Multi-gram scale synthesis of FR180204

Article abstract of DOI:10.1021/jo901835m

(Chemical Equation Presented) A concise synthesis of the ERK inhibitor FR180204 has been developed. The synthesis consists of six operationally simple steps and can be utilized to make multi-gram quantities of FR180204.

Full text of DOI:10.1021/jo901835m

pubs.acs.org/joc
It is well-known that irregularities in the Ras-Raf-MEK-
Multi-Gram Scale Synthesis of FR180204
ERK signal transduction pathway play a crucial part in
many cancers.6 The MAP kinase ERK is the last player in
this pathway controlling the activation of transcription
factors in the nucleus which control cellular processes such
as proliferation, survival, and growth. The inhibition of
ERK also holds promise as a therapy for inflammatory
diseases.7 For instance, the signaling through the TGFβ
pathway, an important cascade controlling inflammation,
may lead to ERK activation.8 Thus, the modulation of this
nodal downstream protein kinase might prove to be extre-
mely beneficial in controlling disease states arising due
to malfunctioning of various upstream proteins. With this
background, it is surprising that a synthetic route to
FR180204 is not available in the literature. To that end, we
set out to devise a simple scalable synthetic route that would
allow access to gram quantities. This will facilitate its use as
an important tool compound for the study of ERK inhibi-
tion in various disease models.
Samarjit Patnaik,*,† Harry C. Dietz,‡ Wei Zheng,†
Christopher Austin,† and Juan J. Marugan*,†
†National Institutes of Health Chemical Genomics Center,
9800 Medical Center Drive, Rockville, Maryland 20850, and
‡Johns Hopkins University School of Medicine, 733 North
Broadway, Baltimore, Maryland 21205
patnaiks@mail.nih.gov
Received August 25, 2009
A concise synthesis of the ERK inhibitor FR180204 has
been developed. The synthesis consists of six operation-
ally simple steps and can be utilized to make multi-gram
quantities of FR180204.
FIGURE 1. Retrosynthesis.
The FR180204 (1) chemical structure consists of two drug-
like motifs, a pyrazolo[1,5-a]pyridine and a pyrazolo[3,4-c]-
pyridazine. The disconnection that we envisioned passes
though the synthesis of oxopyridazine carboxamide 2 and
pyrazolopyridine ketone 3 as key intermediates. The pyrazolo-
pyridine ring system can be accessed via a 3 þ 2 cyclo-
addition of aminopyridium salts and alkynes. We decided to
construct this ring system first, as alternate routes involved
carrying a reactive alkyne throughout the synthesis of the
pyrazolopyridazine ring or its late generation for the pro-
posed cycloaddition. To that end, commercially available
reagents 1-aminopyridinium iodide and 4-phenylbut-3-yn-2-
one were condensed under basic conditions9 to provide the
known ethanone 3 (Scheme 1).10 The latter contained the
methyl ketone which would form a template for the stepwise
construction of the pyrazolopyridazine ring system. An
initial attempt of producing the oxopyridazine carboxamide
intermediate involved the introduction of the remaining
carbon atoms via the displacement of a R-haloketone with
diethylmalonate.11 We found that treatment of 3 with bro-
mine led to poor yields of R-bromoketone 4 with significant
FR180204 is a potent and selective ATP-competitive
inhibitor of extracellular signal-regulated kinases (ERK) 1
and 2.1 Discovered by Ohori and co-workers, it has IC50
values of 0.51 and 0.33 μM in enzymatic assays against
ERK1 and ERK2, respectively.2 It was also shown to inhibit
TGFβ-induced AP-1 activation with an IC50 of 3.1 μM in a
cellular assay. A crystal structure of FR180204 in human
wild-type ERK2 revealed that it is bound to the kinase hinge
region via the 3-NH2 and the 2-N of the pyrazolopyridazine.3
Besides in vitro activity, Ohori has shown that a greater than
60% response can be achieved in a rheumatoid arthritis
mouse model with an ip administration of 100 mg/kg bid
dose of FR180204.4 Recently, Regan et al. have demon-
strated that FR180204 can reduce iron-mediated neuronal
damage caused by hemoglobin breakdown by a likely reduc-
tion in phosphorylation of heme-oxygenase by ERK.5
(1) Ohori, M.; Kinoshita, T.; Okubo, M.; Sato, K; Yamazaki, A.;
Arakawa, A.; Nishimura, S.; Inamura, N.; Nakajima, H.; Neya, M.; Miyake,
H.; Fujii, T. Biochem. Biophys. Res. Commun. 2005, 336, 357–363.
