GS-9973 | Srebp Signal

Orally bioavailable Syk inhibitors with activity in a rat PK/PD model

Gebhard Thoma a,⇑, Siem Veenstra a, Ross Strang a, Joachim Blanz b, Eric Vangrevelinghe a, Jörg Berghausen c, Christian C. Lee e, Hans-Günter Zerwes d

a Global Discovery Chemistry, Novartis Institutes for Biomedical Research, 4056 Basel, Switzerland

b Analytical Sciences & Imaging, Novartis Institutes for Biomedical Research, 4056 Basel, Switzerland

c Metabolism & Pharmacokinetics, Novartis Institutes for Biomedical Research, 4056 Basel, Switzerland

d Autoimmunity, Transplantation and Inflammation Research, Novartis Institutes for Biomedical Research, 4056 Basel, Switzerland

e Genomics Institute of the Novartis Research Foundation, 10675 John Jay Hopkins Drive, San Diego, CA 92121, USA

a r t i c l e i n f o

Article history:

Received 5 August 2015

Revised 12 August 2015

Accepted 13 August 2015

Available online xxxx

Keywords:

Spleen Tyrosine Kinase

Fostamatinib

BIIB-057

GS-9973

Kinase inhibitors

a b s t r a c t

Design and optimization of benzo- and pyrido-thiazoles/isothiazoles are reported leading to the discov-ery of the potent, orally bioavailable Syk inhibitor 5, which was found to be active in a rat PK/PD model. Compound 5 showed acceptable overall kinase selectivity. However, in addition to Syk it also inhibited Aurora kinase in enzymatic and cellular settings leading to findings in the micronucleus assay. As a con-sequence, compound 5 was not further pursued.

2015 Published by Elsevier Ltd.

Spleen Tyrosine Kinase (Syk) is a non-receptor kinase discov-ered in 1991.1 Syk is critical for the transduction of intracellular signal cascades through various immune recognition receptors, such as B cell receptor, Fc receptors, adhesion receptors, or C-type lectin receptors. Following activation Syk phosphorylates a number of substrates and participates in a multi-protein signaling complex, the ‘signalosome’, which leads to the activation of downstream effector pathways such as PKC, MAPK and NFjB.2 Syk is an estab-lished drug target for autoimmune diseases because aberrant acti-vation of immunoreceptor signaling can contribute to the initiation and progression of chronic inflammation and autoimmunity.3

Despite considerable efforts,4 only few Syk inhibitors have been evaluated in clinical trials (for structures and data on clinical can-didates such as compounds 1,5 26 and 37 see Fig. 1 and Table 1). We stopped early work on Syk inhibitors sharing the binding mode of 2 because of insurmountable PK issues.8 Furthermore, we had to abandon the promising Syk inhibitor 4 (see Fig. 1 and Table 1) due to liver findings and a hERG flag leading to an insufficient ther-apeutic index for autoimmune indications.9 In rat compound 4 exhibited >100 fold higher exposure in liver and kidney compared to blood. We hypothesized that the basic, primary amino group required for potency and acceptable PK properties contributed to

⇑ Corresponding author. Tel.: +41 61 3243342; fax: +41 61 3246735. E-mail address: [email protected] (G. Thoma).

http://dx.doi.org/10.1016/j.bmcl.2015.08.037 0960-894X/ 2015 Published by Elsevier Ltd.

both the high tissue exposure (volume of distribution was Vss = 16.4 L/kg) and the safety issues observed in the toxicity stud-ies. Thus, we set out to identify structurally unrelated back up compounds devoid of an amino group.

Here we disclose the discovery of the potent, orally bioavailable Syk inhibitor 5, which is uncharged at physiological pH and struc-turally unrelated to 4. Compound 5 did not inhibit the hERG chan-nel (IC50 >30 lM) and showed substantially reduced tissue exposure compared to 4. Due to good absorption and low in vivo clearance, compound 5 exhibited favorable PK properties in rat and was active in a rat PK/PD model. Unfortunately, compound 5 could not be further pursued because in addition to Syk it also inhibited Aurora kinase in enzymatic and cellular settings leading to findings in the micronucleus assay.

