Luciferin and derivatives as a DYRK selective scaffold for the design of protein kinase inhibitors

Accepted Manuscript Luciferin and derivatives as a DYRK selective scaffold for the design of protein kinase inhibitors Ulli Rothweiler, Jonas Eriksson, Wenche Stensen, Frederick Leeson, Richard A. Engh, John S. Svendsen PII:

S0223-5234(15)00131-2

DOI:

10.1016/j.ejmech.2015.02.035

Reference:

EJMECH 7717

To appear in:

European Journal of Medicinal Chemistry

Received Date: 19 December 2014 Revised Date:

19 January 2015

Accepted Date: 19 February 2015

Please cite this article as: U. Rothweiler, J. Eriksson, W. Stensen, F. Leeson, R.A. Engh, J.S. Svendsen, Luciferin and derivatives as a DYRK selective scaffold for the design of protein kinase inhibitors, European Journal of Medicinal Chemistry (2015), doi: 10.1016/j.ejmech.2015.02.035. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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D-Luciferin is an inhibitor of several protein kinases The DYRK-family is particularly affected by a lucifein-library X-ray structures of CDK2/inhibitor complexes reveal important binding interactions

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Luciferin is an inhibitor scaffold with intrinsic selectivity for DYRK-kinases

ACCEPTED MANUSCRIPT

Luciferin and derivatives as a DYRK selective scaffold for the design of protein kinase inhibitors

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Ulli Rothweiler1, Jonas Eriksson2,†, Wenche Stensen2,3, Frederick Leeson2,3, Richard A.

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Engh1,* and John S. Svendsen2,3,*

The Norwegian Structural Biology Centre, Department of Chemistry, UiT The Arctic

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University of Norway, N-9037 Tromsø, Norway, 2Lytix Biopharma AS, P.O. Box 6447, Tromsø Science Park, N-9294 Tromsø, Norway and 3Department of Chemistry, UiT The Arctic University of Norway, N-9037 Tromsø, Norway.

Corresponding Authors

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AUTHOR INFORMATION

+47 776 44086.

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*Corresponding authors: [email protected] +47 776 44073; [email protected]

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Present Addresses

†Laboratories for Chemical Biology Umeå (LCBU), Chemical Biology Consortium Sweden (CBCS), Department of Chemistry, Umeå University, SE-901 87 Umeå.

Keywords: luciferin, protein kinase, inhibitor profiling, drug design, crystallography

1

ACCEPTED MANUSCRIPT Abstract D-Luciferin

is widely used as a substrate in luciferase catalyzed bioluminescence assays for

in vitro studies. However, little is known about cross reactivity and potential interference of D-luciferin

with other enzymes. We serendipitously found that that firefly inhibited the

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CDK2/Cyclin A protein kinase. Inhibition profiling of D-luciferin over a 103-protein kinase panel showed significant inhibition on a small set of protein kinases, in particular the DYRKfamily, but also other members of the CMGC-group, including ERK8 and CK2. Inhibition

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profiling on a 16-member focused library derived from D-luciferin confirms that D-luciferin represents a DYRK-selective chemotype of fragment-like molecular weight. Thus

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observation of its inhibitory activity the initial SAR information reported here promise to be

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useful for further design of protein kinase inhibitors with related scaffolds.

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ACCEPTED MANUSCRIPT 1. Introduction Novel compounds with protein kinase inhibitory properties may be highly valuable, and screening for such activity is therefore included in many bioprospecting efforts. The kinase

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RR[1] technology ("Reaction Rate") is a popular choice to identify fractions with protein kinase inhibitory activity; it was developed to enable real-time monitoring target peptide/protein phosphorylation by protein kinases.[2] In this assay, luciferase and D-luciferin

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are present during the entire kinase reaction, allowing continuous measurement of light emission, although for high throughput screening, measurement of light emission is required

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only at the beginning and after a pre-set time point. In the late evaluation process prior to the launch of Kinase RR, the assay was tested against a panel of 38 protein kinases selected to represent active site diversity. Although Kinase RR performed well and could be consistent against a reference assay method, there was one protein kinase that repeatedly failed; caseine

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kinase 2 (CK2). During the Kinase RR implementation in a bioprospecting screen, a similar failure was found for CDK2/Cyclin A. The reason for this failure could be attributed to be the direct inhibitory action of D-luciferin on the CDK2/Cyclin A complex. The finding that many

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protein kinases are unaffected by luciferin while a few kinases are inhibited by the compound prompted us to investigate whether D-luciferin inhibited additional protein kinases, and

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whether D-luciferin could serve as a scaffold for the construction of novel selective protein kinase inhibitors, by preparing a luciferin derived focused library and profiling this library against a large protein kinase panel.

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ACCEPTED MANUSCRIPT 2. Results 2.1 Preparation of inhibitors and protein kinase activity studies Kinase profiling assays of D-luciferin (1) at a concentration of 100 µM were performed with

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a 103-kinase panel at the International Centre for Kinase Profiling at the University of Dundee, UK.[3] The profile revealed that 21 kinases were inhibited significantly (≤ 25% remaining activity) by D-luciferin, but also that the majority of kinases were unaffected even

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at this relatively high concentration. Among the most luciferin sensitive kinases were the DYRK family (1a, 2 and 3 tested), Auroras B and A, CK2, PKBβ, VEGF-R GCK, RSK2,

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ERK8 and PIM3. The discovery that several kinases of potential interest as drug targets were inhibited by D-luciferin prompted us to prepare an exploratory structure-activity relationship (SAR) study on D-luciferin as a scaffold for design of kinase inhibitors (Figure 1).

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(Insert Figure 1.)

The SAR-library design was based on the assumption that the functional groups, i.e. the phenolic hydroxyl group on the benzothiazole ring system and the carboxylic acid on the

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dihydrothiazole ring of luciferin, were important for binding interactions with the affected kinases. These groups were substituted by methyl ether and methyl ester derivatives,

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respectively. Further variations included the replacement of the bicyclic benzothiazole by a quinoline, position of the hydroxyl group on the quinoline ring, the introduction of methyl groups in the dihydrothiazole ring, and the absolute configuration of the carboxylic acid substituent of the dihydrothiazole ring. The benzothiazole and quinoline SAR-libraries were prepared in the conventional manner as outlined in Scheme 1 (following White[4]) by condensing a 2-cyanobenzothiazole (or a 2-cyanoquinoline) with (R)- or (S)-cysteine (or (R)or (S)-penicillamine), with subsequent esterification of the carboxylic acid where necessary. This method is used in nearly all preparations of lucifeins today.[5] The advantage of 4

ACCEPTED MANUSCRIPT condensing cysteine derivatives with cyanobenzothiazoles or cyanoquinolines at a late stage in the process is that the process is mild, produces high yields and preserves the stereocenter of the cysteine derivative in the dihydrothiazole ring.[5] The preparative chemistry of Dluciferin has been recently reviewed.[6] phenols,

6-hydroxy-1,3-benzothiazole-2-carbonitrile

and

6-hydroxyquinoline-2-

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The

carbonitrile, were prepared from their corresponding commercially available methyl ethers by treatment with dry pyridinium chloride at 200 °C.[7] The individual members of this SAR-

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library are presented in Table 1.

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(Insert Scheme 1)

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Table 1. Structure and corresponding protein kinase inhibitory activity of compounds 1-16. Inhibitory activity is expressed as % remaining kinase activity at an inhibitor concentration of 100 µM.

R

Y

*

1

OH

H

H

S

2

OH

H

H

R

3

OH

H

4

OH

H

5

DYRK2

DYRK1a

DYRK3

Aurora A

Aurora B

CDK2/ Cyclin A

4

4

5

7

16

6

39

9

14

10

41

64

75

43

CH3 S

13

8

30

45

96

91

29

CH3 R

14

9

32

52

42

84

33

OCH3 H

H

31

24

55

84

76

82

92

6

OCH3 H

H

R

34

20

55

83

81

106

91

7

OCH3 H

CH3 S

15

41

15

66

43

83

33

8

OCH3 H

CH3 R

14

32

18

65

81

85

89

9

OCH3 CH3 H

S

10

27

21

12

57

109

59

10

OCH3 CH3 H

R

3

5

4

10

37

99

46

11

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S

OH

CH3 H

S

4

4

9

41

58

77

29

OH

CH3 H

R

1

2

5

21

26

88

12

6-OH

H

H

S

4

8

5

9

75

7

64

6-OH

H

H

R

17

21

25

34

95

21

66

15

8-OH

H

H

S

5

37

14

14

60

5

45

16

8-OH

H

H

R

27

52

49

52

78

6

45

12 13 14

dark grey: ≤ 15, light grey: ≤25

6

CK2

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Compound

13 - 16

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1 – 12

ACCEPTED MANUSCRIPT 2.2 Kinome Profiling Analysis Finally, the remaining 15 members of the SAR-library were subjected to kinase inhibition profiling using the same 103-kinase panel. All assays were performed with an ATP concentration at or below Km for the particular protein kinase. The complete results from the

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kinome profiling measured as per cent remaining protein kinase activity at a 100 M inhibitor concentration are given in the Supplementary Information (SI) section, and the

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inhibitory activity against a selected set of kinases is presented in Table 1. Strong inhibition was defined as < 15% remaining activity, and significant inhibition was defined as being in

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the range of 16% – 25% remaining activity. The Table in SI is sorted according to the average of the remaining activities for a particular protein kinase over all 16 compounds in the luciferin library. The protein kinases on the top of the Table are thus protein kinases that are strongly inhibited by many members of the luciferin library. The protein kinase that is most strongly inhibited across the luciferin library is DYRK2, with an average residual

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activity of less than 13%. The two other DYRK family kinases represented in the profiling set, DYRK1a and DYRK3, follow next on the list. PIM3, ERK8 and CK2 are three non-

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DYRK protein kinases that are strongly inhibited by many compounds in the luciferin library. Following these six kinases, the inhibition either becomes weaker or more irregular. Aurora

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B is also strongly inhibited by a few compounds in the library, but is virtually unaffected by the rest. Furthermore, a few kinases of established drug targeting interest are strongly inhibited by a single member of the library, such as PKBβ by compound 10 and PKCζ by compound 2. A subset of the full Table (SI), emphasizing the most strongly and broadly inhibited kinases, is reproduced as Table 1 in the main text.

