Toxoflavins and deazaflavins as the first reported selective small molecule inhibitors of tyrosyl-DNA phosphodiesterase II

Article

pubs.acs.org/jmc

Toxoﬂavins and Deazaﬂavins as the First Reported Selective Small

Molecule Inhibitors of Tyrosyl-DNA Phosphodiesterase II

Ali Raoof,*^,†Paul Depledge,^†Niall M. Hamilton,^†Nicola S. Hamilton,^†James R. Hitchin,^†

Gemma V. Hopkins,^†Allan M. Jordan,^†Laura A. Maguire,^†Alison E. McGonagle,^†Daniel P. Mould,^†

Mathew Rushbrooke,^§Helen F. Small,^†Kate M. Smith,^†Graeme J. Thomson,^†Fabrice Turlais,^§

Ian D. Waddell,^†Bohdan Waszkowycz,^†Amanda J. Watson,^†and Donald J. Ogilvie^†

^†Cancer Research UK Drug Discovery Unit, Paterson Institute for Cancer Research, University of Manchester, Wilmslow Road,

Manchester M20 4BX, U.K.

^§Cancer Research Technology Ltd., Wolfson Institute for Biomedical Research, The Cruciform Building, Gower Street, London

WC1E 6BT, U.K.

S

* Supporting Information

ABSTRACT: The recently discovered enzyme tyrosyl-DNA phospho-

diesterase 2 (TDP2) has been implicated in the topoisomerase-mediated

repair of DNA damage. In the clinical setting, it has been hypothesized that

TDP2 may mediate drug resistance to topoisomerase II (topo II)

inhibition by etoposide. Therefore, selective pharmacological inhibition of

TDP2 is proposed as a novel approach to overcome intrinsic or acquired

resistance to topo II-targeted drug therapy. Following a high-throughput screening (HTS) campaign, toxoﬂavins and deazaﬂavins

were identiﬁed as the ﬁrst reported sub-micromolar and selective inhibitors of this enzyme. Toxoﬂavin derivatives appeared to

exhibit a clear structure−activity relationship (SAR) for TDP2 enzymatic inhibition. However, we observed a key redox liability

of this series, and this, alongside early in vitro drug metabolism and pharmacokinetics (DMPK) issues, precluded further

exploration. The deazaﬂavins were developed from a singleton HTS hit. This series showed distinct SAR and did not display

redox activity; however low cell permeability proved to be a challenge.

approximately 100 000 compounds assembled from a variety

of commercial compound suppliers, where 85% of the

compounds were chemically diverse and the remaining 15%

screened were from a commercial kinase inhibitor collection.

The commercially available β-lapachone, RO 08-2750, and

NSC95397 were employed as the positive controls (with IC₅₀’s

of 0.97, 3.5, and 71 μM, respectively). Subsequent in-house

resynthesis and reconﬁrmation of hits resulted in toxoﬂavins

and a singleton deazaﬂavin emerging as conﬁrmed hits.

Selectivity screening against the related phosphodiesterases

tyrosyl-DNA phosphodiesterase 1 (TDP1) and apurinic/

apyrimidinic endonuclease (APE-1) was carried out to facilitate

an initial assessment of selective pharmacological inhibition by

the two chemical series. While TDP1 is mechanistically and

structurally diﬀerent from TDP2, it catalyzes a similar

phosphodiester bond cleavage and is the closest equivalent

enzyme in terms of function and homology.¹⁰However, despite

this perceived similarity, TDP2 and TDP1 actually perform

very diﬀerent cellular roles and as such should be considered as

separate target family classes.²APE-1, like TDP1, is a DNA

repair enzyme and shares the closest known three-dimensional

shape and folding pattern compared with TDP2.¹¹

INTRODUCTION

■

Topoisomerases are nuclear enzymes involved in the move-

ment of DNA within the nucleus or in the opening of the

double helix. These enzymes generate reversible breaks in DNA

thereby allowing DNA decatenation. In order to carry out its

critical physiological functions, topoisomerase generates tran-

sient topoisomerase−DNA cleavage complexes, so-called

cleavable complexes, in DNA. Oxidation, ionizing radiation,

or chemotherapeutic agents can stabilize this complex and

prevent the enzyme from resealing the DNA break it creates,

resulting in topoisomerase enzyme-mediated DNA damage.¹

Topo II poisons such as etoposide can induce abortive DNA

strand breaks in which topo II remains covalently bound to a 5′

DNA strand terminus via a phosphotyrosyl linker (Figure

1).³⁻⁸TDP2 (EC 3.1.4-, aka TTRAP, EAPII)²is a recently

discovered human 5′-tyrosyl-DNA phosphodiesterase, and it

has been proposed that TDP2 may remove degraded topo II

peptides covalently linked to the 5′ terminus, thus playing a

central role in maintaining normal DNA topology in cells

(Figure 1).⁶⁻⁹Cellular depletion of TDP2 has been shown to

result in an increased susceptibility and sensitivity of cells to

topo II-induced DNA double-strand breaks, thereby suggesting

TDP2 as a potentially attractive anticancer target.¹

A chromogenic enzymatic assay for TDP2, using 4-

nitrophenyl phenylphosphonate (NPPP) as the substrate,¹

was developed for a high-throughput screen comprising

Received: January 29, 2013

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Figure 1. The roles of Topo II and TDP2.

a

Previous to our work, the less potent 5-arylidenethioxothia-

zolidininones were reported as TDP1 inhibitors.¹²Although

submicromolar activities for TDP1 inhibition have been

reported, the selectivity and hence potency against TDP2 for

the reported chemical series may require further optimization

in order to establish clear pharmacological inhibition.

Scheme 1

Toxoﬂavins were ﬁrst isolated as toxins produced by bacteria

including Burkholderia gladioli¹³and are precedented in the

literature as potential antibiotics¹⁴and chemotherapeutic

agents.^15,16The use of these compounds in the treatment of

other diseases such as asthma and diabetes has also been

reported in the patent literature.^17,18The toxoﬂavins exhibit

structural features reminiscent of xanthines such as theophyl-

line, a competitive nonselective phosphodiesterase inhibitor,¹⁹

suggesting that toxoﬂavins could inhibit TDP2 activity. Further,

the pyrimidodione moiety of toxoﬂavins may act as a phosphate

mimetic,²⁰hence providing an avenue for scaﬀold hopping.

Deazaﬂavins were ﬁrst discovered as derivatives of the

biochemically signiﬁcant ﬂavins²¹and have recently been

shown to exhibit antitumor and antiproliferative activity.^22,23

In this study, we explore the drug-like nature of both the

toxoﬂavins and deazaﬂavins as the ﬁrst potent and selective

small molecule inhibitors of the TDP2 enzyme.

a

Reagents: (a) (i) MeNHNH₂, EtOH, μW, 100 °C, 10 min; (ii)

RCHO, 50 °C, 10 min (32−100%); (b) NaNO₂, AcOH, H₂O,

ambient temperature, 16 h; (c) 1,4-bis(sulfanyl)butane-2,3-diol,

EtOH, reﬂux, 1 h (1−82%).

Toxoﬂavin R²modiﬁcations for compounds 38−43 were

prepared utilizing a similar procedure as that illustrated in

Scheme 1 where the R²diversity was introduced by varying the

hydrazine building blocks while maintaining the R¹function-

ality as the 4-ethylphenyl (Scheme 2).²⁴

Appropriately substituted ureas were the source of toxoﬂavin

R³variations for compounds 50−52. R³substituted chlorour-

acils 44−46 were reacted in similar manner to 6-chloro-3-

methyluracil 1 to furnish the desired products in respectable

yields (Scheme 3).

Toxoﬂavin heteroatom deletion compounds were prepared

according to the literature (see Figure 4 for structural

formulas). The commercially available R²deletion compound

69 was isolated as a byproduct from the DTT reduction of the

N-oxide 68. Deazatoxoﬂavin 70 was synthesized by reaction of

the hydrazone intermediate 32 with the commercially available

2-bromo-1-(4-ethylphenyl)ethanone (Scheme 4).²⁵

Commercially available 6-amino-1,3-dimethyl-2,4(1H,3H)-

pyrimidinedione 71 was condensed with 4-ethylbenzaldehyde

to furnish the intermediate hydrazone 72, which was treated

with an ethyl formate equivalent to give the pteridine 73

(Scheme 5).²⁶

CHEMISTRY

■

The synthesis of key SAR compounds is summarized in

Schemes 1−12. The toxoﬂavins were prepared following

adaptations of literature protocols.²⁴R¹variations were

introduced by reaction of methyl hydrazine with the

commercially available 6-chloro-3-methyluracil 1, followed by

condensation with a range of aldehydes to furnish the

intermediate hydrazones 2−23 in respectable yields. Triazine

formation was achieved by treatment of the hydrazones 2−23

with sodium nitrite under acidic conditions, followed by DTT

reduction of the N-oxide to give the toxoﬂavins 24−31 and

53−67 (Scheme 1).

Core variation 77 was prepared from the commercially

available 2-amino-6-chloro-1H-pyrimidin-4-one 74 in manner

analogous to the toxoﬂavins (Scheme 6).²⁴

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dx.doi.org/10.1021/jm400568p | J. Med. Chem. XXXX, XXX, XXX−XXX

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a

Scheme 2

a

Reagents: (a) (i) RNHNH₂salt, Et₃N, EtOH, reﬂux, 16 h; (ii) p-ethylbenzaldehyde 27−100%; (b) (i) NaNO₂, AcOH, water, ambient temperature,

4 h; (ii) 1,4-bis(sulfanyl)butane-2,3-diol, ambient temperature, 1 h, 27−58%.

a

Scheme 3

a

Reagents: (a) (i) MeNHNH₂, EtOH, μW, 100 °C, 10 min; (ii) 4-ethylbenzaldehyde, μW, 50 °C, 10 min, 69−86%; (b) (i) NaNO₂, AcOH, water,

ambient temperature 16 h; (ii) 1,4-bis(sulfanyl)butane-2,3-diol, ambient temperature, 10 min, 39−67%.

a

4-(3,4-Dimethoxyphenyl)thiophen-2-amine hydrochloride

95 was derivatized to yield aromatic amides 96−103 via acid

chloride formation or EDCI activation and as a phenyl urea 104

(Scheme 11).³²

Scheme 4

The deazaﬂavins 128−163 were prepared following literature

precedent.³³⁻⁴³The general procedure involved the reactions

of appropriate amines with 6-chlorouracil 47 to aﬀord the

amino uracils 106−127, which were used crude in the

subsequent SNAr and aldehyde condensation steps to furnish

the desired deazaﬂavins (Scheme 12).

a

Reagents: (a) 2-bromo-1-(4-ethylphenyl)ethanone, 2-methoxyetha-

nol, pressure tube, 120 °C, 16 h, 0.6%.

RESULTS AND DISCUSSION

■

The intermediate hydrazone 75 was also heated with ethyl

phenylglyoxylate in ethanol to furnish the amino pyrimido

pyridazine dione 78 (Scheme 7).²⁷

Although the hydroxy pyrimidone 82 could be prepared from

the benzyl ether precursor 81 (Scheme 8) via TFA cleavage of

the benzyl protecting group, chemical instability prevented

isolation of the clean authentic product.²⁸

The commercially available thiophene carboxylic acid 83 was

coupled to ethyl piperidine-3-carboxylate followed by ester

saponiﬁcation in quantitative yields (Scheme 9). Acid chloride

activation and reaction with trimethyl phosphite furnished the

keto phosphate 86.²⁹

The bis arylated thiophene methyl esters 89 and 90 were

prepared from 2,4-dibromothiophene 88. Saponiﬁcation to the

corresponding carboxylic acids 91 and 92 and reaction with

methanesulfonamide furnished the desired sulfonamide amides

93 and 94, respectively (Scheme 10).^30,31

Having identiﬁed preliminary inhibitors of the TDP2 enzyme

from the HTS, the synthesis of further analogues of toxoﬂavins

was initiated. The most potent hit from the screen, the 4-

chlorophenyl derivative 24, was initially investigated. The

importance of the 4-chloro substituent was evident from the

des-chloro compound 25 and the 4-ethylphenyl derivative 26.

The electronics of the 4-chloro substituent had less of an

impact on potency compared with the 4-triﬂuoromethyl

analogue 27. The three points of diversity (R¹, R², and R³)

were subsequently explored (see Table 1). Early SAR for the R¹

position indicated that 4- and 3-substituted phenyls, for

example, 29 and 30, respectively, were superior to the 2-

substituted analogues such as 31. Substituents around the R²

and R³positions were less tolerated compared with those at R¹.

n

Small alkyl variations such as ethyl and propyl, 38 and 40,

respectively, were best tolerated at R². Surprisingly, substitution

a

Scheme 5

a

Reagents: (a) 4-ethylbenzaldehyde, EtOH, reﬂux, 3 h, 63%; (b) triethoxyethane, TsOH (cat.), DMA, 120 °C, 16 h, 2%.

C

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a

Scheme 6

a

Reagents: (a) MeNHNH₂, EtOH, reﬂux, 4 h, 80%; (b) 4-methylthiophene-2-carbaldehyde, EtOH, μW, 50 °C, 10 min, 75%; (c) (i) NaNO₂, AcOH,

water, ambient temperature, 4 h; (ii) 1,4-bis(sulfanyl)butane-2,3-diol, water, 50 °C, 2 h, 3%.

with the cyclopropyl 39 at the R²position proved detrimental

a

Scheme 7

compared with the methylene cyclopropyl 41, and this might

be attributed to the more sp²-like character of the directly

linked cyclopropyl group. This was further supported by the

inactive phenyl derivative 42. Exempliﬁcation at the R³position

proved synthetically challenging. Limited SAR in this area

indicated that the original methyl substituted compounds were

clearly preferable.

a

Reagents: (a) ethyl phenylglyoxylate, EtOH, reﬂux, 22 h, 32%.

Of note, all the toxoﬂavins prepared showed excellent

a

selectivity against the closely related TDP1 and APE-1 enzymes

(IC₅₀> 200 μM for APE-1 inhibition and <30% inhibition at

Scheme 8

100 μM for TDP1).⁴⁴

Synthetic tractability and the resultant incorporation of

substructure diversity at the R¹position was much more facile

and allowed for further elaboration of the toxoﬂavins (Table 2),

with the aim to improve potency, maintain selectivity, and

initiate drug metabolism and pharmacokinetics (DMPK)

investigations. In order to reduce the aromatic ring count,

a

alkyl and cycloalkyl compounds 53−58 were synthesized.

