Selectivity switch between FAK and Pyk2: Macrocyclization of FAK inhibitors improves Pyk2 potency

Article abstract of DOI:10.1016/j.bmcl.2016.10.092

Herein, we describe the synthesis of Pyk2 inhibitors via macrocyclization of FAK and dual Pyk2-FAK inhibitors. We identified macrocycle 25a as a highly potent Pyk2 inhibitor (IC₅₀= 0.7 nM), with ～175-fold improvement in Pyk2 potency as compared to its acyclic counterpart. In many cases, macrocyclization improved Pyk2 potency while weakening FAK potency, thereby improving the Pyk2/FAK selectivity ratio for this structural class of inhibitors. Various macrocyclic linkers were studied in an attempt to optimize Pyk2 selectivity. We observed macrocyclic atropisomerism during the synthesis of 19-membered macrocycles 10a–d, and successfully obtained crystallographic evidence of one atropisomer (10a-AtropB) preferentially bound to Pyk2.

Full text of DOI:10.1016/j.bmcl.2016.10.092

Accepted Manuscript
Selectivity switch between FAK and Pyk2: Macrocyclization of FAK inhibitors
improves Pyk2 potency
Julie Farand, Nicholas Mai, Jayaraman Chandrasekhar, Zachary E. Newby, Josh
Van Veldhuizen, Jennifer Loyer-Drew, Chandrasekar Venkataramani, Juan
Guerrero, Amy Kwok, Ning Li, Yelena Zherebina, Sibylle Wilbert, Jeff
Zablocki, Gary Phillips, William J. Watkins, Robert Mourey, Gregory Notte
PII:
S0960-894X(16)31140-4
DOI:
http://dx.doi.org/10.1016/j.bmcl.2016.10.092
BMCL 24395
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
30 September 2016
30 October 2016
31 October 2016
Please cite this article as: Farand, J., Mai, N., Chandrasekhar, J., Newby, Z.E., Van Veldhuizen, J., Loyer-Drew, J.,
Venkataramani, C., Guerrero, J., Kwok, A., Li, N., Zherebina, Y., Wilbert, S., Zablocki, J., Phillips, G., Watkins,
W.J., Mourey, R., Notte, G., Selectivity switch between FAK and Pyk2: Macrocyclization of FAK inhibitors
improves Pyk2 potency, Bioorganic & Medicinal Chemistry Letters (2016), doi: http://dx.doi.org/10.1016/j.bmcl.
2016.10.092
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Bioorganic & Medicinal Chemistry Letters
Selectivity switch between FAK and Pyk2: macrocyclization of FAK inhibitors
improves Pyk2 potency
Julie Farand a,*, Nicholas Mai a, Jayaraman Chandrasekhar e, Zachary E. Newby b, Josh Van Veldhuizen d,
Jennifer Loyer-Drew d, Chandrasekar Venkataramani a, Juan Guerrero a, Amy Kwok c, Ning Li c, Yelena
Zherebina c, Sibylle Wilbert f, Jeff Zablocki a, Gary Phillips d, William J. Watkins a, Robert Mourey g,
Gregory Notte a
aDepartment of Medicinal Chemistry, bDepartment of Structural Chemistry and cDepartment of Biology, Gilead Sciences, Inc., 333 Lakeside Drive, Foster City,
CA 94404, USA
dDepartment of Medicinal Chemistry, eDepartment of Structural Chemistry, fDepartment of Drug Metabolism and gDepartment of Biology, Gilead Sciences, Inc.,
199 East Blaine Street, Seattle, WA 98102, USA
ARTICLE INFO
ABSTRACT
Herein, we describe the synthesis of Pyk2 inhibitors via macrocyclization of FAK and dual
Article history:
Received
Pyk2-FAK inhibitors. We identified macrocycle 25a as a highly potent Pyk2 inhibitor (IC50
=
0.7 nM), with ~175-fold improvement in Pyk2 potency as compared to its acyclic counterpart.
In many cases, macrocyclization improved Pyk2 potency while weakening FAK potency,
thereby improving the Pyk2/FAK selectivity ratio for this structural class of inhibitors. Various
macrocyclic linkers were studied in an attempt to optimize Pyk2 selectivity. We observed
macrocyclic atropisomerism during the synthesis of 19-membered macrocycles 10a-d, and
successfully obtained crystallographic evidence of one atropisomer (10a-AtropB) preferentially
bound to Pyk2.
