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2D chemical structure of 391210-10-9


Chemical Formula: C16H14F3IN2O4
Molecular Weight: 482.19

PD0325901; PD 0325901; PD-325901; mirdametinib

IUPAC/Chemical Name: (R)-N-(2,3-dihydroxypropoxy)-3,4-difluoro-2-((2-fluoro-4-iodophenyl)amino)benzamide

SpringWorks Therapeutics (a spin out of Pfizer ) is developing mirdametinib, a second-generation, non-ATP competitive, allosteric MEK1 and MEK2 inhibitor derived from CI-1040, for treating type 1 neurofibromatosis (NF1) and advanced solid tumors. In June 2021, a phase I/II trial was initiated in patients with low grade glioma.

  • OriginatorPfizer
  • DeveloperAstraZeneca; BeiGene; BIOENSIS; Pfizer; SpringWorks Therapeutics; St. Jude Childrens Research Hospital; University of Oxford
  • ClassAniline compounds; Anti-inflammatories; Antineoplastics; Benzamides; Immunotherapies; Small molecules
  • Mechanism of ActionMAP kinase kinase 1 inhibitors; MAP kinase kinase 2 inhibitors
  • Orphan Drug StatusYes – Neurofibromatosis 1
  • Phase IINeurofibromatosis 1
  • Phase I/IIGlioma
  • Phase ISolid tumours
  • PreclinicalChronic obstructive pulmonary disease
  • No development reportedCervical cancer
  • DiscontinuedBreast cancer; Cancer; Colorectal cancer; Malignant melanoma; Non-small cell lung cancer
  • 22 Jul 2021SpringWorks Therapeutics receives patent allowance for mirdametinib from the US Patent and Trademark Office for the treatment of Neurofibromatosis type 1-associated plexiform neurofibromas
  • 16 Jun 2021SpringWorks Therapeutics and St. Jude Children’s Research Hospital agree to develop mirdametinib in USA for glioma
  • 15 Jun 2021Efficacy and safety data from the phase IIb RENEU trial for Neurofibromatosis type 1-associated plexiform neurofibromas released by SpringWorks Therapeutics




On July 20, 2021, SpringWorks Therapeutics announced that the United States Patent and Trademark Office (USPTO) has issued US11066358 , directed to mirdametinib , the Company’s product candidate in development for several oncology indications, including as a monotherapy for patients with neurofibromatosis type 1-associated plexiform neurofibromas (NF1-PN) and was assigned to Warner-Lambert Company (a subsidiary of Pfizer ).This patent was granted on July 20, 2021, and expires on Feb 17, 2041. Novel crystalline forms of mirdametinib and compositions comprising them are claimed.

N—((R)-2,3-dihydroxypropoxy)-3,4-difluoro-2-(2-fluoro-4-iodo-phenylamino)-benzamide (“mirdametinib”, or “PD-0325901”) is a small molecule drug which has been designed to inhibit mitogen-activated protein kinase kinase 1 (“MEK1”) and mitogen-activated protein kinase kinase 2 (“MEK2”). MEK1 and MEK2 are proteins that play key roles in the mitogen-activated protein kinase (“MAPK”) signaling pathway. The MAPK pathway is critical for cell survival and proliferation, and overactivation of this pathway has been shown to lead to tumor development and growth. Mirdametinib is a highly potent and specific allosteric non-ATP-competitive inhibitor of MEK1 and MEK2. By virtue of its mechanism of action, mirdametinib leads to significantly inhibited phosphorylation of the extracellular regulated MAP kinases ERK1 and ERK2, thereby leading to impaired growth of tumor cells both in vitro and in vivo. In addition, evidence indicates that inflammatory cytokine-induced increases in MEK/ERK activity contribute to the inflammation, pain, and tissue destruction associated with rheumatoid arthritis and other inflammatory diseases.
      Crystal forms of N—((R)-2,3-dihydroxypropoxy)-3,4-difluoro-2-(2-fluoro-4-iodo-phenylamino)-benzamide have been described previously. WO2002/006213 describes crystalline Forms I and II. U.S. Pat. No. 7,060,856 (“the ‘856 patent”) describes a method of producing Form IV. The ‘856 patent indicates that the material produced by this method was greater than 90% Form IV (The ‘856 patent, Example 1). The ‘856 patent also states that the differential scanning calorimetry (“DSC”) of the material produced shows an onset of melting at 110° C., as well as a small peak with an onset at 117° C., consistent with the material being a mixture of two forms.
      WO 2006/134469 (“the ‘469 PCT publication”) also describes a method of synthesizing N—((R)-2,3-dihydroxypropoxy)-3,4-difluoro-2-(2-fluoro-4-iodo-phenylamino)-benzamide. The ‘469 PCT publication reports the method yields a product conforming to the polymorphic Form IV disclosed in U.S. patent application Ser. No. 10/969,681 which issued as the ‘856 patent.
      Compositions containing more than one polymorphic form are generally undesirable because of the potential of interconversion of one polymorphic form to another. Polymorphic interconversion can lead to differences in the effective dose or physical properties affecting processability of a drug, caused by differences in solubility or bioavailability. Thus, there is a need for a composition containing essentially pure Form IV of N—((R)-2,3-dihydroxypropoxy)-3,4-difluoro-2-(2-fluoro-4-iodo-phenylamino)-benzamide, for use in treatment of a tumor, a cancer, or a Rasopathy disorder.

