Structure-based design and SAR development of 5,6-dihydroimidazolo[1,5-f]pteridine derivatives as novel Polo-like kinase-1 inhibitors

Article abstract of DOI:10.1016/j.bmcl.2016.10.009

Using structure-based drug design, we identified a novel series of 5,6-dihydroimidazolo[1,5-f]pteridine PLK1 inhibitors. Rational improvements to compounds of this class resulted in single-digit nanomolar enzyme and cellular activity against PLK1, and oral bioavailability. Compound 1 exhibits >7 fold induction of phosphorylated Histone H3 and is efficacious in an in vivo HT-29 tumor xenograft model.

Full text of DOI:10.1016/j.bmcl.2016.10.009

Accepted Manuscript
Structure-based design and SAR development of 5,6-Dihydroimidazolo[1,5-f]
pteridine derivatives as novel Polo-Like Kinase-1 inhibitors
Andre Kiryanov, Srinivasa Natala, Benjamin Jones, Christopher McBride,
Victoria Feher, Betty Lam, Yan Liu, Kouhei Honda, Noriko Uchiyama,
Tomohiro Kawamoto, Yuichi Hikichi, Lilly Zhang, David Hosfield, Robert
Skene, Hua Zou, Jeffrey Stafford, Xiaodong Cao, Takashi Ichikawa
PII:
S0960-894X(16)31037-X
http://dx.doi.org/10.1016/j.bmcl.2016.10.009
BMCL 24312
DOI:
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
31 May 2016
4 October 2016
5 October 2016
Please cite this article as: Kiryanov, A., Natala, S., Jones, B., McBride, C., Feher, V., Lam, B., Liu, Y., Honda, K.,
Uchiyama, N., Kawamoto, T., Hikichi, Y., Zhang, L., Hosfield, D., Skene, R., Zou, H., Stafford, J., Cao, X.,
Ichikawa, T., Structure-based design and SAR development of 5,6-Dihydroimidazolo[1,5-f] pteridine derivatives
as novel Polo-Like Kinase-1 inhibitors, Bioorganic & Medicinal Chemistry Letters (2016), doi: http://dx.doi.org/
10.1016/j.bmcl.2016.10.009
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Structure-based design and SAR development of 5,6-Dihydroimidazolo[1,5-f]
pteridine derivatives as novel Polo-Like Kinase-1 inhibitors
Andre Kiryanova,*, Srinivasa Natalaa,†, Benjamin Jonesa, Christopher McBridea, Victoria Fehera,‡ , Betty Lama, Yan
Liua, Kouhei Hondab, Noriko Uchiyamab, Tomohiro Kawamotob, Yuichi Hikichib, Lilly Zhanga, David Hosfielda,§,
Robert Skenea, Hua Zoua, Jeffrey Stafforda,±, Xiaodong Caoa,¶ , and Takashi Ichikawab
aTakeda California, 10410 Science Center Drive, San Diego 92121, USA
bTakeda Pharmaceutical Company, Ltd, 2-26-1, Muraokahigashi, Fujisawa, Kanagawa, 251-855, Japan
Abstract: Using structure-based drug design, we identified a novel series of 5,6-dihydroimidazolo[1,5-f]pteridine PLK1 inhibitors.
Rational improvements to compounds of this class resulted in single-digit nanomolar enzyme and cellular activity against PLK1, and
oral bioavailability. Compound 1 exhibits >7 fold induction of phosphorylated Histone H3 and is efficacious in an in vivo HT-29 tumor
xenograft model.
Keywords:
PLK1 inhibitor
Structure-based drug design
Antitumor activity
Multidrug resistance
Polo-like kinase 1 (PLK1) is a serine/threonine kinase that controls entry into and progression through
mitosis at multiple stages.1 Over-expression of PLK1 is associated with poor prognosis and survival rates in
a number of human cancers.1d,2 Several reports have demonstrated that PLK1 depletion (by α-PLK1
antibody injection or siRNA interference) causes mitotic arrest and apoptosis in cancer cell lines, but not in
normal diploid cells or non-dividing cells.3 PLK1 was therefore considered an attractive target for treatment
of proliferative disease.4 Currently, small molecule PLK1 inhibitors are being evaluated in human clinical
trials.5 Among which, clinical responses have been reported with the investigational IV drug, Volasertib.6
Anti-mitotic agents that target tubulin, such as taxanes, have been clinically validated. However, these
drugs have several drawbacks including development of peripheral neuropathy,7 the need for pretreatment
to reduce anaphylaxis,8 and the potential for tumors to become refractory/resistant due to multidrug
resistance.2b Consequently, there is a need for novel anti-mitotic drugs that target non-microtubule proteins
such as the mitotic kinases.3d Herein, we report the discovery and development of a novel 5,6-
dihydroimidazolo[1,5-f]pteridine series of PLK1 inhibitors.
*
Corresponding author. Tel.: +1 858 731 3613; fax: +1 858 550 0526;
Email address: andre.kiryanov@takeda.com (A. Kiryanov)
Present address: Genomics Institute of the Novartis Research Foundation
Present address: UC San Diego, Department of Chemistry and Biochemistry, USA.
Quanticel Pharmaceuticals Inc.
Present address: Ben May Cancer Institute, University of Chicago, USA.
Present address: Viva Biotech, Ltd., USA.
†
‡
±
§
¶

