Synthesis and cytotoxicity of thieno[2,3-b]pyridine and furo[2,3-b]pyridine derivatives

European Journal of Medicinal Chemistry 86 (2014) 420e437

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European Journal of Medicinal Chemistry

journal homepage: h ttp://www.elsevier.com/locate/ejmech

Original article

Synthesis and cytotoxicity of thieno[2,3-b]pyridine and furo[2,3-b]

pyridine derivatives

a

b

a

ꢀ

Joy M. Hung , Homayon J. Arabshahi , Euphemia Leung , Johannes Reynisson ,

David Barker ^a

,

*

^aSchool of Chemical Sciences, The University of Auckland, Auckland, New Zealand

^bAuckland Cancer Society Research Centre, The University of Auckland, New Zealand

a r t i c l e i n f o

a b s t r a c t

Article history:

Forty seven thieno[2,3-b]pyridines-2-carboxamides, furo[2,3-b]pyridines-2-carboxamides and tetrahy-

drothieno[2,3-b]quinolones-2-carboxamides derivatives were synthesized and tested for their anti-

proliferative activity against the NCI-60 cell lines. The 5-keto-tetrahydrothieno[2,3-b]quinolones-2-

carboxamides (series 17) were found to have the greatest activity, with the compound containing a 3-

methoxyphenylcarboxamide (compound 17d) being the most active, with GI₅₀values in the low nano-

Received 24 March 2014

Received in revised form

27 July 2014

Accepted 1 September 2014

Available online 3 September 2014

molar range against a range of cell lines, in particular the melanoma cell line MDA-MD-435 (GI₅₀

e

23 nM) and the breast cancer cell line MDA-MB-468 (GI₅₀e 46 nM). Molecular modelling of series 17

against phosphoinositide speciﬁc-phospholipase C reveals that the side chains of the amino acids His356,

Glu341, Arg549 and Lys438 are involved in hydrogen bonding with the ligands as well as a lipophilic

pocket is occupied by the phenyl carboxamide moiety.

Keywords:

Thieno[2,3-b]pyridines

Furo[2,3-b]pyridines

Phosphoinositide speciﬁc-phospholipase C

Molecular modelling

1. Introduction

tricyclic analogues containing the motifs found in these compounds

and compare their antiproliferative activity to previously examined

Breast cancer is the most common cancer in women even

though early detection and the introduction of new therapies have

resulted in decline in mortality [1e4]. Hormone-dependent breast

cancer is commonly treated with tamoxifen, whilst trastuzumab is

effective against human epidermal growth factor receptor 2 (HER2)

positive cancers [5]. Tumours lacking the oestrogen receptor (ER),

the progesterone receptor (PR) and HER2 are classiﬁed as triple-

negative breast cancer [6,7], with the MDA-MB-231/468 cell lines

belonging to this type of cancer [8].

compounds in the aim of ﬁnding more potent agents.

Previous molecular modelling studies [10e12] indicated that

the aryl carboxamide and the 3-amino moiety of compounds 1 and

2 have hydrogen bonding interactions within the PLC active site

(Fig. 2). Furthermore, the phenyl ring is embedded in a lipophilic

pocket. Modelling has also indicated the keto group of 1 and sub-

stituents at the 8-position of 3 also positively contribute to binding.

To investigate each of these binding interactions three series of

compounds were prepared. In the ﬁrst compound series, the thieno

and furopyridine-2-carboxamides, different substituted phenyl

rings were designed to investigate the lipophilic contacts of the

binding pocket and also the effect of substitution at C-5 (Fig. 3). In a

second compound series, the pyrimidinone and triazinone ana-

logues, the 3-amino functional group is incorporated into a ring

system, thus eliminating the hydrogen donor ability to the amino

acids GLU341 (Fig. 2), shedding light on the importance of this

putative intermolecular interaction. Thirdly, various N-aryl-tetra-

hydrothieno- and furo[2,3-b]quinoline-2-carboxamides, a com-

pound series which has antiproliferative activity [10e12], would

further investigate the optimal substitution of the phenyl ring and

the alteration of the keto group. Comparison of the activity of 5-

substituted-thienopyridine-2-carboxamides with N-aryl-5-oxo-

Recently, Reynisson et al. [9] identiﬁed a series of potential

phosphoinositide speciﬁc-phospholipase C-

g (PLC-g) inhibitors

using a virtual high throughput screening (vHTS) method and

subsequently assessed them for their antiproliferative activity [10].

Some of the most potent compounds contained a thieno[2,3-b]

pyridine-3-amine scaffold, e.g. 1 and 2, or 3H-pyrimido[5,4-b]

indol-4(5H)-one scaffold, e.g. 3 (Fig. 1). Further analysis of these

classes of compounds revealed their activity against a range of

cancer cell lines including triple-negative breast cancer cell lines

[11,12]. We therefore wished to prepare a series of bicyclic and

* Corresponding author.

E-mail address: d.barker@auckland.ac.nz (D. Barker).

tetrahydrothieno[2,3-b]quinoline-2-carboxamides

(or

furo

http://dx.doi.org/10.1016/j.ejmech.2014.09.001

J.M. Hung et al. / European Journal of Medicinal Chemistry 86 (2014) 420e437

421

in 60% and 90% yields respectively. 5-Halo-substituted thieno[2,3-

b]pyridines 4dei were prepared using the same conditions as for

4aec, reaction of 2,5-dichloronicotinonitrile 7b or 5-bromo-2-

chloro-3-cyano-pyridine 7c with mercaptoacetamides 6aec gave

5-halo-thieno[2,3-b]pyridines 4dei in 40e99% yields.

Derivatization of thieno[2,3-b]pyridines 4aei into thienopyr-

imidinones 8aei was achieved by heating thienopyridine-2-

carboxamides 4aei in excess triethyl orthoformate (Scheme 1).

The addition of acetic acid was found to be essential for successful

conversion [15] and gave thienopyrimidinones 8aei in 41e99%

yields.

Another series of analogues, the thienotriazinones 9aei, were

prepared by diazotization of 4aei using sodium nitrite [15]. These

reactions gave the desired thienotriazinones 9aei in 50e99% yields

(Scheme 1). In total 27 analogues with the thieno[2,3-b]pyridine

core structure were easily prepared with diversity in the aryl car-

boxamide, at the 5-position and in the chemical nature of the 3-

amino position.

Fig. 1. Previously discovered inhibitors of PLC-g.

2.1.2. Furopyridine-2-carboxamide analogues

Following successful synthesis of the thienopyridine series, it

was attempted to prepare furopyridine-2-carboxamides 10aei us-

ing the same method (Scheme 2). 2-Hydroxyacetamides 11aec

were prepared from glycolic acid 12 in the same manner as thio-

acetamides 6, giving amides 11aec in 56e99% yields. Reaction of

11aec with chloropyridines 7aec, using sodium carbonate in

ethanol, however did not give furopyridine-2-carboxamides 10aei

and gave only cyanopyridines 13aei in 19e92% yields, with the

polyhalogenated derivatives 13f and 13i being obtained in the

lowest yields. Using cyanopyridine 13a, a variety of bases were

explored to facilitate the furan ring formation, and whilst NaOH,

NaOMe and Et₃N were all unsuccessful, giving mixtures of amide-

hydrolysed or polymeric material, the use of hindered base KO^tBu

successfully gave the desired furopyridine-2-carboxamide 10a in

quantitative yield. Similarly, reaction of 13b and 13c gave 10b and

10c, though in reduced yields of 75% and 57%, respectively. Reaction

of 13d, which has a non-substituted phenyl amide, gave furopyr-

idine 10d in 74% yield but reaction of all other halogenated

Fig. 2. Predicted interactions of thienopyridine-2-carboxamides in the PLC binding

pocket.

derivatives), to investigate the functional group requirement at this

position, whilst modiﬁcations of the heterocyclic core along with

the addition of halogen substituents, has the potential to further

reﬁne the requirements for strong inhibitory activity.

2. Results and discussion

2.1. Chemistry

2.1.1. Thienopyridine-2-carboxamide analogues

Thieno[2,3-b]pyridines 4aei were prepared in two steps from

thioglycolic acid 5 (Scheme 1). The synthesis began by preparing

the mercaptoacetamides 6aec, with different substituted phenyl

moieties, by heating the corresponding anilines and thioglycolic

acid

5

without solvent. Whilst thieno[2,3-b]pyridine-2-

carboxamides such as 4 are often prepared through initial forma-

tion of 2-(substituted-thio)pyridines [13,14], it was found that re-

action of 2-mercapto-N-phenylacetamide 6a with 2-chloro-3-

pyridine carbonitrile 7a, using sodium carbonate, in reﬂuxing

ethanol, gave the desired thieno[2,3-b]pyridine 4a in 73% yield in a

single step. Analogues 4b and 4c with 4-methoxy or 3-

chlorophenyl moieties were prepared similarly from pyridine 7a

Fig. 3. Proposed heterocyclic inhibitors of the PLC enzyme.

422

J.M. Hung et al. / European Journal of Medicinal Chemistry 86 (2014) 420e437

Scheme 1. Reagents and conditions: (i) 1 equiv. substituted aniline, 130 ^ꢀC, 3e5 h, 6aec 48e99%; (ii) 1 equiv. 7aec, 1.05 equiv. 6aec, Na₂CO₃, EtOH, reﬂux, 18 h, 4aei 40e99%; (iii)

10 equiv. (EtO)₃CH, CH₃CO₂H, 1e15 h, 8aei 41e99%; (iv) 20 equiv. NaNO₂, CH₃CO₂H, H₂O, 0 ^ꢀC, 2 h, 9aei 50e99%.

Scheme 2. Reagents and conditions: (i) 1 equiv. substituted aniline, 130 ^ꢀC, 3e5 h, 11aec 56e99%; (ii) 1 equiv. 7aec, 1.05 equiv. 6aec, Na₂CO₃, EtOH, reﬂux, 18 h, 13aei 19e92%; (iii)

1.2 equiv. KO^tBu, THF, 80 ^ꢀC, 3 h, 10aed 57e99%; (iv) 10 equiv. (EtO)₃CH, CH₃CO₂H, 1e15 h, 8aei 41e99%; (v) Na₂CO₃, EtOH, reﬂux, 15 h, 14a 12%, 14b 20%.

pyridines 13eei, under the same conditions, led to mixtures of

products, even with very short reaction times. Use of the previously

attempted bases on 13eei also led to complex mixtures of products,

indicating the reduced stability of the polyhalogenated derivatives

of both 10 and 13. With similar biological results obtained between

our initial set of furanopyridines 10 and thienopyridines 4 (see

below) it was decided to abandon further optimisation of the

synthesis of analogues 10.

15aec in 53e77% yields, whilst the reaction of 10d gave only a

complex mixture of products with no 14d or 15d isolated. Base-

induced cyclisation of formimidates 15aec, using Na₂CO₃in

ethanol, gave 14a and 14b, albeit in a low yields of 12% and 20%,

respectively, whilst the halogenated derivative 14c was found to be

unstable.

Finally, using furopyridines 10aec it was attempted to prepare a

series of furotriazinones 16, in a similar manner to the synthesis of

An attempt was made to prepare furopyrimidinones 14aed

using the same procedure as used for the synthesis of 8aei. How-

ever, reaction of furopyridines 10aec in this case gave formimidates

thienotriazinones 9aei. Unfortunately, despite a number of

different reaction times and reagent concentrations being

attempted, none of the desired products were obtained.

J.M. Hung et al. / European Journal of Medicinal Chemistry 86 (2014) 420e437

423

2.1.3. Tetrahydrothieno[2,3-b]quinoline-2-carboxamide analogues

2.2. Biology

After determining the lower stability of oxygen-containing

heterocycles 10, 14 and 16 and coupled with the biological data

showing no signiﬁcant difference in antiproliferative activity be-

tween the already prepared oxygen and sulphur heterocycles (see

below), it was decided to focus on the synthesis of N-aryl-tetra-

hydrothieno[2,3-b]quinoline-2-carboxamides rather than their

furan equivalents. Whilst some derivatives of 17 are commercially

available [10] there is almost no synthetic data reported for this

class of compound [13]. The synthesis of these analogues 17aei

began with the preparation of thiohexahydroquinoline 18 using our

modiﬁcation [11] of a literature method (Scheme 3) [16]. Firstly we

Heterocyclic compounds are frequently shown to inhibit the

growth of cancerous cell lines [21e24] with inhibition of selected

cell lines most often reported. A total of 47 analogues were selected

and tested against the National Cancer Institute's human tumour

cell lines (NCI-60), where the growth inhibition of compounds is

compared to untreated cells at 100% [25]. The NCI-60 cancer cell

panel includes leukaemia's, melanomas, and cancers of renal,

ovarian, prostate, colon, lung, central nervous system and breast

origin [26]. Analysis of the NCI-60 results of 17aec, previously

published by Feng et al. [10], and 5-oxo-tetrahydrothieno[2,3-b]

quinoline-2-carboxamides 17dei showed that unsubstituted

analogue 17a and 3-substituted phenyl derivatives 17c, 17d and 17e

showed the greatest activity. The 4-methoxyphenyl derivative 17b

had the lowest inhibition levels, which is in line with previous

ﬁndings that the para-position is not favoured [12]. Analogues with

strong electron-withdrawing groups at the 3-position (17f, 17g) or

3,5-disubstitution (17h, 17i) had reduced activity or were inactive.

