and isomer Rt = 5.29 D) MS spectra in

positive and negative ion mode of Rt = 5.66 exact mass 499 m/z; E)

Chromatogram of of Rt = 5.29 exact mass 499 m/z.

Reaction VIII

Adding of 1-Adamantylamine (1-Aminoadamantane) or further residues

Substrate I – 4-chloraniline

The adding of the different residues was in case of 4-chloraniline following the protocol

described above. The Finkelstein reaction is used to exchange the chlor residue against an

iodide residue as this is a better leaving group. Therefore the adding of the different residues

can be performed with higher efficacy. The reaction is schematically shown in Figure 21. Here

the iodide attacks the antibonding orbital of the chloromethane, whereby a bond between iodide

and chloromethane is built up while simultaneously the chlorine-carbon bond is weakened.

After passing through the transition state in which both halides are partially bonded to the

carbon atom, the chloride ion exits. Since potassium chloride is less soluble in THF with water

than potassium iodide, the equilibrium is shifted by precipitation of the chloride. By now having

an iodide as leaving group the adding of the 4-chloraniline took place.

R CH

2

Cl I CH2

R

Cl

I CH

2

RKI + -

K+ + KCl

Figure 21: Finkelstein Reaction, named after the German chemist Hans Finkelstein (1885-

1938). It describes the replacement of the halogen substituent of a halogenated

hydrocarbon (eg. haloalkane) by an iodide or a fluoride.

4-chloroaniline was added with an equation of 2 1 to the potassium iodide. Adding 8 drops of

water was enough to dissolve the KI in 3 mL of THF. The reaction was kept for 4 hours under

140 rpm at 25 °C. This resulted in a partial conversion of the substrate (Rt = 5.66) and obtaining

the product (Rt = 6.72), which could be optimized by extending the reaction time or increasing

the reaction temperature up to 45 °C.

Figure 22: Reaction VIII – Adding of 4-chloroaniline A) TLC analysis of reaction in

EtOAc/MeOH (7:3) (1) reaction mixture B) reaction design; reaction mixture

after 4 hours, precipitating is slightly seen; C) Chromatogram of reaction mixture

- 4-chloroaniline, Rt = 1.65; 3β-chlor-amine-digitoxigenine Rt = 5.66 and

chloroaniline-amine-digitoxigenine Rt = 6.72 D) MS spectra in positive and

negative ion mode of Rt = 6.72 exact mass 542 m/z.

Reaction VIII

Adding of 1-Adamantylamine (1-Aminoadamantane) and further residues after Villhauer et

al., 2003

In contrast as it was described by Villhauer et al.,2003, the base triethylamin was added to the

reaction system as a catalyst. The favorite base would have been diisopropylamine. Only after

adding the base triethylamin a conversion of the substrate was detected at all. AMANTADIG

could be only obtained in traces so far. This reaction will be performed after the example above.

Figure 23: Synthesis of AMANTADIG. A) Chromatogram of reaction mixture,

AMANTADIG Rt = 5.20 D) MS spectra in positive ion mode of Rt = 5.20 exact

mass 564 m/z.

Figure 24: Synthesis of 1-methylpeperazine-amine-digitoxigenin. A) Chromatogram of

reaction mixture, Rt = 3.75 D) MS spectra in positive and negative ion mode of

Rt = 3.75 exact mass 513 m/z.

Figure 25: Synthesis of 4-phenylpiperidine-amine-digitoxigenin. A) Chromatogram of

reaction mixture, Rt = 4.89 D) MS spectra in positive and negative ion mode of

Rt = 4.89 exact mass 574 m/z.

Determination of the detection limit of AMANTADIG by UPLC/MS

Different concentrations (1000 nM, 100 nM, 10 nM, 1 nM) of AMANTADIG in acetonitrile

were analyzed by UPLC/MS. 1 nM could still be determined by MS. However almost no peak

was visible in the chromatogram. A 100 nM solution is showed in the following chromatogram

including MS spectra anazlyzis in positive and negative ion mode. The exact mass of

AMANTIG is 564 m/z.

Figure 26: UPLC/MS analysis of AMANTADIG. A) Chromatogram of AMANTADIG, Rt

= 5.10; B) MS spectra of AMANTADIG in positive and negative ion mode.

Exact mass 564 m/z.