Revised
Accepted
Available online
Keywords:
Pyk2
FAK
Macrocycle
Atropisomer
Glioblastoma
2009 Elsevier Ltd. All rights reserved.
Proline-rich tyrosine kinase 2 (Pyk2) is a non-receptor
cytoplasmic tyrosine kinase within the focal adhesion kinase
(FAK) subfamily. Both Pyk2 and FAK are involved in multiple
signaling pathways that regulate cell migration, proliferation and
survival.1,2 Over the last decade, genetic knockdown of Pyk2
together with inhibition by small molecules have demonstrated
the potential role of Pyk2 in the treatment of osteoporosis.3,4
Recently, the biological significance of the focal adhesion
kinases in promoting the migration and invasion of malignant
cells has been of increasing interest.5,6,7 Glioblastoma multiforme
is the most common and most aggressive malignant primary
brain tumor due to the propensity of peripheral glioma cells to
migrate and disperse among neighboring normal brain
cells. Combinations of surgical resection, radiotherapy and/or
chemotherapy with temozolomide are not curative treatments.
Over 90% of the estimated new glioblastoma cases in 2015 were
found in adults ≥40 years old. The 5-year relative survival rate
for patients diagnosed with glioblastoma is 5.1%, a statistic
which has shown no significant improvement over the past 30
years, in spite of recent therapeutic advancements.8 Given this
unmet medical need, we sought to selectively target Pyk2 and
evaluate its role in the migration and invasion of malignant
glioblastoma cells.
Unlike FAK, which is ubiquitously
expressed, Pyk2 expression is highly localized in the brain,
hematopoietic and nervous systems.9,10 Radial migration assays
using SF767 glioblastoma cell lines have shown upregulation of
Figure 1. Representative structures of reported diaminopyrimidine Pyk2/FAK inhibitors.
________
* Corresponding author. Tel.: +1-650-372-7668; e-mail: Julie.Farand@gilead.com

Pyk2 in cells at the migratory rim relative to those at the primary
core.11 A direct correlation between the rate of migration and
levels of activated Pyk2 was also observed. Conversely,
silencing Pyk2 expression with siRNA significantly reduced the
migratory potential of glioblastoma cells.12 A relationship has
(Scheme 1).21 The resulting aminopyridine was treated with
mesyl chloride and the nitrile was subsequently reduced to afford
amine 3a. Chloroaminopyrimidine intermediate 7 was heated
with 3a under basic conditions to generate diaminopyrimidine 8a
(Scheme 2).15,22 Following N-Boc deprotection and tert-butyl
ester hydrolysis, crude amino acid 9a was subjected to a HATU-
mediated macrolactamization under dilute conditions to provide
10a in 56% yield over two steps.23 In contrast to PF-562271, 10a
also
been
demonstrated
between
increasing
Pyk2
expression/phosphorylation events and advancing tumor WHO
grade, thus indicating Pyk2 as
glioblastoma progression.13
a potential mediator of
exhibited moderate selectivity for Pyk2 over FAK (Pyk2 IC50
=
3.1 nM, FAK IC50 = 17 nM, Table 1) demonstrating the validity
of the macrocyclization strategy. The excellent cellular
permeability of 10a was also notable (Caco-2: A to B = 30 x 10-6
cm/s and B to A = 32 x 10-6 cm/s) and displayed a moderate
improvement over acyclic inhibitor PF-562271 in the absorptive
direction (Caco-2: A to B = 11 x 10-6 cm/s and B to A = 29 x 10-6
cm/s).