A structure of the early, moderately active Boehringer Ingel-heim Syk inhibitor 611 bound to Syk kinase domain solved at Novartis revealed that the nitrogen in the 1-position of the 1,6-naphthyridine core and the adjacent sp2-carbon form H-bonds with the hinge sequence of Syk (Fig. 2A, Table 2).12 Recently, the Boehringer group has shown that the basic amine of 6 can be replaced with neutral groups, such as lactams and aromatic amides, leading to more potent analogs.13 To mimic the binding mode of 6, we decided to explore benzo- and pyrido-thiazole cores as novel hinge binding motifs and prepared a set of neutral pilot

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H OMe
NN
N OMe
F
HN OMe
N O

HN Me

Me
O
1
active drug
of Fostamatinib
(Rigel)
N NH
N
N N

O3 GS-9973 (Gilead)

N N

O HN

H2N N

NNH

NH2
2

BIIB-057

(Biogen Idec)

O HN

H2N N

N NH

NH2

nature of the bicyclic core similar metabolites were observed across species mainly affecting the pyrazole (hydroxylation, N-demethylation) and the lactam substituent (hydroxylation). However, in contrast to the thiazoles, the isothiazoles formed trace quantities of glutathione conjugates indicating the formation of reactive intermediates. Their molecular masses pointed to an unexpected metabolic pathway involving addition of both GSH and H2O. The trace quantities prevented us from isolation and structure elucidation. As a consequence, we decided not to con-sider the isothiazoles for the selection of potential development compounds.

However, we used the benzisothiazole core for the rapid opti-mization of the PK properties. Replacement of the pyrazole by aryl and 5 or 6-ring heteroaryl groups did not lead to derivatives with reduced in vivo clearance. In addition, the favorable physicochem-ical properties were compromised. Thus, we explored differently substituted pyrazoles and found the difluoromethyl derivative 13 to show improved PK properties (Table 2; for the synthesis see Scheme 3). However, the potency was limited. In efforts to reduce the hydroxylation of the lactam residue we introduced an isosteric cyclic carbamate (for the synthesis of the building block see

Figure 1. Structures of clinical Syk inhibitors 1–3 and reference compound 4.

compounds 7–10 lacking the basic amine of 6 (Table 2; for the syn-theses of 7–10 see Schemes 1–4).14

We were pleased to learn that these compounds are equipotent to our development candidate 4 in enzymatic, cellular and blood assays (Table 2).10 Notably, thiazoles (7, 8) and isothiazoles (9, 10) were equally active. We obtained crystal structures of repre-sentative compounds 11 (thiazole; Table 2; for the synthesis see Scheme 2) and 12 (isothiazole; Table 2; for the synthesis see Scheme 3) bound to Syk kinase domain, which confirmed our bind-ing hypothesis indicating that the novel sulfur containing thiazole and isothiazole scaffolds are bioisosteres of 1,6-naphthyridine (Fig. 2B and C). Similarly to 6, the thiazole 11 forms two H-bonds with the hinge sequence of Syk. Isothiazole 12 cannot form the sec-ond H-bond; however, this is compensated for by a favorable inter-action between sulfur and the backbone carbonyl oxygen of E449.15

Compounds 7–10 exhibited good solubility (>1 mM at pH 6.8), medium permeability (log PAMPA from 4.9 to 5.2 10 6 cm/s), medium plasma protein binding (human: 80–90%) and acceptable stability against rat liver microsomes (half-life from 34–82 min). In vivo rat PK studies revealed medium to high clearance (CL = 45–86 mL/min kg), low volumes of distribution (Vss 2 L/kg) and short mean residence times (MRT <1 h) for all three series tested (Table 2). The medium to high clearance and the short MRT were confirmed in dog PK studies.

To identify metabolic weak spots we investigated the metabo-lism of representative compounds from all four sulfur containing scaffolds in human and rat liver microsomes. Independent of the

Table 1

Key in vitro data on reference compounds10

Compd Syka (enzyme) Kinaseb selectivity Syka (cell) Syka (blood)
1 68 ± 29 18 (79) 457 ± 8 3354 ± 475
2 13 ± 4 2 (77) 178 ± 8 952 ± 70
3 377 ± 90 0 (58) 878 ± 88 19,416 ± 1474
4 35 ± 4 0 (69) 99±7 367 ± 27

a IC50 in nM; n P 3 for Syk enzyme, cell and blood assays (SEM shown).
b Number of kinases with IC50 <100 nM in addition to Syk (number of kinases Figure 2. Crystal structures of 6, 11 and 12 bound to Syk kinase domain (H-bonds
tested). with hinge sequence E449-M450-A451 shown).12

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3

Table 2

Key in vitro data10 and rat PK parameters

O
NH Thiazoles

(R)