In our previous publication9 we reported the discovery of 7,7-difluoropyrimidodiazepinones (2) as PLK1
inhibitors. Based on PLK1 X-ray co-crystal information, 7,7-difluoropyrimidodiazepinones interact with
Lys82 via a bound water molecule (Fig. 1)9. In an effort to provide a novel back up series, we decided to
design compounds that would exhibit a direct interaction with Lys82 and examine how this interaction
affects potency. In order to gain this direct bonding to Lys82, we designed and synthesized a novel 5,6-
dihydroimidazolo[1,5-f]pteridine (3) scaffold.
Figure 1. Co-crystal structure of a representative compound from 7,7-difluoropyrimidodiazepinone series
with PLK1 where the amino pyrimidine is bound to the hinge residue (A) and the carbonyl at the 7-position
makes a water-mediated interaction with Lys82 (B) (PDB: 5TA8).
Compounds (4-9) were prepared according to Scheme 19. The carbonyl of compound 10 was activated
with diethyl phosphorochloridate and reacted with ethyl 2-isocyanoacetate anion. This intermediate
underwent an intramolecular cyclization to provide ester 11 in good yield. An SNAr reaction at the 2-
position of the core with aniline 12 followed by amide formation provided the key intermediate 13, with the
ester moiety serving as a versatile intermediate containing an appropriate vector for diversification. Finally,
7-substituted analogs 4-9 were obtained using conditions outlined in Scheme 1.
Scheme 1. Synthesis of 7-subsituted 5,6-dihydro-imidazolo[1,5-f]pteridines

For compounds with the 7-nitrile functionality on the dihydroimidazolo[1,5-f]pteridine core, we sought a
more direct synthetic route to access a key intermediate for their synthesis. The DMF adduct of
aminoacetonitrile provided a stable synthon for this transformation and was used for larger scale
production. The synthesis of compounds 1 and 14-17 is presented in Scheme 2. (R)-2-Aminobutanoic acid
(18) was esterified under SOCl2/MeOH conditions followed by a reductive alkylation of the amine moiety
with acetone, which provided compound 19 in 96% yield over two steps. An SNAr reaction with 2,4-
dichloro-5-nitropyrimidine (20) then gave predominantly the 4-substitution product 21. The nitro-group in
21 was hydrogenated under carefully controlled conditions to avoid the reduction of aryl chloride and also
to prevent undesired intramolecular cyclization of the intermediate nitroso-compound. Concomitant
intramolecular cyclization afforded (R)-2-chloro-7-ethyl-8-isopropyl-7,8-dihydropteridin-6(5H)-one (22) in
78% yield after two steps. Activation of the amide functionality in 22 with diethyl phosphorochloridate
and subsequent reaction with N'-(cyanomethyl)-N,N-dimethylformimidamide anion was followed by
cyclization under acidic conditions in a single pot to provide the key intermediate 23 in 92% yield. The
chloride in 23 was then replaced by aniline 24 under acidic conditions to give acid 25. These conditions
were not successful for the fluorine-containing aniline 26, however, and led to decarboxylation. Buchwald
Pd-catalyzed coupling with amine 26 did give rise to the desired fluorine-containing acid 27. Amide
formation with HATU as a coupling reagent afforded the desired final compounds 1 and 14-17. Compound
28 was also prepared according to this procedure, using cyclopentanone instead of acetone in the reductive
alkylation step.