Example 1: Production of Essentially Pure Form IV

Lab Scale Production of Essentially Pure Form IV

      2 kg PD-0325901 has been prepared using the below convergent synthesis scheme starting from commercially available 2,3,4-Trifluorobenzoic Acid (TFBA), 2-Fluoro-4-Iodoaniline (FIA) and chiral S-Glycerol Acetonide (SGA)
 (MOL) (CDX)
 (MOL) (CDX)
Step 1: Preparation of “Side Chain”, PD-0337792
      All reactions were performed in toluene other than otherwise stated. Triflic anhydride gave the best yield.



Coupling Agents for Step 1
No. Coupling Agent Yield Notes
1 Mesyl Chloride did not
2 Benzyl chloride 27 Had to heat 70° C.
for 166 hr
3 4-fluorobenzensulfonylchloride 27 Ran 93 hrs. at 70° C.
4 4-chlorobenzensulfonylchloride 35 Complete after 68 hrs.
50° C.
5 Tosyl Chloride 36 Had to heat to 70° C.
for 164 hrs
6 Benzyl chloride 52 study solvent effects:
all similar DMSO
fastest all complete
after 110 hrs., heated
to 70° C. after 66 hrs.
7 Triflic anhydride 91 Cooled to −74° C.
      Recognizing that triflate gave the highest yield, the possibility of eliminating the cryogenic conditions was investigated, set possibly due to stability concerns of the “methanesulfonate” intermediate. The following experiments suggest no significant yield loss for experiments run at −20° C.


Yield of Coupling Reaction
Experimental Hold time after Yield (Alcohol to
Description* TFMSA addn. IPGAP)
1.07 equiv. NHP 15 min. 85%
1.07 equiv. NHP 2 hours 86%
1.77 equiv. NHP 2 hours 72%
1.07 equiv. NHP (reverse 1 hours 91%

* 2 g (1 eq.) SGA in 16 ml toluene was treated with triflic anhydride, trifluoromethanesulfonic acid (TFMSA) (4.2 g, 1.002 equiv.) at −20° C. and then stirred for a prescribed time prior to solid N-hydroxyphthalimide (NIP) addition or transfer to a flask containing solid NHP.

      The data presented above suggest no detrimental effect was observed after prolonged stirring of the “trifluoromethane sulfonate” intermediate prior to the N-hydroxyphthalimide addition. Reverse addition of intermediate mixture to solid NHP appears to give the highest yield.
      An additional advantage of the triflate usage was easy removal of the Et 3N triflate salts side product simply by water wash. This resulted in highly pure N-hydroxyphthalimide-protected alcohol, IPGAP (PD-0333760) in Toluene, which can be isolated as crystals or carried through to the final deprotection reaction.
      Both aqueous and anhydrous ammonia base were examined as deprotecting agents. The results were both successful. The phthalimide side product was simply filtered out from solution of product (PD-0337792) in toluene when anhydrous ammonia was used. Similarly, it was filtered out from the solution after performing azeotropic water removal from toluene when aqueous ammonia (28% solution) was used. Anhydrous ammonia however, requires the reaction to be performed at high-pressure containment. Experiments conducted by sparging the ammonia gas gave acceptable yields; however, they required large volumes and use of a cryogenic condenser (to avoid gas from escaping the reactor headspace).


Yields for base deprotection
Reagent Yield*
Methyl hydrazine 85-95%
Anhydrous NH(sparged) 78-90%
Anhydrous NH(50 psi) 80-92%
Aqueous NH3 90-97%
*from PD-0333760

Step 2: Fluoride Displacement

      Examination of the reaction in an automated reactor reveals that the reaction is essentially dosed-controlled after the initiation period. Increasing the amount of lithium amide and increased agitation rate appear to shorten the induction time. The addition of water was shown to prolong the induction time. However, it is not clear whether it is due to lithium hydroxide formation.
      Induction time is increased when 0.1 equivalent H 2O was added. The trend was reversed however when 0.1 equivalent lithium hydroxide was added. Induction times were decreased upon increasing lithium amide equivalents and agitation.
      CDI-assisted coupling of PD-0315209 acid and sidechain reagent followed by the acid (with aqueous HCl) hydrolysis consistently yielded good results in the laboratory. The development focus of this step was to ensure that impurity levels are within the specification limit. The known impurities in the final isolated diol product are excess PD-0315209 acid, dimeric impurities and chiral impurities. The chiral impurities are controlled by limiting the R-enantiomer in the starting s-glycerol acetonide. Elevated levels of dimeric impurity (d) has been known to cause difficulties in the polymorph transformation step. The dimeric impurity is formed initially by the reaction of imidazole (CDI-activated acid) in the presence of excess acid PD-0315209 forming dimer (a) and possibly (b) which are then carried through in the subsequent IPGA coupling and acid hydrolysis steps forming dimer (c) and (d), respectively. Impurity d is referred to as PF-00191189.
      The reaction can be easily carried out in the laboratory either by charging both solids, FIPFA and CDI, followed by solvent (acetonitrile) or charging solids CDI into a slurry of FIPFA in acetonitrile. None of the solids is initially soluble in acetonitrile. The acid activation reaction was fast (almost instantaneous), forming highly soluble imidazolide product that turned the slurry into a clear homogenous solution while CO gas evolution occurs.
      Lab experiments generally resulted in impurity levels under 3%, which can be completely removed by the subsequent recrystallization from a 3-5% ethanol-toluene system. An additional recrystallization was performed in the few instances where the impurity level was above 0.3%. Table 4 shows selected results of lab experiments where elevated levels of impurities were observed and how they were removed in the subsequent recrystallization. The crude PD-0325901 was obtained using the acetonitrile/toluene system and the purified product was recrystallized from a 5% ethanol/toluene system. Entries no. 4 and 5 used additional solvent to ensure impurity removal with entry 5 requiring two recrystallizations in order to achieve a level of “ND” in the polymorph transformation. The 8-10 ml/g crude crystallization volume was chosen to limit product loss while maintaining a filterable slurry and ensuring removal of impurities.


