An optimized synthesis of the potent and selective Pak1 inhibitor FRAX-1036

Article abstract of DOI:10.1016/j.tetlet.2015.12.059

FRAX-1036 is a p21-activated kinase I inhibitor of significant interest to cancer biologists yet no commercial providers or detailed procedures are available. In this Letter, we chronicle the optimized synthesis of FRAX-1036, one of the most specific Pak1

Full text of DOI:10.1016/j.tetlet.2015.12.059

Accepted Manuscript
An optimized synthesis of the potent and selective Pak1 inhibitor FRAX-1036
Alexander B. Koval, William M. Wuest
PII:
S0040-4039(15)30474-3
DOI:
http://dx.doi.org/10.1016/j.tetlet.2015.12.059
TETL 47106
Reference:
Tetrahedron Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
24 November 2015
7 December 2015
12 December 2015
Please cite this article as: Koval, A.B., Wuest, W.M., An optimized synthesis of the potent and selective Pak1
inhibitor FRAX-1036, Tetrahedron Letters (2015), doi: http://dx.doi.org/10.1016/j.tetlet.2015.12.059
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An optimized synthesis of the potent and selective Pak1 inhibitor FRAX-1036
Alexander B. Koval and William M. Wuest*
Department of Chemistry, Temple University, Philadelphia PA 19122
Abstract: FRAX-1036 is a p21-activated kinase I inhibitor of significant interest to
cancer biologists yet no commercial providers or detailed procedures are available. In
this letter, we chronicle the optimized synthesis of FRAX-1036, one of the most specific
Pak1 inhibitors known. We report a 65-fold increase in the overall yield of the seven-
step sequence from 0.25% to 16.3%. Our improved route has provided gram quantities
of FRAX-1036 for in vivo animal studies by the scientific community.
Keywords: p21-activated kinase, PAK inhibitor, FRAX-1036
TOC Image:
A number of members of the p21-activated kinase (Pak) kinase family have been
shown to promote the proliferation of many human cancers (breast, ovarian, prostate,
and brain) based on activity in signal transduction pathways that promote tumor
proliferation, survival, migration, and invasion.1 Paks are cytosolic serine-threonine
protein kinases that are classified based on their structural and biochemical properties
into two subclasses, group I [Pak1-3] or group II [Pak4-6]). It has been proposed that
the two groups, although similar, target different substrates, are separately regulated,
and have unique cellular functions.2,3 Pak1-dependent phosphorylation contributes to
the activation of Ras/Raf/Mek/Erk,4 PI3K/Akt, and Wnt/β-catenin5,6, and inhibits
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apoptosis in part via phosphorylation of Bcl-2-associated death promoter (BAD).7
Furthermore, it has been demonstrated that inhibition of group I Paks blocks
transformation by oncogenic forms of Kras or ErbB2, or the cancer-inducing virus
Kaposi’s sarcoma herpes virus (KSHV) that would otherwise induce tumor formation.9-11
Based on these and many other studies, Pak kinases are emerging as promising
candidates for inhibition by targeted therapeutic agents. However, despite the strong
correlation between the up-regulation (or increased activity) of Pak1 and the
presence/proliferation of a number of cancers, only a small number of group I Pak
inhibitors have been reported. Among these are CEP1347,12 OSU-03012,13 and
SU11652,14 which have yet to be unequivocally shown to specifically inhibit Pak (Figure
1A). In 2014 Chow et al. demonstrated that the small-molecules FRAX-716 and FRAX-
1036, analogs of the previously known inhibitor FRAX-597, displayed increased
specificity and efficacy for inhibition of group I Paks (Figure 1B).15 Subsequent studies
compared the effects of these group I-directed FRAX compounds with a compound that
inhibited both group I and II Paks (PF3758309) in ovarian cancer cell lines.16 Treatment
of OVCAR-3 cells with FRAX-1036, the most specific Pak inhibitor, resulted in up-
regulation of p53 and p21, while down-regulating cyclin B1. The in vitro and in vivo tests
with FRAX-1036 have shown that the small-molecule inhibitor can both deter
proliferation in ovarian cancer cells with elevated Pak1 levels and affect tumor growth,
respectively.
To date there are neither commercial vendors nor detailed synthetic procedures
available for the production of FRAX-1036 despite the fact that FRAX-1036 is perhaps
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the most specific group I inhibitor. We sought to rectify this issue by developing a
means to readily produce gram-quantities of material in an academic
Figure 1. A) Proposed Pak1 inhibitors and B) known Pak1 inhibitors disclosed by AFRAXIS, Inc.
laboratory setting, thereby providing the scientific community with valuable quantities of
the lead compound. At the onset of this project we identified the seminal disclosure of
the FRAX family of compounds in a patent from AFRAXIS, Inc.17 The described route to
synthesis of FRAX-1036 was presumably founded from a medicinal chemistry focus, as
the chosen retrosynthetic disconnections facilitates the production of a diverse library of
compounds at the expense of late-stage yields. Unfortunately, the reported route lacked
specific details and, more importantly, complete characterization of key
intermediates.17,18 In an effort to expedite production of FRAX-1036 for in vivo testing,
we sought to optimize the published route in an effort to improve on the published
procedures (9 steps, 0.25% yield) on sufficient scale. Herein, we report a detailed and
optimized route to the Pak1 inhibitor, FRAX-1036, providing gram quantities of material
for biological studies.
In our quest to develop an optimized route of FRAX-1036 with the goal of
producing testable quantities of material, we sought to optimize the nine step longest
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linear sequence on scale. The synthesis began with the facile conversion of acid 1 to
the corresponding boronate 4 in six straightforward steps. Slight modifications to the
reaction time, temperature, and purification conditions resulted in an optimized process
that increased the overall yield three-fold (from 27% to 76%). Initial efforts to couple the
commercially available methylchloropyrazine using Suzuki-Miyaura conditions led to
modest yield. Simply increasing the reaction time to 72 hours and cycling the recovered
starting material (three times) reliably provides 5 in improved yield. It should be noted
that the seven-step synthetic sequence is routinely conducted on a twenty-gram scale
without complication proceeding in 47.6% overall yield.
Scheme 1. Optimized synthesis of the group I Pak inhibitor FRAX-1036
At this stage we focused our efforts toward the penultimate coupling of chloro-
ester 5 with known aldehyde 6. Prolonged reaction times significantly increased product
formation; however, initial attempts to purify the resulting amide proved difficult.
Although not ideal, we resorted to column chromatography in lieu of recrystallization (as
previously reported) and, to our delight, isolated 70% of the product as a white solid.
Optimization of the final reaction in the route, the nucleophilic aromatic substitution of
4