There was no signiﬁcant improvement in the potencies of these

compounds compared with 26. The subsequent strategy

involved further extending the R¹position, ﬁrst by introducing

Reagents: (a) EDCI, HOBt, DIPEA, 4-(3,4-dimethoxyphenyl)-

thiophen-2-amine hydrochloride, DMF, 0 °C to ambient temperature,

72 h, 91%.

a

Scheme 9

a

Reagents: (a) (i) CDI, DMF, ambient temperature, 2 h; (ii) ethyl piperidine-3-carboxylate, 100 °C, 16 h, 100%; (b) LiOH, THF, water, ambient

temperature, 2 h, 100%; (c) (COCl)₂, DMF, DCM, ambient temperature, 16 h, 96%; (d) P(OMe)₃, toluene, 0 °C to ambient temperature, 72 h,

16%.

D

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a

Scheme 10

a

Reagents: (a) (i) Pd(PPh₃)₄, Na₂CO₃(aq), methoxycarbonylphenyl boronic acid, DMF, 80−90 °C, 3 h; (ii) 3,4-dimethoxybenzeneboronic acid,

80−90 °C, 16 h, 20−22%; (b) carboxylic acids 91 or 92, LiOH, MeOH (aq), reﬂux, 5 h, 69−77%; (c) methanesulfonamide, EDCI, DMAP, THF,

ambient temperature, 16 h, 2%.

a

cytochrome P450 liabilities of the toxoﬂavin core. These

compounds failed to retain activity compared with the

toxoﬂavin series (Figure 2). More elaborate changes to the

toxoﬂavin core were subsequently investigated. While the

aminopyrimidone replacement 77 exhibited micromolar activity

and low permeability, an eﬄux liability became apparent

compared with the parent toxoﬂavins (Figure 2). The

aminopyrimidone series was further modiﬁed to replace a

triazine nitrogen with a carbonyl functionality (78). Single

point enzyme inhibition data indicated substantial loss of

activity compared with 77 (Figure 2). Additionally, the

commercially available tetrahydrofolate 79, which was reminis-

cent of the amino pyrimidone 77, was also relatively inactive

(Figure 2).

In the absence of a crystal structure for TDP2, it was

postulated that the toxoﬂavin core may be acting as a

nucleotide mimetic stacking against the substrate DNA bases,

or possibly as a phosphate mimetic, with the pyrimidodione

lone pair electrons involved in binding to Mg/Mn metal ions in

the enzyme active site. With this in mind, the thiophene

dimethoxy phenyl fragment of the most potent toxoﬂavin 67

was coupled to a small selection of phosphates and phosphate

mimetics²⁰in an attempt to eliminate the cytochrome P450

liabilities. A small library of replacements for the pyrimidodione

core were modeled using the Maestro software package from

Schrodinger.⁴⁷The overall three-dimensional similarity was

evident by aligning to the parent compound 67 using the Torch

software from Cresset,⁴⁸which assesses similarity in terms of

shape and properties, where a score of 0.7 or greater was

considered a “good” score (Figure 3). Disappointingly, the

hydroxy pyrimidone 82 proved to be chemically unstable, and

the benzyl ether precursor 81 was inactive compared with the

toxoﬂavin 67. Attempts to prepare a phosphate with an

adjacent gem-diﬂuoro functionality, 87, in order to modify the

pK_aof the molecule proved diﬃcult. However, a keto-

phosphate analogue 86 was synthesized, but again also lacked

activity. The 3- and 4-phenyl sulfonamide amides, 93 and 94,

respectively, also resulted in the complete loss of activity

(Figure 3).

Scheme 11

a

Reagents: (a) for 96−102, (i) carboxylic acid, SOCl₂, reﬂux, 5 h, (ii)

4-(3,4-dimethoxyphenyl)thiophen-2-amine hydrochloride 95, DIPEA,

DMAP, THF, ambient temperature, 1 h, 4−47%; For 103, (i) 4-(3,4-

dimethoxyphenyl)thiophen-2-amine hydrochloride 95, DIPEA, DMF,

0 °C, 15 min, (ii) 4,6-dimethyl-2-oxo-2H-pyran-5-carboxylic acid,

EDCI, ambient temperature, 16 h, 16%; for 104, phenyl isocyanate, 4-

(3,4-dimethoxyphenyl)thiophen-2-amine hydrochloride 95, DIPEA,

DCM, ambient temperature, 10 min, 17%.

aliphatic linkers to the phenyl ring, for example, compounds 56

and 57. This disruption of planarity and aromaticity had little

impact on potency compared with the directly linked phenyl

25. Although the heteroaromatic replacement of the phenyl

ring 25 with the 3-thiazole functionality 63 was also detrimental

in terms of potency, improvements were made by further

substitution on the heteroaromatic ring. Optimized potency,

while retaining TDP1 and APE-1 selectivity,⁴⁴was delivered by

increasing the substituent size on the thiophene R¹group from

methyl 64 to phenyl 66 or 3,4-dimethoxyphenyl 67.

At this stage, early in vitro DMPK of the toxoﬂavins was

assessed. Increasing the lipophilicity and hence calculated log

P⁴⁵of the toxoﬂavin core resulted in a decrease in both

solubility and permeability (Table 3). The initial key

compound, 26, exhibited good aqueous solubility and Caco-2

permeability⁴⁶and at ﬁrst glance appeared to demonstrate good

stability in human liver microsomes. However <1 μM inhibition

of the standard cytochrome P450 isoforms (CYPs) inferred

that this high stability was possibly a consequence of the

saturation of the CYP enzymes, preventing metabolic

degradation.

Toxoﬂavin heteroatom deletion compounds 69, 70, and 73

were prepared in an attempt to retain potency and design out

a

Scheme 12

a

Reagents: (a) NaOH (aq), reﬂux, 2 h, 98%; (b) R¹NH₂, n-propanol, μW, 150 °C, 30 min to 10 h, 45−100%; (c) substituted 2-ﬂuorobenzaldehyde,

DMF, μW, 110 °C, 10 min to 1 h, 2−86%.

E

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Table 1. Initial Toxoﬂavins SAR: R¹, R², and R³Modiﬁcations

a

compound

R¹

R²

R³

EC₅₀(μM)

24

25

26

27

28

29

30

31

38

39

40

41

42

43

50

51

52

4-chlorophenyl

phenyl

methyl

ethyl

methyl

hydrogen

phenyl

benzyl

0.43(0.15)

0.78(0.20)

0.28(0.25)

0.33(0.13)

0.26(0.08)

0.29(0.12)

0.40(0.05)

1.07(0.10)

0.30(0.10)

4.63(0.60)

0.28(0)

4-ethylphenyl

4-(triﬂuoromethyl)phenyl

4-(triﬂuoromethoxy)phenyl

4-tolyl

3-tolyl

2-tolyl

4-ethylphenyl

cyclopropyl

ⁿpropyl

methylenecyclopropyl

phenyl

0.36(0.05)

>10(0)

benzyl

7.42(0.65)

0.67(0.09)

1.02(0.28)

0.70(0.07)

methyl

a

Values stated are a geometric mean of at least two independent determinations with standard deviations given in parentheses. The inhibition values

were expressed as EC₅₀values rather than IC₅₀values, because all inhibitory compounds tested showed a maximum inhibition of 75%, reaching a

plateau at this level. All inhibitory compounds show hill slopes approximating unity. Example dose−response curves of representative toxoﬂavins can

be found in the Supporting Information.

met with little success, Cresset Spark software⁴⁹was then

employed to design de novo toxoﬂavin core mimetics. The

highest scoring solutions (all >0.8) were subsequently

synthesized. In comparison to the toxoﬂavin 67, all such core

hops were relatively inactive, exhibiting potencies of >30 μM in

the TDP2 assay (Figure 4).

Table 2. Toxoﬂavin R¹SAR Expansion

a

compound

R¹

EC₅₀(μM)

While these studies were ongoing, we became aware of a

single report that toxoﬂavin derivatives may undertake redox

cycling in the presence of DTT, which was also present in our

assay to enhance TDP2 activity.⁵⁰This report suggested that

high concentrations of hydrogen peroxide could be generated

in situ in the assay system. This may result in misleading assay

interpretations, although the inclusion of DTT present in the

counter screen versus TDP1 and APE-1 showed that the

toxoﬂavins were relatively inactive against these enzymes.⁴⁴To

this end, we conducted an assessment of the redox potential of

these derivatives by measuring hydrogen peroxide generation in

our assay conditions (Figure 5).⁵¹It was evident from this

assessment that the toxoﬂavins were indeed participating in a

redox cycling interaction in the TDP2 enzymatic assay. Further,

the levels of generated peroxide correlated well with apparent

inhibitory activities in the TDP2 assay system, because the

inactive toxoﬂavin deletion compounds 69, 70, and 73 did not

display redox activity (Figure 6). These data supported the

hypothesis that it was the toxoﬂavin core itself exhibiting redox

liabilities, strongly implying that, although the SAR observed

appeared sensible and credible, these data were compromised

in the context of the design of TDP2 inhibitors.

53

54

55

56

57

58

25

59

60

61

62

63

64

65

66

67

methyl

1.16(0)

ⁿpropyl

0.51(0.23)

0.37(0.04)

0.64(0.08)

0.75(0.19)

0.43(0)

cyclopropyl

cyclopentyl

cyclohexyl

4-tetrahydropyran

phenyl

0.78(0.20)

0.96(0.05)

0.68(0.08)

0.15(0.01)

0.87(0.97)

0.77(0.08)

0.25(0)

benzyl

phenethyl

2-napthyl

3,4-dimethoxyphenyl

3-thiazol

4-methyl-2-thienyl

4-bromo-2-thienyl

4-phenyl-2-thienyl

4-(3,4-dimethoxyphenyl)-2-thienyl

0.20(0.03)

0.12(0.01)

0.05(0.01)

a

Values stated are a geometric mean of at least two independent

determinations with standard deviations given in parentheses. The

inhibition values were expressed as EC₅₀values rather than IC₅₀values

because all inhibitory compounds tested showed a maximum

inhibition of 75%, reaching a plateau at this level. All inhibitory

compounds show hill slopes approximating unity. Example dose−

response curves of representative toxoﬂavins can be found in the

Supporting Information.

In summary for this series, the medicinal chemistry design of

exempliﬁed toxoﬂavins led to apparently potent and selective

inhibitors with undesirable cytochrome P450 liabilities. The

redox assay interference was hoped to have been alleviated by

replacing the toxoﬂavin core. However, all synthetic variations

Because replacing the pyrimidodione portion of the

toxoﬂavin core with phosphate or phosphate mimetics was

F

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Table 3. Toxoﬂavins in Vitro DMPK

a

b

c

d

compound

xLogP

aqueous solubility (μM)

P_a→b(10⁻⁶cm s⁻¹

)

P_b→a/P_a→b

T_1/2(min)

CYP inhibition

26

28

52

67

1.26

1.93

2.99

2.35

100

22

0.48

0.46

0.23

0.25

455

325

227

e

1A2 1.5 μM; 2C19, 2C9, 2D6, 3A4 <1 μM

30−100

3−10

18

e

0.7

0.3

e

a

b

Turbidmetric aqueous solubility: concentration of test compound that produces an increase in absorbance above 1% DMSO in buﬀer. P_a→bis the

permeability when applied to the apical side, while P_b→ais the permeability when applied to the basolateral side in a Caco-2 membrane assay. The

c

ratio of these two values is a measure of Pgp eﬄux, while the P_a→bvalue is a measure of permeability. Metabolic stability in human liver microsomes:

d

e

half-life (T_1/2). CYP (cytochrome P450) inhibition of the ﬁve standard isoforms measured as an IC₅₀. Not determined.

Figure 2. Toxoﬂavin heteroatom deletions and core variations.

resulted in the loss of activity consistent with the redox

hypothesis. The subsequent focus of the study was therefore to

explore additional HTS hit matter, in this instance the

deazaﬂavins, as a potential hit series.

The deazaﬂavin series emerged from a singleton HTS hit,

128, exhibiting sub-10 μM potency. The unsubstituted core

129 and close analogues from the HTS (see compounds 129,

131, and 132 (Table 4)) were relatively inactive, inferring steep

SAR. N-Methylation of the pyrimidodione moiety to furnish

133 also resulted in the loss of activity, in contrast to the

optimized R³methyl of the pyrimidodione of toxoﬂavins,

implying that the SAR from the toxoﬂavins did not cross over

to the deazaﬂavins.

respectively (Table 4). Reassuringly, after early assessment, the

deazaﬂavins with a range of potencies did not appear to be

causing redox cycling in the TDP2 enzymatic assay, giving

conﬁdence that the observed SAR was truly pharmacologically

relevant (Figure 7).

Combining the optimal R¹3-hydroxyphenyl with the R³

nitrile proved fruitful; the SAR appeared additive and aﬀorded

compound 147 as a 0.05 μM inhibitor of the TDP2 enzyme.

Further SAR was established for the deazaﬂavins, whereby

hydrogen bonding substituents at the 3- and 4-positions of the

R¹phenyl and nitrile at the R³position of the C ring resulted in

the most potent TDP2 inhibitors (Table 4).

The pharmacological inhibition of the TDP2 enzyme by

deazaﬂavins was further supported by an orthogonal bio-

chemical assay using a more physiologically relevant

oligonucleotide substrate.¹In comparison to the chromogenic

assay employing NPPP as the substrate, the rank order of the

range of potencies for the two assays were in very good

correlation. (Table 5, Figure 9).

Taken together, the use of these two new TDP2 assays has

been successfully employed to identify novel small molecule

inhibitors of this enzyme. Futhermore these compounds were

shown to be selective over both APE-1 and TDP1. For the

latter enzyme, we took advantage of a newly developed assay,

which measured the AP-cleavage activity of TDP1.⁴⁴Although

not directly measuring the 3′-tyrosyl phosphodiesterase activity,

this assay was shown to generate comparable data with

reference compounds to an ALPHA-screen assay using a

more traditional tyrosyl-oligonucleotide substrate. This served

to validate its use as a direct assay for TDP1 activity.⁴⁴

While this paper was in preparation, Schellenberg et al.

reported the X-ray structure of the catalytic domain of mouse

TDP2 bound to DNA,⁵²alongside a publication from Shi et al.