Purification of PD-0325901
Imp. In Final
Tot. isolated Tot. Imp. assay (after
Imp. In Crude Purified polymorph
Entry reaction PD- Recrystallization PD- trans-
No mixture 0325901 Vol (ml/g crude) 0325901 formation)
1  2.4% ND 8 ND 99.8%
2 10.5%  2% 8 ND 99.6%
3    6%  1% 8 ND 99.4%
4   10% 3.2% 15 ND 98.6%
5   20% 12% 13 0.6% 98.4%*
      A scale up procedure that would give tolerable levels of impurities prior to the polymorph transformation (<0.3%), without losing too much product in the recrystallization was developed considering the solid CDI addition rate. Fast addition is preferred to minimize impurity formation; however, the addition needs to be performed at a rate that ensures safely venting of the evolved CO 2.
      A half portion of solid CDI was initially added to the PD-0325901 acid, followed by solvent addition. The remaining CDI was added then through a hopper in less than 30 minutes to ensure that the impurity levels were below 3%.

Pilot Plant Preparation of Essentially Pure Form IV

Step 1: Preparation of “Side Chain”, PD-0337792

      14.4 kg alcohol (chemical purity 99.4%, optical purity 99.6% enantiomeric excess) was converted to 97.5 kg 9.7% w/w PD-0337792 (IPGA) solution in toluene (overall yield ˜60%). The triflate activation was performed in the 200 L reactor by maintaining temperatures under −20° C. during triflic anhydride addition. The resulting activated alcohol was then transferred to a 400 L reactor containing solid N-hydroxypthalimide (NHP) and the reaction was allowed to occur at ambient temperature to completion. The final base de-protection was performed by adding aqueous ammonia (˜28% soln, 5 equiv., 34 kg). After reaction completion, water was removed by distillation from toluene, and the resulting solid side product was filtered out to yield the product solution.

Step 2: Preparation of PD-0315209

      The process yielded 21.4 kg (99.4% w/w assay), which is 80% of theoretical from starting materials 2,3,4-trifluorobenzoic acid (12 kg, 1 eq.) and 2-fluoro-4-iodoaniline (16.4 kg, 1.02 eq.) with lithium amide base (5 kg, 3.2 eq.). The reaction was initiated by adding 5% of total solution of TFBA and FIA into lithium amide slurry at 50° C. This reaction demonstrated a minimal initiation period of ˜10 minutes, which was observed by color change and slight exotherm. The remaining TFBA/FIA solution in THE was slowly added through a pressure can in an hour while maintaining the reaction temperatures within 45-55° C. There was no appreciable pressure rise (due to ammonia gas release) observed during the entire operation.

Step 3: Preparation of PD-0325901

      A modification was made to the CDI charging to mitigate potential gas generation. Two equal portions of CDI were added into solid FIPFA before and after solvent addition (through a shot loader). The timing between the two solid CDI additions (4.6 kg each) should not exceed 30 minutes. Then two intermediate filter cakes were dissolved with ethanol. The excess ethanol was distilled and replaced with toluene to approximately 5% v/v ethanol prior to PD-0325901 recrystallization. Lab studies suggested that the crystallization from toluene and acetonitrile and recrystallization from ethanol in toluene would not be able to reduce impurities which is essential for the polymorph transformation. The presence of a dimeric impurity (PF-00191189) at a level greater than 0.2% has been known to result in the formation of undesired polymorph.
      The crude crystallization from the final reaction mixture reduced dimeric impurity PF-00191189 to approximately 1.9% and the subsequent recrystallization further reduced it to approximately 0.4%. As a consequence, undesired polymorphs were produced. The DSC patterns indicated two different melting points ˜80° C. (low melt Form II) and ˜117° C. (Form I). Also during the processing, the solids crystallized at a much lower temperature than expected (actual ˜10° C., expected ˜40° C.). It is suspected that the unsuccessful recrystallization is due to a change in the solvent composition as a result of incomplete drying of the crude. Drying of the crude wet cake prior to ethanol dissolution was stopped after about 36 hours when the crude product was ˜28 kg (26 kg theoretical).

