Exploration of the nicotinamide-binding site of the tankyrases, identifying 3-arylisoquinolin-1-ones as potent and selective inhibitors in vitro

Bioorganic & Medicinal Chemistry 23 (2015) 5891–5908

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Bioorganic & Medicinal Chemistry

journal homepage: w ww.elsevier.com/locate/bmc

Exploration of the nicotinamide-binding site of the tankyrases,

identifying 3-arylisoquinolin-1-ones as potent and selective

inhibitors in vitro

Helen A. Paine ^a, Amit Nathubhai ^a, Esther C. Y. Woon ^a,b, Peter T. Sunderland ^a, Pauline J. Wood ^a,

Mary F. Mahon ^c, Matthew D. Lloyd ^a, Andrew S. Thompson ^a, Teemu Haikarainen ^d, Mohit Narwal ^d,

Lari Lehtiö ^d, Michael D. Threadgill ^a,

⇑

^aMedicinal Chemistry, Department of Pharmacy & Pharmacology, University of Bath, Claverton Down, Bath BA2 7AY, UK

^bDepartment of Pharmacy, National University of Singapore, Block S4, Science Drive 4, Singapore 117543, Republic of Singapore

^cX-ray Crystallographic Suite, Department of Chemistry, University of Bath, Claverton Down, Bath BA2 7AY, UK

^dBiocenter Oulu and Faculty of Biochemistry and Molecular Medicine, University of Oulu, Oulu, Finland

a r t i c l e i n f o

a b s t r a c t

Article history:

Tankyrases-1 and -2 (TNKS-1 and TNKS-2) have three cellular roles which make them important targets

in cancer. Using NAD⁺as a substrate, they poly(ADP-ribosyl)ate TRF1 (regulating lengths of telomeres),

NuMA (facilitating mitosis) and axin (in wnt/b-catenin signalling). Using molecular modelling and the

structure of the weak inhibitor 5-aminoiso quinolin-1-one, 3-aryl-5-substituted-isoquinolin-1-ones were

designed as inhibitors to explore the structure–activity relationships (SARs) for binding and to deﬁne the

shape of a hydrophobic cavity in the active site. 5-Amino-3-arylisoquinolinones were synthesised by

Suzuki–Miyaura coupling of arylboronic acids to 3-bromo-1-methoxy-5-nitro-isoquinoline, reduction

and O-demethylation. 3-Aryl-5-methylisoquinolin-1-ones, 3-aryl-5-ﬂuoroisoquinolin-1-ones and

3-aryl-5-methoxyisoquinolin-1-ones were accessed by deprotonation of 3-substituted-N,N,2-trimethyl-

benzamides and quench with an appropriate benzonitrile. SAR around the isoquinolinone core showed

that aryl was required at the 3-position, optimally with a para-substituent. Small meta-substituents were

tolerated but groups in the ortho-positions reduced or abolished activity. This was not due to lack of

coplanarity of the rings, as shown by the potency of 4,5-dimethyl-3-phenylisoquinolin-1-one. Methyl

and methoxy were optimal at the 5-position. SAR was rationalised by modelling and by crystal structures

of examples with TNKS-2. The 3-aryl unit was located in a large hydrophobic cavity and the para-

substituents projected into a tunnel leading to the exterior. Potency against TNKS-1 paralleled potency

against TNKS-2. Most inhibitors were highly selective for TNKSs over PARP-1 and PARP-2. A range of

highly potent and selective inhibitors is now available for cellular studies.

Received 27 April 2015

Revised 24 June 2015

Accepted 25 June 2015

Available online 2 July 2015

Keywords:

Tankyrase

TNKS

Naphthyridinone

7-Aryl-1-methyl-1,2,3,4-tetrahydro-1,6-

naphthyridin-5-one

Crystal structure

1. Introduction

the ADP-ribose (ADPr) units, as for other PARPs.⁴TNKS-1 is also

responsible for poly(ADP-ribosyl)ating NuMA at the mitotic

Tankyrase-1 (PARP-5a, TNKS-1, ARTD5) and tankyrase-2 (PARP-

5b, TNKS-2, ARTD6) are members of the poly(ADP-ribose)poly-

merase (PARP) superfamily of enzymes, which attract considerable

current interest owing to their roles at telomeres and involvement

at the mitotic spindle and in the wnt signalling pathway. TNKS-1

was ﬁrst reported in 1998,¹as regulating the lengths of telomeres

through poly(ADP-ribosyl)ation of telomere repeat binding factor-

1 (TRF-1) and autopoly(ADP-ribosyl)ation.^2,3NAD⁺is the source of

spindle, ensuring its correct functioning during mitosis.⁵

Furthermore, TNKS-1 is a component of the wnt signalling system,

where it poly(ADP-ribosyl)ates axin. Poly(ADPr)-axin is thus

tagged for ubiquitinylation and destruction through the protea-

some, increasing the levels of b-catenin, while decreasing the

levels of phosphorylated b-catenin.^6,7Accumulation of b-catenin

leads to a proliferative signal in the nucleus.⁷The closely struc-

turally related isoform TNKS-2 can substitute for TNKS-1 in many,

but not all, of these activities.^2,8,9

Telomeres are protected by the shelterin complex of six telom-

ere-speciﬁc proteins. Its main role is to protect the telomere from

being recognised as a site of damage by the DNA-repair machinery,

⇑

Corresponding author. Tel.: +44 1225 386840; fax: +44 1225 386114.

E-mail address: m.d.threadgill@bath.ac.uk (M.D. Threadgill).

http://dx.doi.org/10.1016/j.bmc.2015.06.061

5892

H. A. Paine et al. / Bioorg. Med. Chem. 23 (2015) 5891–5908

thus preventing inappropriate recombination events.¹⁰Two shel-

terin proteins, TRF-1 and TRF-2, localise to the telomeres,

maintaining t/d-loop structures and binding directly to double-

stranded TTAGGG repeats.¹¹DNA polymerases cannot replicate

fully to the ends of linear DNA, so the telomere acts as a sacriﬁcial

entity; a critical length is eventually reached, eliciting a DNA-dam-

age checkpoint response, through p53, and subsequent replicative

senescence or apoptosis.^12,13Proliferating cells use telomerase to

re-lengthen the telomeric DNA. TRF-1 binds to the telomere pre-

venting access by telomerase. Poly(ADP-ribosyl)ation of TRF-1 by

TNKS-1 causes it to dissociate from telomeric DNA, leading to

ubiquitinylation and destruction of the TRF-1 and allowing telom-

erase access to the DNA.¹TNKS-1 may also have a role in response

to DNA damage at telomeres.¹⁴During mitosis, TNKS-1 is present

at the ends of interphase chromosomes,^5,15bound to nuclear

mitotic apparatus protein (NuMA), which accumulates around

the pericentriolar matrix.¹⁶Under the possible regulation of

GSK3,¹⁷TNKS-1 poly(ADP-ribosyl)ates NuMA. siRNA knock-down

of TNKS-1 causes mitotic arrest in HeLa cells.¹⁸Similar removal

using RNAi caused post-anaphase arrest.¹⁹A critical role of

TNKSs in mitosis is in resolution of the telomeres.^13,18,19

nicotinamide-binding site, as do our 2-arylquinazolin-4-ones

^36,37and ﬂavones, including 4.^38,39Our lead water-soluble inhibi-

tor of PARP-1 and PARP-2, 5-AIQ 11a,^40–44binds only weakly to the

TNKSs, with IC₅₀in the low M range (preliminary data not

3

l

shown). Compound 5 binds with its quinazolin-4-one at the

nicotinamide-binding site and the side-chain extending to make

interactions in the adenosine-binding site of the NAD⁺-binding

cleft.⁴⁵EB-47 6 occupies both the nicotinamide- and adenosine-

binding pockets but with a linker that uses a different path on the

enzyme surface.⁴⁶Compound 7 uses an amino-1,2,4-triazole to

make the requisite H-bonds within the nicotinamide-binding site,⁴⁷

whereas 8 uses a dihydropyranopyrimidine to bind there.⁴⁸IWR1

9⁴⁹and G007-LK 10^35,50bind to the adenine-binding site alone.

We present here our structure-based design of 3-arylisoquino-

lin-1-ones as inhibitors of the TNKSs and our exploration of the

structure–activity relationships (SARs) around this core and bind-

ing, guided by crystallography and studies in silico. Using rational

modiﬁcation of the substituents around the core, the aim of this

study is to explore the detail of the conformational and space-ﬁll-

ing requirements for optimum occupation of a large hydrophobic

cavity in the enzymes adjacent to the nicotinamide-binding site.

TNKSs are over-expressed in several clinical cancers and cancer

cell lines, including breast cancer,^20,21colon cancer,^22,23chronic

myeloid leukaemia,²⁴brain tumours,^25,26and gastric²⁷and bladder

cancers,²⁸pointing to one or more critical roles. Seimiya²⁹pro-

posed them as therapeutic targets on the basis of their roles at

telomeres. Subsequent revelation of the roles of TNKSs in wnt sig-

nalling⁷and the observation of aberrant wnt signalling in many

tumour types³⁰has reinforced their value as targets for anti-cancer

drug design.^31,32One TNKS inhibitor has shown activity in an APC-

mutant mouse model of colon cancer.³³The role of TNKS-1 in the

replication of Herpes simplex virus may also point to applications

in the treatment of viral infections.³⁴These observations and

opportunities point to the need for potent and selective inhibitors

of the TNKSs and for a greater understanding of the detail of the

binding pockets around the NAD⁺-binding site.

2. Results and discussion

2.1. Design of inhibitors

The design of the isoquinolin-1-one inhibitors was approached

from three start-points. Firstly, we solved the crystal structures of

our water-soluble PARP-1 inhibitors 11a,⁴⁰11b,⁵¹11c,⁵¹and 11d⁵²

(Fig. 1) bound into the NAD⁺-binding site of the catalytic domain

of human TNKS-2. These compounds were shown, in preliminary

studies, to be weak inhibitors of the TNKSs (IC₅₀>10 lM) (data not

shown). Secondly, the structureof XAV939 1 bound into thecatalytic

domain of TNKS-2 is available.⁵³Thirdly, we sought to investigate

‘scaffold-hopping’ from our 2-arylquinazolin-4-one inhibitors, from

our preliminary studies,³⁶to the 3-arylisoquinolin-1-ones.

Figure 2 shows images of the crystal structures of 11a–d bound

into the catalytic domain of human TNKS-2. Each shows binding

Figure 1 shows the structures of some known inhibitors of

the TNKSs. XAV939 1⁷and GM244-LM 2³⁵both bind at the

O

N

O

N

O

N

H

N

H

S

N

S

N

SO₂Me

R⁸

N

Me

R^4'

CF₃

1: XAV939

2: G244-LM

3

4

O

N

O

H

N

OH

H

N

H

S

O

N

R³

OH

N

O

HN

NH₂R⁴

O

N

Me

3

: R = R⁴= H

N

HN

O

11a

11b

N

OMe

: R³= Et, R⁴= H

H

N

Me

5

6: EB-47

7

N

11c: R³= pentyl, R⁴= H

11d: R³= H, R⁴= Me

NH₂

O

NC

O

N

S

O

H

F

H

O

Cl

N

SO₂Me

N

O

HN

N

O

N

O

N

8

9: IWR1

10: G007-LK

Figure 1. Structures of recently disclosed inhibitors of the TNKSs 1–10, of our lead inhibitor of PARP-1 11a and of 5-amino-3/4-alkylisoquinolin-1-ones 11b–d used in the

initial crystallographic study.

H. A. Paine et al. / Bioorg. Med. Chem. 23 (2015) 5891–5908

5893

A

B

E

G

C

Ser¹⁰⁶⁸

Gly¹⁰³²

Hydrophobic cavity

Gly¹⁰³²/ Ser¹⁰⁶⁸

H-bond motif

Tunnel

3-C

4-C

11a

D

F

Figure 2. Crystal structures of (A) 11a; (B) 11a; (D) 11b; (E) 11c; (F) 11d bound into the NAD⁺-binding site of the catalytic domain of human TNKS-2. Panels A and B show the

same view with different rendering, with A showing the classical PARP H-bonding motif and B emphasising the hydrophobic cavity and tunnel. Panel C shows a cartoon

representation of the binding of 11a and of the adjacent hydrophobic cavity and tunnel. Panel D shows the ethyl group of 11b in two partly occupied conformations within

the hydrophobic cavity. Panel G shows the corresponding structure of XAV939 1 bound into tankyrase-2.⁴³

into the NAD⁺-binding site, mimicking nicotinamide. The principal

binding contacts are H-bonds from N–H of the isoquinolin-1-one to

the C@O of Gly¹⁰³²and from the O–H of Ser¹⁰⁶⁸and the N–H of

The published crystal structure of 1 bound into the catalytic

domain of TNKS-2 also shows the classical PARP-binding motif of

three H-bonds from the ligand to Gly¹⁰³²and Ser^{1068 53}

.

The 4-tri-

ﬂuoromethylphenyl group projects into the hydrophobic cavity,

forming a

-stack with Tyr¹⁰⁵⁰and hydrophobic interactions with

Pro¹⁰³⁴, Phe¹⁰³⁵and Ile^{1075 34}

This ﬁts well with our predictions

Gly¹⁰³²to the C@O of the isoquinolinone.

p-Stacking with the aro-

matic side-chain of Tyr¹⁰⁷¹is also seen. This is the classical binding

motif for most inhibitors of the PARP isoforms.^54,55The 5-NH₂of

11a–d was accommodated in a small pocket and there was a small

niche to accept the 4-methyl in 11d. There is a large cavity adjacent

to the 3-position of the isoquinolin-1-ones; the small 3-alkyl

groups of 11b,c were located in this cavity near to the entrance.

The cavity is lined with aromatic and hydrophobic residues

(mainly Pro¹⁰³⁴, Phe¹⁰³⁵, Tyr¹⁰⁵⁰, Tyr¹⁰⁷¹), with a small tunnel

leading from the far end of the hydrophobic cavity towards the

exterior. The size and shape of the hydrophobic cavity suggest that

it could accommodate a benzene ring, with modest substituents. A

water molecule was evident in the centre in complexes of the 3-H

isoquinolinones 11a,d, whereas two water molecules were seen

towards the sides of the cavity when it was partly occupied by

ethyl in 11b. The pentyl group in 11c was too large to allow an

ordered water molecule in the cavity. Figure 2C shows a cartoon

of the arrangement of the hydrophobic cavity and the tunnel,

relative to the H-bonding motif anchoring the lactam of 11a.

p

.

from our results on the binding of 11a–d with TNKS-2, in that

the phenyl ring of 1 occupies much of the hydrophobic cavity

and the triﬂuoromethyl group is located at the entrance to the

tunnel.

The SAR from our preliminary study³⁶on the 2-arylquinazolin-

4-ones suggested that the lactam H-bonding motif was essential

and that a para-substituted benzene ring could be accepted by

0

the hydrophobic cavity. One example, (3: R⁴= CH₂NHCbz,

R⁸= Me) projected deep into the tunnel, with only slight loss of

activity. The preliminary SAR also suggested the need for a small

substituent at the 8-position (R⁸in 3, Fig. 1, corresponding to the

5-position in isoquinolin-1-ones (R⁵in 11)). This SAR was

rationalised by modelling studies.