(2) For another enzyme-potent small-molecule inhibitor of ERK, see:
Aronov, A. M.; Baker, C.; Bemis, G. W.; Cao, J.; Chen, G.; Ford, P. J.;
Germann, U. A.; Green, J.; Hale, M. R.; Jacobs, M.; Janetka, J. W.; Maltais,
F.; Martinez-Botella, G.; Namchuk, M. N.; Straub, J.; Tang, Q.; Xie, X.
J. Med. Chem. 2007, 50, 1280–1287.
(6) Hilger, R. A.; Scheulen, M. E.; Strumberg, D. Onkologie 2002, 25,
511–518.
(3) Kinoshita, T.; Warizaya, M.; Ohori, M.; Sato, K.; Neya, M.; Fujii, T.
Bioorg. Med. Chem. Lett. 2006, 16, 55–58.
(7) Hitti, E.; Kotlyarov, A. Anti-Inflamm. Anti-Allergy Agents Med.
Chem. 2007, 6, 85–97.
(4) Ohori, M.; Takeuchi, M.; Maruki, R.; Nakajima, H.; Miyake, H.
Naunyn-Schmiedeberg’s Arch Pharmacol. 2007, 374, 311–316.
(5) Chen-Roetling, J.; Li, Z.; Chen, M.; Awe, O. O.; Regan, R. F.
Neuropharmacology 2009, 56, 922–928.
(8) Mulder, K. M. Cytokine Growth Factor Rev. 2000, 11, 23–35.
(9) Uehling, D. et al. WO 2006/068826.
(10) Ha, H. -H.; Kim, J. S.; Kim, B. M. Bioorg. Med. Chem. Lett. 2000,
18, 653–656.
8870 J. Org. Chem. 2009, 74, 8870–8873
Published on Web 10/26/2009
DOI: 10.1021/jo901835m
r
2009 American Chemical Society

Patnaik et al.
JOCNote
SCHEME 1
ketone 3 was heated with diethyl 2-oxomalonate to execute
an aldol-like process to provide 8. The crude tertiary alcohol
was then dissolved in ethanol and heated with excess hydra-
zine. This led to the precipitation of pure 9, where the
SCHEME 3
R,R-dibromination. Portionwise addition of 1 equiv of tetra-
butylammonium tribromide did not circumvent this issue.
However, this problem could be resolved via formation of
the intermediate trimethylsilyl enol ether and subsequent treat-
ment with NBS. Treatment of 4 with 3 equiv of diethyl
malonate in refluxing acetone under basic conditions led to
good conversion to 5. Reaction of 5 with 1 equiv of hydrazine
led to practically no conversion to the desired ester 7 and
recovery of starting material. Heating with multiple equivalents
of hydrazine allowed for the ring closure but with uncontrol-
lable simultaneous formation of the corresponding hydrazide
6. Heating with lesser equivalents of hydrazine in the micro-
wave or the use of higher boiling solvents such as isoamyl
alcohol did not resolve this problem.
pyridazin-3(2H)-one had formed via elimination of water
and ring closure. Concomitant formation of the hydrazide
had also taken place during the course of the reaction.
Refluxing in concentrated hydrochloric acid led to the initial
dissolution of 9 and the subsequent precipitation of acid 10.
The latter was simply filtered off and washed with water to
provide pure 10. A high yielding amidation of 10 was
possible with isobutylchloroformate and aqueous ammonia.
The pure amide 11 was simply filtered off from the reaction
mixture. 11 had to be refluxed with neat phosphorus oxy-
chloride to simultaneously convert the pyridazinone and the
carboxamide to the chloropyridazine and nitrile, respec-
tively. At this stage, we felt that purification of nonpolar
12 by flash silica gel chromatography would be useful in
providing material with high purity for the last step. Purified
12 was refluxed with hydrazine in ethanol for 2 h and then left
standing for 16 h to cause the precipitation of FR180204 1.
The solid was filtered to provide material which was of at
Ester 7 would have allowed for the introduction of un-
saturation in the dihydropyridazinone 2 via a bromination
and elimination sequence. Amidation followed by treatment
with phosphorus oxychloride and cyclization with hydrazine
would have then provided FR180204 (Scheme 2).
SCHEME 2
1
least 95% purity as determined by H NMR spectroscopy
and reverse-phase HPLC.