Polymorph Transformation

      Approximately 7.4 kg of PD-0325901 (mixed polymorphs) from the final EtOH/Water crystallization and precipitated materials from the earlier EtOH/Toluene filtrate were taken forward to the polymorph transformation. Both crops were separately dried in the filter until constant weights and each was dissolved in EtOH. The combined EtOH solution was analyzed by HPLC and resulted in an estimated amount of 16.4 kg PD-0325901. The recrystallization was started after removing EtOH via vacuum distillation and adjusting the solvent composition to about 5% EtOH in Toluene at 65° C. (i.e., EtOH is added dropwise at 65° C. until complete solids dissolution).
      A slow 4-hour cooling ramp to 5° C. followed by 12 h stirring was performed to ensure satisfactory results. The resulting slurry was filtered and again it was completely dried in the filter until constant weight (approximately 3 days). The purified solid showed 99.8% pure PD-0325901 with not detected level of dimeric impurity PF-00191189.
      The dried solid (15.4 kg) was re-dissolved in exactly 4 volumes of EtOH (62 L) off of the filter, transferred to the reactor and precipitated by a slow (˜3 h) water addition (308 L) at 30-35° C., cooled to 20° C. and stirred for 12 h. The DSC analysis of a slurry sample taken at 2 h shows the solids to be completely Form IV (desired polymorph).
      21.4 kg PD-0315209, 9.7 kg CDI (1.05 equiv.), 91 kg solution of 9.7% PD-0337792 in Toluene (1.1 equiv.) were used and resulted in 12.74 kg of PD-0325901 (assay 99.4%, 100% Form IV, Yield 48%).


WO2006134469 , claiming methods of preparing MEK inhibitor, assigned to Warner-Lambert Co .


The compound Λ/-[(R)-2,3-dihydroxy-propoxy]-3,4-difluoro-2-(2-fluoro-4-iodo-phenylamino)- benzamide represented by formula 1
Figure imgf000002_0001

i is a highly specific non-ATP-competitive inhibitor of MEK1 and MEK2. The compound of formula ± (Compound I) is also known as the compound PD 0325901. Compound I is disclosed in WO 02/06213; WO 04/045617; WO 2005/040098; EP 1262176; U.S. Patent Application Pub. No. 2003/0055095 A1 ; U.S. Patent Application Pub. No. 2004/0054172 A1; U.S. Patent Application Pub. No. 2004/0147478 A1 ; and U.S. Patent Application No. 10/969,681, the disclosures of which are incorporated herein by reference in their entireties.