The structures of the designed isoquinolin-1-ones 12–17 are

shown in Figure 3. All contain the 3-arylisoquinolin-1-one core,

as predicted by the structural studies and comparison with the

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H. A. Paine et al. / Bioorg. Med. Chem. 23 (2015) 5891–5908

2-arylquinazolin-4-ones to be required. All carry a 5-substituent,

as this was better in the quinazolin-4-ones.³⁶However, the nature

of the 5-substituent was not clearly predicted and comparison of

compounds in the series 12–16 may clarify this. Compounds 12

and 16 have H-bond-donor electron-donating groups (EDGs) (the

5-NH₂in 12 is unprotonated at physiological pH), whereas 15

contain a H-bond-acceptor EDG. Analogues 13 and 14 have non-

polar 5-substituents, which is electron-neutral in 13 and elec-

tron-withdrawing in 14. Within each series, especially 12 and 13,

the nature, size and location of the substituent on the 3-aryl group

is explored. Substituents range from polar H-bond-donors (in

12p,t,u), through H-bond-acceptors (in 12d–f, 13d, 14c, 15h) to

non-polar groups and atoms (in 12b,c,g–n, 13b,c,e–j, 14b,d–g,

15b–f, 16b). Strongly electron-donating, electron-neutral and elec-

tron-withdrawing substituents were investigated. Unsubstituted

3-phenyl was examined in 12a, 13a, 14a, 15a, 16a. Weakly basic

and neutral heterocycles (replacing the 3-phenyl) were incorpo-

rated in 13p–r, 14h, 15g–i, some being electron-poor and some

being electron-rich. The optimal location of the substituent

on the 3-aryl ring was tested in ﬁve sets of compounds, 12b,c,

12d–f, 12g–i, 12k–n and 13f–h. The effect of an ortho-substituent

may be to twist the 3-aryl ring out of the plane of the isoquinoli-

none; this potential twist was replicated in 17 with a methyl group

in the 4-position (‘ortho’ to the phenyl).

5-nitroisoquinoline-N-oxide followed by reduction of the nitro

group⁵⁶and selective reduction of the nitrile of methyl 2-cyano-

methyl-3-nitrobenzoate,⁵⁷cyclisation in situ and further

reduction. The current optimum method is condensation of methyl

2-methyl-3-nitrobenzoate with DMFDMA, hydrolysis of the enam-

ine to 5-nitroisocoumarin, conversion to 5-nitroisoquinolin-1-one

and reduction.⁴⁰However, none of these routes can be adapted

readily to the preparation of a library of 3-substituted analogues.

We previously reported some approaches to the target compounds

(Scheme 1) but each suffers from lack of generality and modest

yield and none feature late introduction of the 3-substituent, a

requirement for efﬁcient preparation of

a focussed library.

Sonogashira coupling of methyl 2-iodo-3-nitrobenzoate 18 with

phenylethyne, followed by 6-endo-dig cyclisation of the alkyne

19, gave the isocoumarin 20 (R³= Ph),⁵⁸leading to 22 (R³= Ph)

and hence to 12a (R³= Ph). Similar electrophilic cyclisation of 19

with ICl led to the isocoumarin 21 and hence to 20 (R³= Ph).⁵⁸

The initial Sonogashira reaction was limited to this example.

Reaction of 23 with Cu(I) phenylacetylides under Castro–

Stephens conditions gave the 17 directly but only when R³= Ph,

4-MePh and 4-MeOPh.⁵⁸Hurtley couplings of the corresponding

bromonitrobenzoic acid 24 afforded a wide range of intermediate

isocoumarins 20 directly but the yields were modest and the reac-

tion was limited to R³= alkyl, electron-rich aryl.⁵¹Nevertheless,

samples of 12a,c,f,m were prepared for this study. Finally, treat-

ment of 25 with benzoyl chlorides under Friedel–Crafts conditions

gave modest yields of 20 but this process failed for electron-donat-

ing substituents.⁵⁹There was, therefore, a pressing need to design

a synthetic route which allowed efﬁcient late incorporation of

diverse 3-substituents.

We have previously described Pd-catalysed Suzuki–Miyaura

and Stille couplings to 1-alkoxy-4-bromo-5-nitroisoquinolines as

a method to access 4-substituted analogues of 5-AIQ 11a, which

are inhibitors of PARP-2.⁶⁰Adaptations of this method were

explored to access the required corresponding 3-aryl-5-AIQs.

1,3-Dichloroisoquinoline 26 was nitrated selectively at the

5-position under highly acidic conditions to give 27 (Scheme 2).

A methoxy group at the 1-position would serve as a masked

lactam; the lactam in the target compounds is essential for biolog-

ical activity but it also renders the isoquinolin-1-ones highly insol-

uble in organic solvents used for coupling reactions. This methoxy

group was introduced using methoxide ion in methanol, giving 28;

this reaction is highly selective for displacement of the chlorine at

The preliminary SAR in the quinazolin-4-one series³⁶and the

crystal structures used here gave limited predictive information

on the precise dimensions and the ﬂexibility of the hydrophobic

cavity; thus we tested these with the greater steric bulk of 12n,

13c,i,q, 15h and, in extremis, by the ferrocene in 13s. The dimen-

sions were also tested by inserting a CH₂CH₂spacer between the

isoquinolinone and the phenyl ring in 12s. The tunnel at the end

of the hydrophobic cavity distal from the isoquinolinone-binding

subsite would be occupied by a long rigid hydrophobic group in

13k. This space was also explored in 13l–o. The latter compounds

incorporate a tertiary aliphatic amine to aid water-solubility; a

protonated primary aliphatic amine in this position leads to major

loss of activity in the quinazolin-4-ones³⁶and an aniline may not

be sufﬁciently basic for formation of soluble salts.

2.2. Chemical synthesis

5-AIQ 11a (Fig. 1), has been prepared by

a number of

different routes, including Polonovski rearrangement of

12: a: R³= Ph; b: R³= 3-MePh; c: R³= 4-

MePh; d: R³= 2-MeOPh; e: R³= 3-

13: a: R³= Ph; b: R³= 4-MePh; c: R³= 4-

Bu^tPh; d: R³= 4-MeOPh; e: R³= 4-F₃CPh; f:

O

MeOPh; f: R³= 4-MeOPh; g: R³= 2-

R³= 2-ClPh; g: R³= 3-ClPh; h: R³= 4-ClPh; i:

H

3

F₃CPh; : R = 3-F₃CPh; : R³= 4-F₃CPh;

R³= 2,6-Cl₂Ph; : R³= 4-BrPh; : R = 4-PhC

h

i

j

k

N

: R³= 4-FPh; : R = 2-ClPh; : R³= 3-

CPh; : R = 4-Me₂NCH₂Ph; : R³= 4-

3

j

k

l

m

R³

3

m

n

o

ClPh; : R = 4-ClPh; : R = 2,6-Cl₂Ph;

:

(piperidin-1-yl)CH ₂Ph; : R³= 4-(pyrrolidin-1-

n

R³= 4-BrPh; : R³= 4-HOPh; : R = 3-

yl)CH₂Ph; : R³= 4-(4-methylpiperazin-1-

3

NH₂

Me

p

q

o

NCPh; r: R³= 4-NCPh; s: R³= PhCH₂CH₂;

t: R³= 3-H₂NOCPh; u: R³= 4-H₂NOCPh

yl)CH₂Ph; p: R³= pyridin-4-yl; q: R³= benzo-

1,3-dioxol-5-yl; r: R³= thiophen-3-yl; s: R³

=

ferrocenyl

O

14: a: R³= Ph; b: R³= 4-MePh; c: R³= 4-

MeOPh; d: R³= 4-F₃CPh; e: R³= 4-FPh;

15: a: R³= Ph; b: R³= 4-MePh; c: R³= 4-

H

Bu^tPh; d: R³= 4-F₃CPh; e: R³= 4-ClPh; f: R³

=

N

f: R³= 4-ClPh; g: R³= 4-BrPh; h: R³

=

4-BrPh; g: R³= pyridin-4-yl; h: R³= benzo-1,3-

R³

dioxol-5-yl; : R³= thiophen-3-yl

i

pyridin-4-yl

F

OMe

H

16: a: R³= Ph; b: R³= 4-F₃CPh

17

R³

OH

Me Me

Figure 3. Structures of designed tankyrase-inhibiting isoquinolin-1-ones 12–17.

H. A. Paine et al. / Bioorg. Med. Chem. 23 (2015) 5891–5908

5895

O

Cl

CO₂Me

I

CO₂Me

O

N

i

iii

Ph

21

Cl

NO₂

Ph

NO₂

I

26

iv

v

i

18

19

ii

O

Cl

OMe

N

OMe

N

CO₂H

H

vii

O

ii

iv

R³

I

R³

Cl

NO₂

ix

NO₂

vi

NO₂

23

20

22

27

28

30a,d,e,g-i,k,l,p-r

viii

iii

O

v or vi

H

CO₂H

O

N

OMe

N

O

H

N

iii

R³

Br

NH₂

NO₂

Cl

R³

24

25

12

NO₂

NH₂

12a,d,e,g-i,k,l,p-r

NH₂

29

31a,d,e,g-i,k,l,p-r

Scheme 1. Earlier synthetic routes explores to 3-substituted analogues 12 of 5-AIQ

11a; R³= alkyl, aryl. Each of these suffers from poor yield or lack of generality or

both. Reagents: (i) HC„CPh, (Ph₃P)₂PdCl₂, Prⁱ₂NH, THF; (ii) HgSO₄, H₂SO₄, Me₂CO,

; (iii) ICl, CH₂Cl₂; (iv) HCO₂H, Pd(OAc)₂, Et₃N, DMF; (v) NH₃, MeOCH₂CH₂OH,

(vi) H₂, Pd/C, aq HCl, EtOH or SnCl₂, EtOH; (vii) CuC„CR³, pyridine, (R³Ph,

4-MePh, 4-MeOPh); (viii) (R³CO)₂CH₂or R³COCH₂COMe, Cu powder, KOBu^t, HOBu^t,

(R³= alkyl, electron-rich aryl); (ix) R³COCl, SnCl₄, PhNO₂, , (R³= electron-poor

aryl).

Scheme 2. Synthesis of target 5-amino-3-arylisoquinolin-1-ones 12a,d,e,g–i,k,l,p–r

via Suzuki–Miyaura couplings to 3-chloro-1-methoxy-5-nitroisoquinolin-1-one 28.

R³as for Figure 2. Reagents: (i) HNO₃, H₂SO₄; (ii) NaOMe, MeOH; (iii) HBr, AcOH;

(iv) R³B(OH)₂, Pd₂dba₃, SPhos, K₃PO₄, toluene (DMF for 28 ? 30p); (v) H₂, Pd/C,

EtOH (30a,d,e,g–i,p–r ? 31a,d,e,g–i,p–r); (vi) H₂, Pt/C, EtOH (30k,l ? 31k,l).

D

D;

D

180 °C in a solvent-free reaction gave the imide 33. An attempt to

replace both oxygens with bromines using phosphorus tribromide

gave a modest yield of 34, along with some of the deoxygenated

side-product 35. Clearly, the P(III) reagent is responsible for this

side-reaction and analogous treatment with phosphorus oxybro-

mide, gave only 34. Nitration of 34 under highly acidic conditions

afforded 36 in almost quantitative yield. As in the chloro series

above, the 1-bromine could be replaced selectively with methoxide

to give 37, with no evidence of reaction at the 3-position despite

the increased leaving-group ability of Br versus Cl. The regioiso-

meric identity of 37 was conﬁrmed by a HMBC NMR experiment,

with correlation between the OCH₃protons and isoquinoline 1-C

and by NOE correlation between OCH₃and 8-H. The increased reac-

tivity of the 3-bromoisoquinoline 37 led to more effective Suzuki–

Miyaura couplings with a wide range of arylboronic acids to give

30. As shown in Scheme 3, this coupling tolerated a variety of sub-

stituents on the boronic acid. Steric bulk generally lowered the

yield but the 2,6-dichloro analogue 30n was formed from 37.

Changing the reaction solvent to DMF and raising the reaction

temperature to 135 °C raised the yields. Table 1 shows direct

comparisons between the yields obtained in coupling using the

3-chloroisoquinoline 28 and the 3-bromoisoquinoline 37 as the

haloarene component. In general, yields were improved with 37

and with DMF as reaction solvent. The highly reactive 37 also

permitted coupling with 4-bromophenylboronic acid, without

homo-coupling of the boronic acid. The poor solubility of

4-hydroxyphenylboronic acid in toluene led to very low yields

for couplings in this solvent but DMF enabled preparation of 30p

in good yield from 28 and from 37.

the 1-position, with no reaction at the 3-position even under

prolonged or more forcing conditions.

Pd-catalysed coupling of an appropriate 3-chloroisoquinolin-1-

one would be desirable, as it places the point of introduction of

diversity very late in the sequence, such that each coupled

analogue only required one further step to generate the targets

12. Demethylation of 28 with hydrogen bromide in acetic acid

gave 29 in moderate yield (Scheme 2). However, this

material was very poorly soluble in solvents appropriate for

Suzuki–Miyaura couplings and all attempts to couple it with

arylboronic acids failed.

By contrast, 28 (containing the lactam masked as the 1-meth-

oxy group) had excellent solubility in common organic solvents.

Coupling with arylboronic acids carrying simple substituents at

ortho-, meta- or para-positions, using a Pd₂dba₃/SPhos combination

in boiling toluene was moderately effective, giving 12a,d,e,g–

i,k,l,p–r in moderate yields. Varying amounts of unreacted 28 were

recovered but the unreacted boronic acids decomposed under the

reaction conditions. Repetition of the reactions with excess aryl-

boronic acids, either from the start of the reaction or added por-

tionwise during the procedure, failed to increase the yields. The

yields of isolated 3-arylisoquinolines 30a,d,e,g–i,k,l,p–r were also

lowered by difﬁculties in separation of 30a,d,e,g–i,k,l,p–r from

28. Catalytic hydrogenation reduced the nitro groups of

30a,d,e,g–i,k,l,p–r to the required amines in 31a,d,e,g–i,k,l,p–r.

Palladium on charcoal was a satisfactory catalyst for most of the

reductions but the monochlorophenylisoquinolines 30k,l also suf-

fered reductive loss of the chlorine under these conditions (giving

31a). This dehalogenation was suppressed by the use of platinum

on charcoal as catalyst. Curiously, 30n was not dehalogenated dur-

ing formation of 31n.

There are two possible sequences from 30 to the targets 12,

demethylation then reduction or vice versa. The former was

investigated with three examples. Demethylation of 30a,i,j

with hydrogen bromide in acetic acid gave the corresponding

3-aryl-5-nitroisoquinolin-1-ones 22a,i,j in good yields. However,

the poor solubility of these nitroisoquinolin-1-ones in solvents

appropriate for catalytic hydrogenation of the nitro groups pre-

cluded efﬁcient conversion to 22a,i,j. Thus 30g–n,p–r were ﬁrst

reduced to 31g–n,p–r, using hydrogen and Pd or Pt as appropriate

(Pt was required for some examples to avoid reductive dehalo-

genation). Demethylation was effected with hydrogen bromide in

It was rationalised that increasing the reactivity of the

3-haloisoquinoline would have two beneﬁts: (a) the intrinsic yield

would be higher, (b) separation of starting isoquinoline from pro-

duct isoquinolines would be easier, as less of the former would

be present in the product mixture before chromatography.