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ACCEPTED MANUSCRIPT 2.3 Structure-Activity Relationships Most of the SAR-trends were kinase specific, but masking of the phenol moiety as a methoxy group generally reduced inhibitory activity compared with the corresponding phenolic

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compound across the kinase set (with DYRK1a as an exception, being slightly less affected than the other kinases). Esterification of the carboxylate generally reduced inhibitory activity for the phenolic compounds (e.g. compounds 3 and 4 versus compounds 1 and 2), however

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for the methoxy-compounds esterification increased activity (e.g. compounds 7 and 8 versus compounds 5 and 6), in particular for DYRK2 and DYRK3. Introduction of two methyl

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groups in the dihydrothiazole ring increased inhibitory activity, in particular for the methoxycompounds, rendering the methoxy-gem-dimethyl compounds 9 and 10 almost as effective as their phenolic counterparts 11 and 12. The stereochemistry of the carboxylate moiety is however more important for the methoxy-compounds 9 and 10, whereby the S-enantiomer 9

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is significantly more active than the R-enantiomer 10. This effect is greatly attenuated for the enantiomeric phenols 11 and 12. Otherwise, stereochemistry seems to play a surprisingly small role for the protein kinases that are promiscuously inhibited by members of the

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luciferin library. For the protein kinases that are more sensitive to scaffold modifications, such as CK2 and Aurora B, the effect of carboxylate stereochemistry can be significant (e.g.

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compounds 1 and 2) or irrelevant (compounds 9 and 10). There are only minor changes in the kinase inhibition pattern when the 2-benzothiazole-system is exchanged with a 2-quinoline system (e.g. compound 1 versus compound 13). The effect of carboxylate stereochemistry is however more significant for the 6-hydroxyquinoline derivatives than for corresponding 6hydroxybenzothiazole derivatives. Moving the hydroxyl-group from the 6-position to the 8position in the quinoline ring system created more marked changes. The 8-hydroxyderivatives were generally more active, in particular in relation to DYRK 1a and DYRK3.

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ACCEPTED MANUSCRIPT 2.4 Interaction with CDK2 The inhibitory action of D-luciferin on protein kinases was first discovered on CDK2/Cyclin A. Our laboratory has a high throughput screening of kinase structure by soaking on

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preformed CDK2 crystals, and the initial work and the structural studies were performed on this system. 2.4.1 Thermal shift assay

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A thermal shift assay was performed to see whether a binding of D-luciferin (1) to CDK2 could be detected by a change in melting temperature. The average thermal shift of the

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melting temperature was increased by 5 °C for the CDK2 kinase in the presence of Dluciferin. When the benzothiazole ring was replaced by a quinoline ring system as in compounds 13 and 15, the thermal shift was significantly reduced (Table 2).

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Table 2. Melting temperature (mT) in °C of CDK2 and complexes of CDK2 with inhibitors 1, 13 and 15. Compound

mT

mT

mT(DMSO)

blank

42.7

0

-

41.5

-1.2

0

43.4

+0.7

+1.9

15

43.9

+1.2

+2.4

1

47.7

+5

+6.2

DMSO

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2.4.2 X-ray crystal structures of CDK2 / inhibitor complexes To investigate the binding of D-luciferin and its derivatives to CDK2, a set of crystallization experiments were performed. Crystals of apo-CDK2 were soaked with different derivatives and measured at the BESSY II Synchrotron in Berlin. The statistics of the structures are summarized in Table 3. Complexation with compounds 1 and 15 were clearly visible in the

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ACCEPTED MANUSCRIPT electron density maps. Compound 15 had the best fit in the difference density, as seen in Figure 2. Weaker difference density showed the position of compound 1, which could be modelled into the map based on the quality of fit to the three strong difference density peaks, attributed to the two sulphur atoms and the hinge binding OH-group. Compound 13 showed

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unclear density for the fragment in the ATP pocket of CDK2. Compound 13 has only one sulphur atom and the difference density peak for this sulphur atom is weak compared to compound 1. Inhibitor 13 can still be modelled into the map based on the structure of the

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CDK2/compound 1 complex, but the CDK2/compound 13 complex is not completely covered by electron density even after refinement. The density map suggests rather that the

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ATP pocket in this crystal has mixed occupancy, with two partial occupancy water molecules binding to the hinge in addition to the partial occupancy inhibitor. (Insert Figure 2.)

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Compound 1 and 15 bind in different orientations in the ATP pocket of CDK2, as a result that can be attributed to the difference in the OH location in the benzothiazole and quinoline ring systems, respectively. For compound 15, the shift of the hydroxyl group to the 8-position of

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the quinoline ring results in a rotation of the inhibitor scaffold by approximately 90 degrees relative to D-luciferin (Figures 3 and 4), and a different residue in the hinge region is used for

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hydrogen bonding to the hydroxyl group. Compound 15 creates a hydrogen bond to the carbonyl of the main chain residue L83 (Gatekeeper+3) whereas compound 1 interacts similarly to the carbonyl from residue E81 (Gatekeeper+1). The hydroxyl oxygen atom in both inhibitors accept a hydrogen bond from the amide of L83. (Insert Figure 3.)

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ACCEPTED MANUSCRIPT Compound 1 has a length suitable to form an additional hydrogen bond between the carboxylate group and residue T14 in the glycine rich loop. Compound 15 penetrates deeper into the pocket, due to the change in the overall orientation, and therefore does not bind to the glycine rich loop, but makes a hydrogen bond to D145 instead. In Apo CDK2, D145 shares a

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hydrogen bond with K33 and this H-bond is disrupted by inhibitor 15 binding (Figure 4). (Insert Figure 4.)

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Compound 1 does not displace this interaction, but instead makes hydrogen bonds with both K33 and D145. A second difference between the binding modes of compound 1 and 15 is the

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orientation of the dihydrothiazole ring. For compound 1, the dihydrothiazole sulphur atom is oriented towards the solvent, whereas for compound 15 the dihydrothiazole ring is flipped 180 degrees and the sulphur atom is oriented towards the inside of the ATP pocket (Figure 3). The presence of weak but significant difference density (Figure 2, Panel C) adjacent to the

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sulphur atom in the dihydrothiazole ring of compound 1 might however indicate partial occupancy of the carboxylate also at this position along with an orientation of the dihydrothiazole ring similar to that of 15. Except for the differences in the binding pocket, the

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overall structures of the kinase in complexes CDK2/1 and CDK2/15 are very similar. In the CDK2/15 structure however, a second binding site for compound 15 in the C-lobe exists that

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is not present in the CDK2/1 structure (Figure 5). (Insert Figure 5.)

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DATA COLLECTION PDB code Synchrotron radiation Wavelength, Å Number of crystals used Number of frames Oscillation range / frame

CDK2/1 4D1X BESSY II 0.9184 1 130 1°

CDK2/15 4D1Z BESSY II 0.9184 1 100 1°

P212121 53.31, 72.12, 72.29

P212121 53.83, 72.41, 73.06

1

1

94018 18036

95708 24735

50.0-2.1 (2.15-2.10)

50.0-1.85 (1.89-1.85)

99.7 (98.6) 19.62 / 3.72

99.0 (96.7) 15.9 / 3.4

6.7% / 46.0%

4.9% / 37.8%

REFINEMENT

3. Discussion

30.2-1.85 (1.89-1.85)

16820 99.9 5.1 2162 18 117 0.194/0.238 28.6 Å2 0.004Å / 0.86° 97% / 3%

22727 99.0 5.1 2016 38 145 0.195/0.225 25.2 Å2 0.005 Å / 0.97° 98.4% / 1.6%

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42.9-2.1 (2.15-2.10)

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Resolution limits, Å (final shell) Number of used reflections Percentage observed % of free reflections Number of protein atoms Number of heterogen atoms Number of solvent atoms R factor overall/free Wilson B-factor RMS bonds/angles Ramachandran (favored / allowed)

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Space group Unit cell parameters, Å Protein molecules in asymmetric unit Number of measurements Unique reflections Resolution Range, Å (final shell) Completeness (final shell) I/σ (final shell) Merging R factor observed (final shell)

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DIFFRACTION DATA

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Table 3. Data collection and refinement statistics

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3.1 Structural interpretation of CDK2-SAR CDK2/Cyclin A is only modestly inhibited by the compounds of the SAR-library. There are however clear variations in the inhibitory activities for each member in the library due to the change of structural features (Table 1), and most of these trends coincide, albeit at a more attenuated level, with the trends observed for the more strongly inhibited protein kinases. The crystal structures provide insight into key features of binding the luciferin derivatives to CDK2 (Figures 3 and 4). The general loss of binding efficacy when the methoxy-group

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ACCEPTED MANUSCRIPT replaces the 6-hydroxy group is due to the loss of a hydrogen bond. The phenolic hydroxyl group in D-luciferin (1) group anchors the molecule at the kinase hinge segment via two hydrogen bonds: a hydrogen donor interaction to the carbonyl group of E81 (at the Gatekeeper+1 site) with a close O-O distance of 2.8 Å, and a hydrogen acceptor interaction

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with the amino group of L83 (Gatekeeper+3), with an O-N distance of 3.0 Å. An identical binding orientation for an inhibitor with a methoxy group substitution (e.g. compound 5) would not be possible due to steric repulsion from the methyl group. Furthermore, even a

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displaced methoxy group could function only as a weaker hydrogen bond acceptor.[6] This could explain the weaker binding of compounds 5, 6, and 8 compared to compounds 1-4.