The signiﬁcance of the R³chloro substituent required further

investigation. Monochloro regioisomers around the C-ring

were prepared (134−136); however these all resulted in the

loss of activity, indicating the R³chloro substituent to be

important. Varying the pendant phenyl ring at R¹with alkyl and

cycloalkyl substituents (137−140) also proved detrimental to

maintaining potency. Substituted phenyls were therefore

synthesized in an attempt to establish further SAR. This

approach was successful, because clear SAR of hydroxyphenyls

141, 143, and 144 emerged, with the 3-hydroxyphenyl 143

proving to be the most potent of the three derivatives.

Biochemical assessment against TDP1 and APE-1 enzymes

demonstrated complete selectivity for all derivatives exhibiting

IC₅₀results less than 1 μM in the TDP2 assay (<20% inhibition

at 30 μM for TDP1 and APE-1 enzymes).⁴⁴Replacing the

hydroxyl of 141 with a methoxy to give 142, and thus losing the

potential for hydrogen bonding interactions, resulted in loss of

activity. In order to explore electronics, substitution of the R³

chloro with the bioisosteric triﬂuoromethyl and nitrile moieties

successfully furnished the more potent analogues 145 and 146,

G

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Figure 4. Computationally designed core variations of toxoﬂavins.

Figure 5. Toxoﬂavins: hydrogen peroxide generation as an indicator of

redox activity. Redox cycling compounds can generate H₂O₂in the

presence of reducing reagents such as DTT, which can in turn oxidize

accessible cysteine, tryptophan, methionine, histidine, or selenocys-

teine residues, resulting in the nonspeciﬁc inhibition of enzymatic

activity. Compounds were assayed for redox cycling potential by

measuring generation of H₂O₂in the presence and absence of 1 mM

DTT. This assay detects H₂O₂production using a coupled enzymatic

assay via the H₂O₂-dependent horseradish peroxidase (HRP)-

mediated oxidation of phenol red and measurement of the subsequent

change in absorbance at 610 nM at alkaline pH.⁵¹The known redox

cycling compound β-lapachone was employed as a positive control,

and 1% DMSO was used as the negative control for this assay.

Figure 3. Toxoﬂavin phosphate mimetics. Putative phosphate

mimetics were attached to the thiophene dimethoxyphenyl moiety

of 67 via various linkers, aligned using Torch software from Cresset.⁴⁸

This highlights designs most similar to 67 in terms of shape and

electrostatic properties. Scores of 0.7 or greater were considered

favorable.

10). In order to determine whether the mouse structure was

able to yield any insight into the potential binding mode and

SAR of the deazaﬂavin series, docking studies were performed

using Glide.⁴⁷Of the four X-ray structures reported by

Schellenberg et al., the structure of the TDP2−DNA reaction

product complex (PDB code 4GZ1) was selected as a suitable

starting point for docking studies, being a well-resolved (1.5 Å)

structure with the catalytic magnesium ion in position. The

DNA duplex and solvent were removed prior to docking. A

selection of the most active deazaﬂavin compounds were

detailing both zebraﬁsh and Caenorhabditis elegans homo-

logues.⁵³The Schellenberg disclosure was the ﬁrst structure of a

mammalian TDP2 to be published. Alongside mutational and

functional analyses, the structural data supported a single metal

ion catalytic mechanism for cleavage of the tyrosyl-DNA

adduct. Mouse TDP2 is highly homologous to human TDP2,

with the catalytic site being particularly well conserved (Figure

H

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permeability and high eﬄux still remained an issue with the

deazaﬂavins. However, the unsubstituted core 130, while

inactive, exhibited good solubility and permeability and reduced

eﬄux, which implied that the poor in vitro DMPK properties

were not an intrinsic liability of deazaﬂavins in general. This

encouraging result prompted the proﬁling of further analogues

in an attempt to balance potency, solubility and permeability,

and lipophilicity (compounds 149, 154, and 160). Unfortu-

nately, the permeability of our TDP2-active elaborated

derivatives remained low. However, the potent tetrazole 163

displayed the best balance of potency and physicochemical

properties.

Selectivity for the deazaﬂavins against TDP1 and APE-1 was

maintained in these advanced analogues, and testing additional

analogues in the hydrogen peroxide assessment encouragingly

reinforced the initial ﬁndings for this series of inhibitors as

being devoid of a potential redox cycling liability (Figure 12).

Figure 6. Toxoﬂavins deletion compounds: hydrogen peroxide

generation as an indicator of redox activity. β-Lapachone was

employed as a positive control, and 1% DMSO was used as the

negative control for this assay.

docked, with the pyrimidodione ring A represented in both

protonated (neutral) and deprotonated (anionic) forms.

Several binding modes were consistently observed for

deazaﬂavin analogues, typiﬁed by the pyrimidodione ring

binding to the magnesium ion and gaining one or more

hydrogen bonding interactions to neighboring phosphate-

binding residues. One such binding mode is illustrated in

Figure 11. The tricyclic deazaﬂavin core ﬁts along the shallow

ﬂoor of the DNA binding site, with the pyrimidodione ring A

bound to the magnesium ion and to an “oxyanion hole”

involved in 5′-phosphoryl binding (i.e., side chains of His236

and Ser239, mouse TDP2 numbering). In terms of the SAR of

the deazaﬂavin series, N-methylation of ring A, such as in

compound 133, was seen to abolish activity, which is consistent

with the hypothesis that ring A, possibly in a deprotonated

state, forms critical metal and hydrogen-bonding interactions.

The R¹phenyl group, which in its lowest energy conformation

is predicted to be oriented orthogonal to the plane of the

tricyclic core in a “sailboat” conformation, binds to the

hydrophobic wall in the region of Leu315. Although in this

model the tetrazole substituent forms no speciﬁc interactions, it

may be that a degree of protein ﬂexibility will allow direct or

water-mediated hydrogen-bonding contacts to polar sites in the

region of the hydrophobic wall (e.g., Leu315 backbone, Arg316

side chain) or on the opposite loop (e.g., Thr240, His243).

Electron-withdrawing groups on ring C at R³(e.g., nitrile, 163)

may be important for stabilizing the deprotonation of ring A or

for enhancing aromatic stacking interactions between ring C

and neighboring residues such as Trp307, Phe325, and Arg327

(Figure 11).

While docking studies proved useful for suggesting potential

binding modes, it should be borne in mind that the

conformation of human TDP2 may not be identical to the

available X-ray structures of mouse TDP2, particularly if a

degree of induced ﬁt binding to the ligand should arise in the

absence of bound DNA.

The solubility and permeability of the deazaﬂavins were next

investigated to triage the selection of compounds for cell assay

proﬁling. The potent methanesulfonamide 147 exhibited good

aqueous solubility but low Caco-2 permeability. Compound

147, a close analogue of 157, was less soluble and also displayed

high levels of eﬄux. These DMPK issues may have been

attributed to the increased lipophilicity and hence calculated log

P⁴⁵of compound 159 (Table 6). The eﬄux liability was further

investigated by proﬁling the nitrile and the methanesulfona-

mide deletion compounds 158 and 146, respectively. Low

CONCLUSION

■

The toxoﬂavins were initially identiﬁed as potent and selective

inhibitors of the TDP2 enzyme. Although there is precedence

for the drug-like nature of such compounds, redox activity in

the biochemical assay strongly suggested that the SAR from the

medicinal chemistry design was not representative of toxoﬂavin

pharmacology against TDP2, and with hindsight, this is

consistent with the redox potential of these compounds.

Consistent with this conclusion, core deletions, variations, and

phosphate mimetics all resulted in the loss of activity. Given the

prevalence of these compounds in the medicinal chemistry

literature and their common incorporation into screening

libraries, we feel it is critically important to raise awareness of

this liability in the community and to highlight more widely

these scaﬀolds as demonstrated assay interference compounds.

We hope this report will alert others to the issue we have

experienced despite apparently clear and rational SAR against

our chosen target.

Despite this, a singleton hit from the deazaﬂavin class of

compounds was successfully translated to a potent series with

no observed redox liabilities, and signiﬁcant SAR has been

elaborated that is distinct from the toxoﬂavin series. The

deazaﬂavins were further tested in an orthogonal and more

pharmacologically relevant oligonucleotide biochemical assay.

In comparison to the chromogenic assay, both the rank order of

compound inhibition and potencies were in very good

correlation. The robustness of all data were veriﬁed by the

acceptable standard deviations. There was no evidence of

compound precipitation or aggregation in all assay formats.

This was also supported by hill slopes approximating unity in

all cases. Computational studies based on the close homology

mouse TDP2 crystal structure provided possible explanations

for the observed SAR, and therefore potential mechanism of

inhibition by the deazaﬂavin series. However, generally low

permeability in the Caco-2 assay prevented further progression

of this series. Nevertheless, 163 exhibited the most balanced

proﬁle of all the deazaﬂavins synthesized and therefore may

oﬀer a potential candidate for investigating the proof of

mechanism of TDP2 inhibition in appropriate cell lines,

permeabilized cell systems, or cell lysates. As such, this work

oﬀers useful preliminary tool compounds for the exploration of

the novel biological eﬀects of TDP2 inhibition and may serve as

a starting point for the elaboration of more advanced, cell-

penetrant derivatives in due course. Moreover, should these

agents conﬁrm a critical role of TDP2 in the reversal of

I

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Table 4. Deazaﬂavins SAR

a

compound

R¹

R²

R³

Cl

R⁴

R⁵

EC₅₀(μM)

128

phenyl

H

Cl

H

Cl

H

Cl

H

Cl

H

7.36(0.24)

>30

129

phenyl

H

130

methyl

H

b

>30

>10

>30

131

benzyl

Cl

132

4-tolyl

Cl

133-N-methyl pyrimidodione

phenyl

Cl

134

135

136

137

138

139

140

141

142

143

144

145

146

147

148

149

150

151

152

153

154

155

156

157

158

159

160

161

162

phenyl

H

phenyl

H

phenyl

H

methyl

Cl

cyclopropyl

cyclohexyl

4-piperidyl

Cl

4-hydroxyphenyl

Cl

1.66(0.45)

>30

0.48(0.04)

>30

2.87(1.62)

4-methoxyphenyl

3-hydroxyphenyl

Cl

2-hydroxyphenyl

Cl

phenyl

CF₃

CN

H

phenyl

0.50(0)

3-hydroxyphenyl

0.05(0)

4-hydroxyphenyl

0.09(0.01)

0.25(0.01)

0.47(0.44)

1.04(0.20)

0.32(0.18)

3.39(1.71)

2.74(3.66)

0.09(0.03)

0.72(0.11)

0.03(0.01)

4.66(1.32)

1.37(0.30)

0.29(0.06)

0.64(0.27)

0.37(0.07)

0.04(0)

3-(hydroxymethyl)phenyl

3-methoxyphenyl

4-methoxyphenyl

3-hydroxy-4-methoxyphenyl

3-ﬂuorophenyl

4-bromophenyl

3-aminophenyl

3-phenylacetamide

3-phenylmethanesulfonamide

3-benzenesulfonamide

1H-indazol-6-yl

H

CN

1H-indazol-4-yl

163

3-(1H-tetrazol-5-yl)phenyl

a

Values stated are a geometric mean of at least two independent determinations with standard deviations given in parentheses. The inhibition values

were expressed as EC₅₀values rather than IC₅₀values, as all inhibitory compounds tested showed a maximum inhibition of approximately 75%,

reaching a plateau at this level (see Figure 8 for a typical curve). All inhibitory compounds show hill slopes approximating unity. Dose response

b

curves of all active deazaﬂavins can be found in the Supporting Information. 10% inhibition at 50 μM.

were recorded on a 300 MHz Bruker spectrometer at ambient

temperature. Solutions were typically prepared in either deutero-

chloroform (CDCl₃) or deuterated dimethylsulfoxide (DMSO-d₆)

with chemical shifts referenced to deuterated solvent as an internal

standard. ¹H NMR data are reported indicating the chemical shift (δ),

the multiplicity (s, singlet; d, doublet; t, triplet; q, quartet; m,

multiplet; br, broad; dd, doublet of doublets, etc.), the coupling

constant (J) in Hz, and the integration (e.g., 1H). Deuterated solvents

were obtained from the Sigma-Aldrich Chemical Co., Goss, or

Fluorochem. HRMS results were obtained on a Waters QTof Micro

instrument, using positive and negative polarity electrospray with

sodium formate as standard calibrant. LC-MS spectra with UV

detection were recorded on a Waters Acquity UPLC. Mass

spectrometry was performed on a Waters Acquity SQD quadrupole

spectrometer running in dual ES+ and ES− mode. High pH runs were

conducted at pH 10, and low pH runs were conducted at pH 3, with a

run time of 2 min. The column temperature was 40 °C, and the ﬂow

resistance to commonly used chemotherapeutic agents, such

permeable derivatives may have the potential to deliver

considerable clinical utility.

EXPERIMENTAL SECTION

■

General Methods. All reagents obtained from commercial sources

were used without further puriﬁcation. Anhydrous solvents were

obtained from the Sigma-Aldrich Chemical Co. Ltd. or Fisher

Chemicals Ltd. and used without further drying. Solutions containing

products were either passed through a hydrophobic frit or dried over

anhydrous MgSO₄or Na₂SO₄and ﬁltered prior to evaporation of the

solvent under reduced pressure. Thin layer chromatography (TLC)

was conducted with 5 cm ×10 cm plates coated with Merck type 60

F254 silica gel to a thickness of 0.25 mm. Chromatography was

performed on Biotage SNAP HP-Sil cartridges using a CombiFlash

Companion machine. Proton (¹H) and carbon (¹³C) NMR spectra

J

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Table 5. Oligonucleotide assay versus NPPP assay

a

a,b

compound oligonucleotide assay IC₅₀(μM)

NPPP assay EC₅₀(μM)

c

54

1.51(0.03)

>30

0.51(0.23)

>30

129

130

131

132

137

139

140

141

142

143

144

145

146

147

148

152

154

157

161

162

163

>30

7.04(1.36)

>30

2.41(0.49)

1.66(0.45)

>30

Figure 7. Deazaﬂavins early SAR compounds: hydrogen peroxide

generation as an indicator of redox activity. β-Lapachone was

employed as a positive control, and 1% DMSO was used as the

negative control for this assay.