1,3-Dibromoisoquinoline 34 is not commercially available at an

economical price and had to be synthesised in two steps from inex-

pensive homophthalic acid 32 (Scheme 3). Heating 32 with urea at

5896

H. A. Paine et al. / Bioorg. Med. Chem. 23 (2015) 5891–5908

Br Br

O

CO₂H

H

i

ii or iii

iv

N

O

Br

H

NO₂

v

32

33

34

36

35

OMe

N

OMe

N

OMe

N

O

viii or ix

vi or vii

R³

Ph

NH₂

x

NH₂

NO₂

x

NO₂

xi

12s

31g-n,p-r

30g-n,p-r

37

x

O

OMe

N

H

N

x

viii

N

viii

N

R³

CONH₂

NO₂

Ph NH₂

NH₂

NO₂

12t,u

12a,g-n,p-r

22a,i,j

38

39

Scheme 3. Synthesis of target 3-substituted 5-aminoisoquinolin-1-ones 12a–g,n,p,t,u via Suzuki–Miyaura and Sonogashira couplings to 3-bromo-1-methoxy-5-

nitroisoquinoline 37. R³as for Figure 2. Reagents: (i) urea, 180 °C, no solvent; (ii) PBr₃; (iii) POBr₃. 1,4-dioxan; (iv) HNO₃, H₂SO₄; (v) NaOMe, MeOH; (vi) R³B(OH)₂,

Pd₂dba₃, SPhos, K₃PO₄, toluene; (vii) R³B(OH)₂, Pd₂dba₃, SPhos, K₃PO₄, DMF; (viii) H₂, Pd/C, EtOH; (ix) H₂, Pt/C, EtOH; (x) HBr, AcOH; (xi) PhC„CH, (Ph₃P)₂PdCl₂, CuI, Na

ascorbate, THF, Prⁱ₂NH.

2,3-Dimethylbenzoic acid 40 was converted into its N,N-

Table 1

dimethylamide 41 in the usual way. Treatment with one equiva-

lent of LDA in THF at ꢀ78 °C selectively removed a proton from

the 2-CH₃. The anion reacted with a range of benzonitriles to pro-

vide the corresponding 3-aryl-5-methylisoquinolin-1-ones 13a–j

in poor-to-excellent yields. The lower yields were due not to failure

of reaction but to issues in isolation of these compounds which are

of limited solubility in convenient organic solvents; sufﬁcient

material was obtained in each case for biochemical evaluation.

The reaction was sensitive to steric crowding in the benzonitrile,

with 2-chlorobenzonitrile and 2,6-dichlorobenzonitrile giving low

yields of 13f,i, respectively. No transmetalation was seen with

Comparison of Suzuki–Miyaura coupling yields using 3-chloro-1-methoxy-5-nitroiso-

quinoline 28 and 3-bromo-1-methoxy-5-nitroisoquinoline 37

Boronic acid

3-Aryl-1-MeO-5-

O₂N-isoquinoline

Isolated yield

from 28 (%)

Isolated yield

from 37 (%)

2-F₃CPhB(OH)₂

3-F₃CPhB(OH)₂

4-FPhB(OH)₂

2-ClPhB(OH)₂

3-ClPhB(OH)₂

4-BrPhB(OH)₂

4-HOPhB(OH)₂

3-NCPhB(OH)₂

4-NCPhB(OH)₂

30g

30h

30j

30k

30l

30o

30p

30q

30r

42^a

55^a

0^a

42^b

63^b

40^b

100^a

34^a

0^a

60^b

52^b

24^b

0^a, 64^b

8^a

9^a, 86^b

12^b

28^a

12^b

4-bromobenzonitrile, which gave

a satisfactory yield of 13j.

a

The reaction was also applicable to heterocyclic nitriles, with

4-cyanopyridine, 5-cyanobenzo-1,3-dioxole and 3-cyanothiophene

reacting well with the anion derived from 41 to give the

3-heteroaryl-5-methylisoquinolin-1-ones 13p–r.

Reaction solvent toluene, 110 °C.

Reaction solvent DMF, 135 °C.

b

Isoquinolin-1-ones 13k–o carry extensions at the 4⁰-position of

the 3-phenyl substituent either to probe the far side of the

hydrophobic pocket or to elicit solubility in water but the

corresponding benzonitriles 43,45a–d are not commercially

available. Sonogashira coupling of 4-bromobenzonitrile 42 with

phenylethyne gave 4-phenylethynylbenzonitrile 43 in good yield.

The 4-(aminomethyl)benzonitriles 45a–d were prepared from

4-bromomethylbenzonitrile 44 and secondary amines, under a

variety of basic conditions (Scheme 4). These benzonitriles gave

the corresponding isoquinolinones 13k–o. These analogues 13l–o

were converted to their hydrochloride salts, which showed

much-enhanced aqueous solubility. The 3-ferrocenylisoquinolinone

target 13s tests whether the hydrophobic pocket could accommo-

date three-dimensional steric bulk while still retaining aromatic-

ity, rather than the planar aromatic 3-substituents of 13a–r.

Commercially available ferrocenecarboxylic acid 46 was converted

into its carboxamide, which was dehydrated into the nitrile 47 in

high yield. Reaction with the anion of 41 proceeded in moderate

yield to give 10s, despite the steric bulk of the ferrocene.

acetic acid, a medium in which the 5-aminoisoquinolines were

readily soluble, to furnish the required 12g–n,p–r in good yields

as their HBr salts. There was no evidence of demethylation of the

4⁰-methoxy group in 31f, indicating the selectivity of the condi-

tions chosen. The nitriles in 31q,r were hydrated under these vig-

orous conditions to give the carboxamides 12t,u, rather than 12q,r.

Sonogashira coupling of phenylethyne to the 3-bromoisoquino-

line 37 was also high yielding, giving the 3-phenylethynyl ana-

logue 38. The nitro group of 38 could not be reduced selectively

but catalytic hydrogenation provided 39. Demethylation afforded

12s as the hydrobromide salt.

The 5-methyl-, 5-ﬂuoro-, 5-methoxy- and 5-hydroxy-3-aryliso-

quinolin-1-ones 13a–s, 14a–h, 15a–i and 16a,b were prepared by a

different route (Scheme 4). Poindexter described a route to simple

3-arylisoquinolin-1-ones by double deprotonation of N,2-

dimethylbenzamide with butyllithium and reaction with benzoni-

triles.⁶¹Cho et al. extended this method to removal of a proton

from the Ar-CH₃of the tertiary amide N,N,2-trimethylbenzamide

with lithium diisopropylamide at ꢀ78 °C and subsequent reaction

with benzonitriles lacking acidic functional groups.⁶²We have

extended this process to the synthesis of 3-aryl-5-substituted-

isoquinolin-1-ones from N,N,2,3-tetramethylbenzamide 41,

3-ﬂuoro-N,N,2-trimethylbenzamide 49 and 3-methoxy-N,N,2-

trimethylbenzamide 51. The advantage of this route is that the

diversity in the 3-aryl substituent is introduced at the ﬁnal stage

of the synthesis, through diversity in the benzonitriles.

This one-step assembly of 3-aryl-5-substituted isoquinolin-

1-ones was extended into the 5-ﬂuoro analogues 14a–h and the

5-methoxy compounds 15a–i (Scheme 4). 3-Fluoro-3-methylben-

zoic acid 48 and 3-methoxy-2-methylbenzoic acid 50 were readily

converted into the corresponding N,N-dimethylbenzamides 49, 51,

respectively, via the acid chlorides. Again, the derived anions

reacted smoothly with a range of benzonitriles to give 14a–h and

H. A. Paine et al. / Bioorg. Med. Chem. 23 (2015) 5891–5908

5897

O

CO₂H

CONMe₂

H

NC

i,ii

iii

N

iv

Me

R³

Br

Me

Ph

40

41

13a-s

42

43

HO₂C

NC

v or vi or

vii or viii

NC

a: R = NMe₂; b: R =

ix-xi

c

piperidin-1-yl; : R =

Fe

pyrrolidin-1-yl; d: R = 4-

methylpiperazin-1-yl

CH₂Br

CH₂R

45

44

46

47

O

CO₂H

CONMe₂

H

CO₂H

Me

CONMe₂

Me

i,ii

iii

N

i,ii

Me

R³

F

OMe

48

49

14a-h

50

51

iii

O

CONMe

H

N

²xiii

²xiv

N

xii

Me

Et

Ph

R³

Me

Me Me

OH

OMe

15a-i

41

52

17

16a,b

Scheme 4. Syntheses of 3-aryl-5-methylisoquinolin-1-ones 13, 3-aryl-5-ﬂuoroisoquinolin-1-ones 14, 3-aryl-5-methoxyisoquinolin-1-ones 15, 3-aryl-5-hydroxyisoquinolin-

1-ones 16 and 4,5-dimethyl-3-phenylisoquinolin-1-one 17. Reagents: (i) SOCl₂; (ii) aq, Me₂NH, CH₂Cl₂; (iii) LiNPrⁱ₂, R³CN, THF, ꢀ78 °C; (iv) PhC„CH, (Ph₃P)₂PdCl₂, CuI, Na

ascorbate, Prⁱ₂NH, THF; (v) aq Me₂NH; (vi) piperidine, K₂CO₃, DMF; (vii) pyrrolidine, Et₃N, THF; (viii) 1-methylpiperazine, Et₃N, CH₂Cl₂; (ix) (COCl)₂; (x) NH₃, Et₂O; (xi) POCl₃,

D

; (xii) BBr₃, CH₂Cl₂; (xiii) LiNPrⁱ₂, MeI, THF, ꢀ78 °C; (xiv) sec-BuLi, R³CN, THF, ꢀ78 °C.

15a–i, conﬁrming that neither the deprotonation of the Ar-CH₃nor

the subsequent attack of the carbanion on the nitrile and cyclisa-

tion were adversely affected by the electron-withdrawing ﬂuorine

or the electron-donating methoxy group ortho to the methyl. Two

examples 15a,d were demethylated at the 5-position to provide

the corresponding 5-hydroxyisoquinolin-1-one 16a,b using boron

tribromide.

4-Alkylisoquinolin-1-ones have been prepared by Stille cou-

pling of 4-bromo-1-methoxyisoquinolines⁶⁰and by Pd-catalysed

double-bond migration and cyclisation of N-allyl-2-halobenza-

mides.⁵²However, neither of these methods provides a short

access to 4,5-dimethyl-3-phenylisoquinolin-1-one 17. This

target was approached by twice exploiting the nucleophilic reac-

tivity of the CH₂-carbanion ortho to the lithium-coordinating

dimethylamide. N,N,2,3-Tetramethylbenzamide 41 was deproto-

nated selectively at the 2-CH₃and reaction with iodomethane gave

the 2-ethyl analogue 52. However, treatment of 52 with lithium

diisopropylamide failed to give the corresponding ArC^(ꢀ)HMe

anion, as shown by the lack of incorporation of deuterium upon

quench with deuterium oxide. The stronger base, sec-butyllithium,

was successful in removing one of the methylene protons and

reaction of the anion with benzonitrile gave 17, demonstrating that

this synthesis of isoquinolin-1-ones is applicable to 4-alkyl

analogues.

(Table 2). Many selected compounds were also evaluated for inhi-

bition of full-length human TNKS-1 (using immobilised histones as

co-substrate) and counter-screened against human PARP-1³⁶and

murine or human PARP-2 to assess selectivity (Table 2). The

PARP-1 assays were conducted using a commercial kit; PARP-2

assays were conducted for 11a, 12a,f,i,m with murine PARP-2 as

described previously⁶³and for 13b,k,p, 14b, 15b with human

PARP-2 using a commercial kit.

2.4. Structure–activity relationships

2.4.1. Structure–activity relationships—5-position

Examining the data for inhibition of TNKS-2 (Table 2), a trend is

evident for the 5-substituent. Comparing the six analogues 12i,

13e, 14d, 15d, 16b, 22i where the 3-substituent is kept constant

as 4-triﬂuoromethylphenyl, potency increases in the order

5-F < 5-NO₂= 5-OH < 5-NH₂< 5-Me = OMe. This trend is also

observed for the entire set of 3-arylisoquinolinones. The data sug-

gest that an electron-donating or electron-neutral hydrophobic

group is optimum at this 5-position, with the electron-withdraw-

ing ﬂuorine and nitro groups having diminished activity.

Similarly, H-bond donors (hydroxy, amino) reduce the inhibitory

potency and hydrophobic groups (methyl, methoxy) are preferred.

Finally, modest size improves activity, with the 5-ﬂuoro com-

pounds showing lower potency; C–F occupies similar steric space

as does C–H. Interestingly, this correlates with the observed prefer-

ence for 8-methyl and 8-methoxy in the 2-arylquinazolin-4-one

series (corresponding to the 5-position in isoquinolinones), where

the 5-H compound was signiﬁcantly less active.³⁶This trend can be

rationalised structurally by considering that the space available to

this 5-substituent is expected to be that occupied by the ribose

anomeric carbon and adjacent 2⁰-CHOH and ether oxygen when

NAD⁺is bound. The part of this space immediately adjacent to

the isoquinolinone 5-carbon is of mixed hydrophilic and

hydrophobic character, being bounded by an edge of the aromatic

2.3. Biochemical evaluation

Target compounds were initially screened for inhibition of the

activity of TNKS-2 at 1.0

truncated human TNKS-2 containing the catalytic (NAD⁺-binding)

and adjacent sterile -motif (SAM) domains. This assay measures

lM, 100 nM and 10 nM using immobilised

a

autopoly(ADP-ribosyl)ation of the enzyme with a mixture of

NAD⁺and biotin-labelled NAD⁺; the biotinylated poly(ADP-ribose)

is then quantiﬁed with streptavidin-horseradish peroxidase.³⁶The

data are shown in Table 2. IC₅₀values were then determined for

examples selected for potency at 10 nM and which would give

detailed information about the structural requirements for activity

ring and the CH₂of Tyr¹⁰⁵⁰, an edge of the aromatic ring of Tyr¹⁰⁶⁰

the

-CH₂of Lys¹⁰⁶⁷and the OH of Tyr¹⁰⁷¹. Further away from the

,

e

5898

H. A. Paine et al. / Bioorg. Med. Chem. 23 (2015) 5891–5908

Table 2

Inhibition of the catalytic activities of TNKS-2, TNKS-1, PARP-1 and PARP-2 by isoquinolin-1-ones 11–17, 22 and by positive control 1

O

H

N

TNKS-2

PARP-1 IC₅₀

(nM)

PARP-2 IC₅₀

(nM)

R³

Compd no.

