Indian Patents. 259398:SYNTHESIS OF DEOXYBIOTINYL HEXAMETHYLENEDIAMINE-DOTA

Title of Invention	SYNTHESIS OF DEOXYBIOTINYL HEXAMETHYLENEDIAMINE-DOTA
Abstract	A process for the synthesis of deoxybiotinyl hexamethylenediamine-DOTA is herein described. Said process comprises reacting biotinyl hexamethylenediamine with tri-t-butyl-DOTA in the presence of benzotriazol-1-yl-oxytripyrrolidinophos-phonium hexafluorophosphate, as the condensing agent, and triethylamine, as the base.

Full Text	SYNTHESIS OF DEOXYBIOTINYL HEXAMETHYLENEDIAMINE-DOTA FIELD OF THE INVENTION The invention reported here relates to an improved procedure for the synthesis of deoxybiotinyl hexamethylenediamine-DOTA. BACKGROUND OF THE INVENTION The compound deoxybiotinyl hexamethylenediamine-DOTA has been described in international patent application WO 02066075 under the name of the Applicant. DOTA is 1,4,7,10-tetraazacyclododecanotetra-acetic acid (1,4,7,10-tetraazacyclododecanetetra-acetic acid). The structural formula of deoxybiotinyl hexamethylenediamine-DOTA is reported here for completeness. The process for synthesising this product is also described in the same patent application WO 02066075 and also reported in the example on page 15 of the above-mentioned application. The compound reported here as ST2210 corresponds to compound 4 of the Example of WO 02066075. In particular the intermediate product biotinyl hexamethylenediamine hydrochloride (ST2551 or compound 3 of the example of WO 02066075), 2 was obtained with preparatory chromatography with a yield of 55% and an undefined titre. The final deoxybiotinyl hexamethylenediamine-DOTA product (ST2210 or compound 4 of the example of WO 02066075), was obtained by condensing ST2551 with DOTA as illustrated in diagram 1. The reaction conducted in this way produced low synthetic yields (the presence in DOTA of four unprotected carboxylic functionality equivalents, involved the formation of various by-products) and problems with purification. Indeed to obtain the end product it was necessary to use HPLC on a preparatory scale with a yield of 20%. 3 DESCRIPTION OF THE INVENTION In the scale-up study, in order to improve the yield of the process and make the purification process easier, alternative synthesis methods were examined: The improved synthesis process has the following advantages over the one described earlier: 1. in the coupling step or condensation (described in WO 02066075 as "step d") the use of tri-terf-butyl-DOTA avoids the formation of more condensation products, due to the partial reactivity of the free carboxyls, which occurred even when working under controlled pH conditions; 2. still in the same step, the condensing agent and the base were changed; the improved yield was obtained using benzotriazol-1-yl- oxytripyrrolidinophosphium hexafluorophosphate (PyBOP Benzotriazol-1 -yl-oxytripyrrolidinophosphonium hexafluorophosphate), as the condensing agent and triethylamine (TEA) as the base; 3. biotinyl hexamethylenediamine (ST2251) is used as a free base, having shown that the use of the dihydrochloride caused an increase in the quantity of unwanted products; in particular there was a noticeable formation of a by-product corresponding to the dimer, in which ST2551 reacts with 2 molecules of tri-f-butyl-DOTA; 4. the final step consists of the hydrolysis of the three ter-butyl groups in acid aqueous solution, for example using from 3N to 6N HCI in 4 different