Improved experimental procedure for the synthesis of the potent MEK inhibitor PD184352

Article abstract of DOI:10.1080/00397910500184909

An improved synthesis of the potent MEK inhibitor PD184352 (2-(2-Chloro-4-iodo-phenylamino)-N-cyclopropylmethoxy-3,4-difluoro-benzamide)) is herein reported. This new and reproducible protocol provides a simple and efficient way of generating gram quantities of PD184352 with minimal purification. Copyright Taylor & Francis, Inc.

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Improved Experimental
Procedure for the Synthesis
of the Potent MEK Inhibitor
PD184352
Natalia Shpiro a & Rodolfo Marquez a
a Division of Biological Chemistry and Molecular
Microbiology, School of Life Sciences , University of
Dundee , Dundee, Scotland, UK
Published online: 18 Aug 2006.
To cite this article: Natalia Shpiro & Rodolfo Marquez (2005) Improved Experimental
Procedure for the Synthesis of the Potent MEK Inhibitor PD184352, Synthetic
Communications: An International Journal for Rapid Communication of Synthetic
Organic Chemistry, 35:17, 2265-2269
To link to this article: http://dx.doi.org/10.1080/00397910500184909
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Synthetic Communicationsw, 35: 2265–2269, 2005
Copyright # Taylor & Francis, Inc.
ISSN 0039-7911 print/1532-2432 online
DOI: 10.1080/00397910500184909
Improved Experimental Procedure
for the Synthesis of the Potent MEK
Inhibitor PD184352
Natalia Shpiro and Rodolfo Marquez
Division of Biological Chemistry and Molecular Microbiology,
School of Life Sciences, University of Dundee, Dundee, Scotland, UK
Abstract: An improved synthesis of the potent MEK inhibitor PD184352 (2-(2-
Chloro-4-iodo-phenylamino)-N-cyclopropylmethoxy-3,4-difluoro-benzamide))
is
herein reported. This new and reproducible protocol provides a simple and efficient
way of generating gram quantities of PD184352 with minimal purification.
Keywords: Inhibitor, Hydroxylamines, PD184352
As part of our efforts to study the selectivity and efficacy of some known
kinase inhibitors, we became interested in the MEK inhibitor PD184352.
PD184352, or 2-(2-chloro-4-iodo-phenylamino)-N-cyclopropylmethoxy-3,4-
difluoro-benzamide, was originally reported by Warner Lambert in 1998 as
a potent MEK/ERK kinase cascade inhibitor that could potentially be used
as a treatment for septic shock.[1]
Received in the U.K. December 15, 2004
Address correspondence to Rodolfo Marquez, Division of Biological Chemistry and
Molecular Microbiology, School of Life Sciences, University of Dundee, Dundee DD1
4HN, Dundee, Scotland, UK. E-mail: R.Marquez@dundee.ac.uk
2265

2266
N. Shpiro and R. Marquez
Unfortunately, the reported synthesis of PD184352 requires a number of
nontrivial low-yielding steps, which can seriously limit its availability for
biological studies. Thus, we now report an improved practical procedure
that can generate the desired benzamide 1 in increased yields compared
with those reported thus far.[2]
In our modified procedure, 2,3,4-trifluoro benzoic acid 2 is treated with
4-iodo-2-chloro-aniline 3 under lithium diisopropyl amide (LDA)-promoted
conditions to generate the desired benzoic acid 4 in reasonable yield. Interest-
ingly, despite various procedural modifications, conversion of the trifluroben-
zoic acid 2 to the corresponding methyl benzoate failed to improve the
coupling yield.
Once the desired carboxylic acid unit 4 was available, we were faced with
the challenge of introducing the required cyclopropylmethyl hydroxylamine
unit. After a considerable amount of experimentation in which a variety of
approaches were considered, the most promising one appeared to involve
the direct peptide coupling of a cyclopropylmethyl hydroxylamine unit to car-
boxylic acid 4.
Generation of the required noncommercially available cyclopropyl-
methyl hydroxylamine hydrochloride unit began with ethyl N-hydroxyaceti-
midate 5, which was efficiently deprotonated and alkylated to generate the
desired alkylated N-hydroxyacetimidate 6.[3] Acid hydrolysis of the crude
acetamidate 6 then cleanly generated the desired cyclopropyl hydroxyla-
mine hydrochloride 7 and ethyl acetate in good yield for the whole
sequence.
However, peptide coupling of the newly generated cyclopropylmethyl
hydroxylamine 7 to benzoic acid 4 under the conditions originally
reported by Bridges, namely, through the use of Benzotriazolyloxy tripyrro-
lidinophosphonium hexafluorophosphate PyBOP and diisopropyl ethyl
amine (DIPEA), failed to afford the desired amide 8 in greater than 37%