TNKS-1 IC₅₀(nM)

R⁵

R³

R⁵

% inhibition

(1.0 M)

% inhibition

(100 nM)

% inhibition

(10 nM)

IC₅₀(nM)

l

1

—

H

—

97 10

87 10

53

9

18

84

21 17

11 18

0

2

20

1

15

3

15

39

4

a

11a

12a

12c

12d

12e

12f

12g

12h

12i

NH₂

Me

16

92

100

8

2

8

18

60

7

8

1600 250^b

1070 70^b

880 140^b

1050 150^b,d

480 150^b,d

Ph

4-MePh

2-MeOPh

3-MeOPh

4-MeOPh

2-F₃CPh

3-F₃CPh

4-F₃CPh

4-FPh

2-ClPh

100

0

2

12

4

34 14

48 10

76 12

73 16

54 14

41

8

2

27

6

2

4

7

4

0

29

8

26

900 210^b

330 70^b

730 250^b,d

170 20^b,d

1

82

100

40

89

94

4

100

78

10

1

2

7

2

4

8

7

1

2

0

15 20

33

97

85

15

57

88

0

27

60

55

6

1

12j

12k

12l

10 12

3-ClPh

4-ClPh

36

65

8

58 10

20 12

32 19

80

86

100

6

4

15

12m

12n

12p

12s

12t

12u

13a

13b

13c

13d

13e

13f

13g

13h

13i

570 30^b

160 50^b,d

2,6-Cl₂Ph

4-HOPh

PhCH₂CH₂

3-H₂NOCPh

4-H₂NOCPh

Ph

100

38 22

11 32

20

100

92

94

100

5

3

2

8

3

1

1.8 0.1

1.3 0.5

5.7 2.0

1.6 0.2

4-MePh

21

43

3

6

>10,000^c

>10,000^e

4-Bu^tPh

91 34

88 18

100 15

78 12

4-MeOPh

4-F₃CPh

2-ClPh

3-ClPh

4-ClPh

100

87

5

1

7

90

98

0

22 24

70

5

88 12

71 25

70

61

5

1

14

100

1

9

4

1400 450

17 12

95 10

81

88

1

2

7

26

91 60

32 25

836 95

20

9.6 3.6

181 21

275 32

1340

>10,000^c

>5000^c

98

0

2

21

6

5

2,6-Cl₂Ph

4-BrPh

13j

13k

13l

Me

95 26

100 10

100

1

4-PhC„CPh

4-Me₂NCH₂Ph

4-(Piperidin-1-

yl)CH₂Ph

4-(Pyrrolidin-1-

yl)CH₂Ph

4-(4-Me-piperazin-1-

yl)CH₂Ph

Pyridin-4-yl

Benzodioxol-5-yl

Thiophen-3-yl

Ferrocenyl

Ph

2

>1000

>10,000^c

>10,000^e

100

9

100 20

100 11

3

13m

100 29

9.8 5.1

13n

13o

Me

60 12

41 12

79 12

0

14

8

1200 210 12 ꢁ 10³2 ꢁ 10³>10,000^c

100

6

9

61

13p

13q

13r

13s

14a

14b

14c

14d

14f

14g

14h

15a

15b

15c

15d

15e

15f

15g

15h

15i

Me

F

100 16

98 11

20 20

51 17

49 15

29

19

23

4

5

48 26

74 27

111 23

163 67^e

>10,000^e

95 10

87 17

67 10

77

5

720 230

54 10

100 10

0

1

100

95

3

8

87 12

54

66 14

21 17

83 17

73

15

88

100

74

100 14

100 10

58

14

57 15

4-MePh

4-MeOPh

4-F₃CPh

4-ClPh

4-BrPh

91

98

79

5

7

8

5

3.8 0.5

64

3

>10,000^c

98 19

100 11

100 17

100 22

92

11

2

F

73

7

5

2

8

Pyridin-4-yl

Ph

29 17

26 12

100 13

100 20

90 12

OMe 100

OMe 100 17

OMe 100

OMe 100 25

OMe 100 12

OMe

OH

2

28 0.6

5.1 0.6

3.8 1.2

2.5 0.9

5.5 0.3

4-MePh

30 14

>10,000^c

>10,000^e

4-Bu^tPh

4

4-F₃CPh

4-ClPh

4-BrPh

Pyridin-4-yl

Benzodioxol-5-yl

Thiophen-3-yl

Ph

4-F₃CPh

Ph

100

100 15

65

50 27

2

76

8

2

5

2

73 17

91 26

94 10

87

73

86

69

8

9

5

53

59

50

68 16

44 27

55 14

4

9

16a

16b

17

22i

22j

100 0.5

OH

85

100

5

4

Me^f

90 32

72

70 19

19

4

244

860 210

4-F₃CPh

4-FPh

NO₂

93 13

47 32

9

a

b

c

d

e

f

Table cells without data indicate Not Determined.

Data taken from Ref. 44.

Limited by solubility.

Murine PARP-2.

Human PARP-2.

Also 4-Me.

H. A. Paine et al. / Bioorg. Med. Chem. 23 (2015) 5891–5908

5899

isoquinolinones, the space becomes more polar, with the carboxy-

late of Glu¹¹³⁸and the ammonium of Lys¹⁰⁶⁷

nicotinamide-binding pocket and the hydrophobic cavity of

TNKS-2 showed dihedral angles between the rings for 13h (5°)

and for 13i (60°) in minimised structures (Fig. 5). The dihedral

angles correlate with potency against the activity of the enzyme.

Compound 17, in which dihedral twist between the rings should

be induced by the 4-Me, did not form crystals of suitable quality

for small-molecule X-ray structure determination. However, when

soaked into the crystal of TNKS-2, it bound well and a crystal

structure was determined (Fig. 6). Similarly, a crystal structure

was obtained for 15e bound into TNKS-2 (Fig. 6). These crystal

structures show that the rings are effectively coplanar in 15e,

whereas the aromatic rings are signiﬁcantly twisted relative to each

other in 17. Thus the inhibitory activity towards TNKS-2 does not

correlate well with the dihedral twist angle between the phenyl

and isoquinoline rings, as 17 is only nine-fold less potent than

13a. Loss of inhibitory activity does, however, correlate with the

presence of bulky ortho-substituents on the phenyl ring.

Compounds 12d,k, 13f with only one ortho-substituent on the

.

2.4.2. Structure–activity relationships—exploration of the

hydrophobic cavity

Comparison of 11a with the 3-phenyl analogue 12a shows the

need for a 3-aryl group. The nature and substitution of this group

have a major role in determining potency, according to whether

or not the substituted aryl can be accommodated in the hydropho-

bic cavity. The 3-(unsubstituted-phenyl)isoquinolinones 12a, 13a,

14a, 15a, 16a all have good potency, taking into account the effect

of the nature of the 5-substituent. The location of the groups

attached to the 3-phenyl is seen to have a profound effect on bind-

ing. Within the isomeric set of 5-amino-3-(methoxyphenyl)

isoquinolin-1-ones 12d–f, the methoxy substituent appears to be

moderately well tolerated at the meta- and para-positions of the

3-phenyl (in 12e,f) but not at the ortho-position, leading to

inactivity of 12d below micromolar concentrations. A similar

strong trend is seen for the corresponding isomeric set of

5-amino-3-(chlorophenyl)isoquinolin-1-ones 12k–m but the

para-chlorophenyl compound 12m also appears to be marginally

more active than the meta-analogue 12l. Compound 12n, with

chlorines in both ortho-positions, is completely inactive. The same

trend is also strongly evident in moving to the 5-methyl series, in

that the para-chloro compound 13h is very potent, the meta-chloro

isomer 13g is marginally less potent, with even lower activity for

the ortho-chloro isomer 13f. Again, the 2⁰,6⁰-dichloro analogue

13i completely lacks activity. Thus the trend for optimum

location of substituents on the 3-phenyl ring appears to be

4⁰> 3⁰ꢂ 2⁰>>> 2⁰,6⁰. We also observed this preference for

ﬂavones.³⁹Interestingly, the benzo-1,3-dioxoles 13q and 15h

retain activity; these can be considered as having the 3-phenyl

rings disubstituted at the meta- and para-positions.

There are two possible structural rationalisations for this trend.

Firstly, the hydrophobic cavity may simply be limited in size and

ﬂexibility at the end nearer the isoquinolinone-binding motif and

thus not be able to accommodate bulky ortho-substituents. In this

rationalisation, the hydrophobic cavity is larger and/or more ﬂexi-

ble at the distal end, near the tunnel to the exterior, to accommo-

date substituents in the meta- and para-positions. In an alternative

rationalisation, it may be the dihedral (twist) angle between the

phenyl and the isoquinoline rings that controls binding in the

hydrophobic cavity. The phenyl–isoquinoline system is essentially

a biphenyl-like structure and the two rings may twist dihedrally

about the linking bond to avoid steric clashes between 2-H and

4-H on the isoquinoline and 2⁰-H and 6⁰-H on the phenyl. Of course,

if any of these positions were occupied by a bulky substituent, the

propensity to twist out of the mutual plane would be greater. In

this rationalisation, the hydrophobic cavity may be oval in cross-

section and rigid, allowing the phenyl ring to bind in one plane

but not in others. Resolution of this dichotomy is achieved by

inducing the dihedral twist between the rings without the ortho-

substituents on the phenyl; 17 has a methyl at the 4-position on

the isoquinoline, which should induce the twist to avoid steric

clash with 2⁰-H and 6⁰-H on the phenyl.

3-phenyl retain limited activity at 1.0 lM, whereas those with both

ortho-positions occupied by bulky substituents (12n, 13i) are com-

pletely inactive. This effect can be rationalised by considering

12d,k, 10f as existing as pairs of interconverting enantiomeric

atropisomers, only one of which will bind to the enzyme, with

the single ortho-substituent orientated away from steric clash with

the protein. If there are two ortho-substituents, this is not possible

and the compound cannot bind.

It can be seen that the optimum location for a substituent is the

4⁰-position, where it can interact with residues lining the

hydrophobic cavity and/or project into the narrow tunnel.

Accordingly, the size and nature of the 4⁰-substituent was studied.

Across all series, several trends are evident. Particularly in 12 and

13, the 4⁰-unsubstituted compound is slightly less active, so a

substituent is preferred. Small lipophilic and polar substituents

are tolerated equally, the former illustrated by 12c,i, 13b,d,e,h,

14b–d,f, 15b,d,e and the latter by 12p,u. Interestingly this was

not observed for 5-unsubstituted 3-arylisoquinolin-1-ones,

implying differences between the scaffolds.³⁷The electronic

characteristics of the 4⁰-substituent also appear to be unimportant,

comparing electron-donating (ꢀI, +M) groups (12f,p, 13d, 14c,

15h) with electron-withdrawing groups (ꢀI: 12i, 13e, 14d, 15d,

16b; ꢀM: 12u) and groups with approximately neutral electron-

demand (12c,m, 13b,h,j, 14b,f,g, 15b,e,f).

Thiophene and pyridine were also investigated as aromatic

heterocyclic replacements for the 3-(substituted)phenyl ring. The

thiophene in 13r, 15i maintains the activity, relative to the 3-phe-

nyl parents 12a, 15a, respectively, as expected for this conservative

replacement. However, the isosteric replacement with pyridine in

13p, 14h, 15g produced some striking effects. Potency against

TNKS-2 was dramatically reduced in 14h, 15g, whereas it was

diminished slightly in 12p. However, 12p also inhibited the activity

of PARP-1 strongly (IC₅₀= 111 nM), in contrast to most of the

3-(substituted)phenyl analogues counter-screened against this

isoform and found to have low potency (IC₅₀= 860 nM to

>10,000 nM).

The 3-aryl substituent was extended to test the boundaries and

ﬂexibility of the hydrophobic cavity and to explore the tunnel at

the distal end. Firstly, a short ﬂexible linker (–CH₂CH₂–) was inter-

posed between the isoquinoline and the phenyl ring, taking the

phenyl out of conjugation and pushing it towards the distal end

of the cavity; this caused a marked loss of activity in 12s.

Secondly, diminution in activity was also caused by replacing

simple planar aryl groups at the 3-position with the 3D bulk of

Small-molecule crystal structures were determined for 13i

(carrying a 2⁰,6⁰-dichlorophenyl) and for 15b, which carries only a

para-methylphenyl (Fig. 4). In 13i, the two chlorines force a twist

of 67° between the rings, whereas the rings are almost coplanar

in 15b, with a dihedral angle of only 1°. Unfortunately, the strict

analogue 13h did not form crystals of sufﬁcient quality for X-ray

diffraction. Molecular modelling of 13h and 13i bound into the

5900

H. A. Paine et al. / Bioorg. Med. Chem. 23 (2015) 5891–5908

Figure 4. Crystal structures of 15b (A) and 13i (B). Ellipsoids are represented at 30% probability and only those hydrogens involved in H-bonding are shown for clarity. The

structures show dihedral angles between the rings of 1° (15b) and 67° (13i).

Figure 5. Minimised molecular models generated for 13h (A) and 13i (B) bound into the nicotinamide-binding pocket and adjacent hydrophobic cavity of tankyrase-2. The

calculated dihedral angles between the rings are 60° (13i) and 5° (13h).

Figure 6. (A) Detail of crystal structure of 15e in complex with tankyrase-2. (B) Detail of crystal structure of 17 in complex with tankyrase-2. The dihedral angle between the

rings is 8° for 15e and 50° for 17.

ferrocene in 13s. As with the mono-ortho-substituted phenyl com-

pounds 12d,g,k, 13f, 13s can exist as a pair of interconverting

enantiomeric atropisomers and it is again likely that only one

atropisomer can bind.

2.4.3. Structure–activity relationships—entering the tunnel

The tunnel at the distal end of the cavity was probed with the

rigid hydrophobic 4⁰-phenylethynyl extension in 13k. Modelling

suggested that this group should occupy the whole tunnel, with

H. A. Paine et al. / Bioorg. Med. Chem. 23 (2015) 5891–5908

5901

the distal phenyl starting to project into solvent. This compound

was potent against TNKS-2, showing that the tunnel is directly

aligned with the 4⁰-position on the proximal benzene ring and

can accept a long narrow hydrophobic group.

4UVS, 4UVT, respectively) in TNKS-2, establishing the initial mode

of binding of the core scaffold, as described above. The improved

analogue structures 12a,f,m, 13h,n, 14f, 15e and 17 (PDB codes

4UVZ, 4UVO, 4UVN, 4UVV, 4UVU, 4UVX, 4UVY, 4UVW, respec-

tively) with varied 3- and 5-substituents were used to rationalise

the SARs.

The tunnel was also exploited in designing more water-soluble

analogues. The isoquinolin-1-one pharmacophore usually gives

high-melting solids of low aqueous solubility. A previous attempt

to introduce a water-solubilising basic aliphatic amine to the 2-

phenylquinazolin-4-one core with a 4⁰-H₃N⁺CH₂but abolished

inhibitory activity against the TNKSs.³⁶Modelling suggested that

this primary ammonium would have to be accommodated in the

hydrophobic part of the tunnel, giving unfavorable interactions.

Thus, in the isoquinolinone series, tertiary aliphatic amines were

incorporated at the corresponding 4⁰-position on the 3-phenyl

ring, to provide a solubilising salt but without compromising

lipophilicity severely. (Protonated)-4⁰-dimethylaminomethyl- (13l),

4⁰-(piperidin-1-yl)methyl- (13m) and 4⁰-(4-methylpiperazinyl)

methyl- (13o) groups were accepted without major loss of activity

but, curiously, the 4⁰-(pyrrolidin-1-yl)methyl analogue 13n had

The compounds bind to the nicotinamide-binding site in the

fashion expected for a typical inhibitor of PARPs. They make the

canonical H-bonds with Gly¹⁰³²and Ser¹⁰⁶⁸of TNKS-2 (Fig. 7a),

as well as

p

-stacking with Tyr¹⁰⁷¹. Superposition of the protein

structures reveals that the ligands occupy the same cavity in all

the cases and the isoquinolinone moiety is nearly identically posi-

tioned in each case. Small structural changes in the surrounding

residues induced by the binding of the analogues can be detected,

when the crystal structures are compared. No structural changes

were observed when the structures were compared with the

TNKS-2 structure with nicotinamide bound (PDB code 3U9H).⁴⁹

The initial small inhibitors 11a–d bind very similarly, with no

structural changes in the protein. Binding of the aryl group in the

larger derivatives 12–15, 17 causes Pro¹⁰³⁴and Phe¹⁰³⁵to move

towards the hydrophobic extensions of the compounds (Fig. 7b).