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However, the exceptional tighter binding of compound 7, despite the methoxy substitution, requires further studies for clarification. Increase of the lipophilic steric bulk in the dihydrothiazole ring system as in the penicillamine derivatives 9 – 12 leads to more effective CDK2/Cyclin A inhibitors, a feature that may arise from solvophobic effects introduced by

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the geminal dimethyl moiety.

On the other hand, the gain in inhibitory efficacy by moving from the carboxylates (e.g. compound 1) to an ester moiety (e.g. compound 3) is not trivially explained. In the crystal

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structure of complex CDK2/1, there is an important interaction between the carboxylate and

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the salt bridge K33/D145, and it is expected that an esterification of the carboxylate would diminish this interaction. However, the inhibition profile studies are performed not on CDK2 alone, but on the CDK2/Cyclin A complex. Complexation of CDK2 with Cyclin A changes the conformation of the active site of CDK2 in accord with its function as activating cofactor.[8, 9] Of particular importance is the movement of helix C (the PSTAIRE-helix) into the catalytic cleft. This movement breaks the salt bridge between K33/D145 and introduces a new salt bridge between K33 and E51.[10] The carboxylate moiety of D145 will be left exposed to the carboxylate moiety of the luciferin derivatives and create a potential charge

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ACCEPTED MANUSCRIPT clash. This charge repulsion between D145 and the luciferin ligands will be reduced in the carboxylic ester derivatives, and hence explain their higher efficacy as inhibitors. Based on the understanding of the CDK2 structures and overall SAR data reported here, the

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SAR features for other individual kinases may be analysed based on published structures.

3.2 SAR of DYRK-family inhibition

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The members in the DYRK-family of the protein kinases are the most strongly inhibited

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enzymes by the compounds in the luciferin-library. DYRK2 is the most sensitive kinase in the DYRK-family and will serve as a model for the discussion. DYRK1a and DYRK3 are less affected by the inhibitors, but the inhibition pattern is similar to that of DYRK2, with minor differences. Protein kinases in the DYRK-family are members of the CMGC-kinases, and kinases from other families in the CMGC-group are also significantly inhibited, albeit to

are examples of this.

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a lesser degree, or by fewer members of the library. ERK 8 (a MAP-kinase), CK2 and CDK2

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DYRK2 is promiscuously inhibited by most compounds in the luciferin SAR-library, with only the methoxy carboxylates (compounds 5 and 6) as inactive inhibitors. Many DYRK-

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family kinase inhibitors have been reported in the literature (see e.g. [11] for a recent review). These falls into several diverse molecular frameworks including natural compounds such as β-carbolines[3]

and

acridone[12]

alkaloids,

flavanols,[13]

meridianines,[14]

and

lamellarines.[15] Furthermore, synthetic natural product derivatives[16] and fully synthetic molecular scaffolds[17-22] have been developed as kinase inhibitors. Despite this wealth of inhibitor scaffolds, only a few X-ray crystallographic structures of inhibitor/DYRK kinase complexes are available. These include complexes between DYRK1a and the benzothiazole

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ACCEPTED MANUSCRIPT INDY (3ANQ)[23], the β-carboline harmine[23] as well as two pyrido-pyrimidines (4MQ1 and 4MQ2),[24] and DYRK2 complexes with a bis-indole indigoid (3KVW)[25] or Leucettine L41 (4AZF and 4AZE).[26] A superposition of published DYRK/inhibitor crystal structures with the CDK2/1 complex (Figure 6) highlights several features, in particular that

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the volume of the active site occupied by the inhibitors are distinctly different. As shown in Figure 6, the inhibitors of the published DYRK1a complex structures interact via a phenolic hydroxy- or a methoxy-group from the inhibitor with the hinge region L241 at the Gatekeeper

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+3 position[23] in a manner similar to the interaction found in the CDK2/1 complex (vide supra). Furthermore, the inhibitors interact through a carbonyl or a pyridine nitrogen with the

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conserved ATP-binding K188 of DYRK1a in a manner similar to the interaction between the carboxylate of the luciferin inhibitors and K33 of CDK2. Likewise, in the DYRK2 complex with an indigoid inhibitor, similar interactions with the hinge backbone residues and the conserved lysine are observed. Despite this similarity in interaction sites between the

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published DYRK complexes and the CDK2/1 complex described here, Figure 6 clearly shows that the placement and orientation of the DYRK inhibitors differs significantly from 1 in CDK2. This effect can be related to the different shape of the ATP-pocket in the DYRK

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kinases and CDK2.

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The differences in surfaces surrounding the pocket may be attributed to a combination of sequence differences and possibly resultant conformation differences. At the C-lobe side of the binding pocket (in this orientation, at the base), residues isoleucine (DYRK2) and leucine (DYRK1a) restrict the volume relative to alanine of CDK2. Elsewhere at the base of the binding pocket, mutually conserved residues from the C-lobe adopt different rotamers. On the left, differing volumes and conformations of the Gatekeeper +2 residues (phenylalanine and methionine in CDK2 and Dyrk1A, respectively) restrict the volume in CDK2. This is linked to a structuring edge-face aromat-aromat interaction between the phenylalanine and the

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ACCEPTED MANUSCRIPT Gatekeeper +4 residue (histidine), which also induces a change in the hinge main chain conformation. Other differences in the shape of the ATP pocket arise from different conformations of the glycine rich loop, which may be due to inhibitor interactions and/or sequence differences; DYRK kinases have a phenylalanine as aromatic residue at the beta

differ strongly due to positions of the catalytic lysine.

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hairpin turn of the glycine-rich loop, while CDK2 has a tyrosine. Finally, the binding pockets

In CDK2, the kinase active site lysine K33 shares a salt bridge interaction with D145,

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creating the pillar like surface at the rear of the pocket and blocking contact to the gatekeeper

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phenylalanine. This may be an inhibitor-induced conformation, but seems most likely linked to the absence of a cyclin in the CDK2 structure (vide supra). These differences in the shape of the active site of DYRK2 and CDK2 do not offer an obvious explanation for why DYRK2 is more effectively inhibited by members of the luciferin SAR-library than CDK2/Cyclin A.

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(Insert Figure 6.) 3.3 SAR of CK2 inhibition

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CK2 has been a drug target for well over a decade, and many inhibitors have been identified, one of which has gone to clinical trials.[27] The inhibitors are chemically highly diverse, but

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many do possess a terminating carboxylic acid group reminiscent of the inhibitor series we report here. A superposition of these inhibitors using coordinates deposited in the PDB show that the CK2 carboxylate groups form one predominating cluster as salt bridge partners of the active site lysine (Figure 7). D-luciferin (1) and the quinoline cognate 13 both make an analogous interaction in CDK2, hence all effective inhibitors in the SAR series are carboxylates. Furthermore, adding to the importance of the carboxylate for inhibitor binding is the sensitivity for carboxylate stereochemistry, where the S-enantiomer is favoured over

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ACCEPTED MANUSCRIPT the R-enantiomer. The exception to this pattern is found for the most lipophilic carboxylates (compounds 9 and 10) where both enantiomers bind equally well. One distinguishing characteristic of CK2 is its ability to use GTP as the phosphate donor,[28]

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a feature shared with calcium/calmodulin-dependent protein kinase II,[29] mst3 kinase,[30] protein kinase C[31] and relatively few other protein kinases.[32] The structural mechanism by which this occurs in CK2 is that the guanine base, which has a hydrogen bond acceptor– donor–donor pattern (in contrast to adenine's donor–acceptor–empty pattern) binds to the

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protein kinase hinge slightly shifted in order to align the two acceptor positions. The shift

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creates a cavity, filled by a water molecule to match the adenine donor position. This anomalous property may be related to the anomalously strong CK2 inhibition of the quinoline derivatives. (Insert Figure 7.)

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3.4 SAR of Aurora A and B inhibition

A significant feature apparent from Table 1 is the inhibition of Aurora B by the quinoline-

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derived inhibitors 13, 14, 15, and 16. In contrast, Aurora A is not significantly inhibited by any of these inhibitors, despite its close similarity to Aurora B. This fact may provide key

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information to identify the mechanisms of selectivity that are operative for the quinolinederived inhibitors.

The general insensitivity to the position of the hydroxyl substituent suggests that the quinoline inhibitors bind to Aurora B without hinge binding to the hydroxyl group, unlike either of the CDK2 co-crystal structures described above. However, the precise position of the hydroxyl group does affect the sensitivity of Aurora B inhibition to changes in stereochemistry and the relative orientation of the carboxylate group relative to the thiazole

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ACCEPTED MANUSCRIPT ring. For the benzoimidazole compounds (1 vs 2), there is strong sensitivity to stereochemistry. For the 6-hydroxy quinoline acids (compounds 13 and 14), the sensitivity is weaker, and for the 8-hydroxy quinoline acids (compounds 15 and 16) it is absent entirely. These fundamental differences in SAR between CDK2/Cyclin A and the Aurora kinases are

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not reflected by any obvious differences in the active sites of the proteins, and experimental determination of binding modes in the Aurora kinases seems necessary to understand the

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3.6 Pairwise SAR effects on strongest inhibitors

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origins of the SAR observed for the Aurora kinases.