the piperidine amine, was the most challenging. After significant experimentation, we
identified conditions (DMF, elevated temperature), which resulted in a four-fold increase
in yield (49%). Presumably using an aprotic polar solvent, like DMF, stabilizes the
charged intermediate that develops during the course of the reaction resulting in higher
efficiency. Similar to the above reactions, we have performed the final two steps on a
multi-gram scale; however, a significant drop in yield is observed when the reactions
were performed on >500 mg (up to a 50% decrease in yield). Based on these findings,
and when combined with the cost of the amine, we ultimately resorted to performing the
final coupling on 500 mg batches.
In summary, we have disclosed an optimized synthesis of the potent group I Pak
inhibitor FRAX-1036 that is capable of producing gram quantities of material in two
weeks time. Our route has resulted in a greater than 65-fold improvement in overall
yield and has produced 1.25 g of material for animal studies immediately. The
aforementioned improvements in reaction conditions and purification procedures have
greatly aided in the manufacturing of necessary amounts of FRAX-1036 for in vivo
studies. We anticipate that the disclosed procedures will be of significant interest to both
academic and commercial interests based on the newfound potency and selectivity of
the Pak1 inhibitor.
Author Information
Corresponding Author
*E-mail: wwuest@temple.edu
Notes
The authors declare no competing financial interest.
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Acknowledgments
We thank Temple University and the Fox Chase Cancer Center (NIH core grant
CA06927) for financial support. We also thank Drs. Jonathan Chernoff and Erica
Golemis for helpful discussions.
Supplementary Material
Experimental procedures as well as compound characterization data can be found
within the Supplementary Material.
References
(1) Radu, M.; Semenova, G.; Kosoff, R.; Chernoff, J.; PAK signalling during the
development and progression of cancer. Nat Rev Cancer. 2014, 14, 13–25.
(2) Arias-Romero, L.E.; Chernoff, J. A tale of two Paks. Biol. Cell. 2008, 100, 97–108.
(3) Bagrodia, S.; Taylor, S.J.; Creasy, C.L.; Chernoff, J.; Cerione, R.A. Identification of a
mouse p21(Cdc42/Rac) activated kinase. J. Biol. Chem. 1995, 270, 22731–22737.
(4) Wang, Z.; Pedersen, E.; Basse, A.; Lefever, T.; Peyrollier, K.; Kapoor, S. et al. Rac1
is crucial ꢀ for Ras-dependent skin tumor formation by controlling Pak1-Mek-Erk hyper-
activation and hyperproliferation in vivo. Oncogene. 2010, 29, 3362–3373.
(5) Arias-Romero, L.E.; Villamar-Cruz, O.; Huang, M.; Hoeflich, K.P.; Chernoff, J.; Pak1
kinase links ErbB2 to beta-catenin in transformation of breast epithelial cells. Cancer
Res. ꢀ 2013, 73, 3671–3682.
(6) He, H.; Shulkes, A.; Baldwin, G.S. PAK1 interacts with beta-catenin and is required
for ꢀ the regulation of the beta-catenin signalling pathway by gastrins. Biochim. Biophys.
6