MEK Inhibitor PD184352
2267
yield. A greater amount of the resulting product was composed of the
undesired and difficult to separate pyrrolidine amide 9 (39%). Interestingly,
biological testing of this pyrrolidine amide side product showed much lower
Mitogen activated protein kinase kinase/extracellular signal regulated
kinase (MEK) inhibitory activity when compared to PD184352.[4]
Faced with this disappointing yield, a significant amount of time and
effort was spent on the optimization of the coupling conditions of cyclopro-
pylmethyl hydroxylamine 7 to the difluoro carboxylic acid 4. Finally,
coupling under similar conditions to those reported by Tripathy and
Georg significantly increased the yield as to afford the desired MEK
inhibitor PD184352 in 73% yield.[5]
This reproducible, optimized process is amenable to scaling and has
finally allowed us to generate multigram quantities of PD184352 for all
of our testing needs.
In conclusion, we have managed to modify and improve the yields for the
synthesis of the potent kinase inhibitor PD184352. This reliable protocol
should be able to provide most researchers with a simple way of generating
gram quantities of PD184352 for biological research.
EXPERIMENTAL PROCEDURES
Synthesis of 2-(2-Chloro-4-iodo-phenylamino)-3,4-difluoro-benzoic
Acid, 4
A 2788C solution of commercially available 4-iodo-2-chloro aniline 3 (1.58 g,
6.25mmol) in THF (10 mL) was treated with a 1.8-M solution of lithium diiso-
propyl amide in heptane/tetrahydrofuran/ethylbenzene (6.63 mL, 11.93 mmol).
The resulting suspension was stirred at 2788C for 15min, at which point a
solution of 2,3,4-trifluorobenzoic acid 2 (1g, 5.68mmol) in THF (15 mL)

2268
N. Shpiro and R. Marquez
was added over 15min. The reaction was then allowed to warm up to room
temperature over 4 h and subsequently stirred at room temperature for 4 days.
The reaction was then diluted with diethyl ether (100 mL) and acidified
with 1M HCl, until the aqueous phase reached a pH of 1. The aqueous layer
was then extracted with diethyl ether (3 ꢀ 15 mL), and the combined organic
extracts dried over sodium sulphate. Solvent removal under vacuum afforded a
crude solid, which was recrystallized from dichloromethane to afford 994 mg
(43%) of the pure carboxylic acid 4 and 381 mg of recovered starting
acid (38%). dH (300 MHz, DMSO-d6) 6.73 (1H, dd, J ¼ 8.5, 7.0 Hz), 7.18
(1H, td, J ¼ 9.4, 7.3 Hz), 7.55 (1H, dd, J ¼ 8.6, 1.9 Hz), 7.88–7.82
(2H, m), 9.27 (1H, bs), 13.9 (1H, bs).
Synthesis of O-cyclopropylmethyl-hydroxylamine Hydrochloride, 7
Sodium metal (670 mg, 29.09 mmol) was added to anhydrous methanol
(45 mL), and the resulting suspension stirred at room temperature until the
sodium was completely dissolved. The resulting anhydrous solution of sodium
methoxide was then treated with ethyl N-hydroxyacetamidate (3 g,
29.09 mmol) in small portions. The resulting solution was then allowed to
stir at room temperature for 1 h before the solvent was removed under vacuum
to generate a white solid. The crude white residue was then suspended in ether
(20 mL), filtered under argon, and subsequently dried under vacuum to afford
ethyl N-hydroxy acetamidate sodium salt as a hygroscopic white solid, which
was used without any further purification.
Dry acetonitrile (100 mL) was added to the crude ethyl N-hydroxy-
acetamidate sodium salt, and the resulting suspension was then treated with
neat cyclopropyl methyl bromide (2.15 mL, 22 mmol). The reaction was
then stirred under argon at room temperature for 24 h and then refluxed for
an additional 3 h. The reaction mixture was then allowed to cool down to
room temperature and diluted with diethyl ether (100 mL), causing a white
precipitate to form. The solid was removed through filtration, and the
resulting filtrate concentrated under vacuum to generate a yellowish residue.
The crude residue was then dissolved in 2-M HCl (50 mL), and the
resulting solution refluxed for 1 h. The reaction was then cooled to room temp-
erature and concentrated under vaccum to afford an amorphous solid. The
resulting solid was then dissolved in methanol (30 mL) and slowly reconcen-
trated under vaccum as to induce precipitation of a white solid. Removal of the
last traces of solvent under vacuum finally afforded 1.89 g (70%) of the desired
hydrochloride salt as a white solid. Mp 114–1178C.
Synthesis of PD184352, 8
A solution of benzoic acid 4 (2.01g, 4.91mmol) in a 1 : 1 mixture of anhydrous
THF and anhydrous DMF (40mL total volume) was treated with 1,3-