From the medicinal chemistry perspective, our initial goal was to find compounds that would exhibit a
potent inhibition of PLK1 enzyme in vitro10. Our newly synthesized 5,6-dihydroimidazolo[1,5-f]pteridine
scaffold 2 allowed for incorporation of various polar substituents at the 7-position of the core for the
desired interaction with Lys82. We found that multiple groups were tolerated at this position and the level
of PLK1 inhibitory activity was under 50nM for all compounds (4-9, 13). Compounds 13 and 8 also
demonstrated sub-10nM inhibition of PLK1 in HT-29 cell line,11,12 with compound 8 exhibiting activity of
3 nM. This compound (8) also showed slow dissociation kinetics (T1/2=476 min)13.
Table 1. 7-Position substitution enzymatic and cellular potency data
No
R
PLK1_KD
IC50 (nM)
HT29_ATP
EC50 (uM)
-COOEt
-COOH
2.1
1.4
2.0
45
0.009
0.4
13
4
-CONH2
-CONHEt
-CH2OH
-CN
0.02
0.1
5
6
2.2
1.5
7.0
0.03
0.003
0.06
7
8
-CON(OH)Me
9
We were able to confirm our design concept with a co-crystal structure (Fig. 2). Indeed, the nitrile
functionality extends into the back-pocket of PLK1 active site to replace one bound water molecule and
interacts directly with Lys82.

Figure 2. Direct hydrogen bonding to Lys82 is observed for 5,6-dihydro-imidazolo[1,5-f]pteridine 7-
carbonitrile series of PLK1 inhibitors (PDB: 5TA6).
We chose this nitrile-containing scaffold for further optimization of ADME properties based on the
attractive cellular potency.
While active in enzymatic and cellular assays, compound 8 had minimal bioavailability in rat (0%),
likely a result of efflux (LLC-MDR/LLC ratio = 21)14 and/or high clearance in rat (76 mL/min/kg). During
our studies of the pyrimidodiazepinone series, we found that introduction of a fluorine ortho to the aniline
could lower the MDR1 susceptibility9. As one can see from compound 28, this strategy proved to be
advantageous in this case as well, as we were able to reduce LLC-MDR/LLC ratio to 2.3.

Table 2. Selected R2 and X substituents SAR: solving CYP inhibition and active transport problems.
No.
R2
X
PLK1_KD HT29_ATP
LLC-
%F rat CYP inh.
IC50 (nM) EC50 (nM) MDR/LLC*
(po)
(pIC50>5)
cyclopentyl
cyclopentyl
isopropyl
H
F
1.5
2.2
8.6
1.9
1.3
2.7
1.0
6.0
4.0
2.0
21
2.3
82
0
3A4
8
49
3A4, 2C8
clean
28
14
15
29
H
F
0.5
47
isopropyl
7.6
ND
clean
cyclohexyl
F
ND
3A4, 2C8
Concurrently, this structural change also resulted in lower clearance (28 mL/kg/min), allowing us to gain
bioavailability of 49% following oral administration in rats. However, compound 28 inhibited CYP
isoforms 3A4 and 2C8. We hypothesized that this issue could be alleviated by lowering its cLogD (3.2). To
this end, we replaced the N-9 cyclopentyl group with a less lipophilic isopropyl substituent resulting in
compound 15 (cLogD of 2.4). This change was significant enough to eliminate the undesired CYP
inhibition. Compound 15 possessed a good overall rodent ADME profile, and showed induction of
phosphorylated Histone H3 (pHH3, 6.3 fold at 48 hours following a single IV dose of 30 mg/kg). However,
we wanted to further improve its microsomal stability (HLM Eh=0.39). To accomplish this, we modified
the nature of the amide substituent, leading to identification of compound 1, which showed improved HLM
Eh=0.28. Compound 1 demonstrated pHH3 induction (7.8) and showed high absorption in mouse, which
was the species used for xenograft studies (Table 4).
In summary, we identified and optimized a novel series of 5,6-dihydroimidazolo[1,5-f]pteridine PLK1
inhibitors. Compound 1 showed a marked increase in pHH3 following a single administration (30 mpk po,
9 fold pHH3 increase after 48h) and showed efficacy (ms, 45 mg/kg iv, 1/time/week x 1 cycle: T/C 54.6%,
BWL 5.5%).