0.48(0.04)

>30

27.48(0.74)

12.96(0.62)

3.71(0.07)

0.52(0.11)

1.45(0.12)

2.15(0.46)

12.51(1.88)

0.64(0.29)

3.63(0.70)

3.82(1.04)

0.59(0.09)

2.87(1.62)

0.50(0)

0.05(0)

0.09(0.01)

0.32(0.18)

2.74(3.66)

0.03(0.01)

0.64(0.27)

0.37(0.07)

0.04(0)

a

Values stated are a geometric mean of at least two independent

determinations with standard deviations given in parentheses, and all

b

inhibitory compounds show hill slopes approximating unity. The

inhibition values were expressed as EC₅₀values rather than IC₅₀values

because all inhibitory compounds tested showed a maximum

inhibition of 75%, reaching a plateau at this level. Toxoﬂavin 54

Figure 8. Dose−response curve of a representative deazaﬂavin 163.

c

was tested to show oligo assay robustness against two distinct chemical

series.

rate was 0.6 mL/min. Further details, including solvent gradients, are

given in the Supporting Information. Details of the preparative HPLC

instrument and the solvent gradient used to purify compounds are also

given in the Supporting Information. All compounds were ≥95%

purity as determined by examination of both the LC-MS and ¹H NMR

spectra unless otherwise indicated. Where Cl or Br were present,

expected isotopic distribution patterns were observed.

(3,4-Dimethoxy-phenyl)-thiophen-2-ylamine was purchased from

Princeton BioMolecular Research Inc. Compound 79 was purchased

from Sigma-Aldrich Company Ltd. Compounds 53, 128, 129, and

131−133 were purchased from InterBioScreen Ltd., Moscow, Russia,

and used as received.

24 (orange solid, 223 mg, 53%); LC-MS (high pH) 0.87 min, m/z

270.5 [M + H]⁺, 100% purity.

3-(4-Ethylphenyl)-1,6-dimethyl-pyrimido[5,4-e][1,2,4]triazine-5,7-

dione, 26. Compound 26 was prepared in a manner analogous to that

for 24 (orange solid, 160 mg, 31%). ¹H NMR (CDCl₃): δ 8.23 (d, J =

8.3 Hz, 2H), 7.3 (d, J = 8.3 Hz, 2H), 4.23 (s, 3H), 3.53 (s, 3H), 2.74

(q, J = 7.6 Hz, 2H), 1.29 (t, J = 7.6 Hz, 3H).

1,6-Dimethyl-3-[4-(triﬂuoromethyl)phenyl]pyrimido[5,4-e][1,2,4]-

triazine-5,7-dione, 27. Compound 27 was prepared in a manner

analogous to that for 24 (yellow solid, 26 mg, 5%); LC-MS (high pH)

1.03 min, m/z 338.5 [M + H]⁺, 100% purity.

1,6-Dimethyl-3-[4-(triﬂuoromethoxy)phenyl]pyrimido[5,4-e]-

[1,2,4]triazine-5,7-dione, 28. Compound 28 was prepared in a

manner analogous to that for 24 (orange solid, 36 mg, 7%). ¹H NMR

(CDCl₃): δ 8.37 (d, J = 8.7 Hz, 2H), 7.36 (d, J = 8.7 Hz, 2H), 4.24 (s,

3H), 3.53 (s, 3H).

Compound 69 can be commercially sourced from Princeton

BioMolecular Research, Inc. Compound 54 can be commercially

sourced from Ambinter Stock Screening Collection.

3-(4-Chlorophenyl)-1,6-dimethyl-pyrimido[5,4-e][1,2,4]triazine-

5,7-dione, 24.²⁴In adaptation of the literature procedure,²¹

a

suspension of 2 (480 mg, 1.64 mmol) in AcOH (25 mL) at ambient

temperature was treated with a solution of sodium nitrite (170 mg,

2.46 mmol) in water (2.5 mL) and stirred for 18 h. The reaction

mixture was diluted with Et₂O (50 mL), and the ﬂask was cooled in an

ice bath and scratched. After the mixture was stirred for 30 min, the

precipitate was collected by ﬁltration and washed with Et₂O (2 × 10

mL). The solid was dried to aﬀord a 7:3 mixture of the product 24/N-

oxide (at N-4) as a bright yellow solid, 419 mg. A 200 mg sample of

this was suspended in EtOH (18 mL) and treated with a solution of

1,4-bis(sulfanyl)butane-2,3-diol (145 mg, 0.94 mmol) (5 equiv with

respect to the amount of N-oxide) in water (1.8 mL). The dark orange

mixture was heated to 50 °C for 1 h and then allowed to cool to

ambient temperature. The precipitate was collected by ﬁltration,

washed with EtOH (2 × 5 mL), and dried to aﬀord 24 (orange solid,

125 mg, 25%); LC-MS (high pH) 0.97 min, m/z 304.5 [M + H]⁺,

100% purity.

1,6-Dimethyl-3-(p-tolyl)pyrimido[5,4-e][1,2,4]triazine-5,7-dione,

29.²⁴Compound 29 was prepared in a manner analogous to that for

24 (orange solid, 549 mg, 59%); LC-MS (high pH) 0.96 min, m/z

284.5 [M + H]⁺, 100% purity.

1,6-Dimethyl-3-(m-tolyl)pyrimido[5,4-e][1,2,4]triazine-5,7-dione,

30. Compound 30 was prepared in a manner analogous to that for 24

1

(yellow solid, 297 mg, 73%). H NMR (CDCl₃): δ 8.08−8.14 (m,

2H), 7.33−7.44 (m, 2H), 4.24 (s, 3H), 3.53 (s, 3H), 2.46 (s, 3H).

1,6-Dimethyl-3-(o-tolyl)pyrimido[5,4-e][1,2,4]triazine-5,7-dione,

31. Compound 31 was prepared in a manner analogous to that for 24

(orange solid, 133 mg, 32%); LC-MS (high pH) 0.91 min, m/z 284.5

[M + H]⁺, 100% purity.

3-(4-Ethylphenyl)-6-methyl-8H-pyrimido[5,4-e][1,2,4]triazine-5,7-

dione, 69, and 1-Ethyl-3-(4-ethylphenyl)-6-methyl-pyrimido[5,4-

e][1,2,4]triazine-5,7-dione, 38. A suspension of the commercially

available 32 in AcOH (15 mL) was treated with a solution of sodium

nitrite (93.2 mg, 1.35 mmol) in water (2 mL) at ambient temperature.

The reaction was stirred for 16 h. Et₂O (100 mL) was added, and the

1,6-Dimethyl-3-phenyl-pyrimido[5,4-e][1,2,4]triazine-5,7-dione,

25.⁵⁴Compound 25 was prepared in a manner analogous to that for

K

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Figure 9. Correlation plot of compound inhibition in the two complementary biochemical assays. R²of correlation = 0.97.

Figure 10. Comparison of mouse and human TDP2 active site sequence homology. The X-ray structure of the mouse TDP2/DNA complex (PDB

4GZ1).⁵²(A) Protein solvent-accessible surface colored by electrostatic potential. (B) Detail of the protein/DNA interface, with the protein

backbone colored in terms of homology to human TDP2 (red, identical residue; orange, similar; gray, dissimilar). Figures prepared using Maestro.⁴⁷

mixture was cooled in an ice bath. After the mixture was stirred for 30

min, the solids were collected by ﬁltration and washed with Et₂O (2 ×

20 mL) to aﬀord a 3:2 mixture of 38/68 N-oxide (at N-4) as an orange

solid, 314 mg. A suspension of the mixture in EtOH (20 mL) was

treated with a solution of 1,4-bis(sulfanyl)butane-2,3-diol (75.8 mg,

0.49 mmol) in water (2 mL) and heated to reﬂux for 16 h. The cooled

reaction mixture was diluted with Et₂O, and the resulting precipitate

was collected by ﬁltration, washed with diethyl ether, and dried to give

analogous to that for 41 (orange solid, 841 mg, 27%). ¹H NMR

(DMSO-d₆): δ 8.09 (d, J = 8.2 Hz, 2H), 7.43 (d, J = 8.2 Hz, 2H),

4.57−4.66 (m, 1H), 2.70 (q, J = 7.6 Hz, 2H), 1.41−1.48 (m, 2H),

1.27−1.34 (m, 2H), 1.22 (t, J = 7.6 Hz, 3H).

3-(4-Ethylphenyl)-6-methyl-1-propyl-pyrimido[5,4-e][1,2,4]-

triazine-5,7-dione, 40. Compound 40 was prepared in a manner

analogous to that for 41 (orange solid, 201 mg, 27%). ¹H NMR

(DMSO-d₆): δ 8.11 (d, J = 8.2 Hz, 2H), 7.43 (d, J = 8.2 Hz, 2H),

4.33−4.46 (m, 2H), 2.70 (q, J = 7.6 Hz, 2H), 1.87−2.01 (m, 2H), 1.23

(t, J = 7.6 Hz, 3H), 0.99 (t, J = 7.4 Hz, 3H).

1-(Cyclopropylmethyl)-3-(4-ethylphenyl)-6-methyl-pyrimido[5,4-

e][1,2,4]triazine-5,7-dione, 41. To a solution of 35 (1.0 g, 3.06 mmol)

in AcOH (40 mL) and water (4 mL) at 5 °C under nitrogen was

added NaNO₂(296 mg, 4.29 mmol), and the resulting mixture was

stirred at 5 °C for 10 min before being warmed to ambient

temperature and stirred for a further 4 h. LCMS analysis of the

reaction mixture showed a 1:1 mixture of 41/N-oxide (at N-4). 1,4-

1

69 (orange solid, 16 mg, 11%). H NMR (DMSO-d₆): δ 8.33 (d, J =

8.0 Hz, 2H), 7.45 (d, J = 8.0 Hz, 2H), 3.30 (s, 3H), 2.72 (q, J = 7.3 Hz,

2H), 1.25 (t, J = 7.5 Hz, 3H). The ﬁltrate was concentrated and

puriﬁed by preparative HPLC at high pH to give 38 (yellow solid, 1.97

1

mg, 1%). H NMR (CDCl₃): δ 8.16 (d, J = 8.2 Hz, 2H), 7.28 (d, J =

8.2 Hz, 2H), 4.61 (q, J = 7.3 Hz, 2H), 3.45 (s, 3H), 2.67 (q, J = 7.5 Hz,

2H), 1.54 (t, J = 7.2 Hz, 3H), 1.22 (t, J = 7.7 Hz, 3H).

1-Cyclopropyl-3-(4-ethylphenyl)-6-methyl-pyrimido[5,4-e][1,2,4]-

triazine-5,7-dione, 39. Compound 39 was prepared in a manner

L

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Figure 11. Postulated binding mode of deazaﬂavin 163. Detail of a typical predicted binding mode for deazaﬂavin compound 163 (R¹= 3-(1H-

tetrazol-5-yl)phenyl, R³= CN) docked to the X-ray structure of mouse TDP2. (A) Protein solvent-accessible surface colored by electrostatic

potential, with the magnesium ion shown as a yellow sphere. (B) Same docked pose of compound 163 compared with the binding mode of the DNA

duplex in the original X-ray structure (thick stick, purple carbon atoms, orange phosphorus atoms). Figures prepared using Maestro.⁴⁷

under vacuum, and the residue was suspended in EtOH (20 mL) and

stirred for 16 h. The solid was collected by ﬁltration and washed with

EtOH (2 × 10 mL) and preadsorbed onto silica. The material was

puriﬁed by ﬂash column chromatography, eluting with a gradient of

DCM to 2% MeOH/DCM to aﬀord 41 (orange solid, 600 mg, 58%).

¹H NMR (DMSO-d₆): δ 8.15 (d, J = 8.2 Hz, 2H), 7.47 (d, J = 8.2 Hz,

Table 6. Deazaﬂavins in Vitro DMPK

a

b

aqueous solubility

P_a→b

P

/

^b→ab

compound xLogP

(μM)

(10⁻⁶cm s⁻¹

)

P_a→b

130

146

149

154

157

158

159

160

163

1.04

2.60

1.97

3.40

1.49

1.77

2.39

1.19

2.57

100

12.10

0.44

0.30

c

3.5

11.4

2.6

d

100

2H), 4.35 (d, J = 7.0 Hz, 2H), 3.35 (s, 3H), 2.73 (q, J = 7.6 Hz, 2H),

1.43−1.55 (m, 1H), 1.25 (t, J = 7.6 Hz, 3H), 0.55−0.68 (m, 4H).

3-(4-Ethylphenyl)-6-methyl-1-phenyl-pyrimido[5,4-e][1,2,4]-

triazine-5,7-dione, 42. Compound 42 was prepared in a manner

analogous to that for 41. The product 42 and N-oxide (at N-4) were

isolated crude, after trituration with Et₂O, as an approximate 8:2

mixture before the ﬁnal step reduction was carried out in reﬂuxing

ethanol for 1 h. The crude product was puriﬁed by preparative low pH

1−10

30−100

1−65

10−65

100

0.16

0.41

0.25

1.34

2.4

7.5

13.7

2.0

0.1

100

a

1

HPLC to give 42 (orange solid, 8.8 mg, 4%). H NMR (CDCl₃): δ

Turbidmetric aqueous solubility: concentration of test compound

8.20 (d, J = 8.2 Hz, 2H), 7.72−7.88 (m, 2H), 7.43−7.59 (m, 3H), 7.28

(d, J = 8.2 Hz, 2H), 3.47 (s, 3H), 2.68 (q, J = 7.6 Hz, 2H), 1.24 (t, J =

7.6 Hz, 3H).

that produces an increase in absorbance above 1% DMSO in buﬀer.

P_a→bis the permeability when applied to the apical side, while P_b→ais

b

the permeability when applied to the basolateral side in a Caco-2

membrane assay. The ratio of these two values is a measure of Pgp

eﬄux, while the P_a→bvalue is a measure of permeability.

1-Benzyl-3-(4-ethylphenyl)-6-methyl-pyrimido[5,4-e][1,2,4]-

triazine-5,7-dione, 43.⁵⁴To a solution of 69 (80 mg, 0.28 mmol) in

1,4-dioxane (10 mL) was added K₂CO₃(78 mg, 0.56 mmol) followed

by benzyl bromide (0.1 mL, 0.85 mmol). The reaction mixture was

heated at reﬂux for 5 h and then concentrated under vacuum. The

residue was recrystallized from EtOH to aﬀord 43 (orange solid, 11

mg, 10%); LC-MS (high pH) 1.38−1.40 min, m/z 374.4 [M + H]⁺,

100% purity.

c

d

Undetectable. Not applicable.