ratios with the product for times ranging between 1 hour and 12 hours, with yields ranging between 96% and 98%; 5. elimination from the synthesis process of two rather laborious preparatory chromatography stages; 6. obtaining a product with a titre of from 94% to 96%. Therefore the object of the present invention is a process for synthesising deoxybiotinyl hexamethylenediamine-DOTA which comprises reacting biotinyl hexamethylenediamine with tri-f-butyl-DOTA in the presence of benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate, as the condensing agent, and triethylamine, as the base. The process of the present invention therefore involves a step following the condensation step which is hydrolysis at acid phis. Condensation is preferably carried out in an organic solvent. More preferably this solvent is dichloromethane. Of those analysed, the best synthesis method developed is the one which involved the stages illustrated in diagram 2. In addition, the invention is illustrated with Examples which are in no way limited to the invention itself. 5 6 EXAMPLES (for the numbering of the compounds refer to Diagram 2) Methods The tests carried out were monitored using an analytical HPLC system connected to a diode array detector and to a mass spectrometer with an electrospray. The analytical method developed involves the use of an ACE C-18 analytical column (150 x 4.60 mm, 5\x), a flow rate of 1.0 mL/min and a linear elution gradient of 5-95% of B in 20 min (A= H20 + 0.1% TFA; B= AcCN + 0.1% TFA), or 10-90 % of B in 20 min, or alternatively a 10% isocratic solution of B for 6 min, followed by the gradient 10-90 % of B in 20 min. For the end product ST2210, a method working in an isocratic solution of B at 15% is used for 40 min. The range of wave lengths examined by the detector is 205-400 nm. Rapid monitoring by TLC involves the use of eluents mixtures of the OEt acid type: isopropanol: NH3 30% or DCM : isopropanol:NH3 30% (4:5:1), revealing the products sorted out (not UV visible) by means of b or phosphomolybdic reagent. Preparation of biotinyl hexamethylenediamine (ST2551) as the free base (2.) Product ST2551 [(1), purity of 91%, 400 mg, 1.0 mmol) was dissolved in water (20 mL) and the solution was put into a separating funnel; a solution of NaOH 2M (20mL) was added and the product precipitated was extracted with DCM (40 mL) three times. The combined organic phases were dehydrated 7 with anhydrous Na2S04 and evaporated under reduced pressure. The product was obtained (2) as a white solid (290 mg, yield = 98%). 1H-NMR (200 MHz, CDCI3) 5 (ppm): 1.25-2.05 [m, 16H, CH(CH2)4 and NHCH2(CAY2)4)]; 2.50-3.30 (m, 9H, 2 x HCHS and CHS and 3 x Ctf2N); 4.32 (m, 1H, CHCHNH); 4.51 (m, 1H, CHCHNH); 5.77 (s, 1H, CONH); 6.05 (s, 1H, CONH). ES-MS m/z: 329.5 [M+H]+. Synthesis of deoxybiotinyl hexamethylenediamine - ter- butyl-DOTA (4). A solution of tri-fert-butyl-DOTA [(3), 524 mg, 0.915 mmol, 1 eq], PyBOP (714 mg, 1.37 mmol, 1.5 eq) and triethylamine (166 L, 1.19 mmol, 1.3 eq) in DCM (5 mL) left under agitation at room temperature for 10 minutes, was added drop by drop to a solution of ST2551-free base [(2), 300 mg, 0.915 mmol, 1 eq) in DCM (5 mL), obtained by heating the mixture at 40°C for 5 min. It was left to react at room temperature for 3 hours, monitoring the completeness of the reaction by TLC and LC-MS, according to the methods above. At the end of the reaction the solvent was evaporated