2,3,5-Trisubstituted pyridines as selective AKT inhibitors-Part I: Substitution at 2-position of the core pyridine for ROCK1 selectivity

Article abstract of DOI:10.1016/j.bmcl.2009.11.064

2,3,5-Trisubstituted pyridines have been designed as potent AKT inhibitors that are selective against ROCK1 based on the comparison between AKT and ROCK1 structures. Substitution at the 2-position of the core pyridine is the key element to provide selectivity against ROCK1. An X-ray co-crystal structure of 9p in PKA supports the proposed rationale of ROCK1 selectivity.

Full text of DOI:10.1016/j.bmcl.2009.11.064

Bioorganic & Medicinal Chemistry Letters 20 (2010) 673–678
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
2,3,5-Trisubstituted pyridines as selective AKT inhibitors—Part I: Substitution
at 2-position of the core pyridine for ROCK1 selectivity
a
a
a
a
a
Hong Lin a, , Dennis S. Yamashita , Jin Zeng , Ren Xie , Wenyong Wang , Sirishkumar Nidarmarthy ,
Juan I. Luengo a, Nelson Rhodes b, Victoria B. Knick b, Anthony E. Choudhry b, Zhihong Lai b,
Elisabeth A. Minthorn c, Susan L. Strum d, Edgar R. Wood d, Patricia A. Elkins d, Nestor O. Concha d,
Dirk A. Heerding a
*
a Oncology Medicinal Chemistry, GlaxoSmithKline, 1250 S. Collegeville, Rd., Collegeville, PA 19426, United States
b Oncology Biology, GlaxoSmithKline, 1250 S. Collegeville, Rd., Collegeville, PA 19426, United States
c Oncology Drug Metabolism and Pharmacokinetics, GlaxoSmithKline, 1250 S. Collegeville, Rd., Collegeville, PA 19426, United States
d Molecular Discovery Research, GlaxoSmithKline, 1250 S. Collegeville, Rd., Collegeville, PA 19426, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
2,3,5-Trisubstituted pyridines have been designed as potent AKT inhibitors that are selective against
ROCK1 based on the comparison between AKT and ROCK1 structures. Substitution at the 2-position of
the core pyridine is the key element to provide selectivity against ROCK1. An X-ray co-crystal structure
of 9p in PKA supports the proposed rationale of ROCK1 selectivity.
Received 25 September 2009
Revised 11 November 2009
Accepted 16 November 2009
Available online 20 November 2009
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
AKT kinase inhibitors
Selectivity
Activation of oncogenes, such as Ras, ErbB-2, and Src or loss of
tumor suppressor genes, such as PTEN, can lead to aberrant signal-
ing in the PI3K/AKT signal transduction pathway.1 There are three
to AGC superfamily of Ser/Thr protein kinases, and have Met as
the gate keeper residue (Met229 and Met153, respectively). There
are different amino acids, Leu 204 for AKT and Met 128 for ROCK1,
at the back cleft of the ATP binding pocket, an area in close proxim-
ity with the gate keeper. This subtle difference suggested that the
gate keeper Met of ROCK1 might favor the adoption of a less flex-
ible conformation than that observed for AKT due to a tighter pack-
ing in the ATP back-pocket of ROCK1. Since PKA is a common
surrogate of AKT for structural studies,10 we compared crystal
structures of ROCK and PKA. Similar to that of AKT, the gate keeper
(Met120) of PKA should be more flexible than that of ROCK1
(Met153) by virtue of a smaller Leu95 residue being in close prox-
imity with Met120 in PKA (see Fig. 2). Therefore, it was reasoned
that substitution at the 2-position of the pyridine core might be
tolerated in AKT or PKA but not in ROCK1.
We believed that we could design ROCK1 selective AKT inhibi-
tors by incorporating substitution at C-2 of the core pyridine.
Therefore, we selected the 3,5-disubstituted pyridine series to test
this hypothesis as a means of introducing selectivity against
ROCK1 by rational design. Herein, we report the results of those
efforts.