We evaluated the kinase selectivity of 1 in a panel of 442
kinases at 10 μM and found >99% inhibition of kinase
activity for 12 out of 386 nonmutant kinases (>90% for 29/
386).13 We also measured the dissociation constants against
a few kinases of interest.13 Table 1 discloses some activities in
the range of previously reported values. These studies con-
firm the activity and selectivity reported by Ohori.1
In summary, we have developed a scalable route to
FR180204. This was used to prepare multi-gram quantities
of FR180204 from known ketone 3 in six steps and 30%
overall yield. Silica gel chromatography was done in only one
As an alternative route, we turned our attention to a
related method for the construction of pyrazolopyridazines,
which utilizes 2-oxodiethyl malonate as the source of the
carbon atoms of the ring system (Scheme 3).12 Thus, methyl
(11) (a) Brana, M. F.; Cacho, M.; Garcia, M. L.; Mayoral, E. P.; Lopez, B.;
Pascual-Teresa, B.; Ramos, A.; Acero, N.; Llinares, F.; Munoz-Mingarro, D.;
Lozach, O.; Meijer, L. J. Med. Chem. 2005, 48, 6843–6854. (b) Wermuth,
C. G.; Schlewer, G.; Bourguignon, J. J.; Maghioros, G.; Bouchet, M. J.; Moire,
C.; Kan, J. P.; Worms, P.; Biziere, K. J. Med. Chem. 1989, 32, 528–537.
(c) Pinna, G. A.; Curzu, M. M.; Gavini, E.; Mule, A.; Pirisino, G.; Satta, M.;
Peana, G. Il Farmaco 1993, 48, 1239–1248.
(12) (a) Sircar, I.; Duell, B. L.; Bobowski, G.; Bristol, J. A.; Evans, D. B.
J. Med. Chem. 1985, 28, 1405–1413. (b) Singh, B.; Lesher, G. Y. Heterocycles
1990, 31, 2163–2172.
(13) These data were generated by Ambit Biosciences.
J. Org. Chem. Vol. 74, No. 22, 2009 8871

Patnaik et al.
JOCNote
TABLE 1. Kinase Activity Data
7.79 (s, 1 H), 7.91 (dt, J = 9.0, 1.3 Hz, 1 H), 8.83 (d, J = 7.0 Hz,
1H), 10.46 (br s, 1 H), 11.55 (br s, 1 H); 13C NMR (101 MHz,
DMSO-d6) δ ppm 103.9, 113.9, 117.6, 126.2, 128.2, 128.8, 128.9,
129.0, 132.1, 133.6, 139.1, 141.6, 151.4, 159.3, 159.7; IR (neat,
enzyme
Kd (nM)
ERK1
ERK2
MEK1
EGFR
P38-alpha
160
420
8700
1200
1400
diamond/ZnSe) 3220, 2867, 1683, 1583, 1520, 1450, 1225 cm-1
;
HRMS (m/z) calcd for C18H15N6O2 (M þ H)þ 347.1256, found
347.1253.
Acid 10: Hydrazide 9 (6.30 g, 18.2 mmol) was refluxed in
concd HCl (100 mL). After initial dissolution, a precipitate was
observed to form after 16 h of reflux. The reaction was cooled
step. This route will give access to gram quantities of
FR180204, which will enable its use as a tool compound
for studying the therapeutic effects of ERK inhibition in in
vitro and in vivo disease models.
€
and filtered through a Buchner funnel. The residue was washed
with water and diethyl ether and then air-dried to provide acid
10 (5.18 g, 15.6 mmol, 86% yield): LC-MS rt (min) = 3.31; 1H
NMR (400 MHz, DMSO-d6) δ ppm 7.11 (td, J = 7.1, 1.6 Hz),
7.44-7.52 (m, 4 H), 7.59-7.66 (m, 2 H), 7.70 (s, 1 H), 7.92 (d,
J = 9.0 Hz, 1 H), 8.85 (d, J = 7.0 Hz, 1 H); 13C NMR (101 MHz,
DMSO-d6) δ ppm 103.5, 114.1, 117.6, 126.5, 127.0, 128.9, 128.9,
129.1, 129.1, 132.1, 135.2, 139.2, 142.4, 151.6, 160.8, 163.5; IR
Experimental Section
General Methods: Chromatography on silica gel was per-
formed using forced flow (liquid) of the indicated solvent system
on Biotage KP-Sil prepacked cartridges and using the Biotage
SP-1 automated chromatography system. 1H and 13C NMR
spectra were recorded on a Varian Inova 400 MHz spectro-
meter. Chemical shifts are reported in δ with the solvent
resonance as the internal standard (CDCl3 δ 7.26, 77.0,
DMSO-d6 δ 2.50, 39.5 for 1H and 13C, respectively). Analytical
analysis and retention times reported here were performed on an
Agilent LC/MS (Agilent Technologies, Santa Clara, CA) with a
3 min gradient of 4-100% MeCN (containing 0.025% TFA) in
water (containing 0.05% TFA) and was used with a 4.5 min run
time at a flow rate of 1 mL/min. A Phenomenex Gemini phenyl
column (3 μm, 3 ꢀ 100 mm) was used at a temperature of 50 °C.