under reduced pressure, the residue was dissolved in DCM and was washed twice with NaOH 1M. The organic phase was dehydrated on Na2S04 and evaporated under vacuum. The residue thus obtained underwent chromatography on a silica gel column (ratio in weight of crude product: silica 1:30), using as the 8 eluent system a DCM mixture: isopropanol 5:4 to which is added an increasing concentration of 30% aqueous NH3 (from 0.2 to 1). The column was packed with the initial DCM mixture: isopropanol: NH3 (5:4: 0.2) and the process was continued with the gradient of NH3. [the eluent mixture usually becomes homogeneous after agitation; only in the final phase of the elution, with NH3=1 it may prove to be necessary to add isopropanol to improve miscibility, in this case using the volumetric ratio (4:5:1) (DCM:isopropanol:NH3). A slightly yellow oily product was obtained which tended to produce a foamy solid under vacuum [(4),, 600 mg, yield = 74%). 1H-NMR (300 MHz, DMSO-d6, T=50°C) 8 (ppm): 1.16-1.70 (m, 43H, CH(CH2)4 and NHCH2(CH2)4 and 9 x CH3); 2.40-3.32 (m, 34H, 2 x HCHS and 3 x CH2N, 8 x DOTA-ring CH2 and CHS and 4 x DOTA CH2CO and NH amine); 4.13 (m, 1H, CHCHNH); 4.29 (m, 1H, CHCHNH); 6.18 (s, 1H, NH biotin); 6.21 (s, 1H, NHbiotin); 8.10 (t, 1H, NH amide). 13C-NMR (75 MHz, DMSO-d6) 8 (ppm): 27.22; 27.35; 27.56; 28.50; 28.61; 29.04; 29.23; 30.14; 30.25; 30.39; 39.14; 40.42; 50.07; 52.59; 52.82; 53.24; 54.72; 56.14; 56.92; 57.38; 59.31; 60.05; 61.90; 80.75; 163.34; 170.98; 171.25. ES-MS m/z: 883.4 [M+H]+. Synthesis of deoxybiotinyl hexamethylenediamine -DOTA [ST2210, (5)]. A solution of (4) (210 mg, 0.238 mmol) in HCI 6N [1 mL, 20% w/v solution of (4)] was left under agitation at room temperature for 1 hour. Then the mixture 9 was evaporated at reduced pressure, the residue was dissolved in H20 (approx. 1:50 w/v) and the solution was subjected to lyophilisation. A white solid was obtained (205 mg, 96%).1H-NMR (200 MHz, DMSO-d6) 5 (ppm): 1.30-1.77 [m, 16H, CH(CH2)4 and NHCH2(CH2)4]; 2.59 (d, 1H, HCHS); 2.77- 2.88 (m, 7H, HCHS and 3 x CH2N); 2.98-3.70 (m, 25H, 8 x DOTA-ring CH2 and CHS and 4 x DOTA CH2CO); 4.15 (m, 1H, CHCHNH); 4.31 (m, 1H, CHCHNH); 7.58 (br s, 2H, 2 x biotin NH); 8.86 (t, 1H, NH amide); 9.19 (br s, 2H, NH2+). 13C-NMR (75 MHz, DMSO-d6) 5 (ppm): 25.77; 25.85; 26.32; 26.53; 26.70; 28.74; 29.16; 47.08; 47.16; 48.54; 49.00; 51.35; 53.13; 54.51; 55.38; 56.02; 59.90; 61.61; 163.44; 165.58; 168.90; 172.31. ES-MSm/z: 715.4 [M+H]+. 10 CLAIMS 1. Process for synthesising deoxybiotinyl hexamethylenediamine-DOTA which involves reacting biotinyl hexamethylenediamine with tri-f-butyl- DOTA in the presence of benzotriazol-1-yl- oxytripyrrolidinophosphonium hexafluorophosphate, as the condensing agent, and triethylamine, as the base. 2. Process according to claim 1, in which biotinyl hexamethylenediamine is reacted with tri-f-butyl-DOTA in an organic solvent. 3. Process according to claim 2 in which the organic solvent is dichloromethane. 4. Process according to any one of the preceding claims, involving a subsequent step of hydrolysis in an aqueous environment with an acid pH. A process for the synthesis of deoxybiotinyl hexamethylenediamine-DOTA is herein described. Said process comprises reacting biotinyl hexamethylenediamine with tri-t-butyl-DOTA in the presence of benzotriazol-1-yl-oxytripyrrolidinophos-phonium hexafluorophosphate, as the condensing agent, and triethylamine, as the base.