The exercise of explaining observed inhibitory modulation via pairwise comparisons that focus on individual binding features provides both a test and a summary of SAR hypotheses. 3.6.1. Stereochemistry of the carboxylic acid substituent

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As described above, the observed and mostly likely interaction of the carboxylate substituent in the luciferin derivatives is the active site lysine (see also Figure 7). If this interaction exists in strong inhibitors, as seems likely, changing the stereochemistry would either destroy the

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interaction and weaken the inhibition, or the interaction would be preserved by a combination

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of inhibitor rotation, thiazole rotation, and side chain flexibility. A more constrained ATP site and/or binding mode would thus be more sensitive to the stereochemistry change. Four pairs of inhibitors may be compared to test for these effects. Changing the stereochemistry of Dluciferin (1) produces compound 2, which greatly weakens binding to most kinases strongly inhibited by D-luciferin, however the effect on the DYRK-family is small. The stereochemistry shift between 9 and 10 does not change the strong inhibition of CK2 but stereochemical preference for the DYRK-family changes and now favours the R-enantiomer. The corresponding changes between the quinoline enantiomeric pairs of 13/14 and 15/16

18

ACCEPTED MANUSCRIPT show generally similar effects on DYRK 2, DYRK3, CK2, and Aurora B, with the Sconfiguration giving the strongest inhibition. 3.6.2. Neutralization of the carboxylic acid substituent by esterification

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If an anionic charge at carboxylate position is important (as we assume for active site lysine salt bridge formation), its neutralization via methylation will show weakened binding. Indeed, this is the case for the two pairs that involve strong inhibition of DYRK2 and DYRK3: 1/3

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and 2/4. DYRK1a is unaffected by this change. However, for the methoxy-compounds 5/7 and 6/8, the situation is reversed and the more lipophilic ester derivatives are more active

3.6.3. Modification of lipophilic bulk

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than the free acids.

The addition of methyl groups to the dihydrothiazole ring are seen with pairs 1/11, 2/12, 5/9, and 6/10. The addition of bulk to luciferin (1/11) is fully tolerated by the strongly inhibited

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kinases in the DYRK-family. The other strongly inhibited kinases loose activity upon methylation of the dihydrothiazole ring. The enantiomeric 2/12 with R-configuration at the

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carboxylate pair shows that methylation even increases inhibition of the DYRK-kinases. For the methoxylated pairs 5/9 and 6/10 differing only by the stereochemistry of the carboxylate

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on the dihydrothiazole, methylation is tolerated by the DYRK-kinases while the CK2-kinase is substantially more inhibited. 3.6.4. Modifications of the benzothiazole or quinoline phenol Methylation of the benzothiazole phenol eliminates strong binding when compared to the phenolic benzothiazoles, except for 9 and 10 inhibition of CK2 and DYRK2. This is consistent with most binding modes involving hydrogen bonding of the alcohol to the hinge. Unique characteristics of CK2 and DYRK2 have been discussed above and include GTP

19

ACCEPTED MANUSCRIPT binding to CK2 and the generally strong inhibition of DYRK2 by all compounds. The methoxy derivatives require a carboxylic acid for CK2 inhibition. In striking contrast to benzothiazoles, the quinolines inhibit only Aurora B, CK2 and DYRK2 strongly, as discussed

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above. Binding patterns for these three proteins are qualitatively similar.

4. Conclusion

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The observation that D-luciferin inhibits quite selectively several protein kinases in the CMGC-group has two important consequences. First, D-luciferin and cognates represent a

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useful fragment-like scaffold for protein kinase inhibitor design with an interesting intrinsic selectivity for the DYRK-family of protein kinases complementing the already known molecular scaffolds. Furthermore, the profiling and SAR analysis here also demonstrate good potential for achieving a wide range of selectivity patterns among the other drug target

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kinases described here. With multiple binding geometries at the hinge, and multiple positions for further derivatisation, the scaffolds provide myriad possibilities for drug design. On the other hand, the multiple binding geometries limits the extension of the qualitative

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understanding of the SAR derived from the structures of CDK2 complexes to other kinases.

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Second, the potential for ligand interactions to be shared between protein kinases and luciferases deserves close attention, as luciferin/luciferase based assays are in widespread use. Awareness of this possibility may aid interpretation of apparently anomalous results.

5. Experimental 5.1 Protein Expression, Purification and Crystallization

20

ACCEPTED MANUSCRIPT CDK2 was expressed with an N-terminal His-tag in BL21(DE3) cells. The protein was purified via a His-tag column (50 mM sodium phosphate, 500 mM NaCl, pH 7.0) and eluted via an imidazole gradient (50 mM sodium phosphate, 300 mM NaCl, 500 mM imidazole pH 7.0). The combined fractions with the CDK2 were loaded on a S200 16/60 gelfiltration

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column (10 mM Hepes, 20 mM NaCl, 2 mM DTT, pH 7.4). CDK2 formed crystals spontaneously after concentrating the fractions from the gel filtration above 6 mg/ml at 4 °C.

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5.2 Structure determination

Crystals were soaked with either D-luciferin or the derivatives and cryoprotected with

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glycerol prior to the freezing. The crystals were measured at the Berlin Electron Storage Ring Society for Synchrotron Radiation (BESSY II) in Berlin/Germany. 5.3 Thermal shift assay

20 µl of CDK2 wild-type protein 0.7-0.8 mg/ml (50 mM phosphate buffer pH 7.5, 150 mM

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NaCl) was mixed with 5 µL 6% SyproOrange (Sigma). 1 µL of DMSO or 1µL of 10 mM inhibitor dissolved in DMSO was added. The thermal shift assay was done on a Miniopticom

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(Biorad) by heating the sample slowly from 20 °C to 90 °C with a step interval of 1/3 °C. All experiments were performed in triplicate.

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5.4 Preparation of inhibitors

The inhibitors were prepared by a condensation between cysteine and a benzothiazole nitrile or a quinoline nitrile derivative. Demethylation of the aromatic methoxy group or esterification of the dihydrothiazolecarboxylate group was preformed as needed. 1H-NMR and ESI mass spectral data for all compounds were in accordance with the literature and are given in the Supplementary Information. 5.5 Determination of protein kinase inhibition

21

ACCEPTED MANUSCRIPT The determination of protein kinase inhibition was performed at the International Centre for Kinase Profiling at the University of Dundee, U.K. The method used is a radioactive filter binding assay using 33P ATP.[3, 33] The ATP concentrations were at or below the calculated

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Km for ATP for each particular kinase.

ACKNOWLEDGEMENT

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Provision of beam time at Bessy II, Berlin Germany at BL14.1 is gratefully acknowledged. Special thanks to the Helmholtz center, for supporting the travel expenses for Ulli

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Rothweiler.

REFERENCES

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[1] A. Lundin, J. Eriksson, A real-time bioluminescent HTS method for measuring protein kinase activity influenced neither by ATP concentration nor by luciferase inhibition, Assay and drug development technologies, 6 (2008) 531-541. [2] P. Singh, B.J. Harden, B.J. Lillywhite, P.M. Broad, Identification of kinase inhibitors by an ATP depletion method, Assay and drug development technologies, 2 (2004) 161-169. [3] J. Bain, L. Plater, M. Elliott, N. Shapiro, C.J. Hastie, H. McLauchlan, I. Klevernic, J.S. Arthur, D.R. Alessi, P. Cohen, The selectivity of protein kinase inhibitors: a further update, The Biochemical journal, 408 (2007) 297-315. [4] E.H. White, F. McCapra, G.F. Field, The structure and synthesis of firefly luciferin, J. Am. Chem. Soc., 85 (1963) 337-343. [5] D.C. McCutcheon, M.A. Paley, R.C. Steinhardt, J.A. Prescher, Expedient synthesis of electronically modified luciferins for bioluminescence imaging, J Am Chem Soc, 134 (2012) 7604-7607. [6] G. Meroni, M. Rajabi, E. Santaniello, D-Luciferin, derivatives and analogues: synthesis and in vitro/in vivo luciferase-catalyzed bioluminicent activity, ARKIVOC, 2009 (2009) 265288. [7] H.-J. Böhm, S. Brode, U. Hesse, G. Klebe, Oxygen and Nitrogen in Competitive Situations: Which is the Hydrogen-Bond Acceptor?, Chemistry – A European Journal, 2 (1996) 1509-1513. [8] P.D. Jeffrey, A.A. Russo, K. Polyak, E. Gibbs, J. Hurwitz, J. Massague, N.P. Pavletich, Mechanism of CDK activation revealed by the structure of a cyclinA-CDK2 complex, Nature, 376 (1995) 313-320. [9] A.A. Russo, P.D. Jeffrey, A.K. Patten, J. Massague, N.P. Pavletich, Crystal structure of the p27Kip1 cyclin-dependent-kinase inhibitor bound to the cyclin A-Cdk2 complex, Nature, 382 (1996) 325-331.