The NCI results of the similar alcohol analogues 23aei were similar

to those for the corresponding keto derivative. Again the 3-

substituted phenyl derivatives (23c, 23d, 23e) were the most

active, however in this series the analogues 23f and 23g were more

active than their keto counterparts. The similarity between the

biological results for the keto 17 and alcohol 23 series suggest that

they are inhibiting the same enzyme or a class of highly related

enzymes (Table 1).

performed

a multi-component reaction [17e20] where 1,3-

cyclohexanedione 19 was reacted with dimethylformamide

dimethyl acetal (DMFDMA) [15] to give enamine 20, which was

then immediately reacted with cyanothioacetamide giving thio-

hexahydroquinoline 18 in 82% yield. Bromoacetamides 21aei were

prepared in 51e99% yields from the acylation, at 0 ^ꢀC, of various

substituted anilines 22aei with bromoacetyl bromide. It was found

that reaction at higher temperatures led to further amination at the

a

-bromide [21]. Similar to the synthesis of thieno[2,3-b]pyridine-2-

carboxamides 4aei, we found that reaction of thiohexahy-

droquinoline 18 with sodium carbonate and bromoacetamide

21aei, gave the desired 5-oxo-thieno[2,3-b]pyridines 17aei in

14e99% yields in a single step. Finally reduction of the keto group in

17aei using sodium borohydride, in a mixture of methanol and

THF, gave 5-hydroxy-thieno[2,3-b]pyridines 23aei in 66e99%

yields.

Compounds 17a, 17c, 17d, 17e, 23a, 23c, 23d, 23e, 23f and 23g

were selected for dose response testing based on their bioactivity

and the results are given in Table 2. The Growth Inhibition at 50%

(GI₅₀) was derived from these measurements [27]. Six tumour cell

lines were particularly affected, i.e., MDA-MB-435 (melanoma),

MDA-MB-468 (breast), HS 578T (breast), NCI-H522 (non-small cell

lung cancer), OVCARe3 (ovarian) and RXF 393 (renal). Many other

cell lines also showed good response, e.g., COLO 205 (colon),

SNBe75 (CNS), BT 549 (breast) and MDA-MB-231 (breast). All the

biological data is given in the Supplementary Information section.

It can be seen in Table 2 that meta methoxy substitution on the

phenyl ring causes the most growth inhibition for both the keto 17

and alcohol series 23. These derivatives 17/23d show either the best

or one of the best GI₅₀values for the cell lines shown. When the

averaged GI₅₀values for the meta methoxy derivatives (17/23d) are

correlated an R²of 0.969 is obtained, which is a strong indication

that they are inhibiting the same enzyme. The other series are less

correlated (17/23a e 0.497, 17/23c e 0.325 and 17/23e e 0.534). The

keto methoxy compound 17d is a better inhibitor than its alcohol

23d counterpart for the entire cell lines considered in Table 2.

Indeed, when the other substitution patterns are considered the

trend is that the ketone derivatives are usually more active.

We then compared the activity of the tetrahydrothieno[2,3-b]

quinoline-2-carboxamides 17 and 23 with the synthetic com-

pounds not containing a cyclohexane ring. Compounds 4aed and

10aed were relatively inactive, showing that a furan ring in the

core structure did not provide any advantage over compounds with

more easily synthesizable thiophenes (Table 3). Similar to com-

pounds 17 and 23, analogues with a 4-methoxyphenyl carbox-

amide moiety had the lowest inhibition means with an average of

96.8%, whereas analogues with 3-chlorophenyl carboxamide had

improved activities with an average of 84.9%. Also in the more

active 3-chlorophenyl carboxamide analogues, activity improved as

the size of the substituent at the 5-position increased from H (e.g.

8c,104.1%) to Cl (e.g. 8f, 77.6%) to Br (e.g. 8i, 69.3%) but did not reach

the level of the corresponding 3-chlorophenyl derivatives 17c

(36.3%) and 23c (27.1%) showing that derivatives containing a

cyclohexanone or cyclohexanol ring are much more favourable

Scheme 3. Reagents and conditions: (i) DMFDMA, DMF, 24 h; (ii) cyanothioacetamide,

NaH, DMF, 24 h 82% (2 steps); (iii) 1 equiv. 22aei, 1.1 equiv. bromoacetyl bromide, Et₃N,

CH₂Cl₂, 1 h, 51e99%; (iv) Na₂CO₃, EtOH, reﬂux, 18 h, 14e99%; (v) 1 equiv. NaBH₄,

MeOH/THF, 2 h, 66e99%.

424

J.M. Hung et al. / European Journal of Medicinal Chemistry 86 (2014) 420e437

Table 1

alcohols 23. Interestingly, ChemPLP is reported to give the best

results of the scoring functions available in GOLD [28].

The NCI Mean percentages (%) of growth at 10

(100% growth) for compound series 17 and 23.

mM as compared to untreated cells

Similar to the biological activities there were no signiﬁcant

differences found in the binding scores between compounds 4 and

8, with a thiophene ring, and compounds 10 and 14 with a furan

ring. Furthermore, this series of compounds had lower binding

scores than the more biologically active tetrahydrothieno[2,3-b]

quinolines 17 and 23 (as shown in Tables S1 and S2 in the

Supplementary Information). Pyrimidinones, 8 and 14, and tri-

azinone 9 series showed the molecules had rotated by 180^ꢀin the

binding pocket and showed hydrogen bonding between N-1 and

HIS311 rather than interactions with GLU341, and no interactions

between the amide carbonyl with either HIS311 or HIS356. These

results suggested that the important interaction between GLU341

and the 3-amino group in 4 and 10 is blocked by the formation of

the pyrimidinone (in 8 and 14) and triazinone (in 9) moieties

further explaining the lower bioactivity of these compounds.

When the conﬁgurations of all analogues were scored and

ranked it was noticed that analogues with a 3-substituent on the

aryl carboxamide generally have the highest scores, whereas para-

methoxy phenyl derivatives of 4/8/9/10/14/17b/23b usually had the

lowest scores, which correlated well with the obtained biological

results. The scoring data is given in the Supplementary Information

section.

Thienopyridines and furopyridines with 5-chloro (4d/e/f and

10d) or 5-bromo (4g/h/i) substitution generally have higher, but not

signiﬁcantly higher, scores versus analogues with no substituent

(4a/b/c and 10a/b/c). The binding scores of these compounds when

compared with the corresponding tetrahydrothieno[2,3-b]quino-

lines 17 which have the equivalent of a ketone at the 5-position,

were signiﬁcantly lower. The ketone carbonyl group is predicted to

interact with both ARG549 and LYS438, whilst the halogens at the

5-position have limited contact with these amino acids.

Compound

R¹

R²

NCI-60 mean (%)

17a^a

17b^a

17c^a

17d

17e

17f

17g

17h

17i

23a

23b

23c

23d

23e

23f

23g

23h

23i

H

O

30.2

103.0

36.3

24.9

31.6

66.0

88.3

84.9

94.8

30.0

95.5

27.1

24.0

27.6

50.3

32.8

81.6

96.3

4-OMe

3-Cl

3-OMe

3-Br

3-NO₂

3-CF₃

3,5-di-OMe

3,5-di-Cl

H

4-OMe

3-Cl

3-OMe

3-Br

3-NO₂

3-CF₃

3,5-di-OMe

3,5-di-Cl

O

H/OH

a

Data from Ref. [10].

than the tested halide derivatives. The antiproliferation testing of

the triazinones 9 and pyrimidinones 8 and 14 showed similar re-

sults as the thieno 4 and furopyridine-2-carboxamides 10, although

none of the triazinone and pyrimidinone analogues had directly

comparable inhibition activities, showing that functionalization of

the 3-amino group was detrimental to the bioactivity.

3. Conclusions

A number of thieno[2,3-b]pyridine 4/8/9, furo[2,3-b]pyridines

10/14/16 and tetrahydrothieno[2,3-b]quinolines 17/23 were syn-

thesised and tested for their antiproliferative activity against a

range of cancer cell lines. Tetrahydrothieno[2,3-b]quinolines 17/23

with a non-electron withdrawing substituent at the 3-position

were found to be the most active with 17d having the greatest

activity in NCI testing. Thieno[2,3-b]pyridine-2-carboxamides 4

and furo[2,3-b]pyridines-2-carboxamides 10 and their derivatives

8/9/14/16 were found to were found to have diminished bio-

activities in comparison with analogues 17/23. In both of these

series compounds with substitution at the 5-position were more

active than those without substitution, and in general the larger the

substituent the greater the activity. The biological results correlate

well with the predicted binding of the ligands in the PLC active site,

however further experimental work is required to conﬁrm these

observations, as it is a challenging endeavour to demonstrate

mechanistic action of small molecule inhibitors in complex bio-

logical system.

2.3. Molecular modelling study

Protein-ligand docking was undertaken on all 47 synthetic an-

alogues with the GOLD software using four scoring functions (see

experimental methods 4.8 for details). As the PLC-

g crystal struc-

ture is not available, the known PLC- isoform was used as a model,

d

since the highest amino acid similarity between the isoforms occur

in their substrate binding site [9]. There are predicted hydrogen

bonding interactions between the 3-amino moiety, with GLU341,

and amide carbonyl, with HIS356 and the HIS311 in all of the

thienopyridine- 4, furopyridine- 10 and tetrahydrothieno[2,3-b]

quinoline-2-carboxamide 17 and 23 series. Also predicted is the

insertion of the phenyl ring in a lipophilic pocket. Tetrahydrothieno

[2,3-b]quinolines 17, with a ketone carbonyl group at the 5-

position, are predicted to further interact with both ARG549 and

LYS438. The same is found in the S-enantiomers, but not the R-

enantiomers, of the alcohol series 23. It can be hypothesised that

the S-enantiomers are responsible for the bioactivity of the 23 se-

ries, due to superior hydrogen bonding, since a racemic mixture

was used in the experiments (Fig. 4). These ﬁndings are in line with

previous modelling studies on compounds from series 17 [9e12].

Compound 17b has consistently the worst score for the scoring

functions used, except for Gold Score, which correlates with the

biological results. Furthermore, the NCI mean and the predicted

scores generated by ChemPLP for the 17 series have a reasonable

linear correlation (R²e 0.443). This is also the trend observed for

4. Experimental section

4.1. General experimental

All reactions were carried out under a nitrogen atmosphere in

dry, freshly distilled solvents unless otherwise noted. All optical

rotation measurements were determined at 20 ^ꢀC on the sodium

D line (

l

¼ 589 nm, 0.1 dm cell). All NMR spectra were recorded

on either Bruker Avance DRX 300 MHz or 400 MHz spectrome-

ters at ambient temperatures. Chemical shifts are reported

J.M. Hung et al. / European Journal of Medicinal Chemistry 86 (2014) 420e437

425

Table 2

The GI₅₀(50% growth inhibition) in nanomolar (nM) are shown for six tumour cell lines: MDA-MB-435 (melanoma), MDA-MB-468 and HS 578T (breast), NCI-H522 (non-small

cell lung cancer), OVCAR-3 (ovarian) and RXF 393 (renal). All derivatives were tested twice and both values are given.

Compound

MDA-MB-435

MDA-MB-468

HS 578T

333/825

452/342

342/73

630/566

X/362

831/3150

199/531

395/X

NCI-H522

OVCAR-3

RXF 393

17a^a

17c^a

17d

17e

23a

23c

23d

23e

23f

88/137

45/71

22/23

54/157

66/50

120/123

25/23

53/115

416/391

306/338

173/150

45/171

44/47

247/272

186/290

267/247

55/142

186/303

2030/2200

1790/1600

150/309

190/285

44/57

176/350

196/401

237/793

73/149

369/460

4610/8090

2860/56700

243/287

305/245

58/64

316/408

274/344

298/325

68/164

301/340

3440/5950

3720/4890

192/189

251/913

55/127

265/522

211/349

304/515

133/278

233/558

4140/11100

3150/10100

16100/7500

9040/4300

23g

a

Data from Ref. [10]; X: data not available.

relative to the solvent peak of chloroform (

d

7.26 for ¹H and

d

77.0

4.2. Synthesis of 2-mercaptoacetamides 6 and 2-

hydroxyacetamides 11

for ¹³C) or DMSO ( 2.50 for ¹H and 39.5 for ¹³C). ¹H NMR data is

d d

reported as position (d), relative integral, multiplicity (s, singlet;

d, doublet; t, triplet; q, quartet; m, multiplet; br, broad peak; qd,

4.2.1. 2-Mercapto-N-phenylacetamide 6a

quartet of doublets), coupling constant (J, Hz), and the assign-

Aniline (2.0 g, 21.7 mmol) was added to a solution of thioglycolic

acid 5 (2.0 g, 21.7 mmol) in toluene (10.8 mL) and the mixture

heated at reﬂux, under an atmosphere of nitrogen, using a Dean-

eStark apparatus. After 5 h the resulting mixture was cooled to

room temperature and hexane (11 mL) added. The mixture was

cooled on ice, and the resultant precipitate was ﬁltered to give the

title product 6a (1.75 g, 48%) as a yellow solid. m.p. 122e125 ^ꢀC. lit.

m.p. 112 ^ꢀC [29]. ¹H NMR (400 MHz; CDCl₃) 2.03 (1H, t, J ¼ 9.1 Hz,

ment of the atom. ¹³C NMR data are reported as position (

d) and

assignment of the atom. All NMR assignments were performed

using HSQC and HMBC experiments. High-resolution mass

spectroscopy (HRMS) was carried out by either chemical ioniza-

tion (CI) or electrospray ionization (ESI) on a MicroTOF-Q mass

spectrometer. Unless noted, chemical reagents were used as

purchased.