The catalytic domains of Pyk2 and FAK kinases have 73%
sequence similarity. The similarity is slightly higher (78%) for
residues generally thought to form the ATP binding sites of these
two enzymes.14 Consequently, the discovery of a small molecule
inhibitor selective for Pyk2 has been challenging. We initiated
efforts toward that goal for proof-of-concept studies and focused
our attention on diaminopyrimidine-based inhibitors PF-
4313963,15, PF-56227116 and compound 117 (Figure 1). PF-
562271 was described as a potent, ATP-competitive and
reversible FAK inhibitor (IC50 = 1.5 nM) with a ~9-fold
selectivity over Pyk2 (IC50 = 13 nM). To better understand the
interaction of PF-562271 with Pyk2, we solved a crystal
structure of the complex. A comparison in the binding pose of
PF-562271 complexed with FAK18 versus Pyk219 revealed a
significant conformational difference in the amino-methyl
pyridinyl sulfonamide group (Figures 2a and 2b). The rotamer of
the benzylic CH2-NH bond of PF-562271 observed in the Pyk2-
bound structure induced a conformation of the nearby DFG loop
in Pyk2 (DFG-in) distinct from that observed in FAK (helical
DFG). We hypothesized that a macrocyclic analogue of PF-
562271 favoring the Pyk2-bound conformation shown in Figure
2b would enhance selectivity of the inhibitor over FAK.20
A)
Scheme 1. Reagents and conditions for N-pyridinylmethanesulfonamide and
N-phenylsulfonamides: (a) tBuO2C(CH2)4NH2, EtOH, 85°C (54%); (b)
LiHMDS, MsCl, THF, -78°C (64%): (c) H2, Raney Ni, 50 bar, EtOH, 60°C
(40%); (d) Br(CH2)4CO2tBu, K2CO3, DMF, 125°C (54-82%)24; (e) H2, Raney
Ni, 70 bar, EtOH, 50°C (50%); (f) Boc2O, NaHCO3, THF/H2O (95%); (g)
MeSO2NH2 or EtSO2NH2, CuI, N-methylglycine, K3PO4, DMF, 100°C (40%);
(h) NaH, MeI, THF (85%); (i) 4N HCl in 1,4-dioxane (95%).
Hinge
B)
DFG motif
Figure 2. (A) Co-crystal structure of PF-562271 (pink) bound to FAK (PDB
entry 3BZ3)18 showing the methionine gatekeeper (cyan), the hydrogen
bonding interactions between the aminopyrimidine and the hinge backbone
(orange), as well as the amino-methyl pyridinyl sulfonamide group projecting
toward the DFG motif (residues Asp-Phe-Gly in lavender). (B) Co-crystal
structure of PF-562271 (yellow) bound to Pyk2 (PDB entry 5TOB, resolution
2.2 Å) depicting the amino-methyl pyridinyl sulfonamide group adopting a
unique conformation next to the DFG motif. The macrocyclization strategy is
also depicted (black dotted line).
Macrocycle 10a was synthesized as described in Schemes 1 and
Scheme 2. Reagents and conditions: (a) iPr2NH, PhMe/IPA, 90°C; (b) TFA,
DCM; (c) HATU, 4-methylmorpholine, DMF (0.005M) (10-30% over 3
steps).
2.
Under SNAr conditions, 2-chloronicotinonitrile 2a was
heated with tert-butyl 5-aminopentanoate in refluxing ethanol

Interestingly, a small molecule crystal structure of 10a
revealed the presence of atropisomers 10a-AtropA and 10a-
AtropB in the solid state (Figure 3a).25 For 10a-AtropA, the
linker alkyl chain appeared disordered but was successfully
modeled as 80/20 occupancy. Co-crystallization of macrocycle
10a with Pyk2 confirmed preservation of the binding mode,
inhibitor conformation, protein-inhibitor interactions and
revealed the kinase adopting a DFG-in conformation (Figure
3b).26 An overlay of the Pyk2-bound conformation of 10a with
the solid state conformation of 10a-AtropB indicated that the
latter was the relevant atropisomer for inhibition (Figure 3c).
Attempts to separate the atropisomers in chiral HPLC and SFC
screens were unsuccessful, suggesting that rapid interconversion
was occurring on the HPLC timescale.27,28 On the other hand,
chiral HPLC analysis of the N-phenylmethanesulfonamide
macrocycle 10b (Pyk2 IC50 = 0.9 nM) suggested a 1:1 ratio of
atropisomers, however baseline resolution of the split peak could
not be achieved, presumably due to on-column interconversion.29
suggested the atropisomers may be separable on preparative
scale.27,31 Unfortunately, however, peak resolution for 10c could
not be achieved due to on-column interconversion. Nonetheless,
the rapidly interconverting class 1 atropisomers32 constituting
both 10c and 10d showed >100-fold improvement in Pyk2 IC50
(1.5 nM and 1.9 nM respectively) when compared to their
corresponding acyclic diaminopyrimidines 9c and 9d, together
with 5-fold selectivity over the FAK enzyme (Table 1).