The synthesis of the trisubstituted pyridines is outlined in
Scheme 1. A Mitsunobu reaction of amino alcohol 3 (either com-
mercially available or prepared by BH3 reduction of the
corresponding carboxylic acid) and 3-bromo-2-chloro-5-hydroxy-
pyridine (4)11 afforded pyridine 5. Boronate ester 6, prepared from
isoforms of AKT kinases, known as AKT1 (PKBa), AKT2 (PKBb) and
AKT3 (PKB ). All three are up-regulated in different types of can-
c
cers including NSCLC (non-small cell lung carcinoma), breast and
prostate cancers, making them potential oncology targets. Several
small molecule AKT inhibitors have recently been reported2–5
including the phospholipid perifosine, dual AKT1/2-allosteric
diphenylquinolines, and ATP competitive inhibitors, such as
GSK690693 (1) (see Fig. 1).6,7
Kinase selectivity is important because long term inhibition of
off-target kinases may cause undesired side effects and toxicity.
Following up on our work on the development of GSK690963 (1),
we overlayed 2a, an ATP-competitive AKT inhibitor that belongs
to a novel series of 3,5-disubstituted pyridine analogs having po-
tent in vitro and in vivo activities,8 with compound 1 in an AKT2
homology model. This modeling suggested that a C-2 substitution
of the core pyridine of 2a could occupy the space where the 3-hy-
droxy-3-methyl-butynyl substituent resided in compound 1. The
3-hydroxy-3-methyl-butynyl substituent was observed to function
as an important selectivity element to reduce ROCK1 (Rho kinase)9
inhibitory activity of this chemical class.7 AKT and ROCK1 belong
* Corresponding author.
E-mail address: hong.2.lin@gsk.com (H. Lin).
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.11.064

674
H. Lin et al. / Bioorg. Med. Chem. Lett. 20 (2010) 673–678
OH
for kinase selectivity
H
N
H
2
N
N
R'
H2N
N
N
N
N
N
N
O
N
N
H
O
O
O
R
NH2
R
NH2
NH
R = phenyl, A-674563 (2a)
R = Indole-3-yl, A-443654 (2b)
R' = aryl, heteroaryl
GSK690693 (1)
Figure 1. Lead structures.
Figure 2. Comparison of the gate keeper Met153 in ROCK1 and Met120 in PKA, a common structural surrogate for AKT.
Boc
Cl
O
N
N
Br
Cl
O
N
B
Br
O
N
a
OH
R
6
+
NHBOC
b
OH
R
NHBoc
3
5
4
Boc
Boc
N
N
H
N
N
N
Cl
N
c
d
N
N
N
O
O
O
R
R
R
NH2
NHBOC
NHBoc
7
8
9
Scheme 1. Synthesis of 2,3,5-trisubstituted pyridine analogs. Reagents and conditions:13 (a) Ph3P, DEAD, THF, 1 h (87%); (b) Pd(PPh3)4, 2 M Na2CO3, 1,4-dioxane, 100 °C,
overnight (80%); (c) phenylboronic acid, Pd(PPh3)4, 2 M Na2CO3, 1,4-dioxane, microwave irradiation, 160 °C, 20 min (57%); (d) TFA, DCM, 30 min (78%).
its corresponding bromoindazole,12 was coupled with 5 in a Suzuki
coupling reaction to provide 7. A second Suzuki coupling to install
the phenyl ring at the 2-position of the pyridine core was carried
out under microwave irradiation conditions, furnishing 8 in good
yield. The final de-protection of the BOC group afforded the desired
tri-substituted pyridine derivative 9.

H. Lin et al. / Bioorg. Med. Chem. Lett. 20 (2010) 673–678
675
A variety of analogs with a range of C-2 substituents were pre-
Table 1 (continued)
pared by this route. A Stille or Negishi coupling reaction was also
used if the corresponding organo-tin or organo-zinc reagents were
available. In the cases of 9n and 9o, a Sonogashira reaction was ap-
plied as previously described.7 The representative analogs are
listed in Table 1.