Numerous mitogen-activated protein kinase (MAPK) signaling cascades are involved in controlling cellular processes including proliferation, differentiation, apoptosis, and stress responses. Each MAPK module consists of 3 cytoplasmic kinases: a mitogen-activated protein kinase (MAPK), a mitogen-activated protein kinase kinase (MAPKK), and a mitogen-activated protein kinase kinase kinase (MAPKKK). MEK occupies a strategic downstream position in this intracellular signaling cascade catalyzing the phosphorylation of its MAP kinase substrates, ERK1 and ERK2. Anderson et al. “Requirement for integration of signals from two distinct phosphorylation pathways for activation of MAP kinase.” Nature 1990, v.343, pp. 651-653. In the ERK pathway, MAPKK corresponds with MEK (MAP kinase ERK Kinase) and the MAPK corresponds with ERK (Extracellular Regulated Kinase). No substrates for MEK have been identified other than ERK1 and ERK2. Seger et al. “Purification and characterization of mitogen-activated protein kinase activator(s) from epidermal growth factor-stimulated A431 cells.” J. Biol. Chem., 1992, v. 267, pp. 14373-14381. This tight selectivity in addition to the unique ability to act as a dual-specificity kinase is consistent with MEK’s central role in integration of signals into the MAPK pathway. The RAF-MEK-ERK pathway mediates proliferative and anti-apoptotic signaling from growth factors and oncogenic factors such as Ras and Raf mutant phenotypes that promote tumor growth, progression, and metastasis. By virtue of its central role in mediating the transmission of growth- promoting signals from multiple growth factor receptors, the Ras-MAP kinase cascade provides molecular targets with potentially broad therapeutic applications.
One method of synthesizing Compound I is disclosed in the above-referenced WO 02/06213 and
U.S. Patent Application Pub. No. 2004/0054172 A1. This method begins with the reaction of 2-fluoro-4- iodo-phenylamine and 2,3,4-trifluoro-benzoic acid in the presence of an organic base, such as lithium diisopropylamide, to form 3,4-difluoro-2-(2-fluoro-4-iodo-phenylamino)-benzoic acid, which is then reacted with (R)-0-(2,2-dimethyl-[1,3]dioxolan-4-ylmethyl)-hydroxylamine in the presence of a peptide coupling agent (e.g., diphenylphosphinic chloride) and a tertiary amine base (e.g., diisopropylethylamine). The resulting product is hydrolyzed under standard acidic hydrolysis conditions (e.g., p-TsOH in MeOH) to provide Compound 1. (R)-O-(2,2-dimethyl-[1,3]dioxolan-4-ylmethyl)-hydroxylamine is prepared by reaction of [(4S)-2,2-dimethyl-1,3-dioxolan-4-yl]methanol with N-hydroxyphthalimide in the presence of Ph3P and diethyl azodicarboxylate.
Another method of synthesizing Compound I, which is disclosed in the above-referenced U.S.
Patent Application No. 10/969,681, comprises reaction of 3,4-difluoro-2-(2-fluoro-4-iodo-phenylamino)- benzoic acid with (R)-O-(2,2-dimethyl-[1,3]dioxolan-4-ylmethyl)-hydroxylamine in the presence of N1N1– carbonyldiimidazole. The resulting product is hydrolyzed with aqueous acid and crystallized to provide polymorphic form IV of Compound I.
Although the described methods are effective synthetic routes for small-scale synthesis of Compound I, there remains a need in the art for new synthetic routes that are safe, efficient and cost effective when carried out on a commercial scale.
The present invention provides a new synthetic route including Steps I through Step III to the MEK inhibitor Λ/-[(R)-2,3-dihydroxy-propoxy]-3,4-difluoro-2-(2-fluoro-4-iodo-phenylamino)-benzamide (Compound I).
Step I: Preparation of 0-{r(4RV2.2-dimethyl-1.3-dioxolan-4-ynmethyl}hydroxylanπine (6) The method of the present invention comprises a novel Step I of preparing of 0-{[(4R)-2,2- dimethyl-1 ,3-dioxolan-4-yl]methyl}hydroxylamine (6) from [(4S)-2,2-dimethyl-1 ,3-dioxoIan-4-yl]methanol (1) through the formation of [(4R)-2,2-dimethyl-1 ,3-dioxolan-4-yl]methyl trifluoromethanesulfonate (3) and its coupling with N-hydroxyphthalimide (4) to afford 2-{[(4R)-2,2-dimethyl-1 ,3-dioxolan-4-yl]methoxy}-1 H- isoindole-1 ,3(2H)-dione (5), which is subsequently de-protected to give 6 as shown in Scheme 1.
Scheme 1
Figure imgf000009_0001
Figure imgf000009_0002
Figure imgf000009_0003
The reaction of compound (1) with trifluoromethanesulfonic anhydride (2) is carried out in the presence of a non-nucleophilic base, such as, for example, a tertiary organic amine, in an aprotic solvent at a temperature of from -5O0C to 50C, preferably, at a temperature less than -150C, to form triflate (3). A preferred tertiary organic amine is triethylamine, and a preferred solvent is toluene. Treatment of triflate (3) with N-hydroxyphthalimide (4) furnishes phthalimide (5), which can be isolated if desired. However, in order to minimize processing time and increase overall yield, 0-{[(4R)- 2,2-dimethyl-1,3-dioxolan-4-yl]methyl}hydroxylamine (6) can be prepared in a one-pot process with no phthalimide (S) isolation. Cleavage of the phthalimide function could be achieved by methods known in the art, for example, by hydrazinolysis. However, the use of less hazardous aqueous or anhydrous ammonia instead of methyl hydrazine (CH3NHNH2) is preferred.
Step II: Preparation of 3.4-difluoro-2-(2-fluoro-4-iodophenylamino)-benzoic acid (9) As shown in Scheme 2, Step Il of the method of the present invention provides 3,4-difluoro-2-(2- fluoro-4-iodophenylamino)-benzoic acid (9).
Scheme 2
Figure imgf000010_0001
Preparation of compound (9) can be carried out by reacting compound (7), wherein X is halogen, or O-SC^R^ or 0-P(3O)(OR^, wherein R^ is alkyl or aryl, with compound (8) optionally in a solvent, and in the presence of from about 1 mol equivalent to about 10 mol equivalents of at least one base, wherein the base is selected from: a Group I metal cation hydride or a Group 2 metal cation hydride, including lithium hydride, sodium hydride, potassium hydride, and calcium hydride, a Group I metal cation dialkylamide or a Group 2 metal cation dialkylamide, including lithium diisopropylamide, a Group I metal cation amide or a Group 2 metal cation amide, including lithium amide, sodium amide, potassium amide, a Group I metal cation alkoxide or a Group 2 metal cation alkoxide, including sodium ethoxide, potassium terf-butoxide, and magnesium ethoxide, and a Group I metal cation hexamethyldisilazide, including lithium hexamethyldisilazide; for a time, and at a temperature, sufficient to yield compound (9).
Preferably, preparation of compound (9) is carried out by reacting compound (7), wherein X is halogen, more preferably, X is fluorine, in an aprotic solvent with compound (8) in the presence of from about 3 mol equivalents to about 5 mol equivalents of a Group I metal cation amide at a temperature of from 2O C to 55°C, more preferably, at a temperature from 45°C to 55°C. A catalytic amount of Group I metal cation dialkylamide can be added if necessary. A preferred Group I metal cation amide is lithium amide, a preferred Group I metal cation dialkylamide is lithium diisopropylamide, and a preferred solvent is tetrahydrofuran. Preferably, the reaction is performed by adding a small amount of compound (7) and compound (8) to lithium amide in tetrahydrofuran followed by slow continuous addition of the remaining portion. This procedure minimizes the risk of reactor over-pressurization due to gas side product (ammonia) generation.
Step III: Preparation of N-((RV2.3-dihydroxypropoxy)-3.4-difluoro-2-(2-fluoro-4-iodo-phenylamino)- benzamide (Compound I)
Compound I can be obtained by coupling 0-{[(4R)-2,2-dimethyl-1,3-dioxolan-4- yl]methyl}hydroxylamine (6) with 3,4-difluoro-2-(2-fluoro-4-iodophenylamino)-benzoic acid (9) using a carboxylic acid activating reagent such as, for example, COCI2, S(O)C^, S(O)2Cl2, P(O)Cl3, triphenylphosphine/diethylazodicarboxylate, diphenylphosphinic chloride, N, N’-dicyclohexylcarbodiimide, (benzotriazol-1 -yloxy)tripyrolidinophosphonium hexafluorophosphate, (benzotriazol-1 – yloxy)tris(dimethylamino)phosphonium hexafluorophosphate, N-ethyl-N’-(3- dimethylaminopropyl)carbodiimide hydrochloride, or 1,1′-carbonyldiimidazole (CDI).
A preferred carboxylic acid activating reagent is 1,1′-carbonyldimidazole (CDI) shown in Scheme 3. Preparation of the desirable polymorphic Form IV of Compound I using CDI is described in the above- referenced U.S. Patent Application No. 10/969,681.
Scheme 3
Figure imgf000011_0001
Figure imgf000011_0002
10 11 Compound I
In according to the present invention, the method was modified to include the advantageous procedure for product purification and isolation, which procedure is performed in single-phase systems such as, for example, toluene/acetonitrile for the first isolation/crystallization and ethanol/toluene for the second recrystallization. Water addition, implemented in the previous procedure, was omitted to avoid the two-phase crystallization from the immiscible water-toluene system that caused inconsistent product purity. The one-phase procedure of the present invention provides consistent control and removal of un- reacted starting material and side products. Alternatively, Compound I can be obtained by coupling 0-{[(4R)-2,2-dimethyl-1,3-dioxolan-4- yl]methyl}hydroxylamine (6) with 3,4-difluoro-2-(2-fluoro-4-iodophenylamino)-benzoic acid (9) using thionyl chloride (SOCI2) as shown in Scheme 4.
Scheme 4
Figure imgf000012_0002
Figure imgf000012_0001