Full Text

SYNTHESIS OF DEOXYBIOTINYL HEXAMETHYLENEDIAMINE-DOTA
FIELD OF THE INVENTION
The invention reported here relates to an improved procedure for the
synthesis of deoxybiotinyl hexamethylenediamine-DOTA.
BACKGROUND OF THE INVENTION
The compound deoxybiotinyl hexamethylenediamine-DOTA has been
described in international patent application WO 02066075 under the name
of the Applicant. DOTA is 1,4,7,10-tetraazacyclododecanotetra-acetic acid
(1,4,7,10-tetraazacyclododecanetetra-acetic acid).
The structural formula of deoxybiotinyl hexamethylenediamine-DOTA is
reported here for completeness.

The process for synthesising this product is also described in the same
patent application WO 02066075 and also reported in the example on page
15 of the above-mentioned application. The compound reported here as
ST2210 corresponds to compound 4 of the Example of WO 02066075. In
particular the intermediate product biotinyl hexamethylenediamine
hydrochloride (ST2551 or compound 3 of the example of WO 02066075),

2
was obtained with preparatory chromatography with a yield of 55% and an
undefined titre.
The final deoxybiotinyl hexamethylenediamine-DOTA product (ST2210 or
compound 4 of the example of WO 02066075), was obtained by condensing
ST2551 with DOTA as illustrated in diagram 1.

The reaction conducted in this way produced low synthetic yields (the
presence in DOTA of four unprotected carboxylic functionality equivalents,
involved the formation of various by-products) and problems with purification.
Indeed to obtain the end product it was necessary to use HPLC on a
preparatory scale with a yield of 20%.

3
DESCRIPTION OF THE INVENTION
In the scale-up study, in order to improve the yield of the process and make
the purification process easier, alternative synthesis methods were
examined:
The improved synthesis process has the following advantages over the one
described earlier:
1. in the coupling step or condensation (described in WO 02066075 as
"step d") the use of tri-terf-butyl-DOTA avoids the formation of more
condensation products, due to the partial reactivity of the free
carboxyls, which occurred even when working under controlled pH
conditions;
2. still in the same step, the condensing agent and the base were
changed; the improved yield was obtained using benzotriazol-1-yl-
oxytripyrrolidinophosphium hexafluorophosphate (PyBOP
Benzotriazol-1 -yl-oxytripyrrolidinophosphonium hexafluorophosphate),
as the condensing agent and triethylamine (TEA) as the base;
3. biotinyl hexamethylenediamine (ST2251) is used as a free base,
having shown that the use of the dihydrochloride caused an increase
in the quantity of unwanted products; in particular there was a
noticeable formation of a by-product corresponding to the dimer, in
which ST2551 reacts with 2 molecules of tri-f-butyl-DOTA;
4. the final step consists of the hydrolysis of the three ter-butyl groups in
acid aqueous solution, for example using from 3N to 6N HCI in

4
different ratios with the product for times ranging between 1 hour and
12 hours, with yields ranging between 96% and 98%;
5. elimination from the synthesis process of two rather laborious
preparatory chromatography stages;
6. obtaining a product with a titre of from 94% to 96%.
Therefore the object of the present invention is a process for synthesising
deoxybiotinyl hexamethylenediamine-DOTA which comprises reacting
biotinyl hexamethylenediamine with tri-f-butyl-DOTA in the presence of
benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate, as the
condensing agent, and triethylamine, as the base.
The process of the present invention therefore involves a step following the
condensation step which is hydrolysis at acid phis.
Condensation is preferably carried out in an organic solvent. More preferably
this solvent is dichloromethane.
Of those analysed, the best synthesis method developed is the one which
involved the stages illustrated in diagram 2.
In addition, the invention is illustrated with Examples which are in no way
limited to the invention itself.

5

6
EXAMPLES (for the numbering of the compounds refer to Diagram 2)
Methods
The tests carried out were monitored using an analytical HPLC system
connected to a diode array detector and to a mass spectrometer with an
electrospray. The analytical method developed involves the use of an ACE
C-18 analytical column (150 x 4.60 mm, 5\x), a flow rate of 1.0 mL/min and a
linear elution gradient of 5-95% of B in 20 min (A= H20 + 0.1% TFA; B=
AcCN + 0.1% TFA), or 10-90 % of B in 20 min, or alternatively a 10%
isocratic solution of B for 6 min, followed by the gradient 10-90 % of B in 20
min. For the end product ST2210, a method working in an isocratic solution
of B at 15% is used for 40 min. The range of wave lengths examined by the
detector is 205-400 nm.
Rapid monitoring by TLC involves the use of eluents mixtures of the OEt acid
type: isopropanol: NH3 30% or DCM : isopropanol:NH3 30% (4:5:1), revealing
the products sorted out (not UV visible) by means of b or phosphomolybdic
reagent.
Preparation of biotinyl hexamethylenediamine (ST2551) as the free base
(2.)
Product ST2551 [(1), purity of 91%, 400 mg, 1.0 mmol) was dissolved in
water (20 mL) and the solution was put into a separating funnel; a solution of
NaOH 2M (20mL) was added and the product precipitated was extracted with
DCM (40 mL) three times. The combined organic phases were dehydrated