Compd
R
IC50 (lM)
AKT1
ROCK1
S
9l
0.050a
6.0
Table 1
*
2-Substitution to improve selectivity
N
H
9m
0.015a
10.5c
>10
N
R
N
*
OH
N
9n
>10
>10
O
*
OH
NH2
Compd
2a
R
IC50 (lM)
AKT1
ROCK1
0.079
9o
0.72b
H
0.125a
*
9a
0.050a
0.015a
>10
9.5
a
Values were measured at or below Km for ATP; n P 2.
n = 1.
Data were generated using truncated AKT1 enzyme. For this chemical series,
b
c
*
*
the data generated from the truncated AKT1 (without PH domain) correlate well
with those from the full length AKT1; n P 2.
9b
9c
OH
OH
As shown in Scheme 2, the phenyl group in the amino-aryl side
chain was also replaced by a substituted indole ring, which was
introduced via Larock’s indole synthesis as the key step, starting
from substituted bromoanilines and silylated alkynes.14 Commer-
cially available chiral amino acid 10 was reduced with LiAlH4 to af-
ford alcohol 11, which was coupled with hydroxypyridine under
Mitsunobu reaction conditions to afford compound 12. The TMS
group was introduced onto the terminal alkyne to give 13, which
was coupled with boronate ester 6 under microwave assisted Su-
zuki reaction conditions to afford 14 in good yield. Treatment of
compound 14 with substituted bromoanilines followed by a sec-
ond Suzuki coupling with 3-furanyl boronic acid were carried out
in one pot under microwave irradiation at 170 °C. TFA deprotection
of the Boc group gave the target molecules 17. Additional azaindole
analogs (18a–c, see Table 2) were prepared in a similar manner
from compound 14 and different regioisomers of ortho- amino-
bromo-pyridines.
Consistent with our hypothesis that substitution at the 2-posi-
tion of the core pyridine would diminish ROCK1 inhibition, a >200-
fold improvement in selectivity over ROCK1 was achieved when
comparing compound 9a and compound 2a (see Table 1). In addi-
tion, compound 9a demonstrated nearly 3-fold higher AKT1 po-
tency, while compound 2a was observed to be approximately
equipotent against ROCK1 and AKT1.
0.079a
>10
1.4
*
OH
9d
0.50a
*
*
9e
9f
0.10a
>10
9.8
OMe
F
F
0.010c
OH
*
*
F
9g
9h
0.063b
0.003a
>10
7.6
OH
Cl
OH
Table 1 shows the SAR preferences of the substitution at C-2 of
the pyridine core. Besides phenyl, ortho-phenol of 9b was also tol-
erated, providing excellent selectivity against ROCK1. The haloge-
nated ortho-phenol analogs (compounds 9f-h) were observed as
being potent and selective, especially compound 9h, which dem-
onstrated single digit nanomolar potency against AKT1 and greater
than 2,000-fold selectivity over ROCK1. However, the meta- and
para-phenol derivatives (9c and 9d, respectively) were less potent
AKT1 inhibitors. Likewise, the ortho-anisole 9e, demonstrated re-
duced potency against AKT1.
*
O
O
9i
0.79c
1.29
5.2
*
9j
0.010a
0.079a
*
S
Incorporation of five membered heterocycles at C-2 position
was also investigated. The 3-furanyl derivative 9j was observed
to be much more potent and selective than the 2-furanyl regio iso-
9k
2.5
*

676
H. Lin et al. / Bioorg. Med. Chem. Lett. 20 (2010) 673–678
Cl
Cl
Br
Br
c
N
N
a
NHBoc
OH
b
NHBoc
OH
Boc
Boc
Si
NH
NH
O
O
O
11
10
12
13
O
O
N
Br
H
H
N
R
Cl
O
N
N
NH2
OH
N
B
d
N
16
15
HO
Boc
H
Si
NH
N
e
f, g
NH2
14
O
17
R
Scheme 2. Synthesis of substituted tryptophanol analogs. Reagents and conditions: (a) LiAlH4, THF, rt; (b) 4, Ph3P, DEAD, THF, 0 °C, rt, overnight (82% for 2 steps); (c) EtMgBr,
À36 °C, then TMSCl, (72%); (d) 6, Pd(Ph3P)4, aq Na2CO3, dioxane, microwave irradiation, 160 °C, 30 min (83%); (e) 15, Pd(Ph3P)4, aq Na2CO3, dioxane, microwave irradiation,
170 °C, 30 min; (f) 16, microwave irradiation, 170 °C, 30 min; (g) TFA, DCM, RP-HPLC purification.