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[10] R.A. Engh, D. Bossemeyer, The protein kinase activity modulation sites: mechanisms for cellular regulation - targets for therapeutic intervention, Advances in enzyme regulation, 41 (2001) 121-149. [11] B. Smith, F. Medda, V. Gokhale, T. Dunckley, C. Hulme, Recent advances in the design, synthesis, and biological evaluation of selective DYRK1A inhibitors: a new avenue for a disease modifying treatment of Alzheimer's?, ACS chemical neuroscience, 3 (2012) 857-872. [12] M.A. Beniddir, E. Le Borgne, B.I. Iorga, N. Loaec, O. Lozach, L. Meijer, K. Awang, M. Litaudon, Acridone alkaloids from Glycosmis chlorosperma as DYRK1A inhibitors, Journal of natural products, 77 (2014) 1117-1122. [13] J. Bain, H. McLauchlan, M. Elliott, P. Cohen, The specificities of protein kinase inhibitors: an update, The Biochemical journal, 371 (2003) 199-204. [14] M. Gompel, M. Leost, E.B. De Kier Joffe, L. Puricelli, L.H. Franco, J. Palermo, L. Meijer, Meridianins, a new family of protein kinase inhibitors isolated from the ascidian Aplidium meridianum, Bioorganic & medicinal chemistry letters, 14 (2004) 1703-1707. [15] C. Neagoie, E. Vedrenne, F. Buron, J.Y. Merour, S. Rosca, S. Bourg, O. Lozach, L. Meijer, B. Baldeyrou, A. Lansiaux, S. Routier, Synthesis of chromeno[3,4-b]indoles as Lamellarin D analogues: a novel DYRK1A inhibitor class, European journal of medicinal chemistry, 49 (2012) 379-396. [16] M. Debdab, F. Carreaux, S. Renault, M. Soundararajan, O. Fedorov, P. Filippakopoulos, O. Lozach, L. Babault, T. Tahtouh, B. Baratte, Y. Ogawa, M. Hagiwara, A. Eisenreich, U. Rauch, S. Knapp, L. Meijer, J.P. Bazureau, Leucettines, a class of potent inhibitors of cdc2like kinases and dual specificity, tyrosine phosphorylation regulated kinases derived from the marine sponge leucettamine B: modulation of alternative pre-RNA splicing, Journal of medicinal chemistry, 54 (2011) 4172-4186. [17] P. Kassis, J. Brzeszcz, V. Beneteau, O. Lozach, L. Meijer, R. Le Guevel, C. Guillouzo, K. Lewinski, S. Bourg, L. Colliandre, S. Routier, J.Y. Merour, Synthesis and biological evaluation of new 3-(6-hydroxyindol-2-yl)-5-(Phenyl) pyridine or pyrazine V-Shaped molecules as kinase inhibitors and cytotoxic agents, European journal of medicinal chemistry, 46 (2011) 5416-5434. [18] Y. Loidreau, P. Marchand, C. Dubouilh-Benard, M.R. Nourrisson, M. Duflos, O. Lozach, N. Loaec, L. Meijer, T. Besson, Synthesis and biological evaluation of Narylbenzo[b]thieno[3,2-d]pyrimidin-4-amines and their pyrido and pyrazino analogues as Ser/Thr kinase inhibitors, European journal of medicinal chemistry, 58 (2012) 171-183. [19] Y. Loidreau, P. Marchand, C. Dubouilh-Benard, M.R. Nourrisson, M. Duflos, N. Loaec, L. Meijer, T. Besson, Synthesis and biological evaluation of N-aryl-7methoxybenzo[b]furo[3,2-d]pyrimidin-4-amines and their N-arylbenzo[b]thieno[3,2d]pyrimidin-4-amine analogues as dual inhibitors of CLK1 and DYRK1A kinases, European journal of medicinal chemistry, 59 (2013) 283-295. [20] L. Demange, F.N. Abdellah, O. Lozach, Y. Ferandin, N. Gresh, L. Meijer, H. Galons, Potent inhibitors of CDK5 derived from roscovitine: synthesis, biological evaluation and molecular modelling, Bioorganic & medicinal chemistry letters, 23 (2013) 125-131. [21] E. Deau, Y. Loidreau, P. Marchand, M.R. Nourrisson, N. Loaec, L. Meijer, V. Levacher, T. Besson, Synthesis of novel 7-substituted pyrido[2',3':4,5]furo[3,2-d]pyrimidin-4-amines and their N-aryl analogues and evaluation of their inhibitory activity against Ser/Thr kinases, Bioorganic & medicinal chemistry letters, 23 (2013) 6784-6788. [22] O. Dehbi, A. Tikad, S. Bourg, P. Bonnet, O. Lozach, L. Meijer, M. Aadil, M. Akssira, G. Guillaumet, S. Routier, Synthesis and optimization of an original V-shaped collection of 4-7disubstituted pyrido[3,2-d]pyrimidines as CDK5 and DYRK1A inhibitors, European journal of medicinal chemistry, 80 (2014) 352-363.

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[23] Y. Ogawa, Y. Nonaka, T. Goto, E. Ohnishi, T. Hiramatsu, I. Kii, M. Yoshida, T. Ikura, H. Onogi, H. Shibuya, T. Hosoya, N. Ito, M. Hagiwara, Development of a novel selective inhibitor of the Down syndrome-related kinase Dyrk1A, Nature communications, 1 (2010) 86. [24] K. Anderson, Y. Chen, Z. Chen, R. Dominique, K. Glenn, Y. He, C. Janson, K.C. Luk, C. Lukacs, A. Polonskaia, Q. Qiao, A. Railkar, P. Rossman, H. Sun, Q. Xiang, M. Vilenchik, P. Wovkulich, X. Zhang, Pyrido[2,3-d]pyrimidines: discovery and preliminary SAR of a novel series of DYRK1B and DYRK1A inhibitors, Bioorganic & medicinal chemistry letters, 23 (2013) 6610-6615. [25] M. Soundararajan, A.K. Roos, P. Savitsky, P. Filippakopoulos, A.N. Kettenbach, J.V. Olsen, S.A. Gerber, J. Eswaran, S. Knapp, J.M. Elkins, Structures of Down Syndrome Kinases, DYRKs, Reveal Mechanisms of Kinase Activation and Substrate Recognition, Structure, 21 (2013) 986-996. [26] T. Tahtouh, J.M. Elkins, P. Filippakopoulos, M. Soundararajan, G. Burgy, E. Durieu, C. Cochet, R.S. Schmid, D.C. Lo, F. Delhommel, A.E. Oberholzer, L.H. Pearl, F. Carreaux, J.P. Bazureau, S. Knapp, L. Meijer, Selectivity, cocrystal structures, and neuroprotective properties of leucettines, a family of protein kinase inhibitors derived from the marine sponge alkaloid leucettamine B, Journal of medicinal chemistry, 55 (2012) 9312-9330. [27] G. Cozza, L.A. Pinna, S. Moro, Protein kinase CK2 inhibitors: a patent review, Expert Opin Ther Pat, 22 (2012) 1081-1097. [28] K. Niefind, M. Putter, B. Guerra, O.G. Issinger, D. Schomburg, GTP plus water mimic ATP in the active site of protein kinase CK2, Nature structural biology, 6 (1999) 1100-1103. [29] S.L. Bostrom, J. Dore, L.C. Griffith, CaMKII uses GTP as a phosphate donor for both substrate and autophosphorylation, Biochemical and biophysical research communications, 390 (2009) 1154-1159. [30] K. Schinkmann, J. Blenis, Cloning and characterization of a human STE20-like protein kinase with unusual cofactor requirements, The Journal of biological chemistry, 272 (1997) 28695-28703. [31] M. Gschwendt, W. Kittstein, K. Kielbassa, F. Marks, Protein-Kinase C-Delta Accepts Gtp for Autophosphorylation, Biochemical and biophysical research communications, 206 (1995) 614-620. [32] C.A. Smith, M. Toth, H. Frase, L.J. Byrnes, S.B. Vakulenko, Aminoglycoside 2''phosphotransferase IIIa (APH(2'')-IIIa) prefers GTP over ATP: structural templates for nucleotide recognition in the bacterial aminoglycoside-2'' kinases, The Journal of biological chemistry, 287 (2012) 12893-12903. [33] C.J. Hastie, H.J. McLauchlan, P. Cohen, Assay of protein kinases using radiolabeled ATP: a protocol, Nature protocols, 1 (2006) 968-971.

24

ACCEPTED MANUSCRIPT Captions to the Figures and Schemes. Figure 1. SAR variations applied to the D-luciferin scaffold Scheme 1. Outline of the preparation of the benzothiazole SAR-library. The quinoline library

RI PT

was prepared by the same methods. Reagents and conditions: (a) Na2CO3 (aq), rt, 3h; (b) SOCl2, Methanol, rt, 1h.

Figure 2. ATP binding pocket of CDK2. A and B: electron density of the inhibitors before

SC

the refinement, and C and D: after the refinement. A and C represents the complex between CDK2 and 1, whereas B and D represents the complexes with 15. The electron density is fference density in green and red at 2.5 σ for A and B, and at 3 σ

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shown in blue at 1 σ for C and D.

Figure 3. Orientation of 1 (magenta) and 15 (orange) in the ATP binding pocket of CDK2. Compound 15 is rotated approximately by 90 degrees relative to 1 and penetrates deeper into

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the ATP pocket. The protein part of CDK2 in complex with 1 is represented in green; with 15 using yellow carbon atoms. The position of the OH group on the benzothiazole and quinoline rings defines the orientation and binding mode of the inhibitor. Both inhibitors bind to the

EP

hinge, but use different backbone carbonyl groups for the H-bond.

AC C

Figure 4. Binding pocket of CDK2 bound to compounds 1 (A) and 15 (B).

Figure 5: Superposition of CDK2/1 (green) and CDK2/15 (yellow). The CDK2/15 structure has a secondary binding site for compound 15 in the C-lobe not present in the CDK2/1 structure. Figure 6. DYRK1a kinases (3ANR, 3ANQ, cyan) have a differently shaped ATP site in comparison with the CDK2/1 structure (yellow) of this study, due to conformation or rotamer

25

ACCEPTED MANUSCRIPT differences in combination with differences in sequence. As a result, luciferin occupies a volume distinct from that of the DYRK inhibitors (see text). Figure 7. Carboxylate groups of CK2 inhibitors cluster as salt bridge partners to the active

AC C

EP

TE D

M AN U

SC

RI PT

site lysine.

26

N

S

EP

TE

D

HO

AC C

Hydrogen bond donation

Stereochemistry

M AN U

Ring size and geometry

SC

RI PT

ACCEPTED MANUSCRIPT

O

N

OH H

S

Lipophilic bulk

Removal of acidic proton by esterification

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

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ACCEPTED MANUSCRIPT

To

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Supplementary information

Luciferin and derivatives as a DYRK selective

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scaffold for the design of protein kinase

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inhibitors

Ulli Rothweiler1, Jonas Eriksson2,†, Wenche Stensen2,3, Frederick Leeson2,3, Richard A. Engh1,* and John S. Svendsen2,3,*

1

The Norwegian Structural Biology Centre, Department of Chemistry, UiT The Arctic

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University of Norway, N-47 Tromsø, Norway, 2Lytix Biopharma AS, P.O. Box 6447, Tromsø Science Park, N-9294 Tromsø, Norway and 3Department of Chemistry, UiT

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The University of Tromsø, N-47 Tromsø, Norway.