Table 3. Selected R1-substituent SAR: optimization for efficacy
No.
R1
PLK1_KD
IC50 (nM)
HT29_ATP
EC50 (nM)
LLC-
%F
CYP inh.
Fold pHH3 incr.
(IV) 30 mpk
Notes
MDR/LLC*
rat, po (pIC50>5)
1.9
3.9
1.7
4.0
22
8
2
47
clean
clean
clean
6.3
7.5
1.9
15
1
~100
ND
F~100% (ms)
3.0
ND
16
17
hERG 37%
0.4
3.0
4
ND
clean
5.2
inh. @ 10uM

Table 4. Pharmacokinetic parameters of compound 1 in mouse (female) following IV and PO
administration19
Route
(dose)
Cmax
Tmax
h
T1/2
h
MRT
h
AUCextrap
ng*h/mL
AUC0-t
CL
Vdss
F
ng/mL
ng*h/mL
mL/kg/min
L/kg
%
IV
258
354
-
4.7
8.5
6.5
13
1007
4591
713
22.7
-
8.9
-
-
(1mpk)
Oral
4
3900
101
(5mpk)
Acknowledgments
The authors would like to acknowledge the assistance of Dr. Matt Marx in preparation of this
manuscript, Dr. Andy Jennings for providing the co-crystal figures and Sin-chi Matsumoto and
Nobuyuki Amano for MDR1 data.
References and notes
1.
a) Llamazares, S.; Moreira, A.; Tavares, A. and et al. Genes Dev. 1991, 5, 2153. b) Barr, F.A.; Sillje, H.H.; Nigg, E.A. Nat. Rev. Mol. Cell
Biol. 2004, 5, 429. c) Sunkel, C.E.; Glover, D.M. J. Cell Sci. 1988, 89, 25. d) Strebhardt, K.; Ullrich, A. Nat. Rev. Cancer. 2006, 6, 321. e)
Archambault, V.; Glover, D.M. Nat. Rev. Mol. Cell Biol. 2009, 10, 265. f) Lens, S.M.; Voest, E.E.; Medema, R.H. Nat. Rev. Cancer. 2010,
10, 825.
2.
3.
a) Eckerdt, F.; Yuan, J.; Strebhardt, K. Oncogene. 2005, 24, 267. b) Szakacs, G.; Paterson, J.K.; Ludwig, J.A.; Booth-Genthe, C.;
Gottesman, M.M. Nat. Rev. Drug Discov. 2006, 5, 219.
a) Steegmaier, M.; Hoffmann, M.; Baum, A.; Lénárt, P.; Petronczki, M.; Krššák, M., et al. Current Biology. 2007, 17, 316. b) Liu, X.; Lei,
M.; Erikson, R.L. Mol. Cell Biol. 2006, 26, 2093. c) Guan, R.; Tapang, P.; Leverson, J.D.; Albert, D.; Giranda, V.L.; Luo, Y. Cancer Res.
2005, 65, 2698. d) Jackson, J.R.; Patrick, D.R.; Dar, M.M.; Huang, P.S. Nat. Rev. Cancer. 2007, 7, 107. e) Reagan-Shaw, S.; Ahmad, N.
FASEB J. 2005, 19, 611. f) Renner, A.G.; Dos Santos, C.; Recher, C.; Bailly, C.; Créancier, L.; Kruczynski, A. Blood. 2009, 16, 659.
a) Chopra, P.; Sethi, G.; Dastidar, S.G.; Ray, A. Expert Opin. Investig. Drugs. 2010, 19, 27. b) McInnes, C.; Wyatt, M.D. Drug Discovery
Today. 2011, 16, 619. c) Medema, R.H.; Lin, C.C.; Yang, J.C. Clin. Cancer Res. 2011, 17, 6459. d) Degenhardt, Y.; Lampkin, T. Clin.
Cancer Res. 2010, 16, 384.
4.
5.
6.
a) Murugan, R.N.; Park, J.E.; Kim, E.H. Shin, S.Y.; Cheong, C.; Lee, K.S.; Bang, J.K. Mol. Cells. 2011, 32, 209. b) Schöffski, P. The
Oncologist. 2009, 14, 559.
a) Bug, G.; Schlenk, R.F.; Muller-Tidow, C.; Lubbert, M.; Kramer, A.; Fleischer, F. Blood. 2010, 116, Abstract 3316. b) Schöffski, P.;
Awada, A.; Dumez, H.; Gil, T.; Bartholomeus, S.; Wolter, P.; Taton, M.; Fritsch, H.; Glomb, P.; Munzert, G. Eur. J. Cancer. 2012, 48, 179.
Wolf, S.; Barton, D.; Kottschade, L.; Grothey, A.; Loprinzi, C. Eur. J. Cancer. 2008, 44, 1507.
7.
8.
9.
Hennenfent , K.L.; Govindan, R. Ann. Oncol. 2006, 17, 735.
a) Synthesis of compound 10: Nie, Z.; Feher, V.; Natala, S.; McBride, C.; Kiryanov, A.; Jones, B.; Lam, B.; Liu, Y.; Kaldor, S.; Stafford,
J.; Hikami, K.; Uchiyama, N.; Kawamoto, T.; Hikichi, Yu.; Matsumoto, S.; Amano, N.; Zhang, L.; Hosfield, D.; Skene, R.; Zou, H.; Cao,
X.; Ichikawa, T. Bioorg. Med. Chem. Lett. 2013, 23, 3662. b) Full experimental details for all compounds in this paper can be found in the
following patent application: WO2010025073.
10. PLK1 enzyme assay: The inhibitory activities of the inhibitors were assessed by the TR-FRET assay, which the ATP-dependent
phosphorylation of a biotinylated substrate peptide (1-μM) corresponding to residues 2470 through 2488 of mTOR protein (Biotin-
AGAGTVPESIHSFIGDGLV) is measured.
11. Cell proliferation assay: HT-29 human colon adenocarcinoma cells were seeded into 96-well plates at 3,000 cells/well in DMEM
(Dulbecco's Modified Eagle's Medium) plus 10% fetal calf serum (FCS). After 24 hours, cells were treated with serial dilutions of PLK1
inhibitors, and 72 hours later, the number of viable cells was assessed using the CellTiter-Glo Assay (Promega).
12. Hikichi, Y.; Honda, K.; Hikami, K.; Miyashita, H.; Kaieda, I.; Murai, S.; Uchiyama, N.; Hasegawa, M.; Kawamoto, T.; Sato, T.; Ichikawa,
T.; Cao, S.; Nie, Z.; Zhang, L.; Yang, J.; Kuida, K.; Kupperman, E. Mol. Cancer Ther. 2012, 11, 700.
13. The dissociation kinetics of PLK1 inhibitors was assessed by a method designed to determine the dissociation rate of a compound from an
enzyme. Inhibitors were mixed with PLK1 and incubated at room temperature for 60 min. to allow for sufficient binding. The
PLK1/inhibitor complex was then diluted into reaction buffer containing 1000-M ATP and 1-μM FAM-mTOR peptide, and this initiation