Table 3

The NCI Mean percentages (%) of growth at 10 mM as compared to untreated cells (100% growth) for compounds 4, 8, 9, 10 and 14.

Compound

X

R¹

R²

NCI-60 mean (%)

4a

4b

4c

4d

4e

4f

4g

4h

4i

8b

8c

8e

8f

8g

8h

8i

9a

9b

9c

9e

9f

H

Cl

Br

H

S

O

97.5

96.4

92.8

91.8

95.1

73.4

91.5

96.8

70.7

105.3

104.1

83.8

77.6

84.3

99.6

69.3

102.9

106.5

98.2

97.9

72.9

96.3

97.0

94.4

96.9

98.4

96.1

95.3

87.7

4-OMe

3-Cl

H

4-OMe

3-Cl

H

4-OMe

3-Cl

4-OMe

3-Cl

4-OMe

3-Cl

H

4-OMe

3-Cl

H

4-OMe

3-Cl

4-OMe

3-Cl

H

4-OMe

3-Cl

H

Cl

Br

H

Cl

Br

H

Cl

H

9g

9h

9i

10a

10b

10c

10d

14b

4-OMe

3-Cl

H

4-OMe

426

J.M. Hung et al. / European Journal of Medicinal Chemistry 86 (2014) 420e437

97%) was obtained as a white solid. m.p. 85e86 ^ꢀC. lit. m.p.75 ^ꢀC

[30]. ¹H NMR (400 MHz; CDCl₃) 2.04 (1H, t, J ¼ 9.2 Hz, SH), 3.39 (2H,

d, J ¼ 9.2 Hz, CH₂), 7.12 (1H, d, J ¼ 8.0 Hz, H-4⁰), 7.23e7.28 (1H, m, H-

5⁰), 7.40 (1H, d, J ¼ 8.0 Hz, H-6⁰), 7.66 (1H, s, H-2⁰), 8.57 (1H, br s,

NH). The ¹H and data was in agreement with the literature values

[29].

4.2.4. 2-Hydroxy-N-phenylacetamide 11a

A mixture of aniline (0.36 mL, 3.94 mmol) and glycolic acid 12

(0.30 g, 3.94 mmol) were stirred at 130 ^ꢀC for 5 h. The mixture was

then cooled to room temperature and left to stand for 1 h. Diethyl

ether (20 mL) was added and the resultant solid was ﬁltered to give

the title product 11a (0.31 g, 56%) as a white solid. m.p. 90e92 ^ꢀC. lit.

m.p. 92e94 ^ꢀC [31]. ¹H NMR (400 MHz; d₄-MeOH) 4.13 (2H, s, CH₂),

7.11e7.15 (1H, m, AreH), 7.33 (2H, d, J ¼ 8.0 Hz, AreH), 7.61 (2H, d,

J ¼ 8.0 Hz, AreH). The ¹H NMR data was in agreement with the

literature values [31].

4.2.5. 2-Hydroxy-N-(4⁰-methoxyphenyl)acetamide 11b

A mixture of glycolic acid 12 (0.25 g, 3.25 mmol) and p-anisidine

(0.40 g, 3.25 mmol) were stirred at 130 ^ꢀC for 3 h. The mixture was

then cooled to room temperature and left to stand for 1 h. The

resultant solid was ﬁltered to give the title product 11b (0.45 g, 76%)

as a dark purple solid. m.p. 87e89 ^ꢀC. lit. m.p. 95e96 ^ꢀC [32]. ¹H

NMR (400 MHz; CDCl₃) 3.77 (3H, s, OCH₃), 4.16 (2H, s, CH₂), 6.85

(2H, d, J ¼ 9.0 Hz, H-3⁰and H-5⁰), 7.42 (2H, d, J ¼ 9.0 Hz, H-2⁰and H-

6⁰), 8.38 (1H, s, NH). The ¹H NMR data was in agreement with the

literature values [33].

4.2.6. N-(3⁰-Chlorophenyl)-2-hydroxyacetamide 11c

Using the same procedure as for amide 11b, glycolic acid 12

(0.20 g, 2.63 mmol) and 3-chloroaniline (0.28 mL g, 2.63 mmol)

stirring at 130 ^ꢀC for 5 h. After workup the title product 11c (0.49 g,

99%) was obtained as a light orange solid. m.p. 92e95 ^ꢀC. ¹H NMR

(400 MHz; d₆-acteone) 3.65 (1H, s, OH), 4.14 (2H, s, CH₂), 7.12 (1H, d,

J ¼ 8.0 Hz, H-4⁰), 7.31e7.35 (1H, m, H-5⁰), 7.64 (1H, d, J ¼ 8.0 Hz, H-

6⁰), 7.99 (1H, s, H-2⁰), 9.34 (1H, br s, NH). ¹H NMR (100 MHz; d₆-

acteone) 62.9 (CH₂), 117.0 (C-6⁰), 120.2 (C-2⁰), 124.3 (C-4⁰), 131.0 (C-

Fig. 4. The docked conﬁguration of S-23d in the binding site of PLC-d1 using ChemPLP.

(A) The protein surface is rendered and ligand S-23d is shown. The phenyl group of S-

23d occupies a lipophilic cavity to the left hand side and the methoxy substituent

occupies a pocket. Red depicts a positive partial charge on the surface, blue depicts

negative partial charge and grey shows neutral/lipophilic areas. (B) Hydrogen bonds

are depicted as green lines between ligand S-23d and the amino acids HIS356, GLU431

ARG549 and LYS438. (For interpretation of the references to colour in this ﬁgure

legend, the reader is referred to the web version of this article.)

5⁰), 134.7 (C-3⁰), 140.8 (C-1⁰), 171.8 (C]O). IR: y_max(ﬁlm)/cm^ꢂ1

:

3374, 3280, 3188, 3068, 2924, 1650, 1594, 1551, 1070, 968. m/z

(ESIþ): 210 (³⁷Cl MNa^þ, 31%), 208 (³⁵Cl MNa^þ, 100); HRMS (ESI^þ)

found (³⁷Cl MNa^þ): 210.0106, C₈H₈NO³₂⁷ClNa requires 210.0107.

Found (³⁵Cl MNa^þ): 208.0136, C₈H₈NO³₂⁵ClNa requires 208.0136.

SH), 3.41 (2H, d, J ¼ 9.1 Hz, CH₂), 7.13e7.18 (1H, m, AreH), 7.35e7.37

(2H, m, AreH), 7.54e7.61 (2H, m, AreH), 8.58 (1H, s, NH). The ¹H

NMR data was in agreement with the literature values [29].

4.3. Synthesis of thienopyridine-2-carboxamides 4 and

furopyridine-2-carboxamides 10

4.2.2. 2-Mercapto-N-(4⁰-methoxyphenyl)acetamide 6b

A mixture of thioglycolic acid 5 (0.11 mL, 1.62 mmol) and p-

anisidine (0.20 g, 1.62 mmol) were stirred for 5 h, at 130 ^ꢀC, under

an atmosphere of nitrogen. The mixture was then cooled to room

temperature and diluted with DCM (15 mL), washed with 2 M HCl

(2 ꢁ 15 mL), H₂O (15 mL), and brine (15 mL), dried (Na₂SO₄) and the

solvent was removed in vacuo to give the title product 6b (0.32 g,

99%) as a light purple solid. m.p. 116e118 ^ꢀC. lit. m.p.118 ^ꢀC [30]. ¹H

NMR (400 MHz; CDCl₃) 2.01 (1H, t, J ¼ 9.3 Hz, SH), 3.39 (2H, d,

J ¼ 9.3 Hz, CH₂), 3.80 (3H, s, OCH₃), 6.88 (2H, d, J ¼ 9.0 Hz, H-3⁰and

H-5⁰), 7.45 (2H, d, J ¼ 9.0 Hz, H-2⁰and H-6⁰), 8.40 (1H, s, NH). ¹³C

NMR (400 MHz; CDCl₃) (100 MHz; CDCl₃) 29.0 (C-2), 55.5 (OCH₃),

114.2 (C-3⁰and C-5⁰), 121.8 (C-2⁰and C-6⁰), 130.4 (C-1⁰), 156.8 (C-1⁰),

167.0 (C]O).

4.3.1. General procedure A: synthesis of the thienopyridine-2-

carboxamides 4 and cyanopyridines 13 from pyridines 7

A

mixture of 2-mercaptoacetamides 6aec or 2-

hydroxyacetamides 11aec (1.05 mmol), pyridine 7aec (1 mmol)

and anhydrous sodium carbonate (1.05 mmol) in absolute ethanol

(1 mL) was stirred at reﬂux for 15 h. The mixture was then cooled to

room temperature and the solvent removed in vacuo. The resultant

crude solid was washed with small amounts of water and the

remaining solid was recrystallized, using methanol to give the

thienopyridine-2-carboxamide 4 or cyanopyridine 13.

4.3.1.1. 3-Amino-N-phenylthieno[2,3-b]pyridine-2-carboxamide 4a.

The reaction was carried out according to general procedure A us-

ing acetamide 6a (1.0 g, 5.98 mmol) and pyridine 7a (0.79 g,

5.70 mmol) to give the title product 4a (1.17 g, 73%) as a yellow solid.

m.p. 247e249 ^ꢀC; ¹H NMR (400 MHz; d₆-DMSO) 7.07e7.11 (1H, m,

AreH), 7.33 (2H, t, J ¼ 7.9 Hz, AreH), 7.39 (2H, s, NH₂), 7.48 (1H, dd,

J ¼ 4.5, 8.0 Hz, H-5), 7.71 (2H, d, J ¼ 7.9 Hz, AreH), 8.51 (1H, dd,

4.2.3. N-(3⁰-Chlorophenyl)-2-mercaptoacetamide 6c

Using the same procedure as for amide 6b, using 3-chloroaniline

(0.23 mL, 2.17 mmol) and thioglycolic acid 5 (0.15 mL, 2.17 mmol),

stirring for 3 h at 130 ^ꢀC. After workup the title product 6c (0.43 g,

J.M. Hung et al. / European Journal of Medicinal Chemistry 86 (2014) 420e437

427

J ¼ 1.5, 8.0 Hz, H-4), 8.68 (1H, dd, J ¼ 1.5, 4.5 Hz, H-6), 9.45 (1H, s,

NH); ¹³C NMR (100 MHz; d₆-DMSO) 96.6 (C-2), 119.4 (C-5), 121.1

(AreCH), 123.4 (AreCH), 126.1 (C-3a), 128.4 (AreCH), 130.9 (C-4),

138.9 (AreC), 146.8 (C-3), 150.2 (C-6), 158.7 (C-7a), 163.9 (C]O); IR:

y_max(ﬁlm)/cm^ꢂ1: 3428, 3318, 3227, 3061, 1642, 1588, 1488, 1239; m/

z (ESIþ): 292 (MNa^þ, 100%). HRMS (ESI^þ) found (MNa^þ): 292.0521,

(OCH₃), 99.0 (C-2), 113.6 (C-3⁰and C-5⁰), 123.0 (C-2⁰and C-6⁰), 127.0

(C-3a), 127.1 (C-5), 130.2 (C-4), 131.7 (C-1⁰), 145.2 (C-3), 148.3 (C-6),

155.6 (C-4⁰), 156.5 (C-7a), 163.3 (C]O); IR: y_max(ﬁlm)/cm^ꢂ1: 3424,

3311, 3016, 2927,1638,1594,1502,1342,1109; m/z: (ESIþ): 358 (³⁷Cl

MNa^þ, 36%), 356 (³⁵Cl MNa^þ, 100); HRMS (ESI^þ) found (³⁷Cl MNa^þ):

358.0194, C₁₅H₁₂N₃O₂S³⁷Cl Na requires 358.0202. Found (³⁵Cl

MNa^þ): 356.0222, C₁₅H₁₂N₃O₂S³⁵ClNa requires 356.0231.

C

₁₄H₁₁N₃OSNa requires 292.0515.