Table 1. Pyk2 and FAK IC50 values for diaminopyrimidine
macrocycles and acyclic precursors.
A)
10a-AtropA
10a-AtropB
Pyk2
IC50 (nM)
FAK
IC50 (nM)
Compound
R1
R2
X
*
*
H
Me
Me
Et
N
3.1
0.9
256
1.9
201
1.5
17
*
10a
10b
9c
*
H
H
CH
CH
CH
CH
CH
3.2
3.3
9.2
177
8.6
H
Et
10c
9d
Me
Me
Me
Me
B)
C)
10d
The effect of various macrocycle linkers on Pyk2 and FAK
biochemical potencies was also investigated (Figure 4).
Shortening the 5-carbon linker of 10a to four carbons yielded 11
as an 18-membered macrocyclic dual Pyk2/FAK inhibitor with a
10-fold loss in Pyk2 potency (IC50 = 30 nM). Replacing the
tertiary methyl sulfonamide moiety in 10b with a secondary
sulfonamide within the linker afforded 19-membered macrocycle
12, an equipotent Pyk2 inhibitor (IC50 = 3.5 nM) with a slight
erosion in FAK selectivity compared to 10b.33 Rigidifying
macrocycle 13 with a phenyl-containing linker did not improve
selectivity.
Figure 3. (A) Small molecule X-ray structure (0.75 Å) of macrocycle 10a as
a mixture of atropisomers 10a-AtropA (green) and 10a-AtropB (orange).
Atoms disordered in the crystal are labeled with an asterisk (*). (B) Co-
crystal structure overlay of macrocycle 10a (cyan, PDB entry 5TO8,
resolution 2.0 Å) and PF-562271 (yellow) bound to Pyk2. (C) Overlay of
macrocycle 10a (cyan) bound to Pyk2 and small molecule X-ray structure of
10a-AtropB (orange).
To increase the energy barrier for atropisomer
interconversion, we focused our attention on N-
phenylsulfonamides, prepared the bulkier ethyl sulfonamide 10c,
and also explored the effect of alkylation of the secondary
benzylic amine in the form of the tertiary analogue 10d. 1-
Iodobenzylamine hydrochloride 4 proved useful as a starting
material for both compounds, with copper mediated N-arylation
of the sulfonamide30 and selective alkylation of the sulfonamide
over the carbamate under basic conditions as key steps (Scheme
1). Chiral HPLC analysis of 10c and 10d were qualitatively
similar and showed two peaks separated by a plateau, which
Figure 4. Effect of various linkers on Pyk2 and FAK potencies.

Scheme 3. Reagents and conditions: (a) Boc2O, Et3N, CH2Cl2 (90%); (b) (Allyl)NHSO2Me, CuI, DMEDA, K2CO3, ACN, 70°C (74%)34; (c) TFA/DCM (58%-
quantitative); (d) NaH, MeI, THF (98%); (e) Boc2O, NaHCO3, THF/H2O (91%); (f) MeSO2NH2, CuI, 1,3-di(2-pyridyl)-1,3-propanedione, K2CO3, DMF, 120°C
(89%); (g) AllylBr, K2CO3, ACN, 70°C (66%); (h) 22a or 22b, iPr2NH, PhMe/IPA, 90°C (38-75%).15
Table 2. Intrinsic potencies for acyclic and 15-membered macrocyclic diaminopyrimidines.
Pyk2
IC50 (nM)
FAK
IC50 (nM)
Compound
R1
R2
X
Y
H
H
H
H
H
H
Me
Me
Me
H
H
H
N
N
N
N
N
N
N
CH
CH
CH
CH
CH
CH
CH
CH
CH
N
122
0.51
10.2
1.26
0.51
4.34
3.21
5.75
7496
14.0
23a
24a
25a
23b
24b
25b
24c
23d
24d
2.60
0.67
19.5
0.84
1.31
1.55
6625
2.70
Morpholine
Morpholine
Morpholine
Morpholine
Morpholine
Morpholine
N
(a) For 24a-c: Pd(OAc)2, (tBu)3P, Et3N, ACN, 80°C (14-31%); and for 24d: Pd2(dba)3, (tBu)3P, Cy2NMe, 1,4-dioxane, 90°C (10%); (b) H2, Pd/C,
EtOH/EtOAc (50-67%).