This induced ﬁt is driven by the enhanced hydrophobic interaction

of the closer ﬁt of the compound. This change was not observed

when a bulkier compound, 13n, was bound to the active site. The

long pyrrolidinyl extension towards the solvent is not well ordered

in the crystal structure and the compound also has a lowered

potency (Fig. 7a, Table 2). This contrasts with the retained activity

of 13m and 13o. The dimethylaminomethyl group in 13l does not

extend to the solvent region with a hydrophobic group, as is the

case with 13n, and 13l retains activity.

Based on the differences in potency, the best 5-substituents are

electron-donating or electron-neutral. In addition, compounds

with hydrophobic 4⁰-substituents may interact weakly with the

Tyr¹⁰⁵⁰(Fig. 7b). No large structural changes in the protein were

observed when comparing the co-crystal structures of 12m, 13h,

14f and 15e, which all have a 4-chlorophenyl group at the 3-posi-

tion of the isoquinolinones but which vary in the 5-substituent.

Only 10° rotation of the Tyr¹⁰⁵⁰side chain was observed with the

5-methoxy derivative 15e (Fig. 7c). This methoxy group is in close

contact (ca. 3 Å) to Tyr¹⁰⁷¹hydroxy and to a water molecule on the

protein surface (Fig. 7b). This would indicate that the methoxy

group would not be the best substitution but apparently other

effects overcome these unfavorable interactions. The 5-NH₂in

12m does not make any H-bonds with the water molecules near

the binding site.

very poor potency (IC₅₀= 1.2 lM). The HCl salts of 13l–o were

soluble to >2.5 mg mL^ꢀ1in water.

Table 2 also shows the activities of selected compounds against

TNKS-1 and the results of counter-screening against PARP-1 and

PARP-2. In general, the IC₅₀values against TNKS-1 parallel those

found for TNKS-2. Most compounds were apparently more active

against TNKS-2 than they were against the TNKS-1 isoform, an

effect also seen in the preliminary studies with 2-aryl-8-

methylquinazolin-4-ones,³⁶but the assays use different constructs

of the enzyme and different formats, so detailed comparisons must

be made with caution. Worthy of note, however, is the apparent

extreme selectivity of 13k for inhibiting TNKS-2 (IC₅₀= 10 nM),

whereas no inhibition of the activity of TNKS-1 is seen up to

1.0

PARP-1, with many IC₅₀values exceeding the limits of solubility

at 5.0–10 M. These data demonstrate that selectivity for TNKS-2

lM. Most compounds examined were very poor inhibitors of

l

versus the PARP-1 isoform of up to 2.5 ꢁ 10³—fold can be achieved

(e.g., 14b). Selected compounds were also counter-screened

against another PARP isoform, PARP-2. The 5-amino-3-aryliso-

quinolin-1-ones 12a,f,i,m inhibited PARP-2 moderately with IC₅₀

values parallelling those for inhibition of PARP-1. By contrast, the

5-methyl-, 5-ﬂuoro- and 5-methoxy-3-arylisoquinolin-1-ones

13b,k 14b,15b did not inhibit PARP-2 activity up to 10 mM. The

major exception to this poor inhibition of PARP-1 and PARP-2 is

the 3-(pyridin-4-yl) compound 13p, which is essentially non-selec-

tive between the four isoforms examined and may constitute a

new structural lead for inhibition of PARP-1/2.

The phenyl group of 17 is rotated as described above but

changes in the protein residues can also be observed when this

compound is bound. Tyr¹⁰⁷¹rotates by 26° on complexation with

17 and Tyr¹⁰⁵⁰(including the main chain atoms) moves ꢃ0.5 Å to

accommodate the 4-methyl group of the compound (Fig. 7c).

2.5. Crystallographic studies on mode of binding

Protein crystallography was used during the studies to under-

stand the modes of binding of the compounds to TNKSs. The cat-

alytic domain of TNKS-2 was used in the structural studies and

the structures were solved at 1.6–2.2 Å resolution. The asymmetric

unit of the crystals contained two protein chains, which are almost

identical at the compound-binding site. We will, therefore, only

discuss chain A of the crystal structures. Importantly, the catalytic

domains of TNKS-1 and TNKS-2 are highly homologous and the

residues surrounding the binding sites are completely conserved.⁶⁷

The apo crystal structures of the proteins have differences in the

conformations of the D-loops lining the substrate-binding site,

which could indicate differences in dynamics of the proteins but

may naturally also be caused by the crystal packing.³³The current

structural information does not explain the observed selectivity of

13k between the isoforms of TNKS.

2.6. Antiproliferative activity

In very preliminary experiments, selected examples were eval-

uated for their antiproliferative activity against FEK4 human

ﬁbroblasts⁶⁴and HT29 human colon adenocarcinoma cells.⁶⁵The

effects of some 5-aminoisoquinolinones on the growth of MDA-

MB-231 human breast cancer cells and on LNCaP human prostate

cancer cells were also assessed. The FEK4 cell line is a model for

normal non-malignant cells. HT29 cells have no BRCA mutation

but do have a class-II mutation in APC. It is evident (Table 3) that

there is a wide range of antiproliferative activities shown by the

isoquinolinones and that this activity does not correlate with

potency of inhibition of the TNKSs in cell-free assays.

Nevertheless, some trends can be noted. XAV939 1 showed no

inhibition of growth of either FEK4 cells or of HT29 cells under

In total, twelve crystal structures were solved, including those

of the small initial hit molecules 11a–d (PDB codes 4UVL, 4UVP,

5902

H. A. Paine et al. / Bioorg. Med. Chem. 23 (2015) 5891–5908

Table 3

Antiproliferative activities of isoquinolin-1-ones 11, 3-arylisoquinolin-1-ones 12–15,

17 and known TNKS inhibitor 1

Compd

no.

FEK4 GI₅₀

M)

HT29 GI₅₀

M)

MDA-MB-231 GI₅₀

M)

LNCaP GI₅₀

M)

(

l

(

l

(

l

(l

1

>500^a

>200

>500^a

>500

>200

b

>200

101

23

73 12

11a

11b

11c

11d

12a

12c

12f

12i

12m

13b

13e

13g

13h

13i

13k

13m

13n

13p

13r

14f

15b

17

>200

49 11

34

7

5

23

51

16

5

4

84

2

7

0.4

>200

54

7

8

1

1.4 0.4

28

59 21

0.65 0.2

85 17

>100

4

14

7

11

4

34

16

10

7

3

4

1

18

4

6.0 3.7

1.8 0.4

>100^a

>200^a

>50^a

>100^a

48.9 0.3

>200^a

107

2

38

3

>50^a

>500^a

26.7 3.3

>500^a

>200^a

>500^a

>200^a

8.2 0.4

214 16

12.4 3.5

39.8 8.6

>500^a

>100^a

14

2

a

Limited by solubility.

Table cells without data indicate Not Determined.

b

selectivity for the malignant HT29 cells over ‘‘normal’’ FEK4 cells.

Interestingly, 13b was strongly antiproliferative against both cell

types, with lesser non-selective activity shown by 13h,i,m.

Comparative studies were limited by insolubility.

Compounds 12c, 13b,e,g,h,i,k, 14b, 13b were selected by the

National Cancer Institute for evaluation in their 60-cell line screen

(Supporting information). Most showed no or very modest inhibi-

tion of proliferation of human tumour cells at 10 lM. However, the

3-(4-methylphenyl) analogues 12c and 13b had interesting activity

against MDA-MB-435 melanoma cells and were selected for evalu-

ation in the ﬁve-concentration screen. Compounds 13e, 14b, 15b

were inhibitory towards UO-31 renal cells, whereas 13g,h, 14b

were active against MCF7 breast tumour cells. The ﬁve-concentra-

tion study showed that 12c had GI₅₀= 250 nM for MDA-MB-435

cells and 13b had GI₅₀= 316 nM for these melanoma cells and

GI₅₀= 341 nM for UO-31 renal carcinoma cells.

3. Conclusions

In this paper, we report the design, synthesis and evaluation

in vitro of 3-arylisoquinolin-1-ones as inhibitors of the TNKSs, using

these compounds as probes of the binding site. Compounds were

designed from three structural starting-points: the crystal struc-

tures of our simple PARP-1 inhibitors 11a⁴⁰, 11b,⁵¹11c⁵¹and

11d⁵²bound into the NAD⁺-binding site of human TNKS-2; the crys-

tal structure of known inhibitor 1 bound to TNKS-2;⁵³‘scaffold-hop-

ping’ from our 2-arylquinazolin-4-one inhibitors of the TNKSs.³⁶

3-Arylisoquinolin-1-ones were designed, carrying a variety of

substituents at the 5-position. The position and nature of sub-

stituents on the 3-aryl group was also explored in this library, to

help to deﬁne SAR and to explore the shape and boundaries of

the hydrophobic cavity adjacent to the nicotinamide-binding

Gly¹⁰³²/Ser¹⁰⁶⁸motif. To improve and generalise from our

previous syntheses of 3- or 4-substituted 5-aminoisoquinolin-1-

ones,^{40,51,52,57,60,63}a new route was established in which the

3-aryl group was introduced from an areneboronic acid in a

Suzuki–Miyaura coupling, giving diversity at a late stage. 3-Aryl-

5-Me-isoquinolin-1-ones 13, 3-aryl-5-F-isoquinolin-1-ones 14

Figure 7. Co-crystal structures of tankyrase-2 with the isoquinolin-1-one inhibi-

tors. (A) All twelve structures are superposed using the protein chain. The surface of

the hydrophobic cavity, the main-chain cartoon and selected residues are shown for

the complex of 11a. Tyr¹⁰⁷¹is not shown for clarity. (B) Comparison of the

structures of the complexes of 11a (blue) and 15e (magenta). A water molecule

located close to the methoxy group is shown as a sphere. (C) Comparison of the

structures of the complexes of 11a (blue) and 17 (cyan). The main-chain cartoon for

the protein is shown for the structure with 11a for panels B and C.

normal serum conditions, correlating with the ﬁndings of Huang

et al.⁷for DLD-1 human colon carcinoma cells. As expected from

earlier studies,^40–44,665-AIQ 11a did not inhibit the growth of

any of the cell lines. The 3-ethyl analogue 11b was similarly inac-

tive. However, 5-aminoisoquinolin-1-ones carrying either a more

substantial 3-alkyl group (11c) or, more effectively, a 3-aryl group

(12a,c,f,i,m) did inhibit the growth of all the cell lines at micromo-

lar concentrations, with no selectivity between cell types.

Replacement of the 5-NH₂group with 5-Me diminished antiprolif-

erative activity in many cases. However, 13p,r, 14 showed

H. A. Paine et al. / Bioorg. Med. Chem. 23 (2015) 5891–5908

5903

and 3-aryl-5-MeO-isoquinolin-1-ones 15 were assembled in one

step by deprotonation of 3-substituted N,N,2-trimethylbenzamides

with LDA and quench with benzonitriles; again diversity of the 3-

aryl is introduced at a very late stage.

(PDB accession code 3U9H) or with molecular replacement with

MOLREP⁶⁹using the TNKS2 structure (PDB accession code 3KR7)

as a starting model. REFMAC5⁷⁰was used for reﬁnement and

COOT⁷¹for manual building of the model. Statistics for collection

of data and for reﬁnement are shown in the Supporting

information.

The nature of the 5-substituent in inhibition of TNKS-2 was rel-

atively unimportant, with 5-Me and 5-OMe analogues being

slightly more active than their 5-F, 5-OH or 5-NH₂counterparts.

By contrast, a (substituted)phenyl or heteroaryl group in the 3-po-

sition was essential for activity. Substitution on this 3-phenyl was

also important, with groups in the para-position generally enhanc-

ing binding and bulky substitution in one or both ortho-positions

abrogating activity. The electronic and polar nature of the para-

substituent was less important; long rigid linear groups were also

accepted. Also tolerated in this para-position were extensions car-

rying water-solubilising tertiary aliphatic amine salts. In general,

potency against TNKS-1 paralleled potency against TNKS-2, with

very few exceptions; this is unremarkable, since the NAD⁺-binding

catalytic domains have a high degree of sequence identity. Most of

the 3-arylisoquinolin-1-ones inhibited the TNKSs highly selec-

tively, with much lower potency against a related enzyme, PARP-

1. Many of the most potent inhibitors of the TNKSs showed

>500-fold selectivity for TNKS-2 versus PARP-1, with the most

selective, 14b, 15b, being >2000-fold selective. Interestingly, the

SAR for inhibition of PARP-1 and of PARP-2 by these 3-aryliso-

quinolin-1-ones did not parallel the SAR for inhibition of TNKSs,

indicating that incorporating selectivity by design was feasible.

Interestingly, the 3-(pyridin-4-yl) analogue 13p proved to be a

moderately good inhibitor of PARP-1, (IC₅₀= 111 nM) and of

PARP-2 (IC₅₀= 167 nM).

The SAR was rationalised by structural studies consequent to

determining the crystal structures of several analogues bound into

the TNKS-2 protein. Together, the SAR and the structural studies

allowed good deﬁnition of the capacity of the hydrophobic cavity

to accept substituted aryl groups. The para-extensions projected

into a narrow tunnel towards the exterior, indicating that this is

a direction in which to seek further binding contacts for second-

generation isoquinolin-1-one inhibitors.

Some of the compounds showed antiproliferative activity,

although the link with inhibition of the tankyrases was not clear.

In particular, 12c and 13b showed good inhibition of the growth

of several tumour cell lines in the NCI 60-cell-line panel.

4.2. Biochemical and antiproliferative evaluation

Assays of inhibition of TNKS-1, TNKS-2 and PARP-1 were per-

formed using methods described previously.³⁶Assays of inhibition

of PARP-2 were performed for 11a, 12a,f,i,m using a method

described previously.⁶³Assays of inhibition of PARP-2 were

performed for 13b,k,p, 14b, 15b with human PARP-2 using a

commercial kit (AMS Biosciences PARP2 Chemiluminescent Assay

Kit, Catalogue # 80552, AMS Biosciences, Abingdon, Oxfordshire

OX14 4SE, UK), following the protocol provided. Antiproliferative

assays were conducted as described previously.⁶³IC₅₀values were

estimated using Sigmaplot.

4.3. Chemical synthesis

4.3.1. General

Chemical reagents, solvents and starting materials were pur-

chased from Sigma Aldrich, Goss Scientiﬁc, Alfa Aesar and Fisher

Scientiﬁc and were used without further puriﬁcation. ¹H and ¹³C

NMR spectra were recorded at 400.04 MHz or 500.13 MHz for ¹H

NMR, at 100.59 MHz or 125.76 MHz for ¹³C NMR and at 376 MHz

for ¹⁹F NMR, using CD₃OD, (CD₃)₂SO and CDCl_3,containing SiMe₄.

Reactions were monitored by thin-layer chromatography

(TLC) on silica gel. MS data were obtained using electrospray ioni-

sation on a microTOF instrument (Bruker Daltonics, Germany) and

calibrated using sodium formate. Mps were measured using a hot-

stage microscope (Reichert-Jung) and are uncorrected.