4.3.1.2. 3-Amino-N-(4⁰-methoxyphenyl)thieno[2,3-b]pyridine-2-

carboxamide 4b. The reaction was carried out according to general

procedure A using acetamide 6b (1.20 g, 6.08 mmol) and pyridine

7a (0.80 g, 5.79 mmol) to give the title product 4b (1.09 g, 60%) as a

yellow solid. m.p. 199e201 ^ꢀC; ¹H NMR (400 MHz; d₆-DMSO) 3.75

(3H, s, OCH₃), 6.91 (2H, d, J ¼ 9.0 Hz, H-3⁰and H-5⁰), 7.33 (2H, s,

NH₂), 7.47 (1H, dd, J ¼ 4.5, 8.0 Hz, H-5), 7.58 (2H, d, J ¼ 9.0 Hz, H-2⁰

and H-6⁰), 8.49 (1H, dd, J ¼ 1.5, 8.0 Hz, H-4), 8.67 (1H, dd, J ¼ 1.5,

4.5 Hz, H-6), 9.36 (1H, s, NH); ¹³C NMR (100 MHz; d₆-DMSO) 55.1

(OCH₃), 96.8 (C-2), 113.6 (C-3⁰and C-5⁰), 119.4 (C-5), 122.9 (C-2⁰and

C-6⁰), 126.1 (C-3a), 130.8 (C-4), 131.8 (C-1⁰), 146.4 (C-3), 150.1 (C-6),

155.5 (C-4⁰), 158.6 (C-7a), 163.6 (C]O); IR: y_max(ﬁlm)/cm^ꢂ1: 3515,

3446, 3335, 3051, 1631, 1595, 1502, 1324, 1235, 1177, 1026; m/z

(ESIþ): 322 (MNa^þ, 100%). HRMS (ESI^þ) found (MNa^þ): 322.0629,

4.3.1.6. 3-Amino-5-chloro-N-(3⁰-chlorophenyl)thieno[2,3-b]pyridine-

2-carboxamide 4f. The reaction was carried out according to gen-

eral procedure A using acetamide 6c (0.12 g, 0.61 mmol) and pyr-

idine 7b (0.10 g, 0.58 mmol) to give the title product 4f (0.20 g, 99%)

as a yellow solid. m.p. 258e260 ^ꢀC; ¹H NMR (400 MHz; d₆-DMSO)

7.13 (1H, d, J ¼ 8.1 Hz, H-4⁰), 7.33e7.37 (1H, m, H-5⁰), 7.44 (2H, s,

NH₂), 7.64 (1H, d, J ¼ 8.1 Hz, H-6⁰), 7.91 (1H, s, H-2⁰), 8.71 (2H, s, H-4

and H-6), 9.68 (1H, s, NH); ¹³C NMR (100 MHz; d₆-DMSO) 98.3 (C-

2), 119.2 (C-6⁰), 120.3 (C-2⁰), 123.0 (C-4⁰), 126.9 (C-3a), 127.0 (C-5),

130.0 (C-5⁰), 130.4 (C-4), 132.7 (C-3⁰), 140.6 (C-1⁰), 146.1 (C-3), 148.7

(C-6), 156.6 (C-7a), 163.6 (C]O); IR: y_max(ﬁlm)/cm^ꢂ1: 3460, 3334,

3055, 1657, 1590, 1522, 1420, 1346, 1231, 1113; m/z (ESIþ): 363

(

³⁷Cl₂MNa^þ, 14%), 361 (³⁵Cl³⁷ClMNa^þ, 78%), 359 (³⁵Cl₂MNa^þ, 100%);

HRMS (ESI^þ) found (³⁷Cl₂MNa^þ): 363.9670, C₁₄H₉N₃OS³⁷Cl₂Na

requires 363.9677. Found 361.9704,

³⁵Cl³⁷Cl MNa^þ):

₁₄H₉N₃OS³⁵Cl³⁷ClNa requires 361.9706. Found ³⁵Cl₂MNa^þ):

359.9737, C₁₄H₉N₃OS³⁵Cl₂Na requires 359.9736.

C

₁₅H₁₃N₃O₂SNa requires 322.0621.

(

C

(

4.3.1.3. 3-Amino-N-(3⁰-chlorophenyl)thieno[2,3-b]pyridine-2-

carboxamide 4c. The reaction was carried out according to general

procedure A using acetamide 6c (0.30 g,1.49 mmol) and pyridine 7a

(0.20 g, 1.42 mmol) to give the title product 4c (0.39 g, 90%) as a

yellow solid. m.p. 240e241 ^ꢀC; ¹H NMR (400 MHz; d₆-DMSO) 7.12

(1H, d, J ¼ 8.0 Hz, H-4⁰), 7.33e7.37 (1H, m, H-5⁰), 7.48 (3H, dd, J ¼ 4.5,

8.0 Hz, NH₂, H-5), 7.67 (1H, d, J ¼ 8.0 Hz, H-6⁰), 7.94 (1H, s, H-2⁰),

8.53 (1H, dd, J ¼ 1.5, 8.0 Hz, H-4), 8.69 (1H, dd, J ¼ 1.5, 4.5 Hz, H-6),

9.61 (1H, s, NH); ¹³C NMR (100 MHz; d₆-DMSO) 96.1 (C-2), 119.1 (C-

6⁰), 119.5 (C-5), 120.2 (C-2⁰), 122.9 (C-4⁰), 126.0 (C-3a), 130.0 (C-5⁰),

131.0 (C-4), 132.7 (C-3⁰), 140.6 (C-1⁰), 147.3 (C-3), 150.4 (C-6), 158.8

(C-7a), 164.0 (C]O); IR: y_max(ﬁlm)/cm^ꢂ1: 3455, 3323, 3068, 1643,

1586, 1475, 1303, 1232; m/z (ESIþ): 328 (³⁷Cl MNa^þ, 35%), 326 (³⁵Cl

4.3.1.7. 3-Amino-5-bromo-N-phenylthieno[2,3-b]pyridine-2-

carboxamide 4g. The reaction was carried out according to general

procedure A using acetamide 6a (0.08 g, 0.49 mmol) and pyridine

7c (0.10 g, 0.46 mmol) to give the title product 4g (0.07 g, 42%) as a

yellow solid. m.p. 224e226 ^ꢀC; ¹H NMR (400 MHz; d₆-DMSO)

7.07e7.11 (1H, m, AreH), 7.33 (2H, d, J ¼ 7.9 Hz, AreH), 7.37 (2H, s,

NH₂), 7.69 (2H, d, J ¼ 7.9 Hz, AreH), 8.78 (1H, d, J ¼ 2.0 Hz, H-6), 8.84

(1H, d, J ¼ 2.0 Hz, H-4), 9.54 (1H, s, NH); ¹³C NMR (100 MHz; d₆-

DMSO) 98.4 (C-2), 115.5 (C-5), 121.2 (AreCH), 123.6 (AreCH), 127.6

(C-3a), 128.4 (AreCH), 133.2 (C-4), 138.8 (AreC), 145.6 (C-3), 150.4

(C-6), 156.9 (C-7a), 163.5 (C]O); IR: y_max(ﬁlm)/cm^ꢂ1: 3446, 3412,

3321, 1632, 1588, 1345, 1311, 1103; m/z (ESIþ): 371 (⁸¹BrMNa^þ,

100%), 369 (⁷⁹BrMNa^þ, 87); HRMS (ESI^þ) found: (⁸¹Br MNa^þ)

MNa^þ, 100); HRMS (ESI^þ) found

₁₄H₁₀N₃OS³⁷ClNa requires 328.0096. Found

326.0127, C₁₄H₁₀N₃OS³⁵Cl Na requires 326.0125.

(

³⁷Cl MNa^þ): 328.0103,

C

(

³⁵Cl MNa^þ):

371.9599,

C (

₁₄H₁₀N₃OS⁸¹BrNa requires 371.9600. Found ⁷⁹Br

MNa^þ): 369.9621, C₁₄H₁₀N₃OS⁷⁹BrNa requires 369.9620.

4.3.1.4. 3-Amino-5-chloro-N-phenylthieno[2,3-b]pyridine-2-

carboxamide 4d. The reaction was carried out according to general

procedure A using acetamide 6a (0.10 g, 0.61 mmol) and pyridine

7b (0.10 g, 0.58 mmol) to give the title product 4d (0.07 g, 40%) as a

yellow solid. m.p. 214e216 ^ꢀC; ¹H NMR (400 MHz; d₆-DMSO)

7.09e7.13 (1H, m, AreH), 7.34 (2H, d, J ¼ 8.0 Hz, AreH), 7.36 (2H, s,

NH₂), 7.69 (2H, d, J ¼ 8.0 Hz, AreH), 8.71 (2H, m, H-4 and H-6), 9.54

(1H, s, NH); ¹³C NMR (100 MHz; d₆-DMSO) 98.7 (C-2), 121.2

(AreCH), 123.6 (AreCH), 127.0 (C-3a and C-5), 128.4 (AreCH), 130.2

(C-4), 138.7 (AreC), 145.7 (C-3), 148.5 (C-6), 156.6 (C-7a), 163.5 (C]

O); IR: y_max(ﬁlm)/cm^ꢂ1: 3449, 3419, 3326, 1688, 1591, 1525, 1490,

1237, 1112; m/z (ESIþ): 328 (³⁷ClMNa^þ, 35%), 326 (³⁵ClMNa^þ, 100%);

HRMS (ESI^þ) found (³⁷ClMNa^þ): 328.0095, C₁₄H₁₀N₃OS³⁷ClNa re-

quires 328.0096. Found (³⁵Cl MNa^þ): 326.0128, C₁₄H₁₀N₃OS³⁵ClNa

requires 326.0125.

4.3.1.8. 3-Amino-5-bromo-N-(4⁰-methoxyphenyl)thieno[2,3-b]pyri-

dine-2-carboxamide 4h. The reaction was carried out according to

general procedure A using acetamide 6b (0.10 g, 0.49 mmol) and

pyridine 7c (0.10 g, 0.46 mmol) to give the title product 4h (0.12 g,

71%) as a yellow solid. m.p. 242e243 ^ꢀC; ¹H NMR (400 MHz; d₆-

DMSO) 3.75 (3H, s, OCH₃), 6.91 (2H, d, J ¼ 9.0 Hz, H-3⁰and H-5⁰),

7.31 (2H, s, NH₂), 7.57 (2H, d, J ¼ 9.0 Hz, H-2⁰and H-6⁰), 8.76 (1H, d,

J ¼ 2.0 Hz, H-6), 8.82 (1H, d, J ¼ 2.0 Hz, H-4), 9.44 (1H, s, NH); ¹³C

NMR (100 MHz; d₆-DMSO) 55.1 (OCH₃), 98.7 (C-2), 113.6 (C-3⁰and

C-5⁰), 115.5 (C-5), 123.0 (C-2⁰and C-6⁰), 127.7 (C-3a), 131.7 (C-4),

133.1 (C-1⁰), 145.2 (C-3), 150.3 (C-6), 155.6 (C-4⁰), 156.8 (C-7a), 163.2

(C]O); IR: y_max(ﬁlm)/cm^ꢂ1: 3401, 3281, 3201, 3132, 1649, 1595,

1501, 1346, 1107; m/z (ESIþ): 401 (⁸¹Br MNa^þ, 100%), 399 (⁷⁹Br

MNa^þ, 98%); HRMS (ESI^þ) found

₁₅H₁₂N₃O₂S⁸¹BrNa requires 401.9706. Found

399.9723, C₁₅H₁₂N₃O₂S⁷⁹BrNa requires 399.9726.

(

⁸¹Br MNa^þ): 401.9698,

C

(

⁷⁹Br MNa^þ):

4.3.1.5. 3-Amino-5-chloro-N-(4⁰-methoxyphenyl)thieno[2,3-b]pyri-

dine-2-carboxamide 4e. The reaction was carried out according to

general procedure A using acetamide 6b (0.12 g, 0.61 mmol) and

pyridine 7b (0.10 g, 0.58 mmol) to give the title product 4e (0.14 g,

74%) as a yellow solid. m.p. 214e216 ^ꢀC; ¹H NMR (400 MHz; d₆-

DMSO) 3.74 (3H, s, OCH₃), 6.91 (2H, d, J ¼ 9.0 Hz, H-3⁰and H-5⁰),

7.30 (2H, s, NH₂), 7.57 (2H, d, J ¼ 9.0 Hz, H-2⁰and H-6⁰), 8.69 (2H, m,

H-4 and H-6), 9.45 (1H, s, NH); ¹³C NMR (100 MHz; d₆-DMSO) 55.1

4.3.1.9. 3-Amino-5-bromo-N-(3⁰-chlorophenyl)thieno[2,3-b]pyri-

dine-2-carboxamide 4i. The reaction was carried out according to

general procedure A using acetamide 3c (0.10 g, 0.49 mmol) and

pyridine 6c (0.10 g, 0.46 mmol) to give the title product 4i (0.12 g,

67%) as a yellow solid. m.p. 272e275 ^ꢀC; ¹H NMR (400 MHz; d₆-

DMSO) 7.11 (1H, d, J ¼ 8.0 Hz, H-4⁰), 7.32e7.36 (1H, m, H-5⁰), 7.43

428

J.M. Hung et al. / European Journal of Medicinal Chemistry 86 (2014) 420e437

(2H, s, NH₂), 7.62 (1H, d, J ¼ 8.0 Hz, H-6⁰), 7.91 (1H, s, Hz, H-2⁰), 8.77

(1H, d, J ¼ 2.0 Hz, H-6), 8.83 (1H, d, J ¼ 2.0 Hz, H-4), 9.69 (1H, s, NH);

¹³C NMR (100 MHz; d₆-DMSO) 98.7 (C-2), 115.5 (C-5), 119.4 (C-6⁰),

120.4 (C-2⁰),122.8 (C-4⁰),127.6 (C-3a),130.0 (C-5⁰),132.7 (C-4),133.2

(C-3⁰), 141.2 (C-1⁰), 145.7 (C-3), 150.4 (C-6), 157.0 (C-7a), 163.8 (C]

O); IR: y_max(ﬁlm)/cm^ꢂ1: 3458, 3331, 3053, 1657, 1588, 1420, 1343,

1102; m/z (ESIþ): 385 (⁸¹Br³⁷Cl MH^þ, 25%), 383 (⁸¹Br³⁵Cl MH^þand

⁷⁹Br³⁷Cl MH^þ, 100%), 381 (⁷⁹Br³⁵Cl MH^þ, 75%); HRMS (ESI^þ) found

an orange solid. m.p. 149e152 ^ꢀC; ¹H NMR (400 MHz; CDCl₃) 5.02

(2H, s, CH₂), 7.14e7.18 (1H, m, H-4⁰), 7.36 (2H, d, J ¼ 8.0 Hz, H-3⁰and

H-5⁰), 7.60 (2H, d, J ¼ 8.0 Hz, H-2⁰and H-6⁰), 7.98 (1H, d, J ¼ 2.5 Hz,

H-4⁰⁰), 8.16 (1H, s, NH), 8.56 (1H, d, J ¼ 2.5 Hz, H-6⁰⁰); ¹³C NMR

(100 MHz; CDCl₃) 66.1 (C-2), 98.2 (C-3⁰⁰), 113.4 (CN), 120.0 (C-2⁰and

C-6⁰), 125.0 (C-4⁰), 125.7 (C-5⁰⁰), 129.2 (C-3⁰and C-5⁰), 136.7 (C-1⁰),

142.2 (C-4⁰⁰), 150.2 (C-6⁰⁰), 160.4 (C-2⁰⁰), 164.6 (C-1); IR: y_max(ﬁlm)/

cm^ꢂ1: 3274, 3207, 3147, 3108, 3070, 2235, 1677, 1556, 1449, 1301,

1253; m/z: (ESIþ): 312 (³⁷Cl MNa^þ, 38%), 310 (³⁵Cl MNa^þ, 100%);

HRMS (ESI^þ) found (³⁷Cl MNa^þ): 312.0321, C₁₄H₁₀N₃O³₂⁷ClNa re-

quires 312.0325. Found (³⁵Cl MNa^þ): 310.0354, C₁₄H₁₀N₃O³₂⁵ClNa

requires 310.0354.