Infrared spectra were recorded on a Perkin-Elmer ET-IR Spec-
trum 10 instument. Molecular weight was confirmed using a
TOF mass spectrometer (Agilent Technologies, Santa Clara,
CA). A 3 min gradient from 4 to 100% acetonitrile (0.1% formic
acid) in water (0.1% formic acid) was used with a 4 min run time
at a flow rate of 1 mL/min. A Zorbax SB-C18 column (3.5 μm,
2.1 ꢀ 30 mm) was used at a temperature of 50 °C. Confirmation
of molecular formula was confirmed using electrospray ioniza-
tion in the positive mode with the Agilent Masshunter software
(version B.02).
(neat, diamond/ZnSe) 2739, 1741, 1640, 1591, 1422, 1401 cm-1
;
HRMS (m/z) calcd for C18H13N4O3 (M þ H)þ 333.0988, found
333.0982.
Amide 11: Acid 10 (5.13 g, 15.4 mmol) was taken in DME (200
mL), cooled to 0 °C, treated with 4-methylmorpholine
(20.0 mL, 182 mmol), stirred for 5 min, and then treated with
isobutylchloroformate (5.0 mL, 38 mmol). The mixture was
stirred for 4 h and then treated with excess ammonium hydro-
xide (50 mL, 1.28 mol). The reaction was stirred for 16 h. The
€
yellow precipitate formed was filtered through a Buchner fun-
nel, washed with water and diethyl ether, and air-dried to
provide amide 11 (4.37 g, 13.2 mmol, 85% yield): LC-MS rt
(min) = 3.09; 1H NMR (400 MHz, DMSO-d6) δ ppm 7.09 (td,
J = 6.9, 1.5 Hz, 1 H), 7.39-7.51 (m, 4 H), 7.57-7.65 (m, 2 H),
7.82 (s, 1 H), 7.89 (dt, J = 8.9, 1.3 Hz, 1 H), 7.95 (d, J = 4.1 Hz,
1 H), 8.79-8.89 (m, 2 H), 13.86 (s, 1 H); 13C NMR (101 MHz,
DMSO-d6) δ ppm 103.9, 113.9, 117.5, 126.1, 128.8, 128.8, 128.9,
129.0, 129.3, 132.1, 134.7, 139.1, 141.4, 151.3, 160.1, 162.3; IR
(neat, diamond/ZnSe) 3312, 2836, 1692, 1580, 1519, 1449, 1419,
1362 cm-1; HRMS (m/z) calcd for C18H14N5O2 (M þ H)þ
332.1147, found 332.1142.
Nitrile 12: Amide 11 (4.35 g, 13.1 mmol) was refluxed in
phosphorus oxychloride (30.0 mL, 322 mmol) for 1 h. Dissolu-
tion of the yellow suspension to an orange colored solution was
observed. The reaction was cooled and poured over ice. Pre-
cipitation of a yellow solid was observed. This was filtered. This
was dissolved in DCM/MeOH mixture, adsorbed on silica gel,
and purified by flash silica gel chromatography (0-5% EtOAc/
DCM) to provide nitrile 12 (3.4 g, 10.3 mmol, 78% yield): LC-
Ketone 3: 4-Phenylbut-3-yn-2-one (7.5 g, 52 mmol) and
1-aminopyridinium iodide (13.4 g, 60.4 mmol) were dissolved
in DMSO (105 mL) and treated with K2CO3 (6.47 g, 46.8 mmol)
and KOH (5.84 g, 104 mmol). The mixture was stirred for 3 h.
Then the reaction was poured in water and extracted with
EtOAc. The organic layer was dried (Mg2SO4), filtered, con-
centrated, and purified by flash silica gel chromatography
(10-100% EtOAc/hexanes) to provide ketone 3 (11.1 g, 46.8
mmol, 90% yield): LC-MS rt (min) = 3.48; 1H NMR (400 MHz,
CDCl3) δ ppm 2.15 (s, 3 H), 7.04 (td, J = 6.8, 1.6 Hz, 1 H),
7.47-7.54 (m, 4 H), 7.56-7.63 (m, 2 H), 8.46 (d, J = 9.0 Hz,
1 H), 8.54 (d, J = 6.7 Hz, 1 H); 13C NMR (101 MHz, CDCl3) δ
ppm 30.1, 111.6, 114.7, 120.2, 128.3, 128.4, 128.5, 129.1, 129.7,
133.4, 141.9, 156.8, 193.6; IR (neat, diamond/ZnSe) 3066, 3024,
1641, 1627, 1497, 1411, 1357, 1211 cm-1; HRMS (m/z) calcd for
C15H13N2O (M þ H)þ 237.1028, found 237.1021.