7
with anhydrous Na2S04 and evaporated under reduced pressure. The
product was obtained (2) as a white solid (290 mg, yield = 98%).
1H-NMR (200 MHz, CDCI3) 5 (ppm): 1.25-2.05 [m, 16H, CH(CH2)4 and
NHCH2(CAY2)4)]; 2.50-3.30 (m, 9H, 2 x HCHS and CHS and 3 x Ctf2N); 4.32
(m, 1H, CHCHNH); 4.51 (m, 1H, CHCHNH); 5.77 (s, 1H, CONH); 6.05 (s, 1H,
CONH).
ES-MS m/z: 329.5 [M+H]+.
Synthesis of deoxybiotinyl hexamethylenediamine - ter- butyl-DOTA
(4).
A solution of tri-fert-butyl-DOTA [(3), 524 mg, 0.915 mmol, 1 eq], PyBOP
(714 mg, 1.37 mmol, 1.5 eq) and triethylamine (166 L, 1.19 mmol, 1.3 eq) in
DCM (5 mL) left under agitation at room temperature for 10 minutes, was
added drop by drop to a solution of ST2551-free base [(2), 300 mg, 0.915
mmol, 1 eq) in DCM (5 mL), obtained by heating the mixture at 40°C for 5
min. It was left to react at room temperature for 3 hours, monitoring the
completeness of the reaction by TLC and LC-MS, according to the methods
above.
At the end of the reaction the solvent was evaporated under reduced
pressure, the residue was dissolved in DCM and was washed twice with
NaOH 1M. The organic phase was dehydrated on Na2S04 and evaporated
under vacuum. The residue thus obtained underwent chromatography on a
silica gel column (ratio in weight of crude product: silica 1:30), using as the

8
eluent system a DCM mixture: isopropanol 5:4 to which is added an
increasing concentration of 30% aqueous NH3 (from 0.2 to 1). The column
was packed with the initial DCM mixture: isopropanol: NH3 (5:4: 0.2) and the
process was continued with the gradient of NH3. [the eluent mixture usually
becomes homogeneous after agitation; only in the final phase of the elution,
with NH3=1 it may prove to be necessary to add isopropanol to improve
miscibility, in this case using the volumetric ratio (4:5:1)
(DCM:isopropanol:NH3). A slightly yellow oily product was obtained which
tended to produce a foamy solid under vacuum [(4),, 600 mg, yield = 74%).
1H-NMR (300 MHz, DMSO-d6, T=50°C) 8 (ppm): 1.16-1.70 (m, 43H,
CH(CH2)4 and NHCH2(CH2)4 and 9 x CH3); 2.40-3.32 (m, 34H, 2 x HCHS and
3 x CH2N, 8 x DOTA-ring CH2 and CHS and 4 x DOTA CH2CO and NH
amine); 4.13 (m, 1H, CHCHNH); 4.29 (m, 1H, CHCHNH); 6.18 (s, 1H, NH
biotin); 6.21 (s, 1H, NHbiotin); 8.10 (t, 1H, NH amide).
13C-NMR (75 MHz, DMSO-d6) 8 (ppm): 27.22; 27.35; 27.56; 28.50; 28.61;
29.04; 29.23; 30.14; 30.25; 30.39; 39.14; 40.42; 50.07; 52.59; 52.82; 53.24;
54.72; 56.14; 56.92; 57.38; 59.31; 60.05; 61.90; 80.75; 163.34; 170.98;
171.25.
ES-MS m/z: 883.4 [M+H]+.
Synthesis of deoxybiotinyl hexamethylenediamine -DOTA [ST2210, (5)].
A solution of (4) (210 mg, 0.238 mmol) in HCI 6N [1 mL, 20% w/v solution of
(4)] was left under agitation at room temperature for 1 hour. Then the mixture