(

⁸¹Br³⁷Cl MH^þ): 385.9356, C₁₄H₁₀N₃OS⁸¹Br³⁷Cl requires 385.9361.

Found (⁷⁹Br³⁵Cl MH^þ): 381.9406, C₁₄H₁₀N₃OS⁷⁹Br³⁵Cl requires

381.9411.

4.3.1.10. 2-(3⁰⁰-Cyanopyridin-2⁰⁰-yloxy)-N-phenylacetamide

13a.

The reaction was carried out according to general procedure A us-

ing acetamide 11a (0.15 g, 0.99 mmol) and pyridine 7a (0.13 g,

0.95 mmol) to give the title product 13a (0.12 g, 49%) as a white

solid. m.p. 157e159 ^ꢀC. ¹H NMR (400 MHz; d₆-DMSO) 5.11 (2H, s,

CH₂), 7.04e7.08 (1H, m, H-5⁰⁰), 7.20e7.23 (1H, m, H-4⁰), 7.31 (2H, d,

J ¼ 7.8 Hz, H-3⁰and H-5⁰), 7.57 (2H, d, J ¼ 7.8 Hz, H-2⁰and H-6⁰), 8.32

(1H, d, J ¼ 7.2 Hz, H-4⁰⁰), 8.42 (1H, d, J ¼ 7.2 Hz, H-6⁰⁰), 10.20 (1H, br s,

4.3.1.14. 2-(5⁰⁰-Chloro-3⁰⁰-cyanopyridin-2⁰⁰-yloxy)-N-(4⁰-methox-

yphenyl)acetamide 13e. The reaction was carried out according to

general procedure A using acetamide 11b (0.11 g, 0.61 mmol) and

pyridine 7b (0.10 g, 0.58 mmol) to give the title product 13e (0.10 g,

55%) as a purple solid. m.p. 160e162 ^ꢀC; ¹H NMR (400 MHz; CDCl₃)

3.80 (3H, s, OCH₃), 5.00 (2H, s, CH₂), 6.88 (2H, d, J ¼ 9.0 Hz, H-3⁰and

H-5⁰), 7.50 (2H, d, J ¼ 9.0 Hz, H-2⁰and H-6⁰), 7.94 (1H, d, J ¼ 2.5 Hz,

H-4⁰⁰), 8.06 (1H, s, NH), 8.36 (1H, d, J ¼ 2.5 Hz, H-6⁰⁰); ¹³C NMR

(100 MHz; CDCl₃) 55.5 (OCH₃), 66.1 (C-2), 98.1 (C-3⁰⁰), 113.4 (CN),

114.3 (C-3⁰and C-5⁰), 121.8 (C-2⁰and C-6⁰), 125.6 (C-5⁰⁰), 129.8 (C-1⁰),

142.2 (C-4⁰⁰), 150.2 (C-6⁰⁰), 156.9 (C-4⁰), 160.4 (C-2⁰⁰), 164.4 (C-1); IR:

y_max(ﬁlm)/cm^ꢂ1: 3330, 3073, 2929, 2838, 2232, 1664, 1550, 1507,

1453, 1426, 1241, 1037; m/z: (ESIþ): 342 (³⁷Cl MNa^þ, 39%), 340

13

NH); C NMR (100 MHz; d₆-DMSO) 64.7 (C-2), 95.7 (C-3⁰⁰), 115.2

(CN), 117.8 (C-5⁰⁰), 119.2 (C-2⁰and C-6⁰), 123.5 (C-4⁰), 128.8 (C-3⁰and

C-5⁰), 138.5 (C-1⁰), 144.1 (C-4⁰⁰), 151.5 (C-6⁰⁰), 162.5 (C-2⁰⁰), 165.7 (C-

1); IR: y_max(ﬁlm)/cm^ꢂ1: 3350, 3067, 2954, 2849, 2235, 1668, 1576,

1534, 1444, 1246, 1037; m/z (ESIþ): 276 (MNa^þ, 100%). High Reso-

lution (ESI^þ) found (MNa^þ): 276.0743, C₁₄H₁₁N₃O₂Na requires

276.0743.

(

³⁵ClMNa^þ, 100%); HRMS (ESI^þ) found: (³⁷Cl MNa^þ) 342.0435,

C

₁₅H₁₂N₃O³₃⁷ClNa requires 342.0431. Found (³⁵Cl MNa^þ): 340.0461,

₁₅H₁₂N₃O³₃⁵ClNa requires 340.0459.

4.3.1.11. 2-(3⁰⁰-Cyanopyridin-2⁰⁰-yloxy)-N-(4⁰-methoxyphenyl)acet-

amide 13b. The reaction was carried out according to general

procedure A using acetamide 11b (0.15 g, 0.83 mmol) and pyridine

7a (0.11 g, 0.79 mmol) to give the title product 13b (0.13 g, 57%) as a

purple solid. m.p. 174e175 ^ꢀC; ¹H NMR (400 MHz; CDCl₃) 3.80 (3H,

s, OCH₃), 5.03 (2H, s, CH₂), 6.89 (2H, d, J ¼ 9.0 Hz, H-3⁰and H-5⁰),

7.11e7.14 (1H, m, H-5⁰⁰), 7.52 (2H, d, J ¼ 9.0 Hz, H-2⁰and H-6⁰), 7.98

(1H, dd, J ¼ 2.0, 7.5 Hz, H-4⁰⁰), 8.15 (1H, br s, NH), 8.42 (1H, dd,

J ¼ 2.0, 7.5 Hz, H-6⁰⁰); ¹³C NMR (100 MHz; CDCl₃) 55.5 (OCH₃), 65.7

(C-2), 97.2 (C-3⁰⁰),114.3 (C-3⁰and C-5⁰), 114.7 (CN),118.1 (C-5⁰⁰), 121.8

(C-2⁰and C-6⁰), 130.0 (C-1⁰), 143.0 (C-4⁰⁰), 151.6 (C-6⁰⁰), 156.9 (C-4⁰),

162.0 (C-2⁰⁰), 164.8 (C-1); IR: y_max(ﬁlm)/cm^ꢂ1: 3246, 3044, 2939,

2843, 2230, 1655, 1581, 1539, 1444, 1235, 1031; m/z (ESIþ): 306

(MNa^þ, 100%). HRMS (ESI^þ) found (MNa^þ): 306.0854,

4.3.1.15. 2-(5⁰⁰-Chloro-3⁰⁰-cyanopyridin-2⁰⁰-yloxy)-N-(3⁰-chlor-

ophenyl)acetamide 13f. The reaction was carried out according to

general procedure A using acetamide 11c (0.11 g, 0.61 mmol) and

pyridine 7b (0.10 g, 0.58 mmol) to give the title product 8f (0.03 g,

19%) as a white solid. m.p. Decomp. >350 ^ꢀC; ¹H NMR (400 MHz;

CDCl₃) 5.01 (2H, s, CH₂), 7.14 (1H, d, J ¼ 8.0 Hz, H-4⁰), 7.26e7.30 (1H,

m, H-5⁰), 7.40 (1H, d, J ¼ 8.0 Hz, H-6⁰), 7.77 (1H, s, H-2⁰), 7.96 (1H, d,

J ¼ 2.5 Hz, H-4⁰⁰), 8.17 (1H, s, NH), 8.36 (1H, d, J ¼ 2.5 Hz, H-6⁰⁰); ¹³C

NMR (100 MHz; CDCl₃) 66.0 (C-2), 98.2 (C-3⁰⁰), 113.4 (CN), 118.0 (C-

6⁰), 120.1 (C-2⁰), 125.1 (C-4⁰), 125.8 (C-5⁰⁰), 130.1 (C-5⁰), 134.9 (C-3⁰),

137.8 (C-1⁰), 142.2 (C-4⁰⁰), 150.2 (C-6⁰⁰), 160.2 (C-2⁰⁰), 164.7 (C-1); IR:

y_max(ﬁlm)/cm^ꢂ1: 3460, 3315, 2231, 1677, 1592, 1452, 1297, 1160,

1035; m/z (ESIþ): 347 (³⁷Cl₂MNa^þ, 10%), 345 (³⁵Cl³⁷Cl MNa^þ, 59),

343 (³⁵Cl₂MNa^þ, 100). HRMS (ESI^þ) found (³⁷Cl₂MNa^þ): 347.9908,

C₁₄H₉N₃O³₂⁷Cl₂Na requires 347.9905.

C

₁₅H₁₃N₃O₃Na requires 306.0849.

4.3.1.12. N-(3⁰-Chlorophenyl)-2-(3⁰⁰-cyanopyridin-2⁰⁰-yloxy)acet-

amide 13c. The reaction was carried out according to general pro-

cedure A using acetamide 11c (0.10 g, 0.54 mmol) and pyridine 7a

(0.07 g, 0.51 mmol) to give the title product 13c (0.06 g, 39%) as a

white solid. m.p. 170e171 ^ꢀC; ¹H NMR (400 MHz; CDCl₃) 5.12 (2H, s,

CH₂), 7.12 (1H, d, J ¼ 8.0 Hz, H-4⁰), 7.20e7.23 (1H, m, H-5⁰⁰),

7.32e7.36 (1H, m, H-5⁰), 7.44 (1H, d, J ¼ 8.0 Hz, H-6⁰), 7.77 (1H, s, H-

2⁰), 8.13 (1H, d, J ¼ 7.5 Hz, H-4⁰⁰), 8.42 (1H, d, J ¼ 7.5 Hz, H-6⁰⁰); ¹³C

NMR (100 MHz; CDCl₃) 64.7 (C-2), 95.7 (C-3⁰⁰), 115.1 (CN), 117.6 (C-

6⁰), 117.8 (C-5⁰⁰), 118.7 (C-2⁰), 123.2 (C-4⁰), 130.5 (C-5⁰), 133.1 (C-3⁰),

139.9 (C-1⁰), 144.1 (C-4⁰⁰), 151.4 (C-6⁰⁰), 162.4 (C-2⁰⁰), 166.2 (C-1); IR:

y_max(ﬁlm)/cm^ꢂ1: 3294, 3069, 2949, 2841, 2229, 1672, 1578, 1540,

1421, 1257; m/z (ESIþ): 312 (³⁷Cl MNa^þ, 31%), 310 (³⁵Cl MNa^þ,

100%); HRMS (ESI^þ) found (³⁷Cl MNa^þ): 312.0332, C₁₄H₁₀N₃O³₂⁷ClNa

requires 312.0325. Found (³⁵Cl MNa^þ): 310.0358, C₁₄H₁₀N₃O³₂⁵ClNa

requires 310.0354.