1

ACCEPTED MANUSCRIPT

Table. Protein kinase inhibitory activity of compounds 1-16. Inhibitory activity is expressed as % remaining kinase activity at an inhibitor concentration of 100 µM. 1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

SUM

DYRK2

4

9

13

14

31

34

15

14

10

3

4

1

4

17

5

27

205

DYRK1A

4

14

8

9

24

20

41

32

27

5

4

2

8

21

37

52

307

5

25

14

49

352

9

23

7

13

372

15

19

24

30

381

10

30

32

55

55

15

18

21

4

9

5

16

2

36

37

48

44

36

32

29

10

20

11

ERK8

15

11

24

31

54

52

23

24

19

12

18

11

7

41

45

52

84

83

66

65

12

10

41

21

IRR MAPKA P-K3

36

25

34

42

57

63

54

50

56

42

27

26

33

43

79

41

18

28

50

54

45

16

62

48

MNK2

17

52

59

58

83

88

64

66

30

30

PIM1

33

0

57

66

72

74

58

42

65

33

VEG-FR

14

39

72

64

57

53

64

68

52

26

HIPK2

20

19

55

59

74

85

67

82

40

23

TrkA

34

53

65

68

59

25

28

61

MNK1

23

55

61

95

81

75

BTK CDK2Cyclin A

36

88

53 11 3

62 10 8

54

52

52

73

39

43

29

33

92

91

GSK3b

27

57

37

45

70

NUAK1

28

14

65

65

MINK1

29

30

84

RSK2

15

40

85

CAMK1

52

2

BRSK2

31

GCK

15

PKBb

14

49

79

46

RIPK2

53

27

52

MLK3

37

29

80

35

HER4 S6K1

34

14

52

635

32

44

52

670

8

26

77

54

681

24

13

704

34

24

26

36

23

28

50

28

48

724

33

19

39

49

50

742

30

19

26

49

40

53

743

30

37

13

60

74

47

64

781

30

24

44

24

24

39

22

27

785

60

73

15

21

51

20

29

28

31

795

33

89

59

46

29

12

64

66

45

45

814

66

34

88

56

49

25

16

59

73

68

59

831

78

81

35

76

78

56

39

31

41

57

53

834

64

46

55

53

47

48

26

47

22

33

67

68

37 13 0

49

28

31

66

83

53

52

63

60

23

55

44

32

28

58

74

68

36

48

52

32

60

83 10 1

863

68

78 11 9

874

27

86

73

60

66

74

69

68

46

63

41

50

63

34

36

887

32

67

69

72

73

68

72

69

48

26

16

47

69

78

894

26

29

61

73

62

14

67

44

86

85

73 10 3

80

917

75

88

92

64

72

61

60

52

24

59

66

42

918

88

76

78

62

80

51

22

43

32

32

49

60

30 11 1

83

89

76

89

94

76

44

64

35

36

56

32

45

931

TE D

M AN U

48

EP

PIM2

9

28

44

AC C

CK2

SC

5

PIM3

DYRK3

RI PT

Kinase ID

66

848

931

24

11 10 4

91

55

43

51

58

55

68

69

21

42

48

50

68

85

932

39

15

80

45

33

45

69

80

85

50

62

59

48

65

87

70

933

ROCK 2

16

49

61

60

61

72

71

69

80

55

42

31

43

55

88

92

945

CHK2

28

34

32

56

60

81

71

55

37

81

56

62

78

75

77

946

BRSK1

24

21

71

77

82

63 10 4

88

77

75

49

58

36

48

53

53

952

MELK

49

26

60

72

65

83

68

71

66

49

52

32

40

44

37 10 5

89

972

Aurora A

16

64

96

42

76

81

43

81

57

37

58

26

75

95

60

78

984

IKKe

52

41

74

81

45

58

55

76

52

65

51

59

38

70

88

88

994

TTK

54

33

56

64

71

73

69

78

70

61

56

50

61

68

78

65

1009

LKB1

58

49

62

71

84

85

81

79

72

68

54

36

52

62

50

54

1016

2

Kinase ID

1

2

3

4

5

11

12

13

14

15

16

SUM

Aurora B

6

75

91

84

99

77

88

7

21

5

6

1022

HIPK3

53

31

68

MSK1 SmMLC K

44

24

65

74

93

68

54

55

46

42

62

66

86

1039

76

73

75

63

43

30

73

85

86

81

1044

51

3

85

76

82

64

45

67

57

68

74

78

66

1049

SRPK1

29

PRAK

62

72

70

86

67

51

77

72

30

57

66

66

1050

62

85

95

68

18

79

53

83

85

72

69

30

59 10 8

1051

82

90

92

85

75

65

57

83

61

42

42

29

51

39

42

62

50 11 5

73 11 1

58 13 4

1065

78

44 11 1

26

81

96

73

89

84

52

72

74

59

49

74

57

65

1073

37

18

81

91

90

87 10 0

72

88

70

40

PKD1

49

39

85

76

69

80

95

81

32

AMPK

48

48

62

74

90

75 10 6

87

89

78

48

TAK1

59

8

81

98

89

99

60

75

89

58

70

50

83

81

EPH-A2

96

89

95

93

74

83

89

97

97

67

95

74

30

32

PAK4

39

82

89

70

69

87

76

69

82

81

75

73

84

99

21

32

15

94

85

80

63

39

92 10 0

65

PHK

97 10 1

78 10 8

82

23

70

HIPK1

63

20

75

88

86

82 10 4 10 4 10 2

79

FGF-R1

70 11 3

80

99

87

70

61

NEK2a

41

52

76

92

62

58

84

91

90

61

MARK3

40

65

75

94

85

PRK2

68

7

76

85

IRAK4

60

42

91

IKKb

65

50

RSK1

47

MKK1

51

MST4

75

PLK1

8

83

79

81

80

70

74

81

74

86

74

94

83

67

60

22

74

67

SGK1

31

40

85

IGF-1R CAMKK b

50

19

36

MLK1

TE D

98

1070

66

52

62

68

78

92

1107

67

69

66

85

75

1123

52

39

60

69

80 10 9

83

1139

79

69

1149

18

25

1154

61

39

1154

31

48

48

1162

61

88

57

78

1165

38

66

82

85

54

68

96 10 8

1193

71

80 10 0

1195

M AN U

7

82

6 10 6

RI PT

10

85

9 10 9

SC

ACCEPTED MANUSCRIPT

88

77

47

80

69

83

85

92

72

1204

84

96

89

98

63

42

80

85

91

63

1217

86

90

88

84

95

96

94

95

75

79

76

49

33

1233

76

73

77

70

75

81

83

84

61

70

96

87

62

69

92

87

92

90

87

62

74

44

80

94

90 11 2

1234

29

96 11 5

1235

37

85 10 0

83

95

77

88

88

82

71

60

92

69

89

83

1244

18

92 10 1

92

88

84

95

90

69

78

47

91

92

39

51

49

73

73

66

47

75

67

78 11 0

84 10 8

81 10 9

1245

87

66 11 0

TBK1

64

99

89

87

90

98

76

84

66

60

98

78

67

65

82

53

1255

MARK4

57

88

97

88

81

79

83

84

81

47

85

73

77

80

81

76

1256

PKCa

69

91

91

77

56

65

76

87

80

87

82

67

67

89

48

48

70

82

93

90

82

96

86

76

69

51

62

84

90 11 7

1258

MST2

1263

PKBa

82

28

84

80

68

78

79

78

48

94

88

94

93

84

1268

EF2K

53

32

78

68

83

86

94

93

83

84

89

98

77

1269

43

67

72

86

91

94

93

81 10 0

65

CK1δ

90 10 5 10 5

83 10 7 10 0

97

68

46

84

84

83

67

1282

JAK2

66

69

95

88

87

50

97

96

92

74

61

86

93

68

70

1283

MARK2

59

55

90

90 10 1

82

95

88

89

82

57

82

79

68

85

92

80

1283

AC C

EP

73

87

79 10 4

1247

3

ACCEPTED MANUSCRIPT

1

2

3

4

5

6

7

8

9

10

11

12

13

38

36

92

87

79

74

77

85

93

56

89

74

PKA

80

52

89

86

78

91

82

83

75

83

87

YES1

75

60

94

86

88

96

72

87

86

35 11 4

94 10 2

94

72

PAK5

66

58

87

99

84

93

83

85

89

61

94

87

MARK1

35

71

89

87

96

84

96

91

81

79

75

74

94

95

77

85

82

75 13 7

91

JNK3 p38b MAPK

99 10 0

83

31

81

67

72

88

90

82

35

78

92

95

91

89

95

81

93

32

99

82

84

94

92

84

78

ERK1

48

75

94

96

70

76

75

67

84

99

92

85

38

46

88

93

99

87

50

9

80

96

94

92

98

76

83

ERK2

57

80

93

98 10 6

83

98

86

90

71

SYK

68

86

90

83 10 3 11 1 10 0

85

PKCz

79 10 2

74 11 0

59

EPH-B3

70 11 0

81 10 9 10 6

87

NEK6

84 10 1

96 10 7 10 3

89 10 0

56 10 1

IR p38d MAPK

68 10 0 10 3 10 2

71 10 6 10 2

99

70

50

95