Naphthyridine S

HN N
NH2
N

6 N

O 5: A=N 7: A=CH 8: A=N 11: A=N 15: A=CH
A X=O F X=CH2 X=CH2 X=CH2 X=O F
R= R= R= R= O R=
N N N N
R N F N N O N F

O
NH Isothiazoles

(R) (R)

O 9: A=CH 10: A=N 12: A=CH 13: A=CH 14: A=CH
A X=CH2 X=CH2 X=O X=CH2 F X=O F

S R= R= R= R= R=
N N N N N
N R N N N N F N F

Compd a
Kinase b
selectivity Syk a
(cell) a
(blood) c
c
(L/kg) MRT c
(h) AUC c,d
iv (nM * h) BAV c
c,d
(nM)
Syk (enzyme) Syk CL (mL/min kg) Vss (%) Cmax
4 35 ± 4 0 (69) 99±7 367 ± 27 31 16.4 8.7 1449 60 80
5 38 ± 15 1 (60) 284 ± 20 522 ± 37 4 1.3 6.0 14,791 88 917
6 552 ± 150 0 (86) 951 ± 234 n.t. n.t. n.t. n.t. n.t. n.t. n.t.
7 33 ± 24 4 (40) 89±34 186 ± 31 n.t. n.t. n.t. n.t. n.t. n.t.
8 26 ± 5 0 (61) 284 ± 20 538 ± 157 54 2.3 0.7 951 46 103
9 46 ± 8 2 (63) 161 ± 52 298 ± 43 45 2.4 0.9 1056 30 94
10 40 (n = 1) 0 (20) 247 (n = 1) 290 ± 49 86 2.4 0.5 577 27 109
11 5 ± 2 4 (66) 132 ± 19 517 ± 95 53 1.2 0.4 858 35 178
12 16 ± 3 7 (60) 166 ± 69 737 ± 116 12 1.5 2.0 3887 100 477
13 57 ± 9 0 (54) 340 ± 31 1018 ± 407 18 2.8 2.6 2341 100 263
14 16 ± 2 5 (60) 328 ± 87 197 ± 38 7 1.4 2.8 5440 100 614
15 9 ± 9 11 (60) 107 ± 8 230 ± 51 5 1.3 4.7 10,142 91 803

a IC50 in nM; n P 3 for Syk enzyme, cell and blood assays (SEM shown).

b Number of kinases with IC50 <100 nM in addition to Syk (number of kinases tested).

c Cassette dosing in Sprague Dawley rats; iv 1 mg/kg, NMP/PEG200 (3:7); po 3 mg/kg, CMC/water/Tween (0.5:99:0.5).

d Dose normalized.

O a, b O c O
HO TsO HS
Br 40% O2N Br O2N Br
(2 steps)
O O O
N
Ref. 13 NH
OH
d OH e, f
S
O
44% N 59%
Br S
(2 steps)

N Br

O NH

O B N
g N
O
60% S

N
7 N

Scheme 1. Synthesis of 7 (15 prepared similarly). Reagents and conditions: (a) HNO3, CH3CO2H, 10 LC ? 25 LC, 1 h; (b) TsCl (1.2 equiv), pyridine (2.0 equiv), CH2Cl2, 0 ? 25 LC, 16 h; (c) Na2S 9H2O (2.0 equiv), DMF, 0 LC, 1 h; (d) Zn powder (5.0 equiv), HCOOH, reflux, 16 h; (e) alcohol13 (1.1 equiv), (t-BuO2C)2N2 (2.0 equiv), PPh3 (2.0 equiv), CH2Cl2, 25 LC, 1 h; (f) TFA, microwave, 100 LC, 20 min; (g) boronate ester (3.0 equiv), Pd(PPh3)2Cl2 (0.1 equiv), Na2CO3 (3.0 equiv), dioxane, 100 LC, 16 h.