3-(4-Ethylphenyl)-1-methyl-pyrimido[5,4-e][1,2,4]triazine-5,7-

dione, 50. Compound 50 was prepared in a manner analogous to that

for 52 (orange solid, 140 mg, 67%). ¹H NMR (CDCl₃): δ 8.52 (br. s,

1H), 8.21 (d, J = 8.6 Hz, 2H), 7.34 (d, J = 8.6 Hz, 2H), 4.24 (s, 3H),

2.74 (q, J = 7.7 Hz, 2H), 1.29 (t, J = 7.7 Hz, 3H).

3-(4-Ethylphenyl)-1-methyl-6-phenyl-pyrimido[5,4-e][1,2,4]-

triazine-5,7-dione, 51. Compound 51 was prepared in a manner

analogous to that for 52 (orange solid, 60 mg, 39%). ¹H NMR

(CDCl₃): δ 8.23 (d, J = 8.2 Hz, 2H), 7.42−7.57 (m, 4H), 7.28−7.38

(m, 3H), 4.28 (s, 3H), 2.74 (q, J = 7.6 Hz, 2H), 1.29 (t, J = 7.6 Hz,

3H).

6-Benzyl-3-(4-ethylphenyl)-1-methyl-pyrimido[5,4-e][1,2,4]-

triazine-5,7-dione, 52. In an adaptation of the literature procedure,²⁴

to a mixture of 49 (100 mg, 0.28 mmol) in AcOH (5 mL) and water

(0.5 mL) at 5 °C under an atmosphere of nitrogen was added NaNO₂

(29 mg, 0.41 mmol), and the resulting mixture was stirred at 5 °C for

10 min before being warmed to ambient temperature and stirred for a

Figure 12. Deazaﬂavins hit to lead−hydrogen peroxide generation as

an indicator of redox activity. β-Lapachone was employed as a positive

control, and 1% DMSO was used as the negative control for this assay.

Bis(sulfanyl)butane-2,3-diol (473 mg, 3.06 mmol) was added, and the

resulting mixture was stirred for 1 h. The mixture was concentrated

M

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further 16 h. LC-MS analysis indicated a 4:1 mixture of 52/N-oxide (at

N-4). The reaction mixture was concentrated, and the residue was

dissolved in a 10:1 (v/v) mixture of EtOH/water (10 mL). 1,4-

Bis(sulfanyl)butane-2,3-diol (85 mg, 0.55 mmol) was added, and the

resulting mixture was stirred for 10 min. The mixture darkened

considerably, and a solid formed, which was collected by ﬁltration and

washed with EtOH (2 × 10 mL). The crude material was preadsorbed

onto silica and puriﬁed by ﬂash column chromatography, eluting with

a gradient of DCM to 5% MeOH/DCM to aﬀord 52 (orange solid, 50

3-[4-(3,4-Dimethoxyphenyl)-2-thienyl]-1,6-dimethyl-pyrimido-

[5,4-e][1,2,4]triazine-5,7-dione, 67. Compound 67 was prepared in a

1

manner analogous to that for 24 (purple solid, 128 mg, 38%). H

NMR (CDCl₃): δ 8.25 (d, J = 1.5 Hz, 1H), 7.56 (d, J = 1.5 Hz, 1H),

7.20 (dd, J = 8.2, 2.2 Hz, 1H), 7.12 (d, J = 2.2 Hz, 1H), 6.93 (d, J = 8.2

Hz, 1H), 4.20 (s, 3H), 3.98 (s, 3H), 3.93 (s, 3H), 3.52 (s, 3H).

1-Ethyl-3-(4-ethylphenyl)-6-methyl-pyrimido[4,5-c]pyridazine-

5,7-dione, 70. Compound 70 was prepared according to the

literature²⁴by reaction of the hydrazone intermediate 32 with the

commercially available 2-bromo-1-(4-ethylphenyl)ethanone to furnish

the product 70 (orange solid, 3.1 mg, 0.6% yield). ¹H NMR (CDCl₃):

δ 8.58 (s, 1H), 7.79 (d, J = 8.6 Hz, 2H), 7.30 (d, J = 8.6 Hz, 2H), 4.70

(q, J = 6.7 Hz, 2H), 2.67 (q, J = 7.7 Hz, 2H), 1.51 (t, J = 7.2 Hz, 3H),

1.11−1.25 (m, 6H).

1

mg, 49%). H NMR (CDCl₃): δ 8.20 (d, J = 8.4 Hz, 2H), 7.56−7.61

(m, 2H), 7.24−7.35 (m, 5H), 5.28 (br. s, 2H), 4.21 (s, 3H), 2.73 (q, J

= 7.6 Hz, 2H), 1.28 (t, J = 7.6 Hz, 3H).

1,6-Dimethyl-3-propyl-pyrimido[5,4-e][1,2,4]triazine-5,7-dione,

54. Compound 54 was prepared in a manner analogous to that for 24,

but in this example, the reaction mixture was concentrated and puriﬁed

by ﬂash column chromatography, eluting with a gradient of DCM to

5% MeOH/DCM (yellow solid, 12 mg, 3%); LC-MS (high pH) 0.74

min, m/z 236.5 [M + H]⁺, >95% purity.

3-Cyclopropyl-1,6-dimethyl-pyrimido[5,4-e][1,2,4]triazine-5,7-

dione, 55. Compound 55 was prepared in a manner analogous to that

for 24 using crude hydrazine, but in this example, the reaction mixture

was concentrated and puriﬁed by ﬂash column chromatography,

eluting with a gradient of DCM to 5% MeOH/DCM, followed by

trituration with diethyl ether (yellow solid, 142 mg, 32% yield from

chlorouracil 1); LC-MS (high pH) 0.64 min, m/z 234.5 [M + H]⁺,

>95% purity.

6-(4-Ethylphenyl)-3,8-dimethyl-pteridine-2,4-dione, 73. Com-

pound 73 was prepared according to the literature,²⁶by reaction of

the intermediate hydrazone 72 with the 6-amino-5-[(E)-(4-

ethylphenyl)methyleneamino]-1,3-dimethyl-pyrimidine-2,4-dione 71

1

to aﬀord the product 73 (cream solid, 22.9 mg, 2% yield). H NMR

(DMSO-d₆): δ 9.37 (s, 1H), 8.10 (d, J = 7.8 Hz, 2H), 7.41 (d, J = 7.8

Hz, 2H), 3.36 (s, 3H), 3.32 (s, 3H), 2.69 (q, J = 7.7 Hz, 2H), 1.24 (t, J

= 7.7 Hz, 3H).

7-Amino-1-methyl-3-(4-methyl-2-thienyl)pyrimido[5,4-e][1,2,4]-

triazin-5-one, 77. A suspension of 76 (384 mg, 1.46 mmol) in AcOH

(14 mL) was treated at ambient temperature with a solution of

NaNO₂(151 mg, 2.19 mmol) in water (1.4 mL). After 40 min, a

further 1 equiv of NaNO₂was added, and the mixture was stirred for

an additional 4 h, then cooled to 0 °C and diluted with diethyl ether

(30 mL). The precipitated solid was collected by ﬁltration, washed

with diethyl ether (2 × 5 mL), and dried to give a red/brown solid,

394 mg as a 1:21 mixture of 77/N-oxide (at N-4). This was taken up

in EtOH (39 mL), treated with a solution of 1,4-bis(sulfanyl)butane-

2,3-diol (528 mg, 3.42 mmol) in water (3.9 mL), and heated to 50 °C

for 2 h. The reaction mixture was cooled to ambient temperature, and

solids were collected by ﬁltration and washed with EtOH (2 × 5 mL).

The ﬁltrate was concentrated to give a red solid, which was

preadsorbed onto silica and puriﬁed, eluting with a gradient of

DCM to 40% MeOH/DCM to aﬀord 77 (orange solid, 10 mg, 3%).

¹H NMR (DMSO-d₆): δ 7.63−7.72 (m, 3H), 7.38−7.41 (m, 1H), 3.96

3-Cyclopentyl-1,6-dimethyl-pyrimido[5,4-e][1,2,4]triazine-5,7-

dione, 56. Compound 56 was prepared in a manner analogous to that

for 24, but in this example, the reaction mixture was concentrated and

puriﬁed by ﬂash column chromatography, eluting with a gradient of

1

DCM to 5% MeOH/DCM (yellow solid, 66 mg, 49%). H NMR

(CDCl₃): δ 4.12 (s, 3H), 3.49 (s, 3H), 3.34−3.46 (m, 1H), 1.69−2.18

(m, 8H).

3-Cyclohexyl-1,6-dimethyl-pyrimido[5,4-e][1,2,4]triazine-5,7-

dione, 57.⁵⁵Compound 57 was prepared in a manner analogous to

that for 24, but in this example, the reaction mixture was puriﬁed by

preparative HPLC (yellow solid, 2 mg, 1%); LC-MS (high pH) 0.96

min, m/z 276.5 [M + H]⁺, >95% purity.

1,6-Dimethyl-3-tetrahydropyran-4-yl-pyrimido[5,4-e][1,2,4]-

triazine-5,7-dione, 58. Compound 58 was prepared in a manner

analogous to that for 24, but in this example, the reaction mixture was

concentrated and puriﬁed by ﬂash column chromatography, eluting

with a gradient of DCM to 5% MeOH/DCM (yellow solid, 20 mg,

(s, 3H), 2.27 (s, 3H).

7-Amino-1-methyl-3-phenyl-4aH-pyrimido[4,5-c]pyridazine-4,5-

dione, 78.²⁷A suspension of 75 (500 mg, 3.22 mmol) in EtOH (25

mL) and water (25 mL) was heated to reﬂux and treated with ethyl

phenylglyoxylate (0.77 mL, 4.83 mmol) in one portion. The reaction

mixture was heated at reﬂux for 22 h, then ﬁltered hot, and the

collected solids were washed with 1:1 EtOH/water (2 × 3 mL) and

dried to give 78 (pale brown solid, 276 mg, 32%); LC-MS (high pH)

0.70 min, m/z 270.5 [M + H]⁺, 100% purity.

5-Benzyloxy-N-[4-(3,4-dimethoxyphenyl)-2-thienyl]-1-methyl-6-

oxo-pyrimidine-4-carboxamide, 81. To a solution of 5-benzyloxy-1-

methyl-6-oxo-pyrimidine-4-carboxylic acid (80; 71.8 mg, 0.28 mmol),

1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (63.5

mg, 0.33 mmol), 1-hydroxy-1H-benzotriazol-1-ol hydrate (50.7 mg,

0.33 mmol), and N,N-diisopropylamine (0.2 mL, 1.1 mmol) in DMF

(10 mL) at 0 °C under an atmosphere of nitrogen was added the

commercially available 4-(3,4-dimethoxyphenyl)thiophen-2-amine hy-

drochloride (75 mg, 0.28 mmol) after 1 h. The reaction mixture was

allowed to warm to ambient temperature and stirred for 72 h. The

product mixture was diluted with EtOAc (20 mL), extracted from

water (3 × 10 mL), dried, and concentrated. Puriﬁcation by

preparative HPLC at low pH followed by trituration with Et₂O

furnished 80 (yellow solid, 120 mg, 91%). ¹H NMR (CDCl₃): δ 10.30

(s, 1H), 8.02 (s, 1H), 7.56−7.62 (m, 2H), 7.35−7.46 (m, 3H), 7.05−

7.12 (m, 2H), 6.89−6.98 (m, 2H), 6.68 (d, J = 1.7 Hz, 1H), 5.53 (s,

2H), 3.97 (s, 3H), 3.94 (s, 3H), 3.61 (s, 3H).

1

4%). H NMR (CDCl₃): δ 4.13 (s, 3H), 4.06−4.13 (m, 2H), 3.49−

3.58 (m, 2H), 3.49 (s, 3H), 3.17−3.28 (m, 1H), 1.85−2.06 (m, 4H).

3-Benzyl-1,6-dimethyl-pyrimido[5,4-e][1,2,4]triazine-5,7-dione,

59.⁵⁵Compound 59 was prepared in a manner analogous to that for

24 (brown solid, 47 mg, 19%); LC-MS (high pH) 0.87 min, m/z 284.5

[M + H]⁺, >95% purity.

1,6-Dimethyl-3-phenethyl-pyrimido[5,4-e][1,2,4]triazine-5,7-

dione, 60.⁵⁵Compound 60 was prepared in a manner analogous to

that for 24 (yellow solid, 29 mg, 8%); LC-MS (high pH) 0.92 min, m/

z 298.5 [M + H]⁺, >95% purity.

1,6-Dimethyl-3-thiazol-4-yl-pyrimido[5,4-e][1,2,4]triazine-5,7-

dione, 63. Compound 63 was prepared in a manner analogous to that

for 24, but in this example, the collected solid was puriﬁed by

1

trituration with 10% MeOH/DCM (orange solid, 11 mg, 3%). H

NMR (DMSO-d₆): δ 9.29 (d, J = 1.9 Hz, 1H), 8.53 (d, J = 1.9 Hz,

1H), 4.03 (s, 3H), 3.28 (s, 3H).

1,6-Dimethyl-3-(4-methyl-2-thienyl)pyrimido[5,4-e][1,2,4]-

triazine-5,7-dione, 64. Compound 64 was prepared in a manner

analogous to that for 24 (orange solid, 207 mg, 54%). ¹H NMR

(CDCl₃): δ 7.85 (d, J = 1.5 Hz, 1H), 7.12−7.14 (m, 1H), 4.16 (s, 3H),

3.51 (s, 3H), 2.31 (m, 3H).

1,6-Dimethyl-3-(4-phenyl-2-thienyl)pyrimido[5,4-e][1,2,4]-

triazine-5,7-dione, 66. Compound 66 was prepared in a manner

analogous to that for 24, but in this example, the collected solid was

[4-(3,4-Dimethoxyphenyl)-2-thienyl]-(3-dimethoxyphosphoryl-

carbonyl-1-piperidyl)methanone, 85. Compound 85 was prepared

according to the literature²⁹to furnish the product as a beige gum (83

1

puriﬁed by trituration with DCM (red solid, 14 mg, 11%). H NMR

1

(CDCl₃): δ 8.30 (d, J = 1.5 Hz, 1H), 7.62−7.67 (m, 3H), 7.40−7.47

(m, 2H), 7.35 (tt, J = 7.3, 1.3 Hz, 1H), 4.19 (s, 3H), 3.52 (s, 3H).

mg, 16% yield). H NMR (DMSO-d₆): δ 6.94−8.87 (m, 5H- mixture

of rotomers), 3.55−5.15 (m, 12H- mixture of rotomers), 2.98−3.44

N

dx.doi.org/10.1021/jm400568p | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

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(m, 2H- mixture of rotomers), 2.47−2.53 (m, 3H- mixture of

rotomers), 2.21−2.41 (m, 1H- mixture of rotomers), 1.86−2.08 (m,

1H- mixture of rotomers), 1.21−1.78 (m, 2H- mixture of rotomers).