96 10 2

90

PAK2

87 10 2

85

89

90 10 3

CSK

93

54

76

82

94

97

PAK6

92

67

97

90 10 1

88

JNK2 p38g MAPK

92

64

90

50

87 10 1

98 10 0

EPH-B4

72

91

97

92 11 1 10 3

MEKK1

82 75

JNK1

85

81

97 10 3 10 6

95

PDK1

96 14 5

Lck

83

97

CHK1 p38a MAPK

91

70

97

Src

99

63 18 2

EPH-A4

80

77

90 87 86

96 11 4

99 11 0 11 7 10 5 10 1

82

93

84

93 11 1

95

82

99 10 2

97

91

88

99

84

98

95

92

90

98

90

88

97

97

97 10 7 10 2 12 1

99 89

92 10 3 11 1 10 9

88

96

90

99

EP

90

91

AC C

89 10 3 10 6

98 10 9 83 10 1

80 88 10 8 89 21 9

87 10 6 97 87 98

SUM

67

1288

57

61

78

89

1308

94

99

79

61

1319

1287

89

87

80

1323

95

77

86

1352

92

95

97

71 11 3

1357

59

83

79

84

1359

92

75

85

94

95

86 10 1

97

98

79 10 1 10 3

1399

94

84 11 5

46

92 11 0 11 1 10 6

82 11 8 10 3

1373

58

85 10 7 10 7

85

91

87

95

72

1417

90

89

91

89

68

1419

90

90

81

96

80

1423

87

89

96

99 10 1

95

89

75

1433

87

82

82

99

93

92

86

1434

90 11 1 10 1

89 10 3

86

91

86

97

85

1501

95

92

81

86 11 0

94 11 1

94

90

80

1539

98 10 2

97 11 2 12 7

1552

90 10 5

91 11 4 10 1

1548

84 12 1

94 10 2 10 0

97

98 10 4 10 0 10 9 10 0

99 10 5 10 6

93 10 2 10 4 10 4

88 11 9

1517

86

97 11 1

97

1573

87 10 6

73 10 3

1614

SC

72

M AN U

85

TE D

91 11 2 10 2

91

16 10 7

96

15 10 5 10 1

14 10 1

RI PT

Kinase ID MAPKA P-K2

92

99

97 11 8 87

97 86

92

90

76 10 2 11 5

83

95

99 10 1

83

87

93

94

1378 1399

1404 1410

1534

1561

1638

4

ACCEPTED MANUSCRIPT

Starting materials: 6-Methoxybenzo[d]thiazole-2-carbonitrile, 6-methoxyquinoline-2carbonitrile and 8-hydroxyquinoline-2-carbonitrile were purchased from SigmaAldrich.

RI PT

General Procedure for Demethylation. The methoxycarbonitrile derivative (1 eq) was mixed with pyridinium chloride (5 eq) under argon and heated to 210°C for 30 minutes. The resulting mixture was partitioned between distilled water and dicloromethane, and the organic layers were concentrated under vacuum. The crude

product was dissolved in 5 % Na2CO3 (50 ml) and filtered before addition of HCl

SC

until pH ≈4.0. The aqueous layer was extracted with dichloromethane (50 mL) and

M AN U

the organic layers removed under vacuum yielding pure product (>98%).

6-Hydroxybenzo[d]thiazole-2-carbonitrile.[1] The title compound was prepared in 70% yield from 6-methoxybenzo[d]thiazole-2-carbonitrile according to the general procedure for demethylation. 1H NMR (400 MHz, CD3OD) δ 7.99 (d, J = 9.0 Hz,

TE D

1H), 7.40 (d, J = 2.4 Hz, 1H), 7.17 (dt, J = 9.0, 1.7 Hz, 1H).

6-Hydroxyquinoline-2-carbonitrile.[2] This title compound was prepared in 75% yield from 6-methoxyquinoline-2-carbonitrile according to the general procedure for

EP

demethylation. 1H NMR (400 MHz, CD3OD) δ 8.26 (d, J = 8.5 Hz, 1H), 7.95 (d, J = 9.3 Hz, 1H), 7.71 (dd, J = 8.5, 1.2 Hz, 1H), 7.45 (ddd, J = 9.2, 2.8, 1.2 Hz, 1H), 7.18

AC C

(d, J = 2.6 Hz, 1H).

General Procedure for Condensation Reaction. The hydroxy- or methoxycarbonitrile derivative (1 eq) was added to the cysteine derivative (1.05 eq) and sodium carbonate (3 eq) in 5 ml water. The mixture was stirred at room temperature for three hours before addition of dilute HCl (1 M) to pH ≈ 3.5 – 4.0. The product was isolated by

extraction with diethyl ether, washed by water, followed by evaporation

5

ACCEPTED MANUSCRIPT

(S)-2-(6-Hydroxybenzo[d]thiazol-2-yl)-4,5-dihydrothiazole-4-carboxylic acid (DLuciferin, 1)[1] was prepared from (S)-cysteine and 6-hydroxybenzo[d]thiazole-2carbonitrile according to the general procedure for condensation. ESMS 280.8 (calcd

RI PT

281.00, M + H+). 1H NMR (400 MHz, CD3OD) δ 7.90 (d, J = 8.9 Hz, 1H), 7.35 (d, J = 2.4 Hz, 1H), 7.07 (dd, J = 8.9, 2.4 Hz, 1H), 5.39 (dd, J = 9.5, 8.8 Hz, 1H), 3.79 –

SC

3.73 (m, 2H).

(R)-2-(6-Hydroxybenzo[d]thiazol-2-yl)-4,5-dihydrothiazole-4-carboxylic acid (L-

M AN U

Luciferin, 2)[3] was prepared from (R)-cysteine and 6-hydroxybenzo[d]thiazole-2carbonitrile according to the general procedure for condensation. ESMS 280.7 (calcd 281.00, M + H+). 1H NMR (400 MHz, CD3OD) δ 7.87 (dd, J = 9.0, 2.4 Hz, 1H), 7.32 (d, J = 2.6 Hz, 1H), 7.04 (dt, J = 9.3, 2.5 Hz, 1H), 5.17 (td, J = 9.4, 2.5 Hz, 1H), 3.71

TE D

(dt, J = 10.1, 3.3 Hz, 2H).

(S)-2-(6-Methoxybenzo[d]thiazol-2-yl)-4,5-dihydrothiazole-4-carboxylic acid (5)[4] was prepared from (S)-cysteine and 6-meyhoxybenzo[d]thiazole-2-carbonitrile

EP

according to the general procedure for condensation. ESMS 294.9 (calcd 295.02, M + H+). 1H-NMR (400 MHz, CD3OD): δ 7.92 (d, J = 9.0 Hz, 1H), 7.53 (d, J = 2.5 Hz,

AC C

1H), 7.14 (ddd, J = 9.0, 2.5, 0.9 Hz, 1H), 5.18 (td, J = 9.3, 0.9 Hz, 1H), 3.88 (s, 3H), 3.72-3.68 (m, 2H).

6

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

Figure 1. 1H NMR (400 MHz, CD3OD) spectrum of Compound 5. (R)-2-(6-Methoxybenzo[d]thiazol-2-yl)-4,5-dihydrothiazole-4-carboxylic

acid

TE D

(6)[5] was prepared from (R)-cysteine and 6-meyhoxybenzo[d]thiazole-2-carbonitrile according to the general procedure for condensation. ESMS 294.9 (calcd 295.02, M + H+). 1H NMR (400 MHz, CD3OD) δ 7.95 (dd, J = 9.0, 0.8 Hz, 1H), 7.57 – 7.55 (m,

EP

1H), 7.17 (ddd, J = 9.0, 2.6, 0.9 Hz, 1H), 5.20 (td, J = 9.4, 0.9 Hz, 1H), 3.91 (d, J =

AC C

1.0 Hz, 3H), 3.73 (ddd, J = 9.2, 3.5, 1.0 Hz, 2H).

(S)-2-(6-Methoxybenzo[d]thiazol-2-yl)-5,5-dimethyl-4,5-dihydrothiazole-4-

carboxylic

acid

(9)[4]

was

prepared

from

(S)-penicillamine

and

6-

meyhoxybenzo[d]thiazole-2-carbonitrile according to the general procedure for condensation. ESMS 322.7 (calcd 323.05, M + H+). 1H-NMR (400 MHz, CD3OD): δ 7.92 (d, J = 9.1 Hz, 1H), 7.55 (d, J = 2.5 Hz, 1H), 7.15 (ddd, J = 9.1, 2.5, 1.0 Hz, 1H), 4.83 (s, 1H), 3.89 (s, 3H), 1.79 (s, 3H), 1.54 (s, 3H).

7

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

Figure 2. 1H NMR (400 MHz, CD3OD) spectrum of Compound 9.

carboxylic

acid

TE D

(R)-2-(6-Methoxybenzo[d]thiazol-2-yl)-5,5-dimethyl-4,5-dihydrothiazole-4(10)[6]

was

prepared

from

(R)-penicillamine

and

6-

meyhoxybenzo[d]thiazole-2-carbonitrile according to the general procedure for

EP

condensation. ESMS 322.9 (calcd 323.05, M + H+). 1H NMR (400 MHz, CD3OD) δ 7.93 (d, J = 9.0 Hz, 1H), 7.56 (d, J = 2.2 Hz, 1H), 7.16 (dd, J = 9.0, 2.5 Hz, 1H), 4.83

AC C

(s, 1H), 3.90 (s, 3H), 1.80 (s, 3H), 1.56 (s, 3H).

(S)-2-(6-Hydroxybenzo[d]thiazol-2-yl)-5,5-dimethyl-4,5-dihydrothiazole-4carboxylic

acid

(11)[6]

was

prepared

from

(S)-penicillamine

and

6-

hydroxybenzo[d]thiazole-2-carbonitrile according to the general procedure for condensation. ESMS 308.8 (calcd 309.03, M + H+). 1H-NMR (400 MHz, CD3OD): δ

8

ACCEPTED MANUSCRIPT

7.89 (d, J = 8.9 Hz, 1H), 7.34 (d, J = 2.4 Hz, 1H), 7.07 (dd, J = 8.9, 2.4 Hz, 1H), 4.97

M AN U

SC

RI PT

(s, 1H), 1.82 (s, 3H), 1.53 (s, 3H).