N-Phenylsulfonamide macrocycles 10b-d suffered from
poor solubility (<1.6 µM at pH 7), and short half-lives were
observed for 10a-d in mouse, dog and/or human liver
microsomes (t1/2 < 60 min). For the next generation of
macrocyclic Pyk2 inhibitors, we employed an intramolecular
Heck reaction for the ring closure step. Macrocycles with
morpholine as R2 and/or 3-pyridyl regioisomers were prepared as
a tactic to enhance solubility (Scheme 3). Intermediate 20, used
as a precursor for the synthesis of macrocycle 23d, was prepared
via a copper-catalyzed N-arylation of methanesulfonamide.35
Heck-mediated macrocyclizations of 23a-d under dilute
conditions provided inhibitors 24a-d bearing 3-carbon linkers,
thus decreasing the number of sites for potential metabolism
(Table 2). In some cases, the alkene was reduced with Pd/C
under an atmosphere of hydrogen to yield macrocycles 25a and
25b. Assessment of the activities of the acyclic precursors 23a-b
relative to the macrocycles 24a-b proved further evidence of the
potency switch toward Pyk2 and selectivity improvement in the
latter. Strikingly, the acyclic tertiary amine 23d was markedly
less active against both enzymes but macrocyclization to 24d
provided for ~2,500-fold and 500-fold increases in potency
against Pyk2 and FAK respectively.
Saturation of the
macrocyclic linker in 25a led to a modest improvement in Pyk2
potency, albeit with a relative loss in selectivity. Incremental
improvements in kinetic solubility at physiological pH were
noticed between 24a, 24c and 24d (0.3, 1 and 3 µM
respectively). Macrocycle 24d, bearing an unsaturated 3-carbon
linker, exemplified the most metabolically stable analogue tested
in the human microsomal stability assay (t1/2 = 263 min).

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SAR trends apparent in Table 2. Acyclic precursors 23a-b are
25-50 fold less active than their macrocyclic counterparts 24a-b
against Pyk2. The global minima for 23a-b are conformations in
which the –NHCH2- linker between the pyrimidine and pyridine
rings is staggered, and the calculated energy penalty to adopt the
gauche local minima evident in the bioactive conformation (cf
Figure 3c) is ca. 2.3 kcal/mol.36 In the still more dramatic case of
the tertiary amines, the 2,500-fold Pyk2 potency differential is
largely explained by the same phenomenon coupled with the
need for the N-Me substituent to lie in the same plane and
pointing towards the adjacent o-CF3 substituent – a conformation
that lies 4.3 kcal/mol above the global minimum for the acyclic
analogue 23d. Conformational analysis also confirms that
second generation macrocycles 24a, 24d and 25a can adopt a low
energy conformation (either the global minimum or within 0.5
kcal/mol of it) that overlays closely with the small molecule
crystal structure of 10a-AtropB (Figure 4). The detrimental
conformational effect of N-methylation is effectively overcome
through macrocyclization of 23d to 24d.
5.
6.
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8.
9.
10. Avraham, S.; London, R.; Fu, Y.; Ota, S.; Hiregowdara, D.; Li, J.;
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12. Lipinski, C. A.; Tran, N. L.; Menashi, E.; Rohl, C.; Kloss, J.; Bay,
R. C.; Berens, M. E.; Loftus, J. C. Neoplasia 2005, 7, 435.
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Neuropathol. 2004, 108, 224.
14. Xing, L.; Rai, B.; Lunney, E. A. J. Comput. Aided Mol. Des. 2014,
28, 13.