3-[4-(3,4-Dimethoxyphenyl)-2-thienyl]-N-methylsulfonyl-benza-

mide, 93.³¹A suspension of methanesulfonamide (61.5 mg, 0.65

mmol), 3-[4-(3,4-dimethoxyphenyl)-2-thienyl]benzoic acid (91; 200

mg, 0.59 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide

hydrochloride (135.2 mg, 0.71 mmol), and 4-dimethylaminopyridine

(35.9 mg, 0.29 mmol) in THF (3 mL) was stirred at ambient

temperature for 16 h. The reaction mixture was diluted with water (10

mL) and washed with EtOAc (3 mL). The aqueous layer was acidiﬁed

to pH 1 with 2 N HCl (aq) (5 mL) and extracted into EtOAc (3 × 10

mL). The combined organic layers were washed with brine (5 mL),

dried, and concentrated. The crude product was puriﬁed by

preparative HPLC at high pH to aﬀord 93 (white solid, 5.2 mg,

N-[4-(3,4-Dimethoxyphenyl)-2-thienyl]-1-methyl-pyrazole-3-car-

boxamide, 101. Compound 101 was prepared in a manner analogous

1

to that for 96 (white solid, 18.2 mg, 21%). H NMR (DMSO-d₆): δ

7.87 (d, J = 2.7 Hz, 1H), 7.36 (d, J = 1.8 Hz, 1H), 7.17−7.22 (m, 2H),

7.07−7.14 (m, 1H), 6.98 (d, J = 8.2 Hz, 1H), 6.79 (d, J = 2.2 Hz, 1H),

3.99 (s, 3H), 3.83 (s, 3H), 3.78 (s, 3H).

N-[4-(3,4-Dimethoxyphenyl)-2-thienyl]-4-methyl-thiadiazole-5-

carboxamide, 102. Compound 102 was prepared in a manner

analogous to that for 96 (cream solid, 6.3 mg, 7%). ¹H NMR (DMSO-

d₆): δ 7.31−7.35 (m, 1H), 7.13−7.24 (m, 3H), 6.99 (d, J = 7.9 Hz,

1H), 3.83 (s, 3H), 3.78 (s, 3H), 2.87 (s, 3H).

N-[4-(3,4-Dimethoxyphenyl)-2-thienyl]-2,4-dimethyl-6-oxo-

pyran-3-carboxamide, 103. N,N-Diisopropylamine (0.24 mL, 1.47

mmol) was added to a suspension of the commercially available 4-(3,4-

dimethoxyphenyl)thiophen-2-amine hydrochloride 95 (100 mg, 0.37

mmol) in DMF (5 mL). After being stirred at ambient temperature for

15 min, the solution was cooled to 0 °C, and 4,6-dimethyl-2-oxo-2H-

pyran-5-carboxylic acid (61.9 mg, 0.37 mmol) and 1-(3-dimethylami-

nopropyl)-3-ethylcarbodiimide hydrochloride (84.7 mg, 0.44 mmol)

were added. The solution was allowed to warm to ambient and left to

stir for 16 h. The reaction mixture was concentrated to dryness,

partitioned between EtOAc (20 mL) and water (10 mL), and

separated. The organic layer was washed with 5% Na₂CO₃(aq) (2 ×

30 mL) and saturated brine (30 mL), dried, and concentrated. The

crude product was ﬁltered and washed with Et₂O to aﬀord 103 (pale

1

2%). H NMR (DMSO-d₆): δ 8.16−8.24 (m, 1H), 7.72−8.00 (m,

4H), 7.30−7.46 (m, 3H), 7.00 (d, J = 8.3 Hz, 1H), 3.79 (s, 3H), 2.46−

2.54 (m, 6H).

4-[4-(3,4-Dimethoxyphenyl)-2-thienyl]-N-methylsulfonyl-benza-

mide, 94.³¹A suspension of methanesulfonamide (27.7 mg, 0.29

mmol), acid 92 (90 mg, 0.26 mmol), 1-(3-dimethylaminopropyl)-3-

ethylcarbodiimide hydrochloride (60.8 mg, 0.32 mmol), and 4-

dimethylaminopyridine (16.2 mg, 0.13 mmol) in THF (3 mL) was

stirred at ambient temperature for 16 h. The reaction mixture was

diluted with water (10 mL) and washed with EtOAc (3 mL). The

aqueous layer was acidiﬁed to pH 1 with 2 N HCl (aq) (5 mL) and

extracted into EtOAc (3 × 10 mL). The combined organic layers were

washed with brine (5 mL), dried, and concentrated. The crude

product was puriﬁed by preparative HPLC at high pH to give 94

1

yellow solid, 22.7 mg, 16%). H NMR (DMSO-d₆): δ 13.11 (br. s,

1H), 7.76−7.82 (m, 1H), 7.55−7.63 (m, 1H), 7.21−7.35 (m, 2H),

6.99 (d, J = 8.2 Hz, 1H), 5.68 (s, 1H), 3.84 (s, 3H), 3.78 (s, 3H), 2.61

(s, 3H), 2.41 (s, 3H).

1

1-[4-(3,4-Dimethoxyphenyl)-2-thienyl]-3-phenyl-urea, 104. Com-

pound 104 was prepared according to the literature⁴⁹by reaction of 95

with phenyl isocyanate to furnish the product as a white solid (22.6

(white solid, 2.1 mg, 2%). H NMR (DMSO-d₆): δ 8.33 (s, 1H),

8.02−8.08 (m, 1H), 7.98 (d, J = 8.1 Hz, 2H), 7.78−7.82 (m, 1H), 7.71

(d, J = 8.0 Hz, 2H), 7.31−7.41 (m, 2H), 7.00 (d, J = 8.6 Hz, 1H), 3.80

(s, 3H), 2.42−2.56 (m, 6H).

1

mg, 17% yield). H NMR (DMSO-d₆): δ 9.63 (s, 1H), 8.82 (s, 1H),

7.47 (d, J = 7.9 Hz, 2H), 7.30 (t, J = 7.9 Hz, 2H), 7.10−7.22 (m, 3H),

N-[4-(3,4-Dimethoxyphenyl)-2-thienyl]-3,5-dimethyl-isoxazole-4-

carboxamide, 96. 3,5-Dimethyl-4-isoxazolecarboxylic acid (90.9 mg,

0.64 mmol) was heated to reﬂux in thionyl chloride (2.1 mL, 28.79

mmol) for 5 h. The reaction mixture was evaporated to dryness,

redissolved in THF (3 mL), and added to a cooled (0 °C), stirred

solution of N,N-diisopropylamine (0.13 mL, 0.77 mmol), 4-

dimethylaminopyridine (1.36 mg, 0.01 mmol), and 4-(3,4-

dimethoxyphenyl)thiophen-2-amine hydrochloride 95 (70 mg, 0.26

mmol) in THF (3 mL). The reaction mixture was stirred at ambient

temperature for 1 h before being partitioned between EtOAc (30 mL)

and water (30 mL). The organic layer was washed with 5% Na₂CO₃

(aq) (2 × 30 mL) and saturated brine (30 mL), dried, and

concentrated. The crude product was puriﬁed by preparative HPLC

at high pH to give 96 (cream solid, 23.1 mg, 25%). ¹H NMR (DMSO-

d₆): δ 7.27−7.30 (m, 1H), 7.13−7.22 (m, 3H), 6.98 (d, J = 8.4 Hz,

1H), 3.83 (s, 3H), 3.78 (s, 3H), 2.59 (s, 3H), 2.37 (s, 3H).

6.89−7.04 (m, 3H), 3.83 (s, 3H), 3.78 (s, 3H).

10-Methylpyrimido[4,5-b]quinoline-2,4-dione, 130.³³Prepared

according to the literature (yellow solid, 6.5 mg, 8%). ¹H NMR

(DMSO-d₆): δ 9.00 (s, 1H), 8.19 (d, J = 8.0 Hz, 1H), 7.92−8.00 (m,

2H), 7.55 (dd, J = 7.0, 2.0 Hz, 1H), 4.04 (s, 3H).

9-Chloro-10-phenyl-pyrimido[4,5-b]quinoline-2,4-dione, 134. A

suspension of 3-chloro-2-ﬂuorobenzaldehyde (65.6 mg, 0.41 mmol)

and 6-anilino-1H-pyrimidine-2,4-dione (106; 70.0 mg, 0.34 mmol) in

DMF (2 mL) was heated by microwave irradiation at 110 °C for 30

min. Water (20 mL) was added to the reaction mixture, and the

resulting precipitate was ﬁltered and washed with water. The ﬁltered

solid was dried under vacuum and then puriﬁed by preparative HPLC

at high pH to yield the product 134 as a yellow solid (20.4 mg, 18%).

¹H NMR (DMSO-d₆): δ 11.6, (s, 1H), 9.10 (s, 1H), 8.25 (dd, J = 8.0,

1.6 Hz, 1H), 7.84 (dd, J = 8.0, 1.6 Hz, 1H), 7.38−7.53 (m, 6H).

7-Chloro-10-phenyl-pyrimido[4,5-b]quinoline-2,4-dione, 135.

Compound 135 was prepared in a manner analogous to that for

N-[4-(3,4-Dimethoxyphenyl)-2-thienyl]benzamide, 97. Com-

pound 97 was prepared in a manner analogous to that for 96 (orange

1

134 (yellow solid, 1.56 mg, 2%). H NMR (DMSO-d₆): δ 9.10 (s,

solid, 52.4 mg, 47%). H NMR (DMSO-d₆): δ 7.98−8.07 (m, 1H),

1H), 8.39 (d, J = 2.5 Hz, 1H), 7.64−7.78 (m, 4H), 7.42−7.45 (m,

7.52−7.67 (m, 3H), 7.12−7.30 (m, 4H), 7.00 (d, J = 8.6 Hz, 1H), 3.84

2H), 6.71 (d, J = 9.3 Hz, 1H).

(s, 3H), 3.78 (s, 3H).

6-Chloro-10-phenyl-pyrimido[4,5-b]quinoline-2,4-dione, 136.³⁴

Compound 136 was prepared in a manner analogous to that for

N-[4-(3,4-Dimethoxyphenyl)-2-thienyl]-1H-imidazole-2-carboxa-

mide, 98. Compound 98 was prepared in a manner analogous to that

for 96 (cream solid, 3.2 mg, 4%). ¹H NMR (DMSO-d₆): δ 7.42 (br. s,

1H), 7.08−7.26 (m, 3H), 7.00 (d, J = 8.5 Hz, 1H), 3.83 (s, 3H), 3.78

(s, 3H).

1

134 (yellow solid, 63.5 mg, 57%). H NMR (DMSO-d₆): δ 11.2 (s,

1H), 9.03 (d, J = 0.7 Hz, 1H), 7.64−7.74 (m, 5H), 7.42−7.45 (m,

2H), 6.69 (ddd, J = 7.3, 2.4, 0.9 Hz, 1H). LC-MS m/z 324.5, 326.5 [M

+ H]⁺, 89% purity.

N-[4-(3,4-Dimethoxyphenyl)-2-thienyl]-1-methyl-imidazole-2-

carboxamide, 99. Compound 99 was prepared in a manner analogous

8-Chloro-10-methyl-pyrimido[4,5-b]quinoline-2,4-dione, 137.

1

to that for 96 (cream solid, 8.6 mg, 10%). H NMR (DMSO-d₆): δ

Compound 137 was prepared in a manner analogous to that for

1

7.47−7.49 (m, 1H), 7.37−7.41 (m, 1H), 7.23−7.26 (m, 1H), 7.17−

7.20 (m, 1H), 7.23−7.26 (m, 1H), 7.08−7.14 (m, 2H), 6.99 (d, J = 8.1

Hz, 1H), 4.02 (s, 3H), 3.83 (s, 3H), 3.78 (s, 3H).

134 (yellow solid, 86.9 mg, 67%). H NMR (DMSO-d₆): δ 11.1 (s,

1H), 9.00 (s, 1H), 8.21 (d, J = 8.6 Hz, 1H), 8.04 (d, J = 1.7 Hz, 1H),

7.61 (dd, J = 8.6, 1.7 Hz, 1H), 4.01 (s, 3H).

N-[4-(3,4-Dimethoxyphenyl)-2-thienyl]-3-methyl-isoxazole-5-car-

8-Chloro-10-cyclopropyl-pyrimido[4,5-b]quinoline-2,4-dione,

138. Compound 138 was prepared in a manner analogous to that for

134 (yellow solid, 80.4 mg, 67%). H NMR (DMSO-d₆): δ 11.1 (s,

boxamide, 100. Compound 100 was prepared in a manner analogous

1

to that for 96 (cream solid, 26.9 mg, 30%). H NMR (DMSO-d₆): δ

7.28−7.35 (m, 2H), 7.19−7.22 (m, 1H), 7.10−7.17 (m, 2H), 6.99 (d, J

= 7.8 Hz, 1H), 3.84 (s, 3H), 3.78 (s, 3H), 2.35 (s, 3H).

1H), 8.92 (s, 1H), 8.15−8.18 (m, 2H), 7.57 (dd, J = 8.5, 1.8 Hz, 1H),

1.42−1.49 (m 2H), 1.23 (s, 1H), 0.92−0.97 (m, 2H).

O

dx.doi.org/10.1021/jm400568p | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

8-Chloro-10-cyclohexyl-pyrimido[4,5-b]quinoline-2,4-dione, 139.

Compound 139 was prepared in a manner analogous to that for 134

(yellow solid, 13.4 mg, 12%). H NMR (DMSO-d₆): δ 11.2 (s, 1H),

8.96 (s, 1H), 8.20 (d, J = 8.5 Hz, 1H), 7.59 (d, J = 8.5 Hz, 1H), 1.06−

1.89 (m, 11H).

HRMS (ESI) m/z [M + H]⁺calcd for C₁₈H₁₁N₄O₃: 331.0826. Found:

331.0820.