Table 2
Table 2 (continued)
Side chain modification to improve potency
Compd
R
R’
IC50 (lM)
H
N
AKT1a
0.010
pGSK3 BT474
R
N
N
O
H
N
18c
4.3
*
N
O
*
a
Values were measured at or below Km for ATP; n P 2.
R'
NH2
Compd
9p
R
R’
IC50 (lM)
AKT1a
0.001
pGSK3 BT474
mer 9i. The 2-pyrrolyl analog 9m was also very potent and selec-
tive, while thienyl derivatives 9k and 9l were less potent against
AKT1. The 3-hydroxy-3-methyl-butynyl substituent, which was
present in GSK690693 (1), was introduced at C-2 (9n). However,
this compound was significantly less potent within this compound
class whereas the 4-hydroxyl-butynyl substituted compound (9o)
was better tolerated (see the discussion on X-ray co-crystal struc-
tures of GSK690693 and 9p).
To further improve enzymatic potency of this chemical class
against AKT, the S-phenylalaninol side chain was replaced with
S-tryptophanol following the same chemistry scheme as described
in Scheme 1.15 Compared to compound 9j, compound 9p displayed
a 10-fold increase of potency against AKT1. Further SAR studies re-
vealed that the 2-methyl-3-furan16 group at 2-position of the core
pyridine provided the most significant increase in activity (e.g., 9q
vs 9p in Table 2), wherein compound 9q was highly potent in cel-
lular mechanistic assay (BT474 cell line) measuring the reduction
of pGSK3b.7 As shown in Table 2, the 5-, 6-, and 7-fluorinated in-
dole analogs (17a, 17b, and 17c) were equipotent against AKT1
and in the cellular assay, compared to the parent compound 9p.
However, azaindole modification was less tolerated, as most deriv-
atives lost cellular potency (18a–18c).
O
H
N
0.50
*
*
*
O
H
N
9q
0.0008
0.001
0.084
0.36
*
O
H
N
17a
*
*
F
F
O
O
O
O
H
N
17b
17c
18a
18b
0.0008
0.001
0.008
0.013
0.22
0.26
0.93
6.8
*
*
*
*
*
F
H
N
*
*
*
Compound 9p was successfully co-crystallized with PKA.18,19 As
shown in Figure 3, the binding interactions of 9p were similar to
those reported for 2b. The indazole ring acts as a two-point hinge
binder, the core-pyridine nitrogen interacts with Lys72, and the
charged amino group on the side chain forms H-bond interactions
with both Asn171 and Asp184. As predicted, the furan ring at the
2-position of the core pyridine pointed to the gate keeper
Met120 and pushed its side chain back towards Leu95. Comparison
with the co-crystal structure of compound 1 in PKA showed that
H
N
N
H
N
N

H. Lin et al. / Bioorg. Med. Chem. Lett. 20 (2010) 673–678
677
Figure 3. Co-crystal structure of 9p in PKA and overlay of co-crystal structures of 9p and 2 in PKA. (A) Compound 9p co-crystal structure in PKA. Hydrogen bonds are
indicated as dotted lines. Key interactive residues are shown in stick. (B) Ribbon diagram representation of PKA co-crystallized with 9p. (C) Overlay of co-crystal structures of
9p (green) and 2 (yellow) in PKA.