4.3.1.16. 2-(5⁰⁰-Bromo-3⁰⁰-cyanopyridin-2⁰⁰-yloxy)-N-phenyl-

acetamide 13g. The reaction was carried out according to general

procedure A using acetamide 11a (0.07 g, 0.49 mmol) and pyridine

7c (0.10 g, 0.46 mmol) to give the title product 13g (0.10 g, 65%) as a

white solid. m.p.169e172 ^ꢀC; ¹H NMR (400 MHz; CDCl₃) 5.01 (2H, s,

CH₂), 7.14e7.18 (1H, m, AreH), 7.36 (2H, d, J ¼ 7.8 Hz, AreH), 7.60

(2H, d, J ¼ 7.8 Hz, AreH), 8.07 (1H, d, J ¼ 2.5 Hz, H-4⁰⁰), 8.15 (1H, s,

NH), 8.45 (1H, d, J ¼ 2.5 Hz, H-6⁰⁰); ¹³C NMR (100 MHz; CDCl₃) 66.0

(C-2), 98.7 (C-3⁰⁰), 112.7 (C-5⁰⁰), 113.3 (CN), 120.0 (AreCH), 125.1

(AreCH), 129.2 (AreCH), 136.7 (AreC), 144.8 (C-4⁰⁰), 152.5 (C-6⁰⁰),

160.8 (C-2⁰⁰), 164.5 (C-1); IR: y_max(ﬁlm)/cm^ꢂ1: 3274, 3146, 3108,

3065, 2235, 1679, 1554, 1448, 1152, 1056; m/z (ESIþ): 355 (⁸¹Br

MNa^þ, 96%), 353 (⁷⁹BrMNa^þ, 100%); HRMS (ESI^þ) found (⁸¹Br

MNa^þ): 355.9825, C₁₄H₁₀N₃O₂⁸¹BrNa requires 355.9829. Found (⁷⁹Br

MNa^þ): 353.9842, C₁₄H₁₀N₃O⁷₂⁹BrNa requires 353.9849.

4.3.1.13. 2-(5⁰⁰-Chloro-3⁰⁰-cyanopyridin-2⁰⁰-yloxy)-N-phenyl-

acetamide 13d. The reaction was carried out according to general

procedure A using acetamide 11a (0.09 g, 0.61 mmol) and pyridine

7b (0.10 g, 0.58 mmol) to give the title product 13d (0.15 g, 92%) as

4.3.1.17. 2-(5⁰⁰-Bromo-3⁰⁰-cyanopyridin-2⁰⁰-yloxy)-N-(4⁰-methox-

yphenyl)acetamide 13h. The reaction was carried out according to

general procedure A using acetamide 11b (0.09 g, 0.49 mmol) and

J.M. Hung et al. / European Journal of Medicinal Chemistry 86 (2014) 420e437

429

pyridine 7c (0.10 g, 0.46 mmol) to give the title product 13h (0.08 g,

47%) as a purple solid. m.p. 150e153 ^ꢀC; ¹H NMR (400 MHz; CDCl₃)

3.80 (3H, s, OCH₃), 5.00 (2H, s, CH₂), 6.89 (2H, d, J ¼ 9.0 Hz, H-3⁰and

H-5⁰), 7.50 (2H, d, J ¼ 9.0 Hz, H-2⁰and H-6⁰), 8.06e8.10 (2H, m, H-4⁰⁰

and NH), 8.45 (1H, d, J ¼ 2.4 Hz, H-6⁰⁰); ¹³C NMR (100 MHz; CDCl₃)

55.5 (OCH₃), 66.0 (C-2), 98.7 (C-3⁰⁰), 112.6 (C-5⁰⁰), 113.4 (CN), 114.3

(C-3⁰and C-5⁰), 121.9 (C-2⁰and C-6⁰), 129.8 (C-1⁰), 144.8 (C-4⁰⁰), 152.5

118.9 (C-5), 121.8 (C-2⁰and C-6⁰), 124.3 (C-2), 131.1 (C-4), 131.9 (C-

1⁰), 135.8 (C-3), 147.7 (C-6), 155.3 (C-4⁰), 158.8 (C-7a), 159.2 (C]O);

IR: y_max(ﬁlm)/cm^ꢂ1: 3450, 3217, 3054, 2923, 2839, 1634, 1595,

1406,1349,1234; m/z (ESIþ): 306 (MNa^þ,100%). HRMS (ESI^þ) found

(MNa^þ): 306.0850, C₁₅H₁₃N₃O₃Na requires 306.0849.

4.3.2.3. 3-Amino-N-(3⁰-chlorophenyl)furo[2,3-b]pyridine-2-

carboxamide 10c. The reaction was carried out according to general

procedure B using N-(3-chlorophenyl)-2-(3-cyanopyridin-2-yloxy)

acetamide 13c (0.10 g, 0.35 mmol) to give the title product 10c

(0.06 g, 57%) as a yellow solid. m.p. 230e232 ^ꢀC. ¹H NMR (400 MHz;

d₆-DMSO) 6.53 (2H, s, NH₂), 7.11 (1H, d, J ¼ 8.0 Hz, H-4⁰), 7.32e7.36

(1H, m, H-5⁰), 7.40 (1H, dd, J ¼ 4.8, 7.8 Hz, H-5), 7.79 (1H, d,

J ¼ 8.0 Hz, H-6⁰), 8.08 (1H, s, H-2⁰), 8.41 (1H, dd, J ¼ 1.6, 7.8 Hz, H-4),

8.48 (1H, dd, J ¼ 1.6, 4.8 Hz, H-6), 10.19 (1H, s, NH); ¹³C NMR

(100 MHz; d₆-DMSO) 114.6 (C-3a), 118.4 (C-6⁰), 119.0 (C-5), 119.5 (C-

2⁰), 122.7 (C-4⁰), 123.7 (C-2), 130.1 (C-5⁰), 131.4 (C-4), 132.8 (C-3⁰),

136.9 (C-3), 140.5 (C-1⁰), 148.2 (C-6), 158.9 (C-7a), 159.6 (C]O); IR:

y_max(ﬁlm)/cm^ꢂ1: 3440, 3276, 3173, 3069, 1671, 1590, 1535, 1408,

1346; m/z (ESIþ): 312 (³⁷ClMNa^þ, 32%), 310 (³⁵ClMNa^þ, 100%);

HRMS (ESI^þ) found (³⁷Cl MNa^þ): 312.0326, C₁₄H₁₀N₃O³₂⁷ClNa re-

quires 312.0322. Found (³⁵Cl MNa^þ): 310.0353, C₁₄H₁₀N₃O³₂⁵ClNa

requires 310.0354.

(C-6⁰⁰), 156.9 (C-4⁰), 160.8 (C-2⁰⁰), 164.4 (C-1); IR: y_max(ﬁlm)/cm^ꢂ1

:

3332, 3062, 2995, 2936, 2230, 1668, 1553, 1453, 1304, 1041; m/z

(ESIþ): 385 (⁸¹Br MNa^þ, 100%), 383 (⁷⁹Br MNa^þ, 96); HRMS (ESI^þ)

found: (⁸¹Br MNa^þ) 385.9931, C₁₅H₁₂N₃O₃⁸¹BrNa requires 385.9934.

Found (⁷⁹BrMNa^þ): 383.9949, C₁₅H₁₂N₃O⁷₃⁹BrNa requires 383.9954.

4.3.1.18. 2-(5⁰⁰-Bromo-3⁰⁰-cyanopyridin-2⁰⁰-yloxy)-N-(3⁰-chlor-

ophenyl)acetamide 13i. The reaction was carried out according to

general procedure A using acetamide 11c (0.09 g, 0.49 mmol) and

pyridine 7c (0.10 g, 0.46 mmol) to give the title product 8i (0.07 g,

39%) as a white solid. m.p. 146e148 ^ꢀC; ¹H NMR (400 MHz; CDCl₃)

5.01 (2H, s, CH₂), 7.14 (1H, d, J ¼ 8.0 Hz, H-4⁰), 7.25e7.29 (1H, m, H-

5⁰), 7.40 (1H, d, J ¼ 8.0 Hz, H-6⁰), 7.76 (1H, s, H-2⁰), 8.08 (1H, d,

J ¼ 2.4 Hz, H-4⁰⁰), 8.18 (1H, s, NH), 8.45 (1H, d, J ¼ 2.4 Hz, H-6⁰⁰); ¹³C

NMR (100 MHz; CDCl₃) 66.0 (C-2), 98.7 (C-3⁰⁰), 112.8 (C-5⁰⁰), 113.3

(CN), 118.0 (C-6⁰), 120.1 (C-2⁰), 125.1 (C-4⁰), 130.1 (C-5⁰), 134.9 (C-3⁰),

137.8 (C-1⁰), 144.9 (C-4⁰⁰), 152.5 (C-6⁰⁰), 160.7 (C-2⁰⁰), 164.7 (C-1); IR:

y_max(ﬁlm)/cm^ꢂ1: 3313, 3066, 2937, 2229, 1675, 1592, 1452, 1423,

4.3.2.4. 3-Amino-5-chloro-N-phenylfuro[2,3-b]pyridine-2-

carboxamide 10d. The reaction was carried out according to general

procedure B using 2-(5-chloro-3-cyanopyridin-2-yloxy)-N-phe-

1297, 1150; m/z (ESIþ): 391

(

⁸¹Br³⁷ClMNa^þ, 24%), 389

(

⁸¹Br³⁵ClMNa^þand ⁷⁹Br³⁷ClMNa^þ, 100%), 387 (⁷⁹Br³⁵ClMNa^þ, 69%);

HRMS

₁₄H₉N₃O₂⁸¹Br³⁷ClNa requires 391.9409. Found

387.9458, C₁₄H₉N₃O⁷₂⁹Br³⁵ClNa^þrequires 387.9459.

(ESI^þ)

found

(

⁸¹Br³⁷Cl

MNa^þ):

391.9400,

nylacetamide 13d (0.10 g, 0.35 mmol) to give the title product 10d

(0.074 g, 74%) as a yellow solid. m.p. 229e230 ^ꢀC. ¹H NMR

(400 MHz; d₆-DMSO) 6.43 (2H, s, NH₂), 7.06e7.09 (1H, m, AreH),

7.33 (2H, t, J ¼ 7.4 Hz, AreH), 7.82 (2H, d, J ¼ 7.4 Hz, AreH),

8.51e8.54 (2H, m, H-4 and H-6), 10.04 (1H, s, NH); ¹³C NMR

(100 MHz; d₆-DMSO) 115.9 (C-3a), 120.3 (AreCH), 123.4 (AreCH),

125.5 (C-2), 125.6 (C-5), 128.5 (AreCH), 130.4 (C-4), 135.4 (C-3),

138.6 (AreC), 146.0 (C-6), 157.0 (C-7a), 159.2 (C]O); IR: y_max(ﬁlm)/

cm^ꢂ1: 3430, 3184, 3060, 2849, 1640, 1594, 1532, 1443, 1269; m/z

(ESIþ): 312 (³⁷Cl MNa^þ, 29%), 310 (³⁵Cl MNa^þ, 100); HRMS (ESI^þ)

found (³⁷Cl MNa^þ): 312.0319, C₁₄H₁₀N₃O₂³⁷ClNa requires 312.0325.

Found (³⁵Cl MNa^þ): 310.0348, C₁₄H₁₀N₃O₂³⁵ClNa requires 310.0354.

C

(

⁷⁹Br³⁵ClMNa):

4.3.2. General procedure B: synthesis of the furopyridine-2-

carboxamides 10 from cyanopyridines 13

A mixture of cyanopyridine 13 (1.0 mmol) and potassium tert-

butoxide (1.2 mmol) in THF (10 mL) was heated at 80 ^ꢀC for 3 h,

under an atmosphere of nitrogen. The mixture was cooled and the

solvent removed in vacuo. The crude solid was washed with small

amounts of water and dried under vacuum to give the furopyridine-

2-carboxamide 10.

4.3.2.1. 3-Amino-N-phenylfuro[2,3-b]pyridine-2-carboxamide 10a.

The reaction was carried out according to general procedure B using

2-(3-cyanopyridin-2-yloxy)-N-phenylacetamide 13a (0.1 g,

0.40 mmol) to give the title product 10a (0.10 g, 99%) as a yellow

solid. m.p. 197e200 ^ꢀC. ¹H NMR (400 MHz; d₆-DMSO) 6.43 (2H, s,

NH₂) 7.05e7.09 (1H, m, AreH), 7.32 (2H, d, J ¼ 7.6 Hz, AreH), 7.39

(1H, dd, J ¼ 4.7, 7.8 Hz, H-5), 7.84 (2H, d, J ¼ 7.6 Hz, AreH), 8.39 (1H,

dd, J ¼ 1.7, 7.8 Hz, H-4), 8.47 (1H, dd, J ¼ 1.7, 4.7 Hz, H-6), 9.97 (1H, s,

NH); ¹³C NMR (100 MHz; d₆-DMSO) 114.7 (C-3a) 118.9 (C-5), 120.2

(AreCH), 123.2 (AreCH), 124.1 (C-2), 128.5 (AreCH), 131.2 (C-4),

136.3 (C-3), 138.8 (AreC), 147.9 (C-6), 158.8 (C-7a), 159.5 (C]O); IR:

y_max(ﬁlm)/cm^ꢂ1: 3427, 3310, 2918, 2849, 1640, 1596, 1561, 1403,

1348, 1240; m/z (ESIþ): 276 (MNa^þ, 100%). HRMS (ESI^þ) found

(MNa^þ): 276.0749, C₁₄H₁₁N₃O₂Na requires 276.0743.

4.4. Synthesis of thieneopyrimidinones 8 and furopyrimidinones 14

4.4.1. General procedure C: synthesis of the thieneopyrimidinones 8

and formimidates 15 from amines 4 and 10

A mixture of thieno- 4 or furo-pyridine 10 (1 mmol) and triethyl

orthoformate (10 mmol) were heated at the stated temperature.

Acetic acid (9 mmol) was then added dropwise over 5 min. The

mixture was then cooled to room temperature and the crude solid

product collected by ﬁltration. Recrystallization from methanol

gave thieneopyrimidinones 8 or formimidates 15.