Scheme 5). Compound 12 and its lactam analog 9 showed similar potencies as well as similar key interactions of lactam and cyclic carbamate with the enzyme (Fig. 3). The carbonyl oxygen and the ring nitrogen form H-bonds with K402 and S511, whereas ben-eficial van der Waals contacts were observed between the methyl group and G378 and V385. These lipophilic interactions along with an improved pre-organization of the bioactive conformation might

O
O N S NH2
N c
S O a, b N

N Br 7% 82%
(2 steps) N
N

O O O
N NH

O Ref. 13

S NH OH d,e O

N
N 23% S N
(2 steps)
N
N
N
8
N

Scheme 2. Synthesis of 8 (5, 11 prepared similarly). Reagents and conditions: (a) RC„CH (1.5 equiv), CuI (0.05 equiv), Pd(PPh3)4 (0.07 equiv), NEt3 (3.0 equiv), DMF, 70 LC, 0.5 h; (b) NH3 (12 equiv), MeOH, 70 LC, 16 h; (c) NaOtBu (1.4 equiv), THF,
70 LC, 1 h; (d) alcohol (1.8 equiv), (t-BuO2C)2N2 (2.2 equiv), PPh3 (2.2 equiv), CH2Cl2,

25 LC, 2 h; (d) TFA, 100 LC, 15 min.

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H2N Br 75%

G. Thoma et al. / Bioorg. Med. Chem. Lett. xxx (2015) xxx–xxx

O O O
O S
O NSO
b Ref. 19 O
O
38% O b
O S N H2N a
Br

S 91%
N Br

O N O

OH Ref. 13
OH d, e

S 68%
N Br
(2 steps)
O NH
O
O B N

OH HN 58%
HO 2 steps
Ref. 20

OH
O
O
O O c O N O
O N
91% O

S
N Br

f N
O

45%

N
9 N

Scheme 5. Synthesis of the protected (hydroxyethyl)oxazolidinone. Reagents and conditions. (a) Epoxide20 (1.0 equiv), Amine (1.3 equiv), EtOH, 60 h, 25 LC;
(b) Na2CO3 (2.5 equiv), ClCO2Me 1.2 equiv), CH2Cl2/H2O (2.5:3.0), 5 ? 25 LC, 3 h;

Scheme 3. Synthesis of 9 (12, 13, 14 prepared similarly). Reagents and conditions:

(a) SOCl2 (2.0 equiv), benzene, reflux, 18 h; (b) N-sulfinyl-methanesulfonamide19 (1.6 equiv), pyridine (1.1 equiv), benzene, reflux, 24 h; (c) 48% aq HBr, 140 LC, 2.5 h;
(d) alcohol (1.0 equiv), (t-BuO2C)2N2 (1.5 equiv), PPh3 (1.5 equiv), THF, 0 ? 25 LC, 16 h; (e) TFA, 80 LC, 3.5 h; (f) boronate (1.5 equiv), Pd(DTBPF)Cl2 (0.06 equiv), K3PO4 (2.0 equiv), Tol/EtOH/H2O (7:2:1), 100 LC, 1 h.

explain that related compounds lacking the methyl group were substantially less active.

Carbamate 12 showed improved PK properties compared to its lactam analog 9 (Table 2; for the synthesis see Scheme 3). However, the MRT of 2 h and the kinase selectivity were subopti-mal. Combination of difluoromethylpyrazole and carbamate led to compound 14 (Table 2; for the synthesis see Scheme 3) with

Br Br O O

CN a NH b NH2

S S 2 S
NBr39% N Br 53% NBr

promising PK properties, low clearance, good potency but limited kinase selectivity. Next we transferred the learnings to the thiazole series and prepared 5 and 15 (Table 2; for the syntheses see Schemes 2 and 1, respectively). Both compounds exhibited low clearance, good MRT and promising AUC. Because of its superior kinase selectivity we selected 5 for further profiling against the abandoned development candidate 4.

Compound 5 showed IC50 values above 30 lM in both hERG binding and automated patch clamp assays. Compound 4 did not exhibit substantial hERG channel inhibition in a manual patch clamp assay at 30 lM and showed an IC50 value of 28 lM in an automated patch clamp assay; however, under GLP conditions, an IC50 value of 5 lM was established.9 Thus, the predictability of the in vitro assays can be challenged, but based on the available data the neutral compound 5 appeared promising.

The PK parameters of 4 and 5 differed substantially probably due to the absence of a basic amine in 5 (Table 2). Compound 4 exhibited medium clearance and a very high Vss leading to a long MRT of 8.7 h while compound 5 showed a low Vss and a low clear-ance also resulting in a long MRT of 6 h. In line with the distinct Vss, the compounds showed very different distribution profiles in rat. Comparable concentrations in blood, liver and kidney were

N S

O
NH2
d NH

observed for 5, whereas compound 4 showed 100–200 fold increased concentrations in tissue (Table 3). In light of the liver findings observed with 4, we considered the PK profile of 5 to be

22%

more desirable.