4. Luo, Y.; Shoemaker, A. R.; Liu, X.; Woods, K. W.; Thomas, S. A.; De Jong, R.; Han,
the furan ring and the propargyl alcohol have different trajectories,
so it is not surprising that the latter does not give any benefit in
terms of potency or selectivity in the pyridine series (compounds
9n and 9o in Table 1).
E. K.; Li, T.; Stoll, V. S.; Powlas, J. A.; Oleksijew, A.; Mitten, M. J.; Shi, Y.; Guan, R.;
McGonigal, T. P.; Klinghofer, V.; Johnson, E. F.; Leverson, J. D.; Bouska, J. J.;
Mamo, M.; Smith, R. A.; Gramling-Evans, E. E.; Zinker, B. A.; Mika, A. K.; Nguyen,
P. T.; Oltersdorf, T.; Rosenberg, S. H.; Li, Q.; Giranda, V. L. Mol. Cancer Ther. 2005,
4, 977.
In summary, we have prepared potent AKT inhibitors from
5. Zhao, Z.; Leister, W. H.; Robinson, R. G.; Barnett, S. F.; Defeo-Jones, D.; Jones, R.
E.; Hartman, G. D.; Huff, J. R.; Huber, H. E.; Duggan, M. E.; Lindsley, C. W. Bioorg.
Med. Chem. Lett. 2005, 15, 905.
a
tri-substituted pyridine series. We have successfully im-
proved selectivity against ROCK1 based on rational design,
wherein an X-ray co-crystal structure supported our rationale
for selectivity.
6. Rhodes, N.; Heerding, D. A.; Duckett, D. R.; Eberwein, D. J.; Knick, V. B.; Lansing,
T. J.; McConnell, R. T.; Gilmer, T. M.; Zhang, S. Y.; Robell, K.; Kahana, J. A.; Geske,
R. S.; Kleymenova, E. V.; Choudhry, A. E.; Lai, Z. V.; Leber, J. D.; Minthorn, E. A.;
Strum, S. L.; Wood, E. R.; Huang, P. S.; Kumar, R. Cancer Res. 2008, 68(7), 2366.
7. Heerding, D. A.; Rhodes, N.; Leber, J. D.; Clark, T. J.; Keenan, R. M.; Lafrance, L.
V.; Li, M.; Safonov, I. G.; Takata, D. T.; Venslavsky, J. W.; Yamashita, D. S.;
Choudhry, A. E.; Copeland, R. A.; Lai, Z.; Schaber, M. D.; Tummino, P. J.; Strum, S.
L.; Wood, E. R.; Duckett, D. R.; Eberwein, D.; Knick, V. B.; Lansing, T. J.;
McConnell, R. T.; Zhang, S.-Y.; Minthorn, E. A.; Concha, N. O.; Warren, G. L.;
Kumar, R. J. Med. Chem. 2008, 51, 5663.
8. Zhu, G.-D.; Gandhi, V. B.; Gong, J.; Thomas, S.; Woods, K. W.; Song, X.; Li, T.;
Diebold, R. B.; Luo, Y.; Liu, X.; Guan, R.; Klinghofer, V.; Johnson, E. F.; Bouska, J.;
Olson, A.; Marsh, K. C.; Stoll, V. S.; Mamo, M.; Polakowski, J.; Campbell, T. J.;
Martin, R. L.; Gintant, G. A.; Penning, T. D.; Li, Q.; Rosenberg, S. H.; Giranda, V. L.
J. Med. Chem. 2007, 50(13), 2990.
9. ROCK1 inhibition is known to cause smooth mucle relaxation and to reduce
blood pressure in spontaneously hypertensive rat (SHR) models. We viewed
that long term inhibition of ROCK1 had potential risks due to its important
regulatory function and ubiquitous expression. See reviews and references
therein: (a) Hu, E.; Lee, D. Expert Opin. Ther. Targets 2005, 9(4), 715; (b) Liao, J.