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Zhao, S. X.; Soglia, J. R.; Aspnes, G. E.; Kung, D. W.; Klug-
McLeod, J.; Zawistoski, M. P.; McGlynn, M. A.; Oliver, R.;
Dunn, M.; Li, J.-C.; Richter, D. T.; Cooper, B. A.; Kath, J. C.;
Hulford, C. A.; Autry, C. L.; Luzzio, M. J.; Ung, E. J.; Roberts,
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Han, S.; Guzman-Perez, A. Bioorg. Med. Chem. Lett. 2008, 18,
6071; (b) Han, S.; Mistry, A.; Chang, J. S.; Cunningham, D.;
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G. E.; Walker, D. P.; Brosius, A. D.; Buckbinder, L. J. Biol.
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16. Roberts, W. G.; Ung, E.; Whalen, P.; Cooper, B.; Hulford, C.;
Autry, C.; Richter, D.; Emerson, E.; Lin, J.; Kath, J.; Coleman, K.;
Yao, L.; Martinez-Alsina, L.; Lorenzen, M.; Berliner, M.; Luzzio,
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C)
D)
Figure 4. Calculated low energy conformations of second generation
macrocycles (A) with secondary benzylic amine 24a (cyan), (B) tertiary
benzylic amine 24d (yellow) and (C) highly potent Pyk2 inhibitor 25a
bearing
a
saturated linker (green).
(D) Overlay of the minimized
conformations of 24a, 24d and 25a with the small molecule crystal structure
10a-AtropB (orange).
17. Walker, D. P.; Zawistoski, M. P.; McGlynn, M. A.; Li, J.-C.;
Kung, D. W.; Bonnette, P. C.; Baumann, A.; Buckbinder, L.;
Houser, J. A.; Boer, J.; Mistry, A.; Han, S.; Xing, L.; Guzman-
Perez. Bioorg. Med. Chem. Lett. 2009, 19, 3253.
In summary, the use of a macrocyclization strategy provided
an enhancement in Pyk2 vs FAK selectivity, despite the high
degree of sequence similarity in their active sites. Macrocyclic
atropisomerism was observed with 10a, where the solid state
conformation of 10a-AtropB was found to be remarkably similar
to the Pyk2 bound conformation. The excellent in vitro potencies
of macrocycles 10, 12, 24 and 25 (IC50 = 0.7 - 3.1 nM), combined
with their Pyk2/FAK selectivity (2- to 3-fold), render them
suitable tool compounds for future proof-of-concept studies.
Improving solubility, metabolic stability and ensuring blood
brain barrier permeability will be necessary to enable their utility
in glioblastoma xenograft models.
18. PDB entry 3BZ3 (2.2 Å): Vajdos, F.; Marr, E.
19. Crystallographic data of PF-562271 bound to Pyk2 has been
deposited to the Protein Data Bank (PDB entry 5TOB, resolution
2.2 Å).
20. Conformational control of small molecule inhibitors via
macrocyclization is a well established strategy. For examples, see
Johnson, T. W.; Richardson, P. F.; Bailey, S.; Brooun, A.; Burke,
J. B.; Collins, M. R.; Cui, J. J.; Deal, J. G.; Deng, Y.-L.; Dinh, D.;
Engstrom, L. D.; He, M.; Hoffman, J.; Hoffman, R. L.; Huang, Q.;
Kania, R. S.; Kath, J. C.; Lam, H.; Lam, J. L.; Le, P. T.; Lingardo,
L.; Liu, W.; McTigue, M.; Palmer, C. L.; Sach, N. W.; Smeal, T.;
Smith, G. L.; Stewart, A. E.; Timofeevski, S.; Zhu, H.; Zhu, J.;
Zou, H. Y.; Edwards, M. P. J. Med. Chem. 2014, 57, 4720 and
references 33 therein.
Acknowledgments
The authors wish to thank Professor Arnold L. Rheingold for
providing small molecule X-ray crystallography data, and we
appreciate insightful discussions with Richard Neve during the
review of this manuscript.
21. Tert-butyl 5-aminopentanoate was prepared and used in situ via
reductive deprotection of readily available tert-butyl 5-
(((benzyloxy)carbonyl)amino)pentanoate.
22. For the synthesis of chloroaminopyrimidines and the
regioselective addition of anilines to the 2-position of 2,4-
dichloro-5-trifluoromethylpyrimidine, see Kath, J. C.; Richter, D.
T.; Luzzio, M. J. US Patent 7,122,670, October 16, 2006.