Compound 5 was tested in our rat PK/PD model (Fig. 4).9 Following oral administration, blood samples were taken at differ-ent times for PK determination as well as assessment of inhibition

O O O
N NH
e

Ref. 13 43% O
OH
OMs

f N
S

45% N N
2 steps 10
N

Scheme 4. Synthesis of 10. Reagents and conditions: (a) H2SO4 (concd), 25 LC, 72 h;

(b) Zn powder (5.0 equiv), MeOH/HCOOH (1.3:1), 10 LC, 2 h; (c) RC„CH (1.4 equiv), CuI (0.07 equiv), Pd(PPh3)4 (0.1 equiv), NEt3 (3.0 equiv), DMF, 70 LC, 0.5 h; (d) NaOtBu (2.1 equiv), DMF/THF, 70 LC, 0.5 h; (e) (1) TFA, microwave, 100 LC, 14 min;
(2) MsCl (0.9 equiv), NEt3 (2.7 equiv), CH2Cl2, 0 ? 25 LC, 1 h; (f) mesylate
(2.0 equiv), K2CO3 (3.0 equiv), DMF, microwave, 140 LC, 0.5 h.

of Syk-dependent signaling events. For this, the extent of SLP76 phosphorylation in monocytes in response to stimulation by anti CD32 was quantified. A dose of 3 mg/kg of 5 led to 78%, 79% and 60% inhibition after 2, 4 and 8 h post administration, respectively. No inhibition was observed after 24 h. Blood concentrations were 1930, 1341, 827 and 40 nM, respectively. The low exposure after 24 h was unexpected based on the PK profile (Table 2). A potential explanation is the usage of different rat strains for PK experiment (Sprague Dawley) and PK/PD model (Lewis). Due to the limited AUC of compound 5 (10-fold lower), higher doses were required to achieve substantial inhibition in this model. However, the effects seen with 5 were more sustained.9

Compound 5 showed acceptable overall kinase selectivity (Table 2). However, in addition to Syk it also inhibited Aurora A

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5

Figure 3. Crystal structures of 11 and 12 bound to Syk kinase domain (key interactions of lactam and cyclic carbamate shown; H-bonds with K402 and S511as well as van der Waals contacts with G378 and V385).12

Table 3

Distribution of compounds 4 and 5 in different matrices following 1 mg/kg iv administration in rat

Matrix 4 5

Blood (ng/g) 41 1014
Liver (ng/g) 9175 2134
Kidney (ng/g) 3866 1433

Figure 4. PK/PD experiments with compound 5 (3 mg/kg, po). Single dose treatment of Lewis rats followed by ex vivo assessment of blood compound levels (PK) and FccR-induced P-SLP76 in peripheral blood monocytes (PD). Bars = % pSLP76; line = blood levels.

in our enzymatic assay (IC50 = 57 nM).16 Unfortunately, this was confirmed in a cellular Aurora B assay (IC50 = 110 nM).17 The com-pound was negative in the Ames test but positive in the micronu-cleus assay. This was not unexpected as Aurora A inhibition causes micronuclei formation and aneuploidy, Aurora B inhibition results in a cytokinesis defect leading to binucleate cells, and inactivation of both Aurora A and B causes an abrogation in chromosome seg-regation and mitotic exit with a single quadroploid nucleus.18
We have described the optimization of novel benzo- and pyr-ido-thiazoles/isothiazoles leading to the discovery of the potent, orally bioavailable Syk inhibitor 5, which was found to be active in a rat PK/PD model. However, as a consequence of findings in the micronucleus assay, compound 5 was not further pursued.

Acknowledgments

We gratefully acknowledge the expert assistance of Valerie Caballero, Thierry Délémonté, Mu-Yun Gao, Joanna Grant, Alice Hauchard, Anne-Marie Jutzi, Alexandre Luneau, Patric Neubert, Kenneth Ng, Jürg Peter, Marc Schäfer, Jennifer Shaffer and Phuoc Thanh Thai. We thank Reiner Aichholz and Hong Liu for helpful dis-cussions. The structural work in this Letter is based on experiments conducted at beamline 5.0.3 of the Advanced Light Source (ALS). The ALS is supported by the Director, Office of Science, Office of Basic Energy Sciences, Material Sciences Division of the US Department of Energy under contract DE-AC03-76SF00098 at Lawrence Berkeley National Laboratory.