1

10-(4-Hydroxyphenyl)-2,4-dioxo-pyrimido[4,5-b]quinoline-8-car-

bonitrile, 148. Compound 148 was prepared in a manner analogous

to that for 134 (orange solid, 49.8 mg, 47%). ¹H NMR (DMSO-d₆): δ

11.2 (br. s, 1H), 10.1 (br. s, 1H), 9.14 (s, 1H), 8.39 (d, J = 8.1 Hz,

1H), 7.87 (dd, J = 8.1, 1.4 Hz, 1H), 7.19 (app. d, J = 8.8 Hz, 2H), 7.12

(s, 1H), 7.03 (app. d, J = 8.8 Hz, 2H). ¹³C NMR (DMSO-d₆): δ 161.5,

159.2, 158.3, 156.3, 141.9, 140.9, 132.4, 129.3, 127.7, 126.2, 123.6,

121.1, 118.2, 117.7, 116.7, 115.6. HRMS (ESI) m/z [M + H]⁺calcd

for C₁₈H₁₁N₄O₃: 331.0826. Found: 331.0819.

8-Chloro-10-(4-piperidyl)pyrimido[4,5-b]quinoline-2,4-dione,

140. Compound 140 was prepared in a manner analogous to that for

134, with an additional deprotection step of the N-Boc piperidinyl

precursor with DCM/TFA (3:1 v/v) at ambient temperature (yellow

1

solid, 37.2 mg, 23%). H NMR (DMSO-d₆): δ 8.97 (s, 1H), 8.38 (s,

2H), 8.32 (br. s, 1H), 8.21 (d, J = 8.6 Hz, 1H), 7.60 (dd, J = 8.6, 1.1

Hz, 1H), 3.30 (app. d, J = 9.5 Hz, 2H), 2.61−3.10 (m, 4H), 1.81 (app.

d, J = 9.5 Hz, 2H).

10-[3-(Hydroxymethyl)phenyl]-2,4-dioxo-pyrimido[4,5-b]-

quinoline-8-carbonitrile, 149. Compound 149 was prepared in a

1

manner analogous to that for 134 (yellow solid, 11 mg, 11%). H

8-Chloro-10-(4-hydroxyphenyl)pyrimido[4,5-b]quinoline-2,4-

NMR (DMSO-d₆): δ 11.20 (s, 1H), 9.17 (s, 1H), 8.42 (d, J = 8.6 Hz,

1H), 7.90 (dd, J = 8.2, 1.4 Hz, 1H), 7.67 (t, J = 8.0 Hz, 1H), 7.60 (app.

d, J = 8.0 Hz, 1H), 7.37 (br s, 1H), 7.28−7.32 (m, 1H), 7.00 (s, 1H),

5.42 (t, J = 5.8 Hz, 1H), 4.64 (d, J = 5.8 Hz, 2H). ¹³C NMR (DMSO-

d₆): δ 161.5, 158.9, 156.3, 145.4, 141.3, 141.0, 136.7, 132.5, 130.1,

127.4, 126.5, 126.3, 125.8, 123.6, 120.8, 118.2, 115.6, 66.3. HRMS

(ESI) m/z [M + H]⁺calcd for C₁₉H₁₃N₄O₃: 345.0983. Found:

345.0984.

dione, 141. Compound 141 was prepared in a manner analogous to

1

that for 134 (yellow solid, 81.1 mg, 75%). H NMR (DMSO-d₆): δ

11.1 (s, 1H), 10.0 (s, 1H), 9.10 (s, 1H), 8.26 (d, J = 8.6 Hz, 1H), 7.57

(dd, J = 8.6, 1.9 Hz, 1H), 7.20 (app. d, J = 8.6 Hz, 2H), 7.02 (app. d, J

= 8.6 Hz, 2H), 6.70 (d, J = 1.9 Hz, 1H). ¹³C NMR (DMSO-d₆): δ

161.8, 159.1, 158.1, 156.4, 143.1, 141.5, 139.3, 133.1, 129.3, 128.1,

124.6, 119.8, 116.7, 116.4, 115.9. HRMS (ESI) m/z [M + H]⁺calcd

for C₁₇H₁₁N₃O₃Cl: 340.0484. Found: 340.0482.

10-(3-Methoxyphenyl)-2,4-dioxo-pyrimido[4,5-b]quinoline-8-car-

8-Chloro-10-(4-methoxyphenyl)pyrimido[4,5-b]quinoline-2,4-

bonitrile, 150. Compound 150 was prepared in a manner analogous

dione, 142. Compound 142 was prepared in a manner analogous to

1

to that for 134 (yellow solid, 10 mg, 14%). H NMR (DMSO-d₆): δ

that for 134 (yellow solid, 82.9 mg, 78%). H NMR (DMSO-d₆): δ

11.26 (s, 1H), 9.16 (s, 1H), 8.40 (d, J = 8.6 Hz, 1H), 7.88 (dd, J = 8.6,

1.3 Hz, 1H), 7.62 (t, J = 8.6 Hz, 1H), 7.23 (ddd, J = 8.4, 2.6, 0.8 Hz,

1H), 7.09 (app. t, J = 0.8 Hz, 1H), 7.06 (dd, J = 2.4, 1.8 Hz, 1H), 6.99

(ddd, J = 8.4, 2.6, 0.8 Hz, 1H), 3.82 (s, 3H). LC-MS m/z 343.5, [M −

H]⁻, 91% purity.

10-(4-Methoxyphenyl)-2,4-dioxo-pyrimido[4,5-b]quinoline-8-car-

bonitrile, 151. Compound 151 was prepared in a manner analogous

to that for 134 (yellow solid, 88.6 mg, 86%). ¹H NMR (DMSO-d₆): δ

11.2 (s, 1H), 9.15 (s, 1H), 8.40 (d, J = 8.1 Hz, 1H), 7.88 (dd, J = 8.1,

1.4 Hz, 1H), 7.35 (app. d, J = 8.9 Hz, 2H), 7.23 (app. d, J = 8.9 Hz,

2H), 7.12 (s, 1H), 3.90 (s, 3H). ¹³C NMR (DMSO-d₆): δ 161.5,

159.7, 159.2, 156.3, 141.7, 141.0, 132.4, 129.4, 129.2, 126.2, 123.6,

121.0, 118.2, 117.7, 115.5, 55.5. HRMS (ESI) m/z [M + H]⁺calcd for

C₁₉H₁₃N₄O₃: 345.0983. Found: 345.0989.

11.1 (s, 1H), 9.12 (s, 1H), 8.27 (d, J = 8.6 Hz, 1H), 7.58 (dd, J = 8.6

Hz, 1H), 7.36 (app. d, J = 8.9 Hz, 2H), 7.23 (app. d, J = 8.9 Hz, 2H),

6.68 (d, J = 1.9 Hz, 1H), 3.89 (s, 3H).

8-Chloro-10-(3-hydroxyphenyl)pyrimido[4,5-b]quinoline-2,4-

dione, 143. Compound 143 was prepared in a manner analogous to

1

that for 134 (yellow solid, 25.9 mg, 33%). H NMR (DMSO-d₆): δ

11.1 (br. s, 1H), 10.1 (br. s, 1H), 9.11 (s, 1H), 8.26 (d, J = 8.8 Hz,

1H), 7.58 (dd, J = 8.5, 1.8 Hz, 1H), 7.48 (t, J = 8.0 Hz, 1H), 7.03 (app.

dd, J = 8.3, 2.3 Hz, 1H), 6.79−6.84 (m, 2H), 6.68 (d, J = 1.8 Hz, 1H).

¹³C NMR (DMSO-d₆): δ 161.7, 158.9, 158.6, 156.4, 142.4, 141.6,

139.2, 138.0, 133.1, 131.1, 124.7, 119.8, 118.4, 116.6, 116.3, 115.9,

115.1. HRMS (ESI) m/z [M + H]⁺calcd for C₁₇H₁₁N₃O₃Cl:

340.0484. Found: 340.0482.

8-Chloro-10-(2-hydroxyphenyl)pyrimido[4,5-b]quinoline-2,4-

10-(3-Hydroxy-4-methoxy-phenyl)-2,4-dioxo-pyrimido[4,5-b]-

dione, 144. Compound 144 was prepared in a manner analogous to

quinoline-8-carbonitrile, 152. Compound 152 was prepared in a

1

that for 134 (yellow solid, 85.7 mg, 79%). H NMR (DMSO-d₆): δ

1

manner analogous to that for 134 (orange solid, 12.0 mg, 8%). H

11.2 (s, 1H), 10.0 (s, 1H), 9.1 (s, 1H), 8.27 (d, J = 8.6 Hz, 1H), 7.59

(dd, J = 8.5, 1.8 Hz, 1H), 7.47 (app. t, J = 7.8 Hz, 1H), 7.29 (dd, J =

7.8, 1.5 Hz, 1H), 7.15 (d, J = 8.5 Hz, 1H), 7.08 (t, J = 7.8 Hz, 1H),

6.68 (d, J = 1.8 Hz, 1H).

10-Phenyl-8-(triﬂuoromethyl)pyrimido[4,5-b]quinoline-2,4-dione,

145. Compound 145 was prepared in a manner analogous to that for

134 (yellow solid, 21.7 mg, 25%). ¹H NMR (DMSO-d₆): δ 11.2 (br. s,

1H), 9.21 (s, 1H), 8.48 (d, J = 7.9 Hz, 1H), 7.84 (dd, J = 8.4, 1.4 Hz,

1H), 7.64−7.76 (m, 3H), 7.47−7.50 (m, 2H), 6.82 (s, 1H). ¹³C NMR

(DMSO-d₆): δ 161.5, 159.0, 156.3, 141.5, 141.3, 137.0, 133.0, 130.4,

129.8, 128.3, 123.3, 120.1, 118.0, 117.7, 113.4 (CF₃not observed).

HRMS (ESI) m/z [M + H]⁺calcd for C₁₈H₁₁N₃O₂F₃: 358.0798.

Found: 358.0798.

NMR (DMSO-d₆): δ 11.22 (br. s, 1H), 9.58 (br. s, 1H), 9.13 (s, 1H),

8.38 (d, J = 8.6 Hz, 1H), 7.87 (dd, J = 8.6, 1.3 Hz, 1H), 7.16−7.20 (m,

2H), 6.77−6.81 (m, 2H), 3.90 (s, 3H).

10-(3-Fluorophenyl)-2,4-dioxo-pyrimido[4,5-b]quinoline-8-car-

bonitrile, 153. Compound 153 was prepared in a manner analogous

to that for 134 (yellow solid, 22.0 mg, 14%). ¹H NMR (DMSO-d₆): δ

11.29 (s, 1H), 9.18 (s, 1H). 8.41 (d, J = 8.6 Hz, 1H), 7.90 (dd, J = 8.6,

1.3 Hz, 1H), 7.77 (td, J = 8.1, 6.4 Hz, 1H), 7.53 (tdd, J = 8.6, 1.9, 0.8

Hz, 1H), 7.43 (ddd, J = 9.4, 2.7, 1.5 Hz, 1H), 7.38 (ddd, J = 8.0, 1.4,

0.8 Hz, 1H), 7.22 (s, 1H).

10-(4-Bromophenyl)-2,4-dioxo-pyrimido[4,5-b]quinoline-8-car-

bonitrile, 154. Compound 154 was prepared in a manner analogous

to that for 134 (yellow solid, 22.6 mg, 23%). ¹H NMR (DMSO-d₆): δ

9.17 (s, 1H), 8.41 (d, J = 8.1 Hz, 1H), 7.88−7.92 (m, 3H), 7.42 (app.

d, J = 8.6 Hz, 2H), 7.26 (s, 1H). ¹³C NMR (DMSO-d₆): δ 161.4,

159.0, 156.1, 141.2, 141.1, 136.1, 133.5, 130.7, 126.5, 124.2, 123.6,

123.0, 121.1, 117.7, 115.9. LC-MS m/z 393.4 [M + H]⁺, 92% purity.

HRMS (ESI) m/z [M + H]⁺calcd for C₁₈H₁₀N₄O₂Br: 392.9982.

Found: 392.9979.

2,4-Dioxo-10-phenyl-pyrimido[4,5-b]quinoline-8-carbonitrile,

146. Compound 146 was prepared in a manner analogous to that for

1

134 (yellow solid, 52.7 mg, 68%). H NMR (DMSO-d₆): δ 11.3 (s,

1H), 9.17 (s, 1H), 8.42 (d, J = 8.1 Hz, 1H), 7.89 (dd, J = 8.1, 1.4 Hz,

1H), 7.64−7.76 (m, 3H), 7.43−7.47 (m, 2H), 7.01 (s, 1H). ¹³C NMR

(DMSO-d₆): δ 161.4, 158.9, 156.2, 141.3, 141.1, 136.8, 132.5, 130.4,

129.7, 128.4, 126.3, 123.6, 120.8, 118.2, 117.7, 115.6. HRMS (ESI) m/

z [M + H]⁺calcd for C₁₈H₁₁N₄O₂: 315.0877. Found: 315.0882.

10-(3-Hydroxyphenyl)-2,4-dioxo-pyrimido[4,5-b]quinoline-8-car-

bonitrile, 147. Compound 147 was prepared in a manner analogous

to that for 134 (yellow solid, 31.7 mg, 42%). ¹H NMR (DMSO-d₆): δ

11.2 (br. s, 1H), 10.1 (br. s, 1H), 9.14 (s, 1H), 8.40 (d, J = 8.1 Hz,

1H), 7.88 (dd, J = 8.1, 1.4 Hz, 1H), 7.49 (t, J = 8.1 Hz, 1H), 7.09 (s,

1H), 7.04 (app. d, J = 8.1 Hz, 1H), 6.79−6.83 (m, 2H). ¹³C NMR

(DMSO-d₆): δ 161.5, 158.9, 158.7, 156.3, 141.2, 141.0, 137.7, 132.4,

131.1, 126.2, 123.6, 120.9, 118.4, 118.2, 117.7, 116.7, 115.6, 115.2.