Compound I

The reagents and conditions of the reactions described herein are merely illustrative of the wide variety of starting materials, their amounts and conditions which may be suitably employed in the present invention as would be appreciated by those skilled in the art, and are not intended to be limiting in any way.
HPLC (Conditions A): 10 μL injection volume onto Agilent Zorbax RX-C18 150 mm x 4.6 mm x 3.5 μm column at 30°C column temperature, 1.0 mL/min flow rate and detection at 246 nm. Mobile phase A (v/v): 25 mM Acetate Buffer, pH 6.0; Mobile phase B (v/v): Acetonitrile, and Linear Gradient Table:
Figure imgf000012_0003
Sample Preparation: Dilute 100 μL reaction mixture to 10 mL with acetonitrile. Mix in a vial 200 μL of this sample solution with 300 μL carbonate buffer pH 10.0 and 300 μL solution of 2-mercaptopyridine in acetonitrile (18 mM), heat the vial for 10 minutes at 500C and dilute to 1:1 ratio in mobile phase A.
GC (Conditions B): 1 μL injection onto an RTX-5 column (30 m x 0.25 mm x 0.25 μm) with initial oven temperature of 120°C for 2 min. to final temperature of 250°C in 15°C/minute ramping and a final time of 2.33 min; Flow rate: 1 mL/min.

HPLC (Conditions C): 5 μL injection onto Phenomenex Luna C18(2) 150 mm x 4.6 mm x 3μm column ; flow rate : 1.0 mL/min; detection at 225 nm; mobile phase A: 95/5 v/v Water/Acetonitrile with 0.1% Trifluoroacetic acid (TFA), mobile phase B: 5/95 v/v Water/Acetonitriie with 0.1% TFA; Linear Gradient Table:

Figure imgf000013_0001
Sample preparation: Dilute 1 ml_ reaction mixture to 100 mL with acetonitrile and dilute 1 mL of this solution to 10 mL with 50:50 Water/Acetonitrile.
HPLC (Conditions D): 5 μL injection onto Waters SymmetryShield RP 18, 150 mm x 4.6 mm x 3.5 μm column; flow rate: 1.0 mL/min; detection at 235 nm; mobile phase A: 25 mM Acetate Buffer adjusted to pH 5.5, mobile phase B: Acetonitrile; Linear Gradient Table:
Figure imgf000013_0002
Sample preparation: Dilute 40 μL of reaction mixture in 20 mL acetonitrile.
HPLC (Conditions E): 10 μL sample injection onto YMC ODS-AQ 5 μm, 250 mm x 4.6 mm column; flow rate: 1.0 ml_/min; detection at 280 nm; temperature 30°C; mobile phase : 75/25 v/v Acetonitrile/Water with 0.1% Formic acid.
Sample preparation: Quench reaction mixture sample with dipropylamine and stir for about 5 minutes before further dilution with mobile phase.
DSC measurement was performed using a Mettler-Toledo DSC 822, temperature range 25° to 150°C with 5°C/min heating rate in a 40 μL aluminum pan. Experimental Conditions for Powder X-Rav Diffraction (XRD):
A Rigaku Miniflex+ X-ray diffractometer was used for the acquisition of the powder XRD patterns. The instrument operates using the Cu Ka1 emission with a nickel filter at 1.50451 units. The major instrumental parameters are set or fixed at:
X-ray: Cu / 30 kV (fixed) / 15 mA (fixed)
Divergence Slit: Variable Scattering Slit: 4.2° (fixed) Receiving Slit: 0.3 mm (fixed) Scan Mode: FT Preset Time: 2.0 s Scan Width: 0.050° Scan Axis: 2Theta/Theta Scan Range: 3.000° to 40.000°
Jade Software Version: 5.0.36(SP1) 01/05/01 (Materials Data, Inc.) Rigaku Software: Rigaku Standard Measurement for Windows 3.1 Version 3.6
(1994-1995) Example 1. Preparation of 0-ffl4R)-2.2-dimethyl-1.3-dioxolan-4-vπmethyl}hvdroxylamine (6)
A solution containing [(4S)-2,2-dimethyl-1,3-dioxolan-4-yl]methanol (1) (13.54 ml_, 0.109 mol) (DAISO Co., Ltd., CAS# 22323-82-6) and triethylamine (18.2 ml_, 0.131 mol) in 115 mL toluene was cooled to -15 C, then trifluoromethanesulfonic anhydride (2) (18.34 mL, 30.75 g, 0.109 mol) (Aldrich, Catalog # 17,617-6 ) was added drop wise while maintaining the temperature at less than -15°C. The mixture was then stirred for 2 hours, and transferred to a separate flask containing a mixture (slurry) of N- hydroxyphthalimide (4) (18.99 g, 0.116 mol) (Aldrich, Catalog # H5.370-4) and 18.2 mL (0.13 mol) triethylamine in 95 mL toluene. The resulting mixture was warmed to 20-25°C and stirred for at least 5 hours or until reaction completion (determined by HPLC (Conditions A)). Water (93 mL) was then added to quench the reaction mixture, the phases were separated, and the bottom aqueous layer was discarded. The water quench was repeated two more times resulting in a pale yellow organic layer. The organic layer was heated to 35 C and treated with 36.7 mL ammonium hydroxide solution (contains about 28-29% wt/wt ammonia). The mixture was stirred for at least 12 hours or until the reaction was deemed complete as determined by GC (Conditions B). The water was then removed under reduced pressure by co- distilling it with toluene to about half of the original volume at temperatures around 35-45 C. Toluene (170 mL) was added to the concentrated solution and the distillation was repeated. A sample was drawn for water content determination by Karl Fisher method (using EM Science Aquastar AQV-2000 Titrator with a sample injected to a pot containing methanol and salicylic acid). The distillation was repeated ifl water content was more than 0.1%. The concentrated solution was filtered to remove the white solid side product, and the filtrate was stored as 112mL (98 g) product solution containing 9.7% w/w compound 6 in toluene. This solution was ready for use in the final coupling step (Example 3). Overall chemical yield was 59%. A small sample was evaporated to yield a sample for NMR identification.
1H NMR (400 MHz, CDCI3): δ 5.5 (bs, 2H), 4.35 (m, 1H), 4.07 (dd, 1H), 3.77 (m, 2H), 3.69 (dd, 1H), 1.44 (s, 3H), 1.37 (s, 3H).
Example 2. Preparation of 3.4-difluoro-2-(2-fluoro-4-iodophenylamino)-benzoic acid (9)
A solution of 2-fluoro-4-iodoaniline (8) (16.4 g, 0.069 mol) (Aldrich, Catalog # 30,660-6) and 2,3,4- trifluorobenzoic acid (7) (11.98 g, 0.068 mol) (Aldrich, Cat # 33,382-4) in 38 mL tetrahydrofuran (THF) was prepared and a portion (about 5%) of this solution was added to a stirring slurry of lithium amide (5 g, 0.22 mol) in 40 mL THF at 50-55 C. After about 15-30 min. an exotherm followed by gas release and color change are observed. The remaining portion of the (8) and (7) solution was added slowly over 1-2 hr while maintaining temperatures within 45-55°C. The mixture was stirred until the reaction was deemed complete (by HPLC (Conditions C). The final mixture was then cooled to 20-25°C and transferred to another reactor containing 6 N hydrochloric acid (47 mL) followed by 25 mL acetonitrile, stirred, and the bottom aqueous phase was discarded after treatment with 40 mL 50% sodium hydroxide solution. The organic phase was concentrated under reduced pressure and 57 mL acetone was added. The mixture was heated to 50°C, stirred, and added with 25 mL warm (40-50°C) water and cooled to 25-30°C to allow crystallization to occur (within 1-4 hours). Once the crystallization occurred, the mixture was further cooled to 0 to -5°C and stirred for about 2 hours. The solid product was filtered and the wet cake was dried in vacuum oven at about 55°C. Overall chemical yield was 21.4 g, 80%. 1H NMR (400 MHz, (CD3)2SO): δ 13.74 (bs, 1H), 9.15 (m, 1 H), 7.80 (dd, 1H), 7.62 (d, 1H), 7.41 (d, 1H), 7.10 (q, 1H), 6.81 (m, 1H).
Example 2B. Preparation of 3.4-difluoro-2-(2-fluoro-4-iodophenylamino)-benzoic acid (9) by the solid addition of lithium amide method