4.4.1.1. 3-Phenylpyrido[3⁰,2⁰:4,5]thieno[3,2-d]pyrimidin-4(3H)-one

8a. The reaction was carried out according to general procedure C

using carboxamide 4a (0.20 g, 0.74 mmol) with heating at 130 ^ꢀC to

give the title product 15a (0.14 g, 65%) as a white solid. m.p.

245e246 ^ꢀC. ¹H NMR (400 MHz; CDCl₃) 7.46e7.61 (6H, m, 5 ꢁ AreH

and H-8), 8.29 (1H, s, H-2), 8.59 (1H, dd, J ¼ 1.6, 8.1 Hz, H-9), 8.82

(1H, dd, J ¼ 1.6, 4.6 Hz, H-7); ¹³C NMR (100 MHz; CDCl₃) 120.5 (C-8),

124.1(C-4a), 127.0 (AreCH), 128.4 (C-9a), 129.6 (AreCH), 129.8

(AreCH), 131.7 (C-9), 136.8 (AreC), 148.2 (C-2), 150.2 (C-9b), 151.2

(C-7), 157.1 (C-4), 163.1 (C-5a). The ¹H NMR data was in agreement

with the literature values [33].

4.3.2.2. 3-Amino-N-(4⁰-methoxyphenyl)furo[2,3-b]pyridine-2-

carboxamide 10b. The reaction was carried out according to general

procedure

B

using

2-(3-cyanopyridin-2-yloxy)-N-(4-

methoxyphenyl)acetamide 13b (0.10 g, 0.35 mmol) to give the ti-

tle product 10b (0.08 g, 75%) as a green solid. m.p. 145e148 ^ꢀC. ¹H

NMR (400 MHz; d₆-DMSO) 3.75 (3H, s, OCH₃), 6.37 (2H, s, NH₂) 6.90

(2H, d, J ¼ 8.9 Hz, H-3⁰and H-5⁰), 7.39 (1H, dd, J ¼ 4.8, 7.5 Hz, H-5),

7.74 (2H, d, J ¼ 8.9 Hz, H-2⁰and H-6⁰), 8.38 (1H, d, J ¼ 1.7, 7.5 Hz, H-

4), 8.46 (1H, d, J ¼ 1.7, 4.8 Hz, H-6), 9.88 (1H, s, NH); ¹³C NMR

(100 MHz; d₆-DMSO) 55.1 (OCH₃), 113.6 (C-3⁰and C-5⁰), 114.8 (C-3a)

4.4.1.2. 3-(4⁰-Methoxyphenyl)pyrido[3⁰,2⁰:4,5]thieno[3,2-d]pyr-

imidin-4(3H)-one 8b. The reaction was carried out according to

430

J.M. Hung et al. / European Journal of Medicinal Chemistry 86 (2014) 420e437

general procedure C using carboxamide 4b (0.20 g, 0.67 mmol)

with heating at 130 ^ꢀC for 1 h to give the title product 8b (0.08 g,

40%) as a white solid. m.p. 238e239 ^ꢀC; ¹H NMR (400 MHz; CDCl₃)

3.89 (3H, s, OMe) 7.07 (2H, d, J ¼ 8.8 Hz, H-3⁰and H-5⁰), 7.38 (2H, d,

J ¼ 8.8 Hz, H-2⁰and H-6⁰), 7.50e7.53 (1H, dd, J ¼ 4.3, 8.0 Hz, H-8),

8.27 (1H, s, H-2), 8.59 (1H, dd, J ¼ 1.3, 8.0 Hz, H-9), 8.82 (1H, dd,

J ¼ 1.3, 4.3 Hz, H-7); ¹³C NMR (100 MHz; CDCl₃) 55.7 (OCH₃), 115.0

(C-3⁰and C-5⁰), 120.5 (C-8), 124.0 (C-4a), 128.2 (C-2⁰and C-6⁰), 128.4

(C-9a), 129.5 (C-1⁰), 131.7 (C-9), 148.5 (C-2), 150.1 (C-9b), 151.1 (C-7),

157.4 (C-4), 160.3 (C-4⁰), 163.1 (C-5a); IR: y_max(ﬁlm)/cm^ꢂ1: 3051,

2985, 2848, 1680, 1585, 1514, 1387, 1239, 1015; m/z (ESIþ): 332

(MNa^þ, 100%). HRMS (ESI^þ) found (MNa^þ): 332.0467,

MNa^þ, 100%); HRMS (ESI^þ) found

(

³⁷Cl₂MNa^þ): 373.9513,

C

₁₅H₇N₃OS³⁷Cl₂Na requires 373.9520. Found ³⁵Cl³⁷ClMNa^þ):

(

371.9542, C₁₅H₇N₃OS³⁵Cl³⁷ClNa requires 371.9550. Found (³⁵Cl₂

MNa^þ): 369.9572, C₁₅H₇N₃OS³⁵Cl₂Na requires 369.9579.

4.4.1.7. 8-Bromo-3-phenylpyrido[3⁰,2⁰:4,5]thieno[3,2-d]pyrimidin-

4(3H)-one 8g. The reaction was carried out according to general

procedure C using carboxamide 4g (0.15 g, 0.43 mmol) with heating

at 70 ^ꢀC for 15 h to give the title product 8g (0.03 g, 20%) as a white

solid. m.p. 259e260 ^ꢀC; ¹H NMR (400 MHz; CDCl₃) 7.46e7.48 (1H,

m, H-4⁰), 7.55e7.7.62 (4H, m, H-2⁰, H-6⁰, H-3⁰and H-5⁰), 8.28 (1H, s,

H-2), 8.72 (1H, d, J ¼ 2.3 Hz, H-9), 8.85 (1H, d, J ¼ 2.3 Hz, H-7); IR:

y_max(ﬁlm)/cm^ꢂ1: 3049, 1685, 1568, 1526, 1490, 1455, 1382, 1234; m/

z (ESIþ): 381 (⁸¹Br MNa^þ, 93%), 379 (⁷⁹Br MNa^þ, 100%); HRMS

(ESI^þ) found (⁸¹Br MNa^þ): 381.9437, C₁₅H₈N₃OS⁸¹BrNa requires

381.9443. Found (⁷⁹Br MNa^þ): 379.9459, C₁₅H₈N₃OS⁷⁹BrNa requires

379.9464.

C

₁₆H₁₁N₃O₂SNa^þrequires 332.0464.

4.4.1.3. 3-(3⁰-Chlorophenyl)pyrido[3⁰,2⁰:4,5]thieno[3,2-d]pyrimidin-

4(3H)-one 8c. The reaction was carried out according to general

procedure C using carboxamide 4c (0.10 g, 0.33 mmol) with heating

at 150 ^ꢀC for 2 h to give the title product 8c (0.07 g, 72%) as a white

solid. m.p. 228e229 ^ꢀC; ¹H NMR (400 MHz; CDCl₃) 7.37e7.41 (1H,

m, ArH) 7.50e7.55 (4H, m, ArH and H-8), 8.25 (1H, s, H-2), 8.60 (1H,

dd, J ¼ 1.6, 8.0 Hz, H-9) 8.83 (1H, dd, J ¼ 1.6, 5.0 Hz, H-7); ¹³C NMR

(100 MHz; CDCl₃) 120.6 (C-8),124.0 (C-4a),125.3 (C-4⁰),127.5 (C-5⁰),

128.3 (C-9a), 129.9 (C-2⁰), 130.7 (C-6⁰), 131.8 (C-9), 135.4 (C-3⁰), 137.8

(C-1⁰), 147.7 (C-2), 150.2 (C-9b), 151.3 (C-7),156.8 (C-4), 163.1 (C-5a);

IR: y_max(ﬁlm)/cm^ꢂ1: 3060, 1688, 1582, 1541, 1471, 1218, 1380, 1068;

m/z (ESIþ): 337 (³⁷Cl MNa^þ, 32%), 335 (³⁵Cl MNa^þ, 100); HRMS

(ESI^þ) found (³⁷Cl MNa^þ): 337.9941, C₁₅H₈N₃OS³⁷ClNa requires

337.9940. Found (³⁵Cl MNa^þ): 335.9972, C₁₅H₈N₃OS³⁵ClNa requires

335.9969.

4.4.1.8. 8-Bromo-3-(4⁰-methoxyphenyl)pyrido[3⁰,2⁰:4,5]thieno[3,2-d]

pyrimidin-4(3H)-one 8h. The reaction was carried out according to

general procedure C using carboxamide 4h (0.15 g, 0.40 mmol) with

heating at 70 ^ꢀC for 15 h to give the title product 8h (0.07 g, 43%) as a

white solid. m.p. 287e290 ^ꢀC; ¹H NMR (400 MHz; CDCl₃) 3.89 (3H,

s, OMe), 7.07 (2H, d, J ¼ 9.0 Hz, H-3⁰and H-5⁰), 7.37 (2H, d, J ¼ 9.0 Hz,

H-2⁰and H-6⁰), 8.26 (1H, s, H-2), 8.71 (1H, d, J ¼ 2.4 Hz, H-9), 8.84

(1H, d, J ¼ 2.4 Hz, H-7); IR: y_max(ﬁlm)/cm^ꢂ1: 3047, 2929, 2836,1672,

1571, 1507, 1239, 1051; m/z (ESIþ): 411 (⁸¹Br MNa^þ, 92%), 409 (⁷⁹Br

MNa^þ, 100%), HRMS (ESI^þ) found

(

⁸¹Br MNa^þ): 411.9554,

⁷⁹Br MNa^þ):

C

₁₆H₁₀N₃O₂S⁸¹BrNa requires 411.9549. Found

409.9571, C₁₆H₁₀N₃O₂S⁷⁹BrNa requires 409.9569.

(

4.4.1.4. 8-Chloro-3-phenylpyrido[3⁰,2⁰:4,5]thieno[3,2-d]pyrimidin-

4(3H)-one 8d. The reaction was carried out according to general

procedure C using carboxamide 4d (0.02 g, 0.07 mmol) with

heating at 130 ^ꢀC for 6 h to give the title product 8d (0.01 g, 24%) as a

white solid. m.p. 255e258 ^ꢀC; ¹H NMR (400 MHz; CDCl₃) 7.46e7.48

(2H, m, H-3⁰and H-5⁰), 7.55e7.62 (3H, m, H-4⁰, H-2⁰and H-6⁰), 8.28

(1H, s, H-2), 8.57 (1H, d, J ¼ 2.5 Hz, H-9), 8.76 (1H, d, J ¼ 2.5 Hz, H-7);

IR: y_max(ﬁlm)/cm^ꢂ1: 3059, 3015, 1668, 1560, 1526, 1488, 1453, 1233,

1214, 1096; m/z (ESIþ): 337 (³⁷Cl MNa^þ, 33%), 335 (³⁵Cl MNa^þ, 100);

HRMS (ESI^þ) found (³⁷Cl MNa^þ): 337.9947, C₁₅H₈N₃OS³⁷ClNa re-

quires 337.9940. Found (³⁵Cl MNa^þ): 335.9978, C₁₅H₈N₃OS³⁵ClNa

requires 335.9969.

4.4.1.9. 8-Bromo-3-(3⁰-chlorophenyl)pyrido[3⁰,2⁰:4,5]thieno[3,2-d]

pyrimidin-4(3H)-one 8i. The reaction was carried out according to

general procedure C using carboxamide 4i (0.15 g, 0.39 mmol) with

heating at 70 ^ꢀC for 15 h to give the title product 15i (0.13 g, 82%) as a

white solid. m.p. 287e290 ^ꢀC; ¹H NMR (400 MHz; CDCl₃) 7.36e7.39

(1H, m, ArH), 7.50e7.54 (3H, m, ArH), 8.25 (1H, s, H-2), 8.72 (1H, d,

J ¼ 2.2 Hz, H-9), 8.85 (1H, d, J ¼ 2.2 Hz, H-7); IR: y_max(ﬁlm)/cm^ꢂ1

:

3095, 3064, 1683, 1563, 1528, 1423, 1369, 1218; m/z (ESIþ): 417

(

⁸¹Br³⁷Cl MNa^þ, 28%), 415 (⁸¹Br³⁵Cl MNa^þand ⁷⁹Br³⁷Cl MNa^þ,

100%), 413 (⁷⁹Br³⁵Cl MNa^þ, 65); HRMS (ESI^þ) found (⁸¹Br³⁷Cl

MNa^þ): 417.9023, C₁₅H₇N₃OS⁸¹Br³⁷ClNa requires 417.9024. Found

(

⁷⁹Br³⁵Cl MNa^þ): 413.9074, C₁₅H₇N₃OS⁷⁹Br³⁵ClNa requires

4.4.1.5. 8-Chloro-3-(4⁰-methoxyphenyl)pyrido[3⁰,2⁰:4,5]thieno[3,2-d]

pyrimidin-4(3H)-one 8e. The reaction was carried out according to

general procedure C using carboxamide 4e (0.10 g, 0.30 mmol) with

heating at 130 ^ꢀC for 4 h to give the title product 8e (0.10 g, 97%) as a

white solid. m.p. 287e288 ^ꢀC; ¹H NMR (400 MHz; CDCl₃) 3.89 (3H,

s, OMe), 7.07 (2H, d, J ¼ 8.9 Hz, H-3⁰and H-5⁰), 7.37 (2H, d, J ¼ 8.9 Hz,

H-2⁰and H-6⁰), 8.27 (1H, s, H-2), 8.56 (1H, d, J ¼ 2.4 Hz, H-9), 8.75

(1H, d, J ¼ 2.4 Hz, H-7); IR: y_max(ﬁlm)/cm^ꢂ1: 3051, 3017, 2923, 2837,

1671, 1571, 1532, 1507, 1377, 1243, 1102; m/z (ESIþ): 368 (³⁷Cl MNa^þ,

36%), 366 (³⁵Cl MNa^þ, 100%); HRMS (ESI^þ) found (³⁷Cl MNa^þ):

413.9074.