ꢀ Acta. 2008, 1783, 1943–1954.
(7) Schurmann, A.; Mooney, A.F.; Sanders, L.C.; Sells, M.A.; Wang, H.G.; Reed, J.C. et
al. p21-activated kinase 1 phosphorylates the death agonist bad and protects cells from
ꢀ apoptosis. Mol. Cell Biol. 2000, 20, 453–461.
(8) Arias-Romero, L.E.; Villamar-Cruz, O.; Pacheco, A.; Kosoff, R.; Huang, M.;
Muthuswamy, S.K. et al. A Rac-Pak signaling pathway is essential for ErbB2-mediated
transformation ꢀ of human breast epithelial cancer cells. Oncogene. 2010, 29, 5839–
5849.
(9) Dadke, D.; Fryer, B.H.; Golemis, E.A. Field J. Activation of p21-activated kinase 1-
nuclear factor kappaB signaling by Kaposi's sarcoma-associated herpes virus G protein-
ꢀ coupled receptor during cellular transformation. Cancer Res. 2003, 63, 8837–8847.
(10) Tang, Y.; Chen, Z.; Ambrose, D.; Liu, J.; Gibbs, J.B.; Chernoff, J. et al. Kinase-
deficient Pak1 mutants inhibit Ras transformation of Rat-1 fibroblasts. Mol. Cell Biol.
1997, 17, ꢀ 4454–4464.
(11) Ong, C.C.; Jubb, A.M.; Haverty, P.M.; Zhou, W.; Tran, V.; Truong, T. et al.
Targeting p21-activated kinase 1 (PAK1) to induce apoptosis of tumor cells. Proc Natl
Acad Sci.ꢀ 2011, 108, 7177–7182.
(12) Nheu, T.V.; He, H.; Hirokawa, Y.; Tamaki, K.; Florin, L.; Schmitz, M.L.; Suzuki-
Takahashi, I.; Jorissen, R.N.; Burgess, A.W.; Nishimura, S.; Wood, J.; Maruta, H. The
K252a derivatives, inhibitors for the PAK/MLK kinase family selectively block the growth
of RAS transformants. Cancer J. 2002, 8, 328–336.
7

(13) Porchia, L.M.; Guerra, M.; Wang, Y.C.; Zhang, Y.; Espinosa, A.V.; Shinohara, M.;
Kulp, S.K.; Kirschner, L.S.; Saji, M.; Chen, C.S.; Ringel, M.D. 2-amino-N-{4-[5-(2-
phenanthrenyl)-3- (trifluoromethyl)-1H-pyrazol-1-yl]-phenyl} acetamide (OSU-03012), a
celecoxib derivative, directly targets p21-activated kinase. Mol. Pharmacol. 2007, 72,
1124–1131.
(14) Eswaran, J.; Lee, W.H.; Debreczeni, J.E.; Filippakopoulos, P.; Turnbull, A.;
Fedorov, O.; Deacon, S.W.; Peterson, J.R.; Knapp, S. Crystal Structures of the p21-
activated kinases PAK4, PAK5, and PAK6 reveal catalytic domain plasticity of active
group II PAKs. Structure. 2007, 15, 201–213.
(15) Chow, H.Y.; Dong, B.; Duron, S.G.; Campbell, D.A.; Ong, C.C.; Hoeflich, K..P et al.
Group I Paks as therapeutic targets in NF2-deficient meningioma. Oncotarget. 2014, 6,
1981–1994.
(16) Prudnikova, T.Y.; Villamar-Cruz, O.; Rawat, S.J.; Cai, K.Q.; Chernoff, J. Effects of
p21-activated kinase 1 inhibition on 11q13-amplified ovarian cancer cells. Oncogene.
2015, 1-8.
(17) Campbell, D.; Duron, S.G.; Pak Inhibitors for the Treatment of Cell Proliferative
Disorders. International Patent WO 2013/067423 Al, May 10, 2013.
(18) Buckman, B. O.; Nicholas, J.B. et al. Lysophosphatidic Acid Receptor Antagonists.
International Patent WO 2013/025733, February 21, 2013.
8

(19) Brown, W.; Johnstone, S.; Labrecque, D. Benzimidazole Derivatives as Vanilloid
Receptor Antagonists, their Preparation, Pharmaceutical Compositions, and use in
Therapy. International Patent WO 2008018827 A1, February 14, 2008.
(20) Wurtz, N.R.; Priestley, E.S. et al. Preparation of Macrocyclic Amino Acid
Derivatives as Factor VIIa Inhibitors Useful as Anticoagulants. International Patent
WO 2008079836 A2, July 3, 2008.
(21) Bayne, C.D.; Johnson, A.T. et al. Preparation of (Hetero)aryl Substituted Pyridones
as Modulators of LXR. US Patent US 20050080111 A1, July 24, 2004.
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