10-(3-Aminophenyl)-2,4-dioxo-pyrimido[4,5-b]quinoline-8-car-

bonitrile, 155. Compound 155 was prepared in a manner analogous

to that for 134, with an additional deprotection step of the N-Boc

aminophenyl precursor with DCM/TFA (3:1 v/v) (11.5 mL) at

1

ambient temperature for 2 h (orange solid, 17.2 mg, 17%). H NMR

(DMSO-d₆): δ 11.2 (br. s, 1H), 9.13 (s, 1H), 8.39 (d, J = 8.1 Hz, 1H),

7.87 (dd, J = 8.1, 1.4 Hz, 1H), 7.31 (t, J = 7.8 Hz, 1H), 7.01 (s, 1H),

6.80 (app. d, J = 8.1 Hz, 1H), 6.45−6.50 (m, 2H), 5.56 (br. s, 2H). ¹³C

NMR (DMSO-d₆): δ 161.5, 158.5, 156.3, 150.7, 141.2, 140.9, 137.5,

132.3, 130.7, 126.1, 123.5, 121.0, 118.2, 115.5, 114.8, 114.4, 112.6.

P

dx.doi.org/10.1021/jm400568p | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

HRMS (ESI) m/z [M + H]⁺calcd for C₁₈H₁₂N₅O₂: 330.0986. Found:

330.0979.

before being stopped with the addition of 33 mM ethylenediaminete-

traacetic acid (EDTA). The signal generated during the assay was

quantiﬁed by absorbance at 405 nm using a BioTek Synergy 2

multimode microplate reader.

N-[3-(8-Cyano-2,4-dioxo-pyrimido[4,5-b]quinolin-10-yl)phenyl]-

acetamide, 156. Compound 156 was prepared in a manner analogous

to that for 134 (yellow solid, 12.0 mg, 17%). ¹H NMR (DMSO-d₆): δ

11.24 (s, 1H), 10.31 (s, 1H), 9.15 (s, 1H), 8.41 (d, J = 8.1 Hz, 1H),

7.90 (dd, J = 8.1, 1.4 Hz, 1H), 7.78−7.80 (m, 1H), 7.67−7.72 (m,

1H), 7.65 (t, J = 8.0 Hz, 1H), 7.06−7.10 (m, 2H) 3.03 (s, 3H). HRMS

(ESI) m/z [M + H]⁺calcd for C₂₀H₁₄N₅O₃: 372.1092. Found:

372.1097.

TDP2 Oligonucleotide Assay. A 50-phosphotyrosyl oligonucleotide

(50-Y-TCCGTTGAAGCCTGCTTT-30) was purchased from Mid-

land Certiﬁed Reagent (USA). Assays were assembled in a ﬁnal

volume of 25 μL in 384-well clear plates in a buﬀer composed of 25

mM Hepes (pH 8.0), 10 mM MgCl₂, 130 mM KCl, 1 mM DTT,

0.03% BSA, 1% DMSO with or without inhibitory compound, 1.5 U of

calf intestinal alkaline phosphatase (CIP, Roche), and 0.36 nM TDP2

protein. The reaction was initiated by the addition of 25 μL of 1 M 50-

phosphotyrosyl oligonucleotide, and the assay was allowed to proceed

at 25 °C for 60 min. Then, 25 μL of Biomol Green reagent (Enzo Life

Sciences) was added to stop and develop the reaction. Following

further incubation for 45 min, the absorbance was read at 620 nm

using a BioTek Synergy 2 multimode microplate reader.

TDP1 Assay. The assay was performed in 384-well black plates in a

25 μL ﬁnal volume with a reaction buﬀer composed of 50 mM Tris

HCl, pH 7.5, 2 mM MgCl₂, 25 mM NaCl, 1 mM DTT, 0.01% Tween-

20, 1% DMSO, 5 nM TDP1, and 50 nM labeled double-stranded AP-

site containing DNA substrate. The oligonucleotides 5′-GAG TCG

TAC GAG GGT GA-[BHQ2]-3′, where BHQ2 is Black Hole

Quencher-2, and 5′-[TAM]-TCA CCΦ TCG TAC GAC TC-3′,

where TAM is TAMRA and Φ is dSpacer, were annealed together

to create the double-stranded DNA substrate. The assay was allowed

to proceed at 25 °C for 30 min before stopping with the addition of

0.08% SDS. The ﬂuorescent signal generated during the assay was

quantiﬁed with excitation at 540 nm and emission at 590 nm using a

BioTek Synergy 2 multimode microplate reader.

N-[3-(8-Cyano-2,4-dioxo-pyrimido[4,5-b]quinolin-10-yl)phenyl]-

methanesulfonamide, 157. Compound 157 was prepared in a

1

manner analogous to that for 134 (yellow solid, 28.0 mg, 20%). H

NMR (DMSO-d₆): δ 11.16 (s, 1H), 10.15 (s, 1H), 9.14 (s, 1H). 8.40

(d, J = 8.6 Hz, 1H), 7.89 (dd, J = 8.2, 1.4 Hz, 1H), 7.66 (t, J = 8.0 Hz,

1H), 7.43 (ddd, J = 8.2, 2.4, 1.0 Hz, 1H), 7.23 (dd, J = 2.4, 1.4 Hz,

1H), 7.20 (app. t, J = 0.6 Hz, 1H), 7.14 (ddd, J = 8.2, 2.4, 1.0 Hz, 1H),

3.11 (s, 3H).

N-[3-(2,4-Dioxopyrimido[4,5-b]quinolin-10-yl)phenyl]-

methanesulfonamide, 158. Compound 158 was prepared in a

manner analogous to that for 134 (yellow solid, 5.0 mg, 4%). ¹H NMR

(DMSO-d₆): δ 11.09 (s, 1H), 10.26 (br. s, 1H), 9.12 (s, 1H), 8.24 (d, J

= 8.0 Hz, 1H), 7.76 (t, J = 8.0 Hz, 1H), 7.64 (t, J = 8.0 Hz, 1H), 7.52

(t, J = 8.0 Hz, 1H), 7.42 (d, J = 8.2 Hz, 1H), 7.19 (s, 1H), 7.13 (d, J =

8.2 Hz, 1H), 6.81 (d, J = 8.2 Hz, 1H), 3.09 (s, 3H).

N-[3-(7-Chloro-2,4-dioxo-pyrimido[4,5-b]quinolin-10-yl)phenyl]-

methanesulfonamide, 159. Compound 159 was prepared in a

1

manner analogous to that for 134 (yellow solid, 12.0 mg, 28%). H

NMR (DMSO-d₆): δ 10.94 (s, 1H), 9.99 (s, 1H), 8.86 (s, 1H), 8.16

(d, J = 2.4 Hz, 1H), 7.54 (dd, J = 9.2, 1.1 Hz, 1H), 7.44 (t, J = 8.0 Hz,

1H), 7.21 (app. d, J = 8.2 Hz, 1H), 6.99 (t, J = 2.0 Hz, 1H), 6.92 (app.

d, J = 8.2 Hz, 1H), 6.60 (d, J = 9.2 Hz, 1H), 2.89 (s, 3H). LC-MS m/z

417.5 [M + H]⁺, 94% purity. HRMS (ESI) m/z [M + Na]⁺calculated

for C₁₈H₁₃N₄O₄NaSCl: 439.0239. Found: 439.0231.

3-(8-Cyano-2,4-dioxo-pyrimido[4,5-b]quinolin-10-yl)-

benzenesulfonamide, 160. Compound 160 was prepared in a manner

analogous to that for 134 (yellow solid, 8.0 mg, 6%). ¹H NMR

(DMSO-d₆): δ 11.28 (br. s, 1H), 9.17 (s, 1H), 8.43 (d, J = 8.6 Hz,

1H), 8.12 (app. d, J = 8.0 Hz, 1H), 7.89−7.97 (m, 3H), 7.71 (app. d, J

= 8.0 Hz, 1H), 7.61 (br. s, 2H), 7.05 (s, 1H).

APE-1 Assay. APE-1 enzyme was obtained from New England

Biolabs Inc. The assay was performed in 384-well black plates in a 25

μL ﬁnal volume with a reaction buﬀer composed of 50 mM Tris HCl,

pH 7.5, 2 mM MgCl₂, 25 mM NaCl, 1 mM DTT, 0.01% Tween-20,

1% DMSO, 50 nM labeled double-stranded AP-site containing DNA

substrate, as described above, and 0.03 nM APE1. The assay was

allowed to proceed for 30 min at 25 °C before being stopped with the

addition of 18 mM EDTA. Fluorescence was measured with excitation

at 540 nm and emission at 590 nm.

10-(1H-Indazol-6-yl)-2,4-dioxo-pyrimido[4,5-b]quinoline-8-car-

bonitrile, 161. Compound 161 was prepared in a manner analogous

to that for 134 (orange solid, 26.2 mg, 26%). ¹H NMR (DMSO-d₆): δ

13.46 (s, 1H), 11.18 (br. s, 1H), 9.10 (s, 1H), 8.42 (d, J = 8.6 Hz, 1H),

8.29 (s, 1H), 8.06 (dd, J = 8.6, 0.4 Hz, 1H), 7.88 (dd, J = 8.2, 1.4 Hz,

1H), 7.69 (s, 1H), 7.17 (s, 1H), 7.10 (dd, J = 8.6, 0.4 Hz, 1H). ¹³C

NMR (DMSO-d₆): δ 161.5, 159.2, 156.3, 141.6, 141.1, 140.2, 134.6,

133.9, 132.3, 126.3, 123.5, 123.2, 122.6, 121.2, 120.2, 118.2, 115.7,

110.6. HRMS (ESI) m/z [M + H]⁺calcd for C₁₉H₁₁N₆O₂: 355.0938.

Found: 355.0942.

ASSOCIATED CONTENT

* Supporting Information

■

S

Summary of purity data, LC-MS methods and solvent

gradients, preparative HPLC instrument and solvent gradients,

preparative methods and spectroscopic data for intermediates

3−23, 32−68, 71,72, 74−76, 80, 83−85, 89−92, and 106−

127, ¹H NMR spectra of target compounds, additional

references, dose−response curves of selected toxoﬂavins and

active deazaﬂavins for the TDP2 chromogenic assay, and

method for generating computational docking poses for the

deazaﬂavins. This material is available free of charge via the

Internet at http://pubs.acs.org.

10-(1H-Indazol-4-yl)-2,4-dioxo-pyrimido[4,5-b]quinoline-8-car-

bonitrile, 162. Compound 162 was prepared in a manner analogous

1

to that for 134 (yellow solid, 4.0 mg, 4%). H NMR (DMSO-d₆): δ

13.53 (s, 1H), 11.18 (s, 1H), 9.23 (s, 1H). 8.42 (d, J = 8.6 Hz, 1H),

7.82−7.90 (m, 3H), 7.64 (t, J = 8.0, 1H), 7.21 (d, J = 7.3 Hz, 1H), 6.99

(s, 1H).

AUTHOR INFORMATION

Corresponding Author

*Telephone: +44 (0)161-918-7447. E-mail: araoof@picr.man.

ac.uk.

2,4-Dioxo-10-[3-(1H-tetrazol-5-yl)phenyl]pyrimido[4,5-b]-

■

quinoline-8-carbonitrile, 163. Compound 163 was prepared in a

1

manner analogous to that for 134 (yellow solid, 15.1 mg, 11%). H

NMR (DMSO-d₆): δ 11.28 (s, 1H), 9.19 (s, 1H), 8.43 (d, J = 8.6 Hz,

1H), 8.27 (ddd, J = 8.0, 1.6, 1.0 Hz, 1H), 8.14 (s, 1H), 7.91 (dd, J =

8.2, 1.4 Hz, 1H), 7.87−7.92 (m, 2H), 7.59 (ddd, J = 8.0, 2.0, 1.0 Hz,

1H), 7.23 (s, 1H).

Notes

The authors declare no competing ﬁnancial interest.

Biological Assay Protocols. TDP2 Chromogenic Assay. The

assay was performed in 384-well clear plates in a ﬁnal volume of 25 μL

comprised of reaction buﬀer (25 mM Hepes (pH 8.0), 10 mM MgCl₂,

130 mM KCl, 1 mM dithiothreitol (DTT)), 0.03% bovine serum

albumin (BSA), 1% dimethyl sulfoxide (DMSO) with or without

inhibitory compound, and 36 nM TDP2 protein. The reaction was

initiated by the addition of 20 mM 4-nitrophenyl phenylphosphonate

(NPPP), and the assay was allowed to proceed at 25 °C for 60 min

ACKNOWLEDGMENTS

■

This work was supported by Cancer Research UK (Grant

C480/A11411). The TDP2 enzyme was cloned and puriﬁed by

E. McKenzie at the Protein Expression Facility, Manchester

Interdisciplinary Biocentre, U.K. HRMS spectra were generated

by R. Sung, School of Chemistry, University of Manchester,

Q

dx.doi.org/10.1021/jm400568p | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

Sham, Y. Y.; Pommier, Y.; Wang, Z. 5-Arylidenethioxothiazolidinones

as Inhibitors of Tyrosyl−DNA Phosphodiesterase I. J. Med. Chem.

2012, 55, 8671−8684.

M13 9PL, U.K. In vitro DMPK data were provided by Cyprotex

Discovery, Macclesﬁeld, U.K. JChem for Excel was used for

structure−property prediction and calculation, and general data

handling (JChem for Excel, version 5.4.0.411, 2008−2009,

ChemAxon, http://www.chemaxon.com). We thank James

Smith (Cancer Research Technology Ltd.) for triaging HTS

output, Andy Barker for providing input and advice for

alternative scaﬀolds to the toxoﬂavins, and H. Nikki March

(Cancer Research UK Drug Discovery Unit, Paterson Institute

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ABBREVIATIONS USED

■

APE1, apurinic/apyrimidinic endonuclease; app, apparent;

Caco-2, heterogeneous human epithelial colorectal adenocarci-

noma cells; DIPEA, diisopropylethylamine; EDCI, 1-ethyl-3-(3-

dimethylaminopropyl)carbodiimide; ES−, negative ion electro-

spray ionization; ES+, positive ion electrospray ionization;

HOBt, hydroxybenzotriazole; HRP, horseradish peroxidase;

NPPP, 4-nitrophenyl phenylphosphonate; SQD, single quadro-

pole detector; TAM, tetramethylrhodamine; TAMRA, tetra-

methylrhodamine; TDP1, tyrosyl-DNA phosphodiesterase I;

TDP2, tyrosyl-DNA phosphodiesterase II; UPLC, ultra

performance liquid chromatography; μW, microwave; EAPII,

ETS1-associated protein II; TTRAP, TRAF and TNF receptor-

associated protein

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dx.doi.org/10.1021/jm400568p | J. Med. Chem. XXXX, XXX, XXX−XXX

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Toxoflavins and deazaflavins as the first reported selective small molecule inhibitors of tyrosyl-DNA phosphodiesterase II

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