9
was evaporated at reduced pressure, the residue was dissolved in H20
(approx. 1:50 w/v) and the solution was subjected to lyophilisation. A white
solid was obtained (205 mg, 96%).1H-NMR (200 MHz, DMSO-d6) 5 (ppm):
1.30-1.77 [m, 16H, CH(CH2)4 and NHCH2(CH2)4]; 2.59 (d, 1H, HCHS); 2.77-
2.88 (m, 7H, HCHS and 3 x CH2N); 2.98-3.70 (m, 25H, 8 x DOTA-ring CH2
and CHS and 4 x DOTA CH2CO); 4.15 (m, 1H, CHCHNH); 4.31 (m, 1H,
CHCHNH); 7.58 (br s, 2H, 2 x biotin NH); 8.86 (t, 1H, NH amide); 9.19 (br s,
2H, NH2+).
13C-NMR (75 MHz, DMSO-d6) 5 (ppm): 25.77; 25.85; 26.32; 26.53; 26.70;
28.74; 29.16; 47.08; 47.16; 48.54; 49.00; 51.35; 53.13; 54.51; 55.38; 56.02;
59.90; 61.61; 163.44; 165.58; 168.90; 172.31.
ES-MSm/z: 715.4 [M+H]+.

10
CLAIMS
1. Process for synthesising deoxybiotinyl hexamethylenediamine-DOTA
which involves reacting biotinyl hexamethylenediamine with tri-f-butyl-
DOTA in the presence of benzotriazol-1-yl-
oxytripyrrolidinophosphonium hexafluorophosphate, as the
condensing agent, and triethylamine, as the base.
2. Process according to claim 1, in which biotinyl hexamethylenediamine
is reacted with tri-f-butyl-DOTA in an organic solvent.
3. Process according to claim 2 in which the organic solvent is
dichloromethane.
4. Process according to any one of the preceding claims, involving a
subsequent step of hydrolysis in an aqueous environment with an acid
pH.

A process for the synthesis of deoxybiotinyl hexamethylenediamine-DOTA is herein described. Said process comprises reacting biotinyl hexamethylenediamine with tri-t-butyl-DOTA in the presence of benzotriazol-1-yl-oxytripyrrolidinophos-phonium hexafluorophosphate, as the condensing agent, and triethylamine, as the base.

Documents:

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04129-kolnp-2007-correspondence others.pdf

04129-kolnp-2007-description complete.pdf

04129-kolnp-2007-form 1.pdf

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4129-KOLNP-2007-(19-09-2012)-ANNEXURE TO FORM 3.pdf

4129-KOLNP-2007-(19-09-2012)-ASSIGNMENT.pdf

4129-KOLNP-2007-(19-09-2012)-CORRESPONDENCE.pdf

4129-KOLNP-2007-(19-09-2012)-FORM-1.pdf

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4129-KOLNP-2007-(19-09-2012)-PETITION UNDER RULE 137.pdf

4129-KOLNP-2007-(27-02-2014)-CORRESPONDENCE.pdf

4129-KOLNP-2007-(27-02-2014)-OTHERS.pdf

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4129-KOLNP-2007-CORRESPONDENCE OTHERS 1.1.pdf

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Patent Number

259398

Indian Patent Application Number

4129/KOLNP/2007

PG Journal Number

11/2014

Publication Date

14-Mar-2014

Grant Date

11-Mar-2014

Date of Filing

26-Oct-2007

Name of Patentee

SIGMA-TAU INDUSTRIE FARMACEUTICHE RIUNITE S.P.A.

Applicant Address

VIALE SHAKESPEARE, 47, I-00144 ROME

Inventors:

#	Inventor's Name	Inventor's Address
1	MARZI MAURO	VIA CINA, 451 , I-00144, ROME
2	DI MARZO MARIA	VIA NAZIONALE, 214,, I-81028, SANTA MARIA A VICO-CASERTA

PCT International Classification Number

C07D 495/04

PCT International Application Number

PCT/EP2006/062800

PCT International Filing date

2006-05-31

PCT Conventions:

#	PCT Application Number	Date of Convention	Priority Country
1	RM2005A000345	2005-06-30	Italy