K.; Minoru, S.; Kensuke, N. J. Cardiovasc. Pharmacol. 2007, 50(1), 17; (c)
Stavenger, R. A. Annu. Rep. Med. Chem. 2008, 43, 87.
10. (a) Gabel, M.; Breitenlechner, C. B.; Ruger, P.; Jucknischke, U.; Schneider, T.;
Huber, R.; Bossemeyer, D.; Engh, R. J. Mol. Biol. 2003, 329, 1021. and references
cited therein; (b) Davies, T. G.; Verdonk, M. L.; Graham, B.; Saalau-Bethell, S.;
Hamlett, C. C. F.; McHardy, T.; Collins, I.; Garrett, M. D.; Workman, P.;
Woodhead, S. J.; Jhoti, H.; Barford, D. J. Mol. Biol. 2007, 367, 882.
Acknowledgments
The authors thank Dr. Arthur Shu for preparing the critical
intermediate (B in note 15) in large scale to support the SAR study,
and Dr. Sharad Verma for carefully reviewing this manuscript. The
authors also thank Edward Dul, Tia S. Lewis and Hongwei Qi for
protein cloning, fermentation and purification.
References and notes
1. (a) Lindsley, C. W.; Barnett, S. F.; Layton, M. E.; Bilodeau, M. T. Curr. Cancer Drug
Targets 2008, 8, 7; (b) Li, Q. Expert Opin. Ther. Patents 2007, 17(9), 1077; (c)
Bellacosa, A.; Kumar, C. C.; Di Cristofano, A.; Testa, J. R. Adv. Cancer Res. 2005,
94, 29.
2. Lin, X.; Murray, J. M.; Rico, A. C.; Wang, M. X.; Chu, D. T.; Zhou, Y.; Del Rosario,
M.; Kaufman, S.; Ma, S.; Fang, E.; Crawford, K.; Jefferson, A. B. Bioorg. Med. Chem.
Lett. 2006, 16(16), 4163.
3. Collins, I.; Caldwell, J.; Fonseca, T.; Donald, A.; Bavetsias, V.; Hunter, L.-J. K.;
Garrett, M. D.; Rowlands, M. G.; Aherne, G. W.; Davies, T. G.; Berdini, V.;
Woodhead, S. J.; Davies, D.; Seavers, L. C. A.; Wyatt, P. G.; Workman, P.;
McDonald, E. Bioorg. Med. Chem. 2006, 14(4), 1255.
11. Koch, V.; Schnatterer, S. Synthesis 1990, 499.

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H. Lin et al. / Bioorg. Med. Chem. Lett. 20 (2010) 673–678
12. Reagents and conditions for this transformation: Pd2dba3, PCy3,
bis(pinacolato)diboron, KOAc, dioxane, 80 °C, 24h (74%).
13. The yields are for the synthesis of compound 9a.
14. Larock, R. C.; Yum, E. K. J. Am. Chem. Soc. 1991, 113(17), 6689.
15. This modification did not affect ROCK1 selectivity. For example, compound 9p
16. 2-Methylfuran-3-yl was introduced via
a
Suzuki coupling reaction with
sequence of halogen- metal
boronate ester B, which was prepared by
a
exchange and quenching the anion with 4,4,5,5-tetramethyl-2-[(1-
methylethyl)oxy]-1,3,2-dioxaborolane on about 150 mmol scale of 3-bromo-
2-methylfuran (A).17 This boronate ester is volatile and can be purified either
by flash column chromatography or by vacuum distillation.
was a very weak ROCK1 inhibitor with IC50 of 1.58
lM.
17. Alvarez-Ibarra, C.; Quiroga, M. L.; Toledano, E. Tetrahedron 1996, 52(11), 4065.
18. Crystallographic data for compound 9p has been deposited to the RSBC Protein
Data Bank with deposition code 3kkv.
19. Distorsion around Phe54 was observed in the co-crystal structure of compound
1 in PKA indicating the flexibility of this residue.
O
O
B
O
O
Br
A
B