MEK Inhibitor PD184352
2269
dicyclohexylcarbodiimide (DCC) (1.52 g, 7.36 mmol), which after stirring for
15 min caused a white precipitate to form. The suspension was then treated
with 1-hydroxybenzotriazole hydrate (HOBT) (0.99 g, 7.36 mmol) and
4-dimethylamino pyridine (DMAP) (120 mg, 0.98 mmol). The resulting
mixture was then stirred at room temperature for 1.5 h, after which freshly
prepared cyclopropyl methyl hydroxylamine hydrochloride
7 (1.21 g,
9.81mmol) and diisopropyl ethyl amine (2.56mL, 14.72 mmol) were sequen-
tially added. The complete reaction mixture was then stirred at room temperature
for 48 h.
Solvent removal under vacuum afforded a crude residue, which was
purified by flash column chromatography (silica gel, 50% diethyl ether in
40–60 petroleum ether) to produce a whitish solid, which was subsequently
recrystallized from ethanol and methanol respectively to provide 1.72 g
(73%) of PD184352, 8, as a white solid. dH (300 MHz, CDCl3) 0.34–0.28
(2H, m), 0.65–0.59 (2H, m), 1.28–1.11 (1H, m), 3.78 (2H, d, J ¼ 7.3 Hz),
6.48 (1H, dd, J ¼ 8.5, 5.5 Hz), 6.96 (1H, appq, J ¼ 8.9 Hz), 7.44–7.38
(2H, m), 7.70 (1H, d, J ¼ 1.9 Hz), 8.17 (1H, bs), 8.87 (1H, bs). Mp 175.5–
176.58C (lit. 172.5–173.58C).
NMR Data for [2-(2-Chloro-4-iodo-phenylamino)-3,4-difluoro-
phenyl]-pyrrolidin-1-yl-methanone, 9
dH (300 MHz, CDCl3) 1.95–1.82 (4H, m), 3.40 (2H, appt, J ¼ 6.3 Hz), 3.55
(2H, appt, J ¼ 6.8 Hz), 6.52 (1H, dd, J ¼ 8.6, 5.8 Hz), 6.94 (1H, td, J ¼ 9.0,
7.0 Hz), 7.17 (1H, ddd, J ¼ 8.6, 5.4, 2.0 Hz), 7.40 (1H, dd, J ¼ 8.6, 1.9 Hz),
7.64 (1H, d, J ¼ 2.0 Hz), 7.72 (1H, bs). dC (75 MHz, CDCl3) 24.72, 26.61,
46.61, 49.91, 81.61, 111.46 (d, J ¼ 17.8 Hz), 117.82 (d, J ¼ 5.3 Hz),
123.36, 123.88, 126.43, 129.99 (d, J ¼ 6.9 Hz), 136.32, 137.64, 139.60,
144.70 (dd, J ¼ 250.9, 14.5 Hz), 152.36 (dd, J ¼ 250.6, 11.5 Hz), 167.02.
IR (Vmax) film/cm21 3344, 2924, 1630, 1464, 1105, 912, 743, 650, 503.
m/z (CI) 463.0 (100%, MHþ), 465.0 (27).
REFERENCES
1. Bridges, A. J. Method of Treating or Preventing Septic Shock by Administering a
MEK inhibitor. Intl. Patent WO98/37881, September 3, 1998.
2. Barrett, S.; Bridges, A. J.; Doherty, A. M.; Dudley, D. T.; Saltiel, A. R. 4-Bromo or
4-Iodo Phenylamino Benzhydroxamic Acid Derivatives and their use as MEK
Inhibitors. Intl. Patent WO99/01426, January 14, 1999.
3. Johnson, G.; Boxer, P. A.; Drummond, J. T.; Boyd, D. K.; Anderson, R. J. Identifi-
cation and evaluation of o-alkyl susbtituted hydroxamic acids as potent in vitro
inhibitors of the hepatic glycine cleavage system and investigation of their action
on in vivo central nervous system glycine concentration. Arzneim.-Forsch./Drug
Res. 1989, 39 (1), 432–437.

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4. Bain, J.; Cohen, P. The MEK inhibitory activity shown by pyrrolidine amide 9 was
ten fold lower than that exhibited by PD184352. Personal communication.
5. Tripathy, N. K.; Georg, G. I. RCM approach for the total synthesis of cryptophy-
cin-24 (arenastatin A). Tetrahedron Lett. 2004, 45 (27), 5309–5311.