Experiments were conducted at ambient temperature, unless

otherwise noted. Solutions in organic solvents were dried with

MgSO₄. Pd₂dba₃refers to tris(dibenzylideneacetone)dipalladium;

SPhos refers to 2-dicyclohexylphosphino-2⁰,6⁰-dimethoxybiphenyl.

The brine was saturated. Compounds for biological evaluation

were shown by HPLC and ¹H NMR to be >97% pure.

Compounds 11a–d,^40,51,5212a,c,f,m⁵¹were prepared as

described previously. The syntheses of 12e,g,h–l,n,p,s,u, 13c–k,m–

r, 14b–d,f–h, 15b–i, 16b, 27, 30b,d,e,h–n,q,r, 31e,g–l,n,p,r,s, 33,

34, 36i,j, 43, 45a–d, 47, 49, 51 are reported in the Supporting

information.

Thus the 3-arylisoquinolin-1-ones are shown to represent a

class of highly potent and selective inhibitors of the TNKSs, with

IC₅₀of some examples (13a–d) in the 1–2 nM range. Adding

water-solubilising substituents is only marginally deleterious to

activity. This core is therefore identiﬁed as the foundation on

which to build more complex inhibitors with optimised pharma-

ceutical and pharmacological properties.

4.3.2. 5-Amino-3-(2-methoxyphenyl)isoquinolin-1-one

hydrobromide (12d)

Compound 31d (33.5 mg, 120 lmol) was stirred with HBr in

4. Experimental

AcOH (33%, 1.1 mL) at 65 °C for 5 h. Evaporation yielded 12d

(31 mg, 72%) as a pale buff solid: mp 172–175 °C; ¹H NMR

(CD₃OD) d 3.91 (3H, s, Me), 6.79 (1H, d, J = 0.5 Hz, 4-H), 7.11 (1H,

td, J = 7.5, 0.9 Hz, Ph 5-H), 7.18 (1H, d, J = 8.3 Hz, Ph 3-H), 7.52

(1H, td, J = 7.6, 1.7 Hz, Ph 4-H), 7.56 (1H, dd, J = 7.6, 1.7 Hz, Ph 6-

H), 7.62 (1H, t, J = 7.9 Hz, 7-H), 7.82 (1H, dd, J = 7.7, 1.2 Hz, 6-H),

8.44 (1H, d, J = 7.3 Hz, 8-H); ¹³C NMR (CD₃OD) (HSQC/HMBC) d

56.20 (Me), 99.86 (4-C), 112.74 (Ph 3-C), 122.06 (Ph 5-C), 124.11

(Ph 1-C), 127.25 (8a-C), 127.33 (4a-C), 127.54 (7-C), 128.60 (6-C),

129.33 (8-C), 131.26 (Ph 6-C), 132.91 (Ph 4-C), 134.01 (5-C),

142.77 (3-C), 158.49 (Ph 2-C), 163.82 (1-C); MS m/z 267.1126

(M+H)⁺(C₁₆H₁₅N₂O₂requires 267.1135).

4.1. Protein crystallography

Protein crystallography experiments were carried out using

human TNKS-2. The catalytic domain was expressed, puriﬁed and

crystallised as previously reported.⁴⁹Inhibitors were soaked for

24 h to 2 months into the crystals in a well solution (0.2 M Li₂SO₄,

0.1 M Tris–HCl pH 8.5, 24–26% PEG 3350) supplemented with the

test compound (100 lM or 1 mM) and NaCl (0.25 M). Before collec-

tion of data, the crystals were brieﬂy soaked in a well solution sup-

plemented with 20% glycerol and ﬂash frozen in liquid N₂.

Data were collected at the Diamond Light Source on beamlines

I038 and I04-1 and at ESRF on beamlines ID14-1, ID23-1 and ID23-

2. Diffraction data were processed and scaled with the XDS pack-

age.⁶⁸The structures were solved using the Difference Fourier

method, with the starting phases derived from the TNKS2 structure

4.3.3. 5-Amino-3-(3-aminocarbonylphenyl)isoquinolin-1-one

hydrobromide (12t)

Compound 31q (11.2 mg, 40

lmol) was stirred with HBr in

AcOH (33%, 1.5 mL) at 65 °C for 16 h. Evaporation yielded 12t

5904

H. A. Paine et al. / Bioorg. Med. Chem. 23 (2015) 5891–5908

(13.5 mg, 98%) as an amber solid: mp >360 °C; IR (KBr) m_max3351,

3173, 1664 cm^ꢀ1; ¹H NMR ((CD₃)₂SO) d 4.2 (3H, br, ⁺NH₃), 7.11 (1H,

s, 4-H), 7.43 (2H, m, Ph 4,5-H₂), 7.55 (1H, br, CONHH), 7.61 (1H, t,

J = 7.7 Hz, 7-H), 7.95 (3H, m, 6,8-H₂+ Ph 6-H), 8.03 (1H, br,

CONHH), 8.32 (1H, s, Ph 2-d), 11.63 (1H, s, 2-H); ¹³C NMR

((CD₃)₂SO) (HSQC/HMBC) d 98.16 (4-C), 121.83 (Ph 3-C), 122.64

(Ph 5-C), 125.83 (Ph 2-C), 126.00, 126.85 (Ph 4-C), 128.26 (8-C),

128.87 (7-C), 129.29 (Ph 6-C), 133.85, 134.93, 139.18 (3-C),

162.33 (1-C), 167.52 (CONH₂); MS m/z 278.0947 (MꢀH)^ꢀ

(C₁₆H₁₂N₃O₂requires 278.0930).

((CD₃)₂SO) d 2.17 (6H, s, NMe₂), 2.56 (3H, s, 5-Me), 3.44 (2H, s,

CH₂), 6.85 (1H, s, 4-H), 7.36 (1H, t, J = 7.7 Hz, 7-H), 7.40 (2H, d,

J = 8.3 Hz, Ph 3,5-H₂), 7.55 (1H, d, J = 7.2 Hz, 6-H), 7.78 (2H, d,

J = 8.3 Hz, Ph 2,6-H₂), 8.06 (1H, d, J = 8.0 Hz, 8-H), 11.50 (1H, br,

NH); ¹³C NMR ((CD₃)₂SO) (HSQC/HMBC/DEPT) d 18.80 (5-Me),

45.05 (NMe₂), 62.97 (CH₂), 99.76 (4-C), 124.59 (8-C), 124.95

(8a-C), 125.84 (7-C), 126.66 (Ph 2,6-C₂), 129.00 (Ph 3,5-C₂),

132.78 (Ph 4-C), 133.21 (6-C), 133.73 (4a-C), 136.69 (5-C), 139.76

(Ph 1-C), 140.33 (3-C), 162.97 (1-C); MS m/z 595.3227 (2M+H)

(C₃₈H₄₂N₄O₂requires 595.3230), 293.1668 (M+H)⁺(C₁₉H₂₁N₂O

requires 293.1654).

4.3.4. 5-Methyl-3-phenylisoquinolin-1-one (13a)

BuLi (1.6 M in hexanes, 0.90 mL, 1.4 mmol) was added to dry

Prⁱ₂NH (172 mg, 1.7 mmol) in dry THF (2.0 mL) at ꢀ78 °C and the

mixture was stirred at ꢀ78 °C for 10 min. Compound 41 (250 mg,

1.4 mmol) in dry THF (2.0 mL) was added and the mixture was stir-

red for 1 h at ꢀ78 °C. PhCN (144 mg, 1.4 mmol) in dry THF (2.0 mL)

was added at ꢀ78 °C and the mixture was stirred for 1 h at ꢀ78 °C,

then at 20 °C for 2 h. Water (1.0 mL) was added. The mixture was

extracted with CH₂Cl₂. The extract was washed thrice with brine

and dried. Evaporation gave 13a (68 mg, 20%) as white crystals:

mp 214–215 °C; ¹H NMR ((CD₃)₂SO) (COSY/NOESY) d 2.56 (3H, s,

Me), 6.86 (1H, s, 4-H), 7.37 (1H, t, J = 7.7 Hz, 7-H), 7.49 (3H, m,

Ph 3,4,5-H₃), 7.56 (1H, d, J = 7.1 Hz, 6-H), 7.82 (2H, dd, J = 6.6,

1.6 Hz, Ph 2,6-H₂), 8.07 (1H, d, J = 8.0 Hz, 8-H), 11.60 (1H, s,

N-H); ¹³C NMR ((CD₃)₂SO)) (HSQC/HMBC) d 18.76 (Me), 100.00

(4-C), 124.57 (8-C), 125.00 (8a-C), 125.90 (7-C), 126.86 (Ph 2,6-C₂),

128.72 (Ph 3,5-C₂), 129.21 (Ph 4-C), 133.20 (6-C), 133.75 (4a-C),

134.20 (Ph 1-C), 136.61 (5-C), 139.87 (3-C), 162.94 (1-C); MS m/z

258.0834 (M+Na)⁺(C₁₆H₁₃NNaO requires 258.0889).

4.3.7. 3-Ferrocenyl-5-methylisoquinolin-1-one (13s)

BuLi (2.5 M in hexanes, 0.46 mL, 1.1 mmol) was stirred with dry

Prⁱ₂NH (127 mg, 1.3 mmol) in dry THF (2.0 mL) at ꢀ78 °C for

10 min. Compound 41 (200 mg, 1.1 mmol) in dry THF (2.0 mL)

was added and the mixture was stirred for 1 h at ꢀ78 °C.

Ferrocenenitrile 47 (238.5 mg, 1.1 mmol) in dry THF (2.0 mL) was

added and the mixture was stirred for 1 h at ꢀ78 °C, then at

20 °C for 16 h. Water (1.0 mL) was added. The mixture was diluted

with CH₂Cl₂and washed (brine, 3ꢁ). Drying and evaporation gave

13s (80.5 mg, 21%) as a dark orange solid: mp >360 °C; IR m_max

3454, 1633 cm^{ꢀ1 1}H NMR ((CD₃)₂SO) (COSY) d 2.50 (3H, s, Me),

;

4.16 (5H, s, Fc⁰-H₅), 4.45 (2H, s, Fc 3,4-H₂), 5.11 (2H, s, Fc 2,5-H₂),

6.72 (1H, s, 4-H), 7.30 (1H, t, J = 7.3 Hz, 7-H), 7.50 (1H, d,

J = 6.6 Hz, 6-H), 8.02 (1H, d, J = 7.7 Hz, 8-H), 11.08 (1H, br, NH);

¹³C NMR ((CD₃)₂SO) (HSQC/HMBC) d 18.81 (Me), 66.33 (Fc 2,5-

C₂), 69.59 (Fc⁰-C₅), 69.74 (Fc 3,4-C₂), 78.61 (Fc 1-C), 97.03 (4-C),

124.44 (8a-C), 124.69 (8-C), 124.91 (7-C), 132.74 (4a-C), 133.12

(6-C), 136.96 (5-C), 140.01 (3-C), 162.74 (1-C); MS m/z 366.0552

(M+Na)⁺(C₂₀H₁₇⁵⁶FeNNaO requires 366.0557).

4.3.5. 5-Methyl-3-(4-methylphenyl)isoquinolin-1-one (13b)

BuLi (1.6 M in hexanes, 0.9 mL, 1.4 mmol) was added to dry

Prⁱ₂NH (172 mg, 1.7 mmol) in dry THF (2.0 mL) at ꢀ78 °C and the

mixture was stirred at ꢀ78 °C for 10 min. Compound 41 (250 mg,

1.4 mmol) in dry THF (2.0 mL) was added and the mixture was stir-

red for 1 h at ꢀ78 °C. 4-MePhCN (164 mg, 1.4 mmol) in dry THF

(2.0 mL) was added at ꢀ78 °C and the mixture was stirred for 1 h

at ꢀ78 °C, then at 20 °C for 16 h. Water (1.0 mL) was added. The

mixture was diluted with CH₂Cl₂, washed (brine, 3ꢁ) and dried.

Evaporation and recrystallisation (EtOH) gave 13b (90 mg, 26%)

as off-white crystals: mp 205–207 °C; ¹H NMR ((CD₃)₂SO) (COSY)

d 2.36 (3H, s, Ph-Me), 2.55 (3H, s, 5-Me), 6.82 (1H, s, 4-H), 7.30

(2H, d, J = 8.0 Hz, Ph 3,5-H₂), 7.35 (1H, t, J = 7.6 Hz, 7-H), 7.54

(1H, d, J = 7.2 Hz, 6-H), 7.72 (2H, d, J = 8.2 Hz, Ph 2,6-H₂), 8.06

(1H, d, J = 8.0 Hz, 8-H), 11.54 (1H, br, N-H); ¹³C NMR ((CD₃)₂SO)

(HSQC/HMBC) d 18.86 (Ph-Me), 20.85 (5-Me), 99.48 (4-C), 124.62

(8-C), 124.89 (8a-C), 125.79 (7-C), 126.76 (Ph 2,6-C₂), 129.36 (Ph

3,5-C₂), 131.38 (Ph 1-C), 133.23 (6-C), 133.69 (4a-C), 136.77 (5-

C), 138.94 (Ph 4-C), 139.92 (3-C), 163.05 (1-C); MS m/z 250.1226

(M+H)⁺(C₁₇H₁₆NO requires 250.1232).

4.3.8. 5-Fluoro-3-phenylisoquinolin-1-one (14a)

BuLi (1.6 M in hexanes, 0.9 mL, 1.4 mmol) was stirred with dry

Prⁱ₂NH (172 mg, 1.7 mmol) in dry THF (2.0 mL) at ꢀ78 °C for

10 min. Compound 49 (250 mg, 1.4 mmol) in dry THF (2.0 mL)

was added and the mixture was stirred for 1 h at ꢀ78 °C. PhCN

(142 mg, 1.4 mmol) in dry THF (2.0 mL) was added at ꢀ78 °C and

the mixture was stirred for 1 h at ꢀ78 °C, then at 20 °C for 16 h.

Water (1.0 mL) was added. The mixture was diluted with CH₂Cl₂,

washed (brine, 3ꢁ) and dried. Evaporation and recrystallisation

(EtOH) gave 14a (113 mg, 34%) as an off-white solid: mp 216–

217 °C; ¹H NMR ((CD₃)₂SO) (COSY) d 6.91 (1H, s, 4-H), 7.55 (4H,

m, 7-H + Ph 3,4,5-H₃), 7.66 (1H, m, 6-H), 7.87 (2H, m, Ph 2,6-H₂),

8.10 (1H, d, J = 7.9 Hz, 8-H), 11.84 (1H, br, NH); ¹³C NMR

((CD₃)₂SO) (HSQC/HMBC) d 94.80 (d, J = 5.3 Hz. 4-C), 117.64 (d,

J = 19.3 Hz, 6-C), 122.78 (d, J = 3.4 Hz, 8-H), 126.61 (d, J = 3.3 Hz,

8a-C), 126.82 (d, J = 7.9 Hz, 7-C), 126.95 (d, J = 16.4 Hz, 4a-C),

126.96 (Ph 2,6-C₂), 128.83 (Ph 3,5-C₂), 129.64 (Ph 4-C), 133.59

(Ph 1-C), 141.40 (3-C), 157.31 (d, J = 247.8 Hz, 5-C), 161.78 (d,

J = 2.8 Hz, 1-C); ¹⁹F NMR ((CD₃)₂SO) d -121.96 (dd, J = 9.9, 5.2 Hz,

F); MS m/z 238.0670 (MꢀH)^ꢀ(C₁₅H₉FNO requires 238.0668).