1
MS rt (min) = 3.77; H NMR (400 MHz, DMSO-d6) δ ppm
7.18 (td, J = 6.8, 1.6 Hz, 1 H), 7.44-7.53 (m, 3 H), 7.53-7.59
(m, 1 H), 7.59-7.65 (m, 2 H), 8.02 (s, 1 H), 8.16 (d, J = 9.0 Hz,
1 H), 8.91 (d, J = 7.0 Hz, 1 H); 13C NMR (101 MHz, CDCl3) δ
ppm 103.5, 112.6, 113.8, 114.5, 119.7, 127.6, 128.9, 129.1, 129.4,
129.4, 129.9, 131.9, 140.4, 150.1, 154.0, 155.7; IR (neat,
diamond/ZnSe) 3077, 3030, 2240, 1733, 1629, 1577, 1511,
1467, 1419, 1351, 1327, 1189 cm-1; HRMS (m/z) calcd for
C18H11ClN5 (M þ H)þ 332.0703, found 332.0700.
FR108204 1: Nitrile 12 (4.80 g, 14.5 mmol) was taken in
ethanol (150 mL) and treated with excess hydrazine hydrate
(7.00 mL, 143 mmol) and then heated to reflux. The suspension
dissolved to an orange solution. After refluxing for 3 h, the
reaction was cooled and left standing for 2.5 days. A yellow
Hydrazide 9: Ketone 3 (4.30 g, 18.20 mmol) and diethyl
2-oxomalonate (6.34 g, 36.4 mmol) were taken in a microwave
vial and heated at 200 °C for 2 h at a “high” setting. The vessel’s
septum was carefully punctured with a needle to release pres-
sure. The crude brown residue (tertiary alcohol) was diluted with
EtOH, transferred to a RB flask, treated with excess hydrazine
hydrate (10.0 g, 200 mmol), and refluxed overnight. The reac-
tion was cooled, and the precipitate formed was filtered through
€
solid had precipitated out. This was filtered via a Buchner
funnel. The solid was washed with EtOH and water and dried
to provide FR180204 1 (4.00 g, 12.2 mmol, 84% yield): LC-MS
rt (min) = 2.98; 1H NMR (400 MHz, DMSO-d6) δ ppm
5.90 (br s, 2 H), 7.04 (td, J = 6.8, 1.4 Hz, 1 H), 7.22-7.46
(m, 4 H), 7.47-7.64 (m, 2 H), 7.76 (dt, J = 8.8, 1.2 Hz, 1 H),
€
a Buchner funnel, washed with ethanol, and air-dried to hydra-
zide 9 (6.30 g, 18.2 mmol, quantitative): LC-MS rt (min) = 2.92;
1H NMR (400 MHz, DMSO-d6) δ ppm 4.80 (br s, 2 H), 7.09 (td,
J = 6.8, 1.4 Hz, 1 H), 7.42-7.50 (m, 4 H), 7.57-7.65 (m, 2 H),
7.96 (s, 1 H), 8.82 (dt, J = 6.9, 1.1 Hz, 1 H), 12.67 (s, 1 H); 13
C
8872 J. Org. Chem. Vol. 74, No. 22, 2009

Patnaik et al.
JOCNote
Acknowledgment. S.P. would like to thank Dr. Craig
Thomas and Dr. Jingbo Xiao for useful suggestions.
NMR (101 MHz, DMSO-d6) δ ppm 107.3, 108.6, 113.3, 117.4,
119.5, 125.1, 128.3, 128.5, 128.5 (two signals overlapped at
128.5), 128.8, 132.7, 139.8, 144.8, 148.2, 150.6, 154.0; IR (neat,
diamond/ZnSe) 3302, 3161, 1630, 1529, 1458, 1422, 1352,
1108 cm-1; HRMS (m/z) calcd for C18H14N7 (M þ H)þ
328.1311, found 328.1309.
Supporting Information Available: 1H and 13C NMR data
for 3, 9, 10, 11, 12, and 1. This material is available free of charge
via the Internet at http://pubs.acs.org.
J. Org. Chem. Vol. 74, No. 22, 2009 8873