of the enzymatic reaction was considered time zero (t0). The dissociation rate was then measured on a Caliper LabChip 3000 (LC-3000)
from Caliper Life Sciences Inc. with off-chip assays using a TC372 chip.
14. LLC – pig kidney cell line; LLC-MDR: MDR1 transfectant.
15. Cheguillaume, A.; Lacroix, S.; Marchand-Brynaert, J. Tetrahedron. Lett. 2003 , 44, 2375.
16. a) Zhang, d.; Bleasdale, c.; Golding, B.T.; Watson, W.P. Chem. Commun. 2000, 1141. b) Davis, F.A.; Qi, H.; Sundarababu, G. Tetrahedron.
2000, 56, 5303.
17. Copeland, R.A.; Pompliano, D.L.; Meek, T.D. Nat. Rev. Drug Discov. 2006, 5, 730. (Drug-target residence time and its implications for
lead optimization)
18. a) Kirk, K.L. J. Fluorine Chem. 2006, 127, 1013. b) Hagmann, W.K. J. Med. Chem. 2008, 51, 4359. c) Müller, K.; Faeh, C.; Diederich, F.
Science. 2007, 317, 1881.
19. Compounds were administered intravenously (IV vehicle: 30% b-cyclodextrin in 0.05M MSA, pH3) and orally (oral vehicle: 0.5% MC) at
approximately 1.0/5.0 mg/kg for rat.

Graphical abstract