4.3.6. 3-(4-Dimethylaminomethylphenyl)-5-methylisoquinolin-

1-one hydrochloride (13l)

4.3.9. 5-Methoxy-3-phenylisoquinolin-1-one (15a)

BuLi (1.6 M in hexanes, 0.7 mL, 1.1 mmol) was stirred with dry

Prⁱ₂NH (142 mg, 1.4 mmol) in dry THF (2.0 mL) at ꢀ78 °C for

10 min. Compound 41 (200 mg, 1.1 mmol) in dry THF (2.0 mL)

was added and the mixture was stirred for 1 h at ꢀ78 °C.

Compound 45a (181 mg, 1.1 mmol) in dry THF (2.0 mL) was added

and the mixture was stirred for 1 h at ꢀ78 °C, then at 20 °C for 16 h.

Water (1.0 mL) was added. The mixture was diluted with CH₂Cl₂.

This mixture was washed (brine, 3ꢁ) and dried. The evaporation

residue was washed (EtOH) to give 13l (15 mg, 4%) as a white solid:

mp 169–170 °C. The solid was then treated for 16 h with aq HCl

(6.0 M, 2.0 mL). Evaporation and drying gave the HCl salt as a white

BuLi (1.6 M in hexanes, 0.8 mL, 1.3 mmol) was stirred with dry

Prⁱ₂NH (156 mg, 1.55 mmol) in dry THF (2.0 mL) at ꢀ78 °C for

10 min. Compound 51 (250 mg, 1.3 mmol) in dry THF (2.0 mL)

was added and the mixture was stirred for 1 h at ꢀ78 °C. PhCN

(133 mg, 1.3 mmol) in dry THF (2.0 mL) was added and the mixture

was stirred for 1 h at ꢀ78 °C, then at 20 °C for 16 h. Water (1.0 mL)

was added. The mixture was diluted with CH₂Cl₂, washed (brine,

3ꢁ) and dried. Evaporation and recrystallisation (EtOH) gave 15a

(97 mg, 30%) as pale peach-coloured crystals: mp 219–220 °C; ¹H

NMR ((CD₃)₂SO) (COSY) d 3.94 (3H, s, Me), 6.94 (1H, s, 4-H), 7.27

(1H, dd, J = 8.0, 0.8 Hz, 6-H), 7.43 (1H, t, J = 8.0 Hz, 7-H), 7.47 (3H,

m, Ph 3,4,5-H₃), 7.77 (3H, m, 8-H + Ph 2,6-H₂), 11.59 (1H, br, N-

solid: mp 292–293 °C; IR m_max3426, 1634 cm^ꢀ1

;

¹H NMR

H. A. Paine et al. / Bioorg. Med. Chem. 23 (2015) 5891–5908

5905

H); ¹³C NMR ((CD₃)₂SO) (HSQC/HMBC) d 55.94 (Me), 96.95 (4-C),

112.28 (6-C), 118.23 (8-C), 125.81 (4a-C), 126.68 (Ph 2,6-C₂),

126.85 (7-C), 128.40 (8a-C), 128.83 (Ph 3,5-C₂), 129.23 (Ph 4-C),

134.07 (Ph 1-C), 139.62 (3-C), 154.39 (5-C), 162.50 (1-C); MS m/z

274.0846 (M+Na)⁺(C₁₆H₁₃NaNO₂requires 274.0844).

(Me), 108.70 (C-4), 119.44 (C-8a), 125.00 (C-7), 129.32 (C-6),

131.49 (C-8), 131.95 (C-4a), 147.40 (C-3), 160.86 (C-1); MS m/z

239.0216 (M+H)⁺(C₁₀H₈³⁵ClN₂O₃requires 239.0223).

4.3.14. 3-Chloro-5-nitroisoquinolin-1-one (29)

Compound 28 (505 mg, 2.1 mmol) was stirred with HBr/AcOH

(33%, 1.0 mL) at 65 °C for 16 h. Evaporation and recrystallisation

(EtOAc) yielded 29 (211 mg, 44%) as yellow needles: mp 271–

272 °C; ¹H NMR (CDCl₃) d 7.25 (1H, s, 4-H), 7.68 (1H, t, J = 8.0 Hz,

7-H), 8.48 (1H, dd, J = 8.0, 1.5 Hz, 6-H), 8.53 (1H, ddd, J = 7.0, 1.5,

0.5 Hz, 8-H), 12.82 (1H, s, N-H); ¹³C NMR (CDCl₃) (HSQC/HMBC) d

102.65 (4-C), 126.29 (7-C), 130.19 (6-C), 130.76 (4a,8a-C₂),

133.63 (8-C), 143.63 (5-C), 146.61 (3-C), 160.84 (1-C); MS m/z

222.9870 (MꢀH)^ꢀ(C₉H₄³⁵ClN₂O₃requires 222.9910).

4.3.10. 5-Hydroxy-3-phenylisoquinolin-1-one (16a)

Compound 15a (25 mg, 0.10 mmol) was heated with BBr₃in

CH₂Cl₂(1.0 M, 2.0 mL) at reﬂux for 16 h. The evaporation residue

was treated with aq NaOH (2.5 M, 3.5 mL) at 0 °C and the mixture

was stirred at 20 °C for 3 h. The solution was acidiﬁed with aq

HCl (2 M). The solid was collected by ﬁltration. Chromatography

(EtOAc/petroleum ether 2:3 ? 1:1) gave 16a (20 mg, 84%) as a pale

yellow solid: mp 248–249 °C; IR m_max3410, 3318, 1677 cm^{ꢀ1 1}H

;

NMR (CD₃OD) d 7.11 (1H, dd, J = 7.8, 1.0 Hz, 6-H), 7.19 (1H, d,

J = 0.6 Hz, 4-H), 7.33 (1H, t, J = 7.9 Hz, 7-H), 7.45 (1H, t, J = 8.5 Hz,

Ph 4-H), 7.51 (2H, t, J = 8.6 Hz, Ph 3,5-H₂), 7.72 (2H, d, J = 8.4 Hz,

Ph 2,6-H₂), 7.79 (1H, dt, J = 8.1, 0.8 Hz, 8-H); ¹³C NMR (CD₃OD)

(HSQC/HMBC) d 100.75 (4-C), 117.44 (6-C), 118.52 (8-C), 126.97

(4a-C), 127.65 (Ph 2,6-C₂), 128.25 (7-C), 129.73 (8a-C), 130.10 (Ph

3,5-C₂), 130.30 (Ph 4-C), 136.05 (Ph 1-C), 139.93 (3-C), 154.58

(5-C), 165.74 (1-C); MS m/z 497.1477 (2M+Na) (C₃₀H₂₂N₂NaO₄

requires 497.1478), 260.0689 (M+Na) (C₁₅H₁₁NNaO₂requires

260.0689), 238.0883 (M+H) (C₁₅H₁₂NO₂requires 238.0868); MS

m/z 236.0705 (MꢀH)^ꢀ(C₁₅H₁₀NO₂requires 236.0712).

4.3.15. 1-Methoxy-5-nitro-3-phenylisoquinoline (30a)

Degassed PhMe (3.5 mL) was added to 28 (144 mg, 0.60 mmol),

Pd₂dba₃(57 mg, 60 lmol), SPhos (57 mg, 120 lmol), PhB(OH)₂

(115 mg, 0.91 mmol) and K₃PO₄(266 mg, 1.2 mmol) in a dry ﬂask.

The mixture was stirred at 100 °C for 2 d. The evaporation residue,

in CHCl₃, was ﬁltered. Chromatography (EtOAc/petroleum ether

1:24), followed by chromatography (EtOAc/petroleum ether 1:29)

gave 30a (142 mg, 84%) as a bright yellow solid: mp 160–166 °C;

¹H NMR (CDCl₃) d 4.28 (3H, s, Me), 7.49 (4H, m, Ph 3,4,5-H₃, 7-

H), 8.20 (2H, dd, J = 8.5, 1.5 Hz, Ph 2,6-H₂), 8.47 (1H, dd, J = 7.8,

1.3 Hz, 6-H), 8.51 (1H, s, 4-H), 8.60 (1H, dt, J = 8.2, 1.1 Hz, 8-H);

¹³C NMR (CDCl₃) (HSQC/HMBC) d 54.43 (Me), 105.41 (4-C),

120.29 (8a-C), 124.72 (7-C), 127.42 (Ph 2,6-C₂), 128.85 (6-C),

129.02 (Ph 3,5-C₂), 129.65 (Ph 4-C), 131.49 (8-C), 131.84 (4a-C),

138.96 (Ph 1-C), 145.28 (5-C), 152.23 (3-C), 160.81 (1-C); MS m/z

281.0918 (M+H)⁺(C₁₆H₁₃N₂O₃requires 281.0928).

4.3.11. 4,5-Dimethyl-3-phenylisoquinolin-1-one (17)

sec-BuLi (1.4 M in cyclohexane, 0.47 mL, 0.66 mmol) was added

to 52 (115 mg, 0.6 mmol) in dry THF (1.0 mL) at ꢀ78 °C and the

mixture was stirred at ꢀ78 °C for 30 min. PhCN (62 mg, 0.6 mmol)

in dry THF (1.0 mL) was added and the mixture was stirred for 1 h at

ꢀ78 °C, then at 20 °C for 16 h. The mixture was then cooled to 0 °C

and quenched with D₂O (0.11 mL, 6.0 mmol) and stirred for 10 min.

The mixture was diluted with CH₂Cl₂and washed thrice with brine.

Drying, evaporation and washing (EtOH) gave 17 (19.5 mg, 13%) as

4.3.16. 1-Methoxy-5-nitro-3-(2-triﬂuoromethylphenyl)isoquino-

line (30g). Method A

Degassed PhMe (3.0 mL) was added to 28 (103 mg, 0.43 mmol),

Pd₂(dba)₃(42.2 mg, 46 mmol), SPhos (41.9 mg, 102 lmol), 2-

a white solid: mp >360 °C; IR m_max3453, 1644 cm^ꢀ1

;

¹H NMR

((CD₃)₂SO) (COSY) d 2.25 (3H, s, 4-Me), 2.76 (3H, s, 5-Me), 7.37

(1H, t, J = 6.8 Hz, 7-H), 7.47 (5H, m, Ph-H₅), 7.53 (1H, d, J = 7.1 Hz,

6-H), 8.18 (1H, d, J = 6.9 Hz, 8-H), 11.21 (1H, br, NH); ¹³C NMR

((CD₃)₂SO) (HSQC/HMBC) d 18.98 (4-Me), 24.54 (5-Me), 108.26

(4-C), 125.52 (8-C), 125.86 (7-C), 127.04 (8a-C), 128.33 (Ph 3,5-

C₂), 128.65 (Ph 4-C), 129.74 (Ph 2,6-C₂), 134.92 (4a-C), 135.65 (Ph

1-C), 136.41 (6-C), 138.17 (5-C), 138.34 (3-C), 161.39 (1-C); MS

m/z 250.1219 (M+H)⁺(C₁₇H₁₆NO requires 250.1232).

F₃CPhB(OH)₂(160.2 mg, 0.84 mmol) and K₃PO₄(179 mg, 0.84 mmol)

and the mixture was stirred at 100 °C for 16 h. The evaporation resi-

due, in CHCl₃, was ﬁltered. Chromatography (Et₂O/petroleum ether

1:199) gave 30g (63 mg, 42%) as a yellow solid.

4.3.17. 1-Methoxy-5-nitro-3-(2-triﬂuoromethylphenyl)isoquino-

line (30g). Method B

Dry DMF (5.0 mL) was added to 37 (175 mg, 620

(57 mg, 60 mol), SPhos (58 mg, 120 mol), 2-F₃CPhB(OH)₂

(176 mg, 930 mol) and K₃PO₄(394 mg, 1.9 mmol). The mixture

lmol), Pd₂dba₃

l

4.3.12. 5-Nitro-3-phenylisoquinolin-1-one (22a)

l

Compound 30d (38.5 mg, 140

l

mol) was stirred with HBr in

was stirred at 135 °C for 16 h. The evaporation residue, in CHCl₃,

was ﬁltered (Celite^Ò). Chromatography (EtOAc/petroleum ether

1:99 ? 1:19) gave 30g (90 mg, 42%) as a yellow solid: mp 95–

99 °C; ¹H NMR (CDCl₃) d 4.18 (3H, s, Me), 7.57 (1H, tt, J = 7.0, 1.4 Hz,

Ph 5-H), 7.64 (3H, m, Ph 4,6-H₂and 7-H), 7.83 (1H, d, J = 7.9 Hz, Ph

3-H), 8.18 (1H, s, 4-H), 8.51 (1H, dd, J = 7.8, 1.2 Hz, 6-H), 8.66 (1H,

dt, J = 8.3, 1.0 Hz, 8-H); ¹³C NMR (CDCl₃) (HSQC/HMBC) d 54.44

(Me), 109.28 (4-C), 119.80 (8a-C), 124.15 (q, J = 270.4 Hz, CF₃),

125.11 (7-C), 126.89 (q, J = 5.0 Hz, Ph 3-C), 128.56 (Ph 5-C), 128.58

(6-C), 128.76 (q, J = 30.6 Hz, Ph 2-C), 130.86 (4a-C), 131.29 (8-C),

131.62 (Ph 6-C), 131.75 (Ph 4-C), 139.69 (Ph 1-C), 144.96 (5-C),

153.10 (3-C), 160.05 (1-C); ¹⁹F NMR (CDCl₃) d –56.75 (s, CF₃); MS

m/z 349.0798 (M+H)⁺(C₁₇H₁₂F₃N₂O₃requires 349.0802).

AcOH (33%, 1.0 mL) at 65 °C for 16 h. Evaporation yielded 22a

(23 mg, 63%) as yellow needles: mp >340 °C; ¹H NMR ((CD₃)₂SO)

d 7.22 (1H, s, 4-H), 7.51 (3H, m, Ph 3,4,5-H₃), 7.64 (1H, t,

J = 8.0 Hz, 7-H), 7.76 (2H, m, Ph 2,6-H₂), 8.46 (1H, d, J = 8.0 Hz, 6-

H), 8.57 (1H, d, J = 8.2 Hz, 8-H), 12.03 (1H, s, NH); ¹³C NMR

((CD₃)₂SO) (HSQC/HMBC) d 97.46 (4-C), 125.73 (7-C), 126.61 (8a-

C), 127.21 (Ph 2,6-C₂), 128.95 (Ph 3,4,5-C₃), 130.19 (6-C), 131.60

(4a-C), 133.54 (8-C), 144.39 (5-C), 155.90 (1-C); MS m/z 265.0619

(MꢀH)^ꢀ(C₁₅H₉N₂O₃requires 265.0613).

4.3.13. 3-Chloro-1-methoxy-5-nitroisoquinoline (28)

Na (700 mg, 31.5 mmol) was added to 27 (6.0 g, 25 mmol) in

dry MeOH (90 mL). The mixture was boiled under reﬂux for 16 h.