23. 1H NMR (400 MHz, Acetonitrile-d3) δ 11.08 (s, 1H), 8.44 (dd, J =
4.8, 1.8 Hz, 1H), 8.29 (s, 1H), 7.58 – 7.45 (m, 2H), 7.37 (dd, J =
7.8, 4.7 Hz, 1H), 7.05 – 6.86 (m, 3H), 4.79 (s, 1H), 4.68 (dd, J =
16.9, 5.0 Hz, 1H), 3.99 (t, J = 8.2 Hz, 2H), 3.84 – 3.73 (m, 1H),
3.17 – 3.01 (m, 4H), 2.97 – 2.81 (m, 1H), 2.82 – 2.66 (m, 2H),
References and Notes
1.
Schlaepfer, D. D.; Hauck, C. R.; Sieg, D. J. Prog. Biophys. Mol.
Bio. 1999, 71, 435.
2.
Parsons, J. T. J. Cell Sci. 2003, 116, 1409.

2.58 – 2.47 (m, 1H), 1.63 – 1.32 (m, 4H). LC-MS (ESI+) m/z:
calcd C25H26F3N7O3S (M+H) 562.2, found 562.2 (M+H).
24. For the synthesis of tert-butyl 5-bromopentanoate, see Riva, R.;
Banfi, L.; Basso, A.; Zito, P. Org. Biomol. Chem. 2011, 9, 2107.
25. CCDC 1505469 contains the supplementary small molecule
crystallographic data. The data can be obtained free of charge
from The Cambridge Crystallographic Data Centre via
www.ccdc.cam.ac.uk/structures.
26. Crystallographic data of macrocycle 10a bound to Pyk2 has been
deposited to the Protein Data Bank (PDB entry 5TO8, resolution
2.0 Å).
27. Welch, C. J.; Gong, X.; Schafer, W.; Chobanian, H.; Lin, L.; Biba,
M.; Liu, P.; Guo, Y.; Beard, A. Chirality 2009, 21, E105.
28. Elleraas, J.; Ewanicki, J.; Johnson, T. W.; Sach, N. W.; Collins,
M. R.; Richardson, P. A. Angew. Chem. Int. Ed. 2016, 55, 3590.
29. Atropisomers of macrocycle 10b were detected using a Chiralpak
IC analytical chiral column (250 mm x 4.6 mm, particle size 5.0
µm) with 1:1 MeOH/EtOH.
30. Deng, W.; Liu, L.; Zhang, C.; Liu, M.; Guo, Q.-X. Tetrahedron
Lett. 2005, 46, 7295.
31. Atropisomer peaks separated by a plateau were detected with
macrocycles 10c and 10d using a Chiralcel AZ-H analytical
column (250 mm x 4.6 mm, particle size 5.0 µm) with 1:1
MeOH/EtOH.
32. LaPlante, S. R.; Fader, L. D.; Fandrick, K. R.; Fandrick, D. R.;
Hucke, O.; Kemper, R.; Miller, S. P. F.; Edwards, P. J. J. Med.
Chem. 2011, 54, 7005.
33. Macrocycle 12 was prepared in
a similar fashion to 10b.
Reaction between 2-cyanobenzenesulfonyl chloride and tert-butyl
γ-aminobutyrate hydrochloride was performed according to Biron,
E.; Kessler, H. J. Org. Chem. 2005, 70, 5183. The nitrile was
subsequently reduced using H2, Raney Ni and EtOH at 70°C (70
bar).
34. Wang, X.; Guram, A.; Ronk, M.; Milne, J. E.; Tedrow, J. S.; Faul,
M. M. Tetrahedron Lett. 2012, 53, 7.
35. Han, X. Tetrahedron Lett. 2010, 51, 360.
36. Maestro, V10.5; Schrödinger, Inc.: Portland, OR, 2014.

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Macrocyclization of FAK inhibitors improves Pyk2
potency
Julie Farand, Nicholas Mai, Jayaraman Chandrasekhar, Zachary E. Newby, Josh Van Veldhuizen, Jennifer
Loyer-Drew, Chandrasekar Venkataramani, Juan Guerrero, Amy Kwok, Ning Li, Yelena Zherebina, Sibylle
Wilbert, Jeff Zablocki, Gary Phillips, William J. Watkins, Robert Mourey, Gregory Notte