To a stirring solution of 2,3,4-trifluorobenzoic acid (13) (5.0 g, 28.4 mmol) and 2-fluoro-4- iodoaniline (14) (6.73 g, 28.4 mmol) in MeCN (100 mL), under N2 atmosphere was added lithium amide (2.61 g, 113.6 mmol) in small portions. The reaction mixture was heated to reflux for 45 minutes, cooled to ambient temperature and quenched with 1 N HCI and then water. The yellowish white precipitate was filtered, washed with water. The solid was triturated in CH2CI2 (30 mL) for 1h, filtered and dried in a vacuum oven at 45°C for 14 hours to give 8.Og (72%) of compound (9) as an off-white solid, mp 201.5-203 °C.
Example 3. Preparation of N-((R)-2.3-dihvdroxypropoxy)-3.4-difluoro-2-(2-fluoro-4-iodo-phenylamino)- benzamide (Compound \)
3,4-Difluoro-2-(2-fluoro-4-iodophenylamino)-benzoic acid (9) (20 g, 0.051 mol) in 100 mL acetonitrile was treated with 1,1′-carbonyldiimidazole (CDI) (8.66 g, 0.053 mol) (Aldrich, Cat # 11,553-3) and stirred for about 2 hours at 20-25°C until the reaction was deemed complete by HPLC (Conditions D). 94 mL (84.9 g) of 9.7% w/w solution of O-{[(4R)-2,2-dimethyl-1,3-dioxolan-4-yl]methyl}hydroxylamine (6) in toluene was then added and stirred for about 4 hours or until the reaction was deemed complete by HPLC (Conditions D). To this mixture was added 66 mL of 5.6 % hydrochloric acid solution, and after stirring, the bottom aqueous phase was discarded. Again 66 mL of 5.6 % hydrochloric acid solution was added to the organic phase and stirred at 20-25°C for 12-18 hours or until the reaction was deemed complete by HPLC (Conditions D). The bottom layer was then discarded and the remaining organic layer was concentrated under reduced pressure to remove about 10-20% solvent, and the volume was adjusted to about 9-11 mL/g with toluene (80 mL). Crude product was then crystallized at 10-15°C. The slurry was allowed to stir for about 2 hours and the crude solid product was filtered, and dried. The dried crude product was recharged to the reactor and dissolved into 150 mL of 5% v/v ethanol/toluene mixture at 55- 67°C. The solution was then clarified at this temperature through filter (line filter) to remove any remaining particulate matter. The solution was then cooled slowly to 5°C to crystallize and stirred for at least 2 h, filtered and dried. The dried solid product was redissolved in EtOH (60 mL) at 35°C, and product was precipitated out by adding water (300 mL) at 35°C followed by cooling to 200C. The slurry was stirred for at least 2 hours to transform the crystals to the desired polymorphic Form IV as determined by DSC and Powder X-ray Diffraction pattern (PXRD). The slurry was filtered and dried under vacuum oven at 70- 90°C to yield the final N-((R)-2,3-dihydroxypropoxy)-3,4-difluoro-2-(2-fluoro-4-iodo-phenylamino)- benzamide (Compound I) product. Overall chemical yield was 13 g, 53%. Melting point (DSC): 112+1° C. Appearance: White to off-white crystals.
Shown in Figure 1, PXRD conforms to polymorphic crystal Form IV disclosed in the above mentioned U.S. Patent Application No. 10/969,681 1H NMR (400 MHz, (CD3)2SO): δ 11.89 (bs, 1H), 8.71 (bs, 1H), 7.57 (d, 1H), 7.37 (m, 2H), 7.20 (q, 1H), 6.67 (m, 1H), 4.84 (bs, 1H), 4.60 (m, 1H), 3.87 (m, 1 H), 3.7 (m, 2H), 3.34 (m, 2H).
Example 4. Preparation of N-((R)-2.3-dihydroxypropoxyV3.4-difluoro-2-(2-fluoro-4-iodo-phenylanrιinoV benzamide (Compound \)
To a stirring solution of 3,4-difluoro-2-(2-fluoro-4-iodophenylamino)-benzoic acid (9) (120 g, 0.30 mol) in a mixture of 1 mL N,N-dimethylformamide and 1000 mL toluene was added thionyl chloride (55 g, 0.462 mol). The mixture was heated to 50-65 C and stirred for 2 hours or until reaction completion as determined by HPLC (Conditions E). The final reaction mixture was then cooled and concentrated under reduced pressure to a slurry keeping the temperature below 35°C. Toluene (600 mL) was added to dissolve the slurry and vacuum distillation was repeated. Additional toluene (600 mL) was added to the slurry dissolving all solids and the solution was then cooled to 5° -10°C. The solution was then treated with O-{[(4R)-2,2-dimethyl-1,3-dioxolan-4-yl]methyl}hydroxylamine (6) (63 g, 0.43 mol) solution in 207 mL toluene followed by potassium carbonate (65 g) and water (200 mL), stirred for at least 2 hours at 20- 25°C. The stirring was stopped to allow phase separation and the bottom phase was discarded. The remaining organic layer was treated with hydrochloric acid solution (7.4%, 240 mL) until pH was less than 1 and stirred for 2 hours. The final reaction mixture was slightly concentrated under vacuum collecting about 100 mL distillate and the resulting organic solution was cooled to 5°C to crystallize the product and filtered. The filter cake was washed with toluene (1000 mL) followed by water (100 mL) and the wet cake (crude product Compound I) was charged back to the flask. Toluene (100 mL), ethanol (100 mL) and water (100 mL) are then added, stirred at 30-35°C for about 15 min, and the bottom aqueous phase was discarded. Water (200 mL) was then added to the organic solution and the mixture was stirred at about 3O C to allow for crystallization. The stirring was continued for 2 hours after product crystallized, then it was further cooled to about 0°C and stirred for at least 2 hours. The slurry was filtered and wet cake was dried under reduced pressure at 55-85°C to yield the final product N-((R)-2,3-dihydroxypropoxy)-3,4- difluoro-2-(2-fluoro-4-iodo-phenylamino)-benzamide (Compound I) product. Overall chemical yield was 86 g, 58%.


WO2002/006213 describes crystalline Forms I and II. U.S. Pat. No. 7,060,856 (“the ‘856 patent”)


////////MIRDAMETINIB, Orphan Drug Status, Neurofibromatosis 1, PHASE 2, PD0325901, PD 0325901, PD-325901, 


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