The solvent was evaporated until 20 mL remained. CHCl₃was

added and the solution was washed (water, 3ꢁ). Drying and evap-

oration gave 28 (4.8 g, 82%) as ﬁne yellow needles: mp 177–179 °C;

¹H NMR (CDCl₃) d 4.20 (3H, s, Me), 7.62 (1H, t, J = 7.9 Hz, 7-H), 8.14

(1H, s, 3-H), 8.52 (1H, dd, J = 7.8, 1.3 Hz, 6-H), 8.59 (1H, dt,

J = 8.3 Hz, 1.2 Hz, 8-H); ¹³C NMR (CDCl₃) (HSQC/HMBC) d 55.06

4.3.18. 3-(4-Hydroxyphenyl)-1-methoxy-5-nitroisoquinoline

(30p). Method A

To 28 (102 mg, 360

lmol) was added Pd₂dba₃(32 mg, 36

lmol),

SPhos (33.5 mg, 70 mol), 4-HOPhB(OH)₂(74 mg, 540

l

mol) and

K₃PO₄(229 mg, 1.1 mmol). Degassed PhMe (3.0 mL) was added

and the mixture was stirred at 100 °C for 16 h. The evaporation

5906

H. A. Paine et al. / Bioorg. Med. Chem. 23 (2015) 5891–5908

residue, in CHCl₃, was ﬁltered (Celite^Ò). Chromatography

(EtOAc/petroleum ether 1:49 ? 1:3) gave 30p (10 mg, 9%) as a

bright red solid: mp >230 °C; ¹H NMR ((CD₃)₂SO) d 4.23 (3H, s,

Me), 6.95 (2H, dd, J = 8.8, 2.0 Hz, Ph 3,5-H₂), 7.72 (1H, t,

J = 8.0 Hz, 7-H), 8.08 (2H, dd, J = 8.8, 2.0 Hz, Ph 2,6-H₂), 8.24 (1H,

s, 4-H), 8.59 (2H, m, 6,8-H₂). 9.87 (1H, br, OH); ¹³C NMR

((CD₃)₂SO) (HSQC/HMBC) d 54.05 (O-Me), 102.51 (4-C), 115.73

(Ph 3,5-C₂), 118.51 (8a-C), 125.13 (7-C), 128.30 (Ph 2,6-C₂),

128.79 (4a-C), 129.11 (6-C), 130.81 (8-C), 144.43 (5-C), 150.95

(3-C/Ph 1-C), 159.06 (Ph 4-C), 159.93 (1-C); MS m/z 297.0861

(M+H)⁺(C₁₆H₁₃N₂O₄requires 297.0877).

added to the cooled mixture and the solution was washed (brine,

3ꢁ). Evaporation and recrystallisation (MeOH) gave 33 (516 mg,

74%) as a yellow solid: mp 188–192 °C; IR m_max1563, 1356 cm^ꢀ1

;

¹H NMR (CDCl₃) (NOESY)

d 4.15 (3H, s, Me), 7.58 (1H, t,

J = 8.1 Hz, 7-H), 8.26 (1H, s, 4-H), 8.46 (1H, dd, J = 7.8, 1.2 Hz, 6-

H), 8.53 (1H, dd, J = 8.2, 0.9 Hz, 8-H); ¹³C NMR (CDCl₃)

(HSQC/HMBC) d 55.06 (Me), 112.64 (4-C), 119.42 (4a-C), 125.11

(7-C), 129.17 (6-C), 131.49 (8-C), 131.71 (8a-C), 137.45 (3-C),

143.75 (5-C), 160.25 (1-C).

4.3.24. 1-Methoxy-5-nitro-3-(2-phenylethynyl)isoquinoline (38)

To 37 (100 mg, 350

(12.6 mg, 18 mol), CuI (6.7 mg, 35

(4.0 mg, 20

mol). Degassed THF (3.0 mL) and Prⁱ₂NH (2.0 mL)

l

mol) in a dry ﬂask was added (Ph₃P)₂PdCl₂

4.3.19. 1-Methoxy-5-nitro-3-(4-hydroxyphenyl)isoquinoline

(30p). Method B

l

lmol) and sodium ascorbate

To 28 (300 mg, 1.26 mmol) was added Pd₂(dba)₃(115 mg,

130 lmol), SPhos (117 mg, 250 lmol), 4-HOPhB(OH)₂(260 mg,

1.9 mmol) and K₃PO₄(800 mg, 3.8 mmol). Dry DMF (7.0 mL) was

added and the mixture was stirred at 135 °C for 16 h. The evapora-

tion residue, in CHCl₃, was ﬁltered (Celite^Ò). Evaporation and chro-

matography (EtOAc/petroleum ether 1:49 ? 2:3) gave 30p

(200 mg, 54%) as a bright red solid.

were added and the mixture was stirred at 50 °C for 30 min, after

which phenylethyne (72 mg, 0.35 mmol) was added. The mixture

was stirred for 16 h at 50 °C. The solvents were evaporated. The

residue, in CHCl₃, was ﬁltered (Celite^Ò). Chromatography

(EtOAc/petroleum ether 1:99 ? 1:19) gave 38 (100 mg, 93%) as a

bright yellow solid: mp 153–154 °C; ¹H NMR (CDCl₃) d 4.22 (3H,

s, Me), 7.38 (3H, m, Ph 3,4,5-H₃), 7.60 (1H, t, J = 8.0 Hz, 7-H), 7.64

(2H, m, Ph 2,6-H₂), 8.32 (1H, s, 4-H), 8.48 (1H, dd, J = 7.8, 1.2 Hz,

6-H), 8.59 (1H, dt, J = 7.1, 1.0 Hz, 8-H); ¹³C NMR (CDCl₃)

(HSQC/HMBC) d 54.63 (Me), 89.26 (ethyne 2-C), 90.14 (ethyne 1-

C), 113.94 (4-C), 120.27 (4a-C), 122.19 (Ph 1-C), 125.51 (7-C),

128.39 (Ph 3,4,5-C₃), 128.82 (6-C), 130.52 (8a-C), 131.30 (8-C),

132.13 (Ph 2,6-C₂), 137.55 (3-C), 144.15 (5-C), 160.76 (1-C); MS

m/z 305.0901 (M+H)⁺(C₁₈H₁₃N₂O₃requires 305.0928).

4.3.20. 3-(4-Hydroxyphenyl)-1-methoxy-5-nitroisoquinoline

(30p). Method C

To 37 (101 mg, 0.36 mmol) in a dry ﬂask was added Pd₂(dba)₃

(32.5 mg, 36 lmol), SPhos (33 mg, 70 lmol), 4-HOPhB(OH)₂

(73.5 mg, 0.53 mmol) and K₃PO₄(226.1 mg, 1.07 mmol). Dry DMF

(3.0 mL) was added and the mixture was stirred at 125 °C for

16 h. The evaporation residue, in CHCl₃, was ﬁltered (Celite^Ò).

Chromatography (EtOAc/petroleum ether 1:39 ? 3:17) gave 30p

(90 mg, 86%) as a bright red solid.

4.3.25. 5-Amino-1-methoxy-3-(2-phenylethyl)isoquinoline (39)

Compound 38 (65 mg, 210 lmol) was stirred vigorously with

Pd/C (10%, 71 mg) in EtOH (5.0 mL) under H₂for 6 h. Filtration

(Celite^Ò) and evaporation gave 39 (51 mg, 87%) as a pale buff oil;

¹H NMR (CDCl₃) d 3.15 (4H, m, CH₂CH₂), 4.15 (Me), 6.88 (1H, dd,

J = 7.5, 1.0 Hz, 6-H), 6.90 (1H, s, 4-H), 7.26 (6H, m, 7-H + Ph-H₅),

7.69 (1H, d, J = 8.5 Hz, 8-H); ¹³C NMR (CDCl₃) (HSQC/HMBC) d

35.65 (ethyl 1-C), 39.92 (ethyl 2-C), 53.47 (Me), 105.65 (4-C),

113.64 (6-C), 114.38 (8-C), 118.79 (4a-C), 125.72 (Ph 4-C), 125.85

(7-C), 128.24 (Ph 2,6-C₂), 128.30 (8a-C), 128.51 (Ph 3,5-C₂),

140.79 (5-C), 142.17 (Ph 1-C), 150.72 (3-C), 160.69 (1-C); MS m/z

279.1560 (M+H)⁺(C₁₇H₁₆N₂O requires 279.1499).

4.3.21. 5-Amino-1-methoxy-3-(2-methoxyphenyl)isoquinoline

(31d)

Compound 30d (40.3 mg, 140 lmol) was stirred vigorously

with Pd/C (10%, 44 mg) in EtOH (9 mL) under H₂for 5 h.

Filtration (Celite^Ò) and evaporation yielded 31d (33.5 mg, 88%) as

a pale yellow solid: mp 119–121 °C; ¹H NMR (CDCl₃) d 3.93 (3H,

s, PhOMe), 4.18 (3H, s, 1-OMe), 6.92 (1H, dd, J = 7.5, 0.8 Hz, 6-H),

7.04 (1H, d, J = 8.2 Hz, Ph 3-H), 7.12 (1H, td, J = 7.4, 1.0 Hz, Ph 5-

H), 7.30 (1H, t, J = 7.8 Hz, 7-H), 7.35 (1H, td, J = 8.3, 1.8 Hz, Ph 4-

H), 7.69 (1H, d, J = 8.2 Hz, 8-H), 7.93 (1H, s, 4-H), 8.14 (1H, dd,

J = 7.7, 1.7 Hz, Ph 6-H); ¹³C NMR (CDCl₃) (HSQC/HMBC) d 53.55

(1-OMe), 55.79 (PhOMe), 109.00 (4-C), 111.67 (Ph 3-C), 113.74

(6-C), 114.28 (8-C), 119.15 (8a-C), 120.95 (Ph 5-C), 126.60 (7-C),

128.15 (4a-C), 128.91 (3-C), 129.04 (Ph 4-C), 131.05 (Ph 6-C),

141.42 (5-C), 144.22 (Ph 1-C), 157.20 (Ph 2-C), 160.29 (1-C); MS

m/z 303.1119 (M+Na)⁺(C₁₇H₁₆N₂NaO₂requires 303.1110).

4.3.26. 2,3,N,N-Tetramethylbenzamide (41)

SOCl₂(3.0 g, 25 mmol) was added to 2,3-dimethylbenzoic acid

40 (1.00 g, 6.7 mmol) at 0 °C. The mixture was heated at reﬂux

for 16 h. The evaporation residue, in CH₂Cl₂(1.0 mL), was added

dropwise to Me₂NH in water (40%, 3.7 mL) at 0–20 °C. The mixture

was stirred at 20 °C for 1 h, diluted with CH₂Cl₂, washed (water,

3ꢁ) and dried. Evaporation gave 41 (990 mg, 84%) as a pale yellow

oil: ¹H NMR (CDCl₃) (COSY/NOESY) d 2.16 (3H, s, 2-Me), 2.27 (3H, s,

3-Me), 2.81 (3H, s, N-Me), 3.12 (3H, s, N-Me⁰), 6.99 (1H, dd, J = 7.2,

1.8 Hz, 4-H), 7.10 (1H, t, J = 7.5 Hz, 5-H), 7.13 (1H, dd, J = 7.6,

1.8 Hz, 6-H); ¹³C NMR (CDCl₃) (HSQC/HMBC) d 16.00 (2-Me),

20.03 (3-Me), 34.48 (N-Me⁰), 38.37 (N-Me), 123.40 (4-C), 125.82

(5-C), 130.01 (6-C), 132.25 (2-C), 137.02 (1-C), 137.42 (3-C),

171.99 (C@O); MS m/z 178.1226 (M+H)⁺(C₁₁H₁₆NO requires

178.1232).

4.3.22. 1,3-Dibromo-5-nitroisoquinoline (36)

Aq HNO₃(67%, 78 mg, 0.87 mmol) in concd H₂SO₄(93 mg,

0.95 mmol) at 0 °C was added dropwise to 35 (251 mg, 0.87 mmol)

in concd H₂SO₄(3.0 mL) at 0 °C. The mixture was stirred at 0 °C for

2H, then at 20 °C for 1H, then poured onto ice. The solid was col-

lected by ﬁltration, washed (H₂O) and dried to yield 36 (285 mg,

98%) as a yellow solid: mp 226–228 °C; IR m_max1516, 1334,

730 cm^ꢀ^{1 1}H NMR ((CD₃)₂SO) d 8.00 (1H, t, J = 7.9 Hz, 7-H), 8.54

;

(1H, s, 4-H), 8.64 (1H, d, J = 8.6 Hz, 6-H), 8.73 (1H, dd, J = 7.8,

0.9 Hz, 8-H); ¹³C NMR ((CD₃)₂SO) (HSQC/HMBC) d 119.57 (4-C),

126.19 (4a-C), 128.93 (7-C), 130.41 (8a-C), 130.64 (8-C), 135.28

(6-C), 135.58 (3-C), 144.09 (1-C), 144.76 (5-C).

4.3.27. 2-Ethyl-N,N,3-trimethylbenzamide (52)

BuLi (1.6 M in hexanes, 1.65 mL, 2.6 mmol) was stirred with dry

Prⁱ₂NH (463 mg, 3.3 mmol) in dry THF (2.0 mL) at ꢀ78 °C for

10 min. Compound 41 (390 mg, 2.2 mmol) in dry THF (3.0 mL)

was added and the mixture was stirred for 1 h at ꢀ78 °C. MeI

(343 mg, 2.4 mmol) in dry THF (2.0 mL) was added and the mixture

was stirred for 1 h at ꢀ78 °C, then at 20 °C for 16 h. Water (1.0 mL)

was added. The mixture was diluted with CH₂Cl₂. This mixture was

4.3.23. 3-Bromo-1-methoxy-5-nitroisoquinoline (37)

Na (403 mg, 17.5 mmol) was dissolved in dry MeOH (10 mL);

this solution was added to 36 (969 mg, 2.9 mmol) in dry MeOH

(10 mL). The mixture was boiled under reﬂux for 24 h. EtOAc was

H. A. Paine et al. / Bioorg. Med. Chem. 23 (2015) 5891–5908

5907

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(EtOAc/petroleum ether 1:9 ? 1:1) gave 52 (100 mg, 24%) as a

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J = 7.6 Hz, CH₂Me), 2.34 (3H, s, 3-Me), 2.60 (2H, m, CH₂), 2.81

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Acknowledgements

We are very grateful to the Association for International Cancer

Research (now Worldwide Cancer Research) for generous funding

of this project (10-0104). Protein crystallography work was funded

by Biocenter Oulu and Academy of Finland (Grant No. 251314 for

L.L. and Grant No. 266922 for T.H.). We also thank Dr. Timothy J.

Woodman (University of Bath) for help with the NMR spectra

and Ciara O’Mahony (School of Pharmacy

& Pharmaceutical

Sciences, Trinity College, Dublin, Ireland) for improving the synthe-

ses of key intermediates 30, 31, 34. Protein crystallography exper-

iments were performed on the ID14-1, ID23-1 and ID23-2

beamlines at the European Synchrotron Radiation Facility (ESRF),

Grenoble, France and at the Diamond Light Source (Didcot, UK)

beamlines I038 and I04-1. We are grateful to local contacts at

ESRF and at Diamond for providing assistance in using beamlines.

The research leading to these results has received funding from the

European Community’s Seventh Framework Programme

(FP7/2007-2013) under BioStruct-X (Grant agreement N° 283570).

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Exploration of the nicotinamide-binding site of the tankyrases, identifying 3-arylisoquinolin-1-ones as potent and selective inhibitors in vitro

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