TE D

Figure 3. 1H NMR (400 MHz, CD3OD) spectrum of Compound 11.

(R)-2-(6-Hydroxybenzo[d]thiazol-2-yl)-5,5-dimethyl-4,5-dihydrothiazole-4acid

(12)[6]

was

prepared

from

(R)-penicillamine

and

6-

EP

carboxylic

hydroxybenzo[d]thiazole-2-carbonitrile according to the general procedure for

AC C

condensation. ESMS 308.9 (calcd 309.03, M + H+). 1H NMR (400 MHz, CD3OD) δ 7.97 (d, J = 8.9 Hz, 1H), 7.37 (d, J = 2.1 Hz, 1H), 7.09 (dd, J = 9.0, 2.4 Hz, 1H), 4.69 (s, 1H), 1.60 (s, 3H), 1.47 (s, 3H).

(S)-2-(6-hydroxyquinolin-2-yl)-4,5-dihydrothiazole-4-carboxylic acid (13)[7] was prepared from (S)-cysteine and 6-hydroxyquinoline-2-carbonitrile according to the general procedure for condensation. ESMS 274.8 (calcd 275.04, M + H+). 1H NMR

9

ACCEPTED MANUSCRIPT

(400 MHz, CD3OD) δ 8.13 (q, J = 8.7 Hz, 2H), 7.96 (d, J = 9.2 Hz, 1H), 7.35 (dd, J = 9.1, 2.7 Hz, 1H), 7.15 (d, J = 2.7 Hz, 1H), 5.26 (t, J = 9.5 Hz, 1H), 3.62 (ddd, J =

TE D

M AN U

SC

RI PT

32.3, 11.0, 9.6 Hz, 2H).

Figure 4. 1H NMR (400 MHz, CD3OD) spectrum of Compound 13.

EP

(R)-2-(6-hydroxyquinolin-2-yl)-4,5-dihydrothiazole-4-carboxylic acid (14)[7] was prepared from (R)-cysteine and 6-hydroxyquinoline-2-carbonitrile according to the

AC C

general procedure for condensation. ESMS 274.9 (calcd 275.04, M + H+). 1H NMR (400 MHz, CD3OD) δ 8.12 (s, 2H), 7.96 (d, J = 9.3 Hz, 1H), 7.34 (s, 1H), 7.15 (s,

1H), 5.33 (d, J = 9.8 Hz, 1H), 3.72 – 3.47 (m, 2H).

(S)-2-(8-hydroxyquinolin-2-yl)-4,5-dihydrothiazole-4-carboxylic acid (15)[8] was prepared from (S)-cysteine and 8-hydroxyquinoline-2-carbonitrile according to the general procedure for condensation. ESMS 274.9 (calcd 275.04, M + H+). 1H-NMR

10

ACCEPTED MANUSCRIPT

(400 MHz, CD3OD): δ 8.25 (d, J = 8.6 Hz, 1H), 8.07 (d, J = 8.6 Hz, 1H), 7.44 (t, J = 7.9 Hz, 1H), 7.33 (dd, J = 8.2, 1.2 Hz, 1H), 7.08 (dd, J = 7.6, 1.2 Hz, 1H), 5.36 (t, J =

TE D

M AN U

SC

RI PT

9.3 Hz, 1H), 3.68-3.58 (m, 2H).

Figure 5. 1H NMR (400 MHz, CD3OD) spectrum of Compound 15.

EP

(R)-2-(8-hydroxyquinolin-2-yl)-4,5-dihydrothiazole-4-carboxylic acid (16)[8] was prepared from (R)-cysteine and 8-hydroxyquinoline-2-carbonitrile according to the

AC C

general procedure for condensation. ESMS 274.9 (calcd 275.04, M + H+). 1H NMR (400 MHz, CD3OD) δ 8.28 (t, J = 5.8 Hz, 1H), 8.06 (d, J = 8.6 Hz, 1H), 7.46 (t, J =

8.0 Hz, 1H), 7.35 (dd, J = 8.2, 1.1 Hz, 1H), 7.09 (dd, J = 7.6, 1.2 Hz, 1H), 5.40 (dd, J = 9.7, 8.8 Hz, 1H), 3.76 – 3.59 (m, 2H).

(S)-Methyl-2-(6-hydroxybenzo[d]thiazol-2-yl)-4,5-dihydrothiazole-4-carboxylate (3)[9] was prepared from 1 according to the general procedure for esterification.

11

ACCEPTED MANUSCRIPT

ESMS 294.7 (calcd 295.02, M + H+). 1H-NMR (400 MHz; CD3OD): δ 7.89 (dd, J = 8.9, 2.0 Hz, 1H), 7.33 (d, J = 2.2 Hz, 1H), 7.06 (dt, J = 8.9, 2.2 Hz, 1H), 5.41 (td, J =

TE D

M AN U

SC

RI PT

9.2, 1.9 Hz, 1H), 3.82 (s, 3H), 3.79-3.70 (m, 2H).

Figure 6. 1H-NMR (400 MHz; CD3OD) spectrum of Compound 3. General Procedure for Esterification. The condensation product (1,3-thiazole-4-

EP

carboxylic acid derivative) was dissolved in methanol before thionyl chloride was added. The reaction mixture was stirred for 1h before the product was recovered

AC C

under vacuum.

(R)-Methyl-2-(6-hydroxybenzo[d]thiazol-2-yl)-4,5-dihydrothiazole-4-carboxylate (4)[9] was prepared from 2 according to the general procedure for esterification.

ESMS 294.7 (calcd 295.02, M + H+). 1H-NMR (400 MHz; CD3OD): δ 7.89 (dd, J =

8.9, 2.0 Hz, 1H), 7.33 (d, J = 2.2 Hz, 1H), 7.06 (dt, J = 8.9, 2.2 Hz, 1H), 5.41 (td, J = 9.2, 1.9 Hz, 1H), 3.82 (s, 3H), 3.79-3.70 (m, 2H)..

12

ACCEPTED MANUSCRIPT

(S)-Methyl-2-(6-methoxybenzo[d]thiazol-2-yl)-4,5-dihydrothiazole-4-carboxylate (7)[9] was prepared from 5 according to the general procedure for esterification. ESMS 308.8 (calcd 309.03, M + H+). 1H-NMR (400 MHz,

RI PT

CD3OD): δ 7.95 (d, J = 9.0 Hz, 1H), 7.56 (d, J = 2.3 Hz, 1H), 7.17 (dd, J = 9.0, 2.3 Hz, 1H), 5.42 (t, J = 9.1 Hz, 1H), 3.90 (s, 3H), 3.83 (s, 3H), 3.76 (dd, J = 9.2, 4.1 Hz,

EP

TE D

M AN U

SC

2H).

AC C

Figure 6. 1H-NMR (400 MHz; CD3OD) spectrum of Compound 7.

(R)-Methyl-2-(6-methoxybenzo[d]thiazol-2-yl)-4,5-dihydrothiazole-4-carboxylate

(8)[9] was prepared from 6 according to the general procedure for esterification.

ESMS 308.9 (calcd 309.03, M + H+). 1H-NMR (400 MHz, CDCl3): δ 8.02 (d, J = 9.1 Hz, 1H), 7.35 (d, J = 2.2 Hz, 1H), 7.13 (dd, J = 9.1, 2.2 Hz, 1H), 5.37 (dd, J = 9.2 Hz, 1H), 3.90 (s, 4H), 3.86 (s, 3H), 3.79 (dd, J = 11.1, 8.9 Hz, 1H), 3.71 (t, J = 10.5 Hz, 1H).

13

ACCEPTED MANUSCRIPT

References

AC C

EP

TE D

M AN U

SC

RI PT

[1] E.H. White, F. McCapra, G.F. Field, The structure and synthesis of firefly luciferin, J. Am. Chem. Soc., 85 (1963) 337-343. [2] A.G.S. Hoegberg, K. Madan, C. Moberg, B. Sjoeberg, M. Weber, M. Muhammed, Selective reagents for solvent extraction of metals-I. Quinaldic acids, Polyhedron, 4 (1985) 971-977. [3] Y. Ohmiya, System for biosynthesis of firefly luminescence substrates with naturel l-cysteine or derivatives thereof and luminescence substrate solutions containing the system., in: PCT (Ed.), 2006. [4] E.H. White, H. Worther, G.F. Field, W.D. McElroy, Analogs of firefly luciferin, J. Org. Chem., 30 (1965) 2344-2348. [5] Z. Yao, H. Bai, C. Li, G. Shi, Colorimetric and fluorescent dual probe based on a polythiophene derivative for the detection of cysteine and homocysteine, Chem. Commun. (Cambridge, U. K.), 47 (2011) 7431-7433. [6] E.H. White, M.G. Steinmetz, J.D. Miano, P.D. Wildes, R. Morland, Chemi- and bioluminescence of firefly luciferin, J. Am. Chem. Soc., 102 (1980) 3199-3208. [7] B.R. Branchini, M.M. Hayward, S. Bamford, P.M. Brennan, E.J. Lajiness, Naphthyl- and quinolylluciferin: green and red light emitting firefly luciferin analogs, Photochem. Photobiol., 49 (1989) 689-695. [8] W. Daily, E. Hawkins, D. Klaubert, J. Liu, P. Meisenheimer, M. Scurria, J.W. Shultz, J. Unch, K.V. Wood, W. Zhou, M.P. Valley, J.J. Cali, Luminogenic and fluorogenic compounds and methods to detect molecules or conditions, Promega Corporation, USA . 2006, pp. 328 pp. [9] J.Q. Wang, K.E. Pollok, S. Cai, K.M. Stantz, G.D. Hutchins, Q.H. Zheng, PET imaging and optical imaging with D-luciferin [11C]methyl ester and D-luciferin [11C]methyl ether of luciferase gene expression in tumor xenografts of living mice, Bioorganic & medicinal chemistry letters, 16 (2006) 331-337.

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