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6 G. Thoma et al. / Bioorg. Med. Chem. Lett. xxx (2015) xxx–xxx

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8. Thoma, G.; Blanz, J.; Buhlmayer, P.; Druckes, P.; Kittelmann, M.; Smith, A. B.; van Eis, M.; Vangrevelinghe, E.; Zerwes, H.-G.; Che, J.; He, X.; Jin, Y.; Lee, C. C.; Michellys, P.-Y.; Uno, T.; Liu, H. Bioorg. Med. Chem. Lett. 2014, 24, 2278.

9. Thoma, G.; Smith, A. B.; van Eis, M. J.; Vangrevelinghe, E.; Blanz, J.; Aichholz, R.;

Littlewood-Evans, A.; Lee, C. C.; Liu, H.; Zerwes, H.-G. J. Med. Chem. 2015, 58, 1950.

10. Potency and kinase selectivity of Syk inhibitors were assessed in enzymatic assays based on the Caliper microfluidic mobility shift technology. Cellular activity was measured in Ramos B-cells upon BCR stimulation with anti-IgM which leads to phosphorylation of the adaptor protein BLNK (B cell linker protein), a direct Syk substrate. Inhibition of Syk in presence of 90% human blood was monitored in monocytes following FccR stimulation with an anti CD32 antibody. This leads to the phosphorylation of the adaptor protein SLP-76 which is also a direct substrate of Syk. For details on the assays see Ref. 9.

11. Cywin, C. L.; Zhao, B.-P.; McNeil, D. W.; Hrapchak, M.; Prokopowicz, A. S.; Goldberg, D. R.; Morwick, T. M.; Gao, A.; Jakes, S.; Kashem, M.; Magolda, R. L.; Soll, R. M.; Player, M. R.; Bobko, M. A.; Rinker, J.; DesJarlais, R. L.; Winters, M. P. Bioorg. Med. Chem. Lett. 2003, 13, 1415.

12. Structures deposited in the RCSB Protein Data Bank under PDB ID: 5CXZ (6),

PDB ID: 5CXH (11) and PDB ID: 5CY3 (12).

13. (a) Fiegen, D.; Handschuh, S.; Hobbie, S.; Hoffmann, M.; Kono, T.; Sato, Y.; Schnapp, A.; Schuler-Metz, A. PCT Int. Appl. WO 2,010,015,518.; (b) Bouyssou, T.; Dahmann, G.; Engelhardt, H.; Fiegen, D.; Handschuh, S.; Hobbie, S.; Hoffmann, M.; Kono, T.; Reiser, U.; Sato, Y.; Schnapp, A.; Schuler-Metz, A. PCT Int. Appl. WO 2,010,015,520.; (c) Hoffmann, M.; Dahmann, G.; Fiegen, D.; Handschuh, S.; Klicic, J.; Linz, G.; Schaenzle, G.; Schnapp, A.; East, S. P.; Mazanetz, M.P.; Scott, J.; Walker, Edward PCT Int. Appl. WO 2,011,092,128.; (d) Hoffmann, M.; Bischoff, D.; Dahmann, G.; Klicic, J.; Schaenzle, G.; Wollin, S. L. M.; Convers-Reignier, S. G.; East, S. P.; Marlin, F. J.; McCarthy, C.; Scott, J. PCT Int. Appl. WO 2,013,014,060.

14. We also explored benzo[c][1,2,5]thiadiazoles and [1,2,5]thiadiazolo[3,4-c] pyridines but the corresponding analogs were found to be less potent. Only the R,R-configuration of the chiral lactam residue led to highly active compounds.

15. Beno, B. R.; Yeung, K.-S.; Bartberger, M. D.; Pennington, L. D.; Meanwell, N. A. J. Med. Chem. 2015, 58, 4383.
16. Compounds 7, 11, 12, 14 and 15 also inhibited Aurora A with IC50 values of 6100 nM. Compounds 9 and 10 were less potrnt showing IC50 values of 576 and 800 nM, respectively.

17. The Aurora B cell assay was performed by ProQinase GmbH, Freiburg, Germany; http://proqinase.com/.
18. Hochegger, H.; Hegarat, N.; Pereira-Leal, J. B. Open Biol. 2013, 3. 120185.

19. Singerman, G. M. J. Heterocycl. Chem. 1975, 12, 877.

20. Trost, B. M.; Ball, Z. T.; Laemmerhold, K. M. J. Am. Chem. Soc. 2005, 127. 100028.