4.4.1.10. Ethyl N-(2-(phenylcarbamoyl)furo[2,3-b]pyridin-3-yl)for-

mimidate 15a. The reaction was carried out according to general

procedure C using carboxamide 10a (0.10 g, 0.40 mmol) with stir-

ring at room temperature for 1 h to give the title product 15a (0.09 g,

77%) as a white solid. m.p. 238e240 ^ꢀC; ¹H NMR (400 MHz; CDCl₃)

1.50 (3H, t, J ¼ 7.1 Hz, CH₃), 4.53 (2H, q, J ¼ 7.1 Hz, OCH₂), 7.13e7.17

(1H, m, AreH), 7.34 (1H, dd, J ¼ 4.7, 7.7 Hz, H-5), 7.35e7.39 (2H, m,

AreH), 7.66e7.68 (2H, m, AreH), 8.06 (1H, dd, J ¼ 1.6, 7.7 Hz, H-4),

8.47 (1H, s, NCH), 8.51 (1H, dd, J ¼ 1.6, 4.7 Hz, H-6), 9.28 (1H, s, NH);

IR: y_max(ﬁlm)/cm^ꢂ1: 3247, 3100, 3069, 2978, 1674, 1619, 1595, 1541,

1443, 1277, 1245, 1149; m/z (ESIþ): 332 (MNa^þ, 100%); HRMS (ESI^þ)

found (MNa^þ): 332.1002, C₁₇H₁₅N₃O₃Na^þrequires 332.1006.

368.0056,

C (

₁₆H₁₀N₃O₂S³⁷ClNa requires 368.0046. Found ³⁵Cl

MNa^þ): 366.0083, C₁₆H₁₀N₃O₂S³⁵ClNa requires 366.0074.

4.4.1.6. 8-Chloro-3-(3⁰-chlorophenyl)pyrido[3⁰,2⁰:4,5]thieno[3,2-d]

pyrimidin-4(3H)-one 8f. The reaction was carried out according to

general procedure C using carboxamide 4f (0.10 g, 0.30 mmol) with

heating at 130 ^ꢀC for 4 h to give the title product 8f (0.10 g, 99%) as a

white solid. m.p. 268e270 ^ꢀC; ¹H NMR (400 MHz; CDCl₃) 7.37e7.39

(1H, m, ArH), 7.51e7.54 (3H, m, ArH), 8.25 (1H, s, H-2), 8.57 (1H, d,

4.4.1.11. Ethyl N-(2-((4⁰-methoxyphenyl)carbamoyl)furo[2,3-b]pyr-

idin-3-yl)formimidate 15b. The reaction was carried out according

to general procedure C using carboxamide 10b (0.10 g, 0.35 mmol)

with stirring at room temperature for 1 h to give the title product

15b (0.63 g, 53%) as a green solid. m.p. 255e257 ^ꢀC; ¹H NMR

(400 MHz; CDCl₃) 1.49 (3H, t, J ¼ 7.1 Hz, CH₃), 3.81 (3H, s, OMe), 4.50

(2H, q, J ¼ 7.1 Hz, CH₂), 6.90 (2H, d, J ¼ 8.8 Hz, H-3⁰and H-5⁰), 7.31

J ¼ 2.4 Hz, H-9), 8.77 (1H, d, J ¼ 2.4 Hz, H-7); IR: y_max(ﬁlm)/cm^ꢂ1

:

3673, 3077, 2978, 2901, 1669, 1561, 1525, 1212, 1096, 1055; m/z

(ESIþ): 373 (³⁷Cl₂MNa^þ, 13%), 371 (³⁵Cl³⁷Cl MNa^þ, 67%), 369 (³⁵Cl₂

J.M. Hung et al. / European Journal of Medicinal Chemistry 86 (2014) 420e437

431

(1H, dd, J ¼ 4.8, 7.8 Hz, H-5), 7.56 (2H, d, J ¼ 8.8 Hz, H-2⁰and H-6⁰),

8.04 (1H, dd, J ¼ 1.4, 7.8 Hz, H-4), 8.46 (1H, s, NCH⁰), 8.47 (1H, dd,

J ¼ 1.4, 4.8 Hz, H-6), 9.19 (1H, s, NH); IR: y_max(ﬁlm)/cm^ꢂ1: 3249,

3071, 2980, 2937, 2838, 1666, 1623, 1545, 1410, 1238, 1148; m/z

(ESIþ): 362 (MNa^þ, 100%). HRMS (ESI^þ) found (MNa^þ): 362.1106,

procedure E using carboxamide 4a (0.10 g, 0.37 mmol) to give the

title product 19a (0.08 g, 78%) as a white solid. m.p. 210e212 ^ꢀC; ¹H

NMR (400 MHz; CDCl₃) 7.52e7.71 (6H, m, AreH and H-8), 8.84 (1H,

d, J ¼ 8.0 Hz, H-9), 8.90 (1H, d, J ¼ 8.0 Hz, H-7); ¹³C NMR (100 MHz;

CDCl₃) 121.6 (C-8), 126.1 (AreCH), 126.9 (C-4a), 128.8 (C-9a) 129.2

(AreCH), 129.6 (AreCH), 132.2 (C-9), 138.3 (AreC), 146.3 (C-9b),

151.9 (C-7), 153.1 (C-5a), 162.8 (C-4); IR: y_max(ﬁlm)/cm^ꢂ1: 3052,

1680, 1585, 1512, 1368, 1269, 1041; m/z (ESIþ): 303 (MNa^þ, 100%);

HRMS (ESI^þ) found (MNa^þ): 303.0309, C₁₄H₈N₄OSNa requires

303.0311.

C

₁₈H₁₇N₃O₄Na requires 362.1111.

4.4.1.12. Ethyl N-(2-((3⁰-chlorophenyl)carbamoyl)furo[2,3-b]pyridin-

3-yl)formimidate 15c. The reaction was carried out according to

general procedure C using carboxamide 9c (0.10 g, 0.35 mmol) with

heating at 80 ^ꢀC for 1 h to give the title product 16c (0.07 g, 58%) as a

white solid. m.p. 239e241 ^ꢀC; ¹H NMR (400 MHz; CDCl₃) 1.52 (3H, t,

J ¼ 7.2 Hz, CH₃), 4.54 (2H, q, J ¼ 7.2 Hz, OCH₂), 7.12 (1H, d, J ¼ 8.0 Hz,

H-4⁰), 7.27e7.31 (1H, m, H-5⁰), 7.34 (1H, dd, J ¼ 4.8, 8.0 Hz, H-5), 7.49

(1H, d, J ¼ 8.0 Hz, H-6⁰), 7.80 (1H, s, H-2⁰), 8.07 (1H, dd, J ¼ 1.8,

8.0 Hz, H-4), 8.47 (1H, s, NCH), 8.52 (1H, dd, J ¼ 1.8, 4.8 Hz, H-6),

9.32 (1H, s, NH); IR: y_max(ﬁlm)/cm^ꢂ1: 3098, 3070, 2977, 1681, 1622,

1593,1542,1478, 1410,1276,1242, 1150; m/z (ESIþ): 368 (³⁷ClMNa^þ,

31%), 366 (³⁵ClMNa^þ, 100%). HRMS (ESI^þ) found (³⁷Cl MNa^þ):

368.0583, C₁₇H₁₄N₃O³₃⁷ClNa requires 368.0588. Found (³⁵Cl MNa^þ):

366.0614, C₁₇H₁₄N₃O³₃⁵ClNa requires 366.0616.

4.5.1.2. 3-(4⁰-Methoxyphenyl)pyrido[3⁰,2⁰:4,5]thieno[3,2-d][1,2,3]tri-

azin-4(3H)-one 9b. The reaction was carried out according to

general procedure E using carboxamide 4b (0.10 g, 0.33 mmol) to

give the title product 19b (0.10 g, 99%) as a white solid. m.p.

238e240 ^ꢀC; ¹H NMR (400 MHz; CDCl₃) 3.90 (3H, s, OMe) 7.08 (2H,

d, J ¼ 8.9 Hz, H-3⁰and H-5⁰), 7.61 (2H, d, J ¼ 8.9 Hz, H-2⁰and H-6⁰),

7.65 (1H, dd, J ¼ 4.7, 8.0 Hz, H-8), 8.84 (1H, dd, J ¼ 1.6, 8.0 Hz, H-9),

8.89 (1H, dd, J ¼ 1.6, 4.7 Hz, H-7); IR: y_max(ﬁlm)/cm^ꢂ1: 3075, 3009,

2969, 2940, 2833, 1673, 1588, 1503, 1365, 1241, 1174; m/z (ESIþ):

333 (MNa^þ, 100%); HRMS (ESI^þ) found (MNa^þ): 333.0416,

C₁₅H₁₀N₄O₂SNa requires 333.0417.

4.4.2. General procedure D: synthesis of furopyrimidinones 14 from

formimidates 15

4.5.1.3. 3-(3⁰-Chlorophenyl)pyrido[3⁰,2⁰:4,5]thieno[3,2-d][1,2,3]tri-

azin-4(3H)-one 9c. The reaction was carried out according to gen-

eral procedure E using carboxamide 4c (0.10 g, 0.33 mmol) to give

the title product 9c (0.10 g, 98%) as a white solid; m.p. 273e274 ^ꢀC;

¹H NMR (400 MHz; CDCl₃) 7.52e7.53 (2H, m, H-4⁰and H-5⁰),

7.64e7.68 (2H, m, H-5 and H-6⁰), 7.75 (1H, s, H-2⁰), 8.85 (1H, d,

A mixture of formimidates 15 (1.0 mmol) and anhydrous sodium

carbonate (1.05 mmol) in absolute ethanol (3 mL) was stirred at

reﬂux for 18 h. The mixture was then cooled to room temperature

and the solvent removed in vacuo. The resultant crude solid was

washed with small amounts of water and the remaining solid was

recrystallized, using methanol to give the furopyrimidinone 14.

J ¼ 7.5 Hz, H-9), 8.92 (1H, d, J ¼ 7.5 Hz, H-7); IR: y_max(ﬁlm)/cm^ꢂ1

:

3089, 3055, 1687, 1587, 1511, 1472, 1370, 1044; m/z (ESIþ): 338 (³⁷Cl

4.4.2.1. 3-Phenylpyrido[3⁰,2⁰:4,5]furo[3,2-d]pyrimidin-4(3H)-one

14a. The reaction was carried out according to general procedure D

using formimidate 15a (0.10 g, 0.32 mmol) to give the title product

14a (0.01 g, 12%) as a white solid. m.p. 240e242 ^ꢀC; ¹H NMR

(400 MHz; CDCl₃) 7.46e7.62 (6H, m, AreH and H-8), 8.25 (1H, s, H-

2), 8.48 (1H, dd, J ¼ 1.7, 7.7 Hz, H-4), 8.67 (1H, dd, J ¼ 1.7, 4.7 Hz, H-

6); IR: y_max(ﬁlm)/cm^ꢂ1: 3060, 2988, 2919, 1700, 1564, 1527, 1490,

1397, 1347, 1185, 1112; m/z (ESIþ): 286 (MNa^þ, 100%); HRMS (ESI^þ)

found (MNa^þ): 286.0584, C₁₅H₉N₃O₂Na requires 286.0587.

MNa^þ, 33%), 336 (³⁵Cl MNa^þ,100); HRMS (ESI^þ): Found (³⁷Cl MNa^þ)

338.9894,

C (

₁₄H₉N₄OS³⁷ClNa^þrequires 338.9892. Found ³⁵Cl

MNa^þ) 336.9915, C₁₄H₉N₄OS³⁵ClNa requires 336.9921.

4.5.1.4. 8-Chloro-3-phenylpyrido[3⁰,2⁰:4,5]thieno[3,2-d][1,2,3]triazin-

4(3H)-one 9d. The reaction was carried out according to general

procedure E using carboxamide 4d (0.02 g, 0.07 mmol) to give the

title product 9d (0.01 g, 43%) as a white solid; m.p. 219e222 ^ꢀC; ¹H

NMR (400 MHz; CDCl₃) 7.53e7.62 (3H, m, H-3⁰, H-4⁰and H-5⁰),

7.68e7.70 (2H, m, H-2⁰and H-6⁰), 8.83e8.86 (2H, m, H-7 and H-9);

IR: y_max(ﬁlm)/cm^ꢂ1: 3671, 3333, 2987, 2903,1684,1406,1393,1252,

1056, 864; m/z (ESIþ): 338 (³⁷Cl MNa^þ, 33%), 336 (³⁵Cl MNa^þ,

4.4.2.2. 3-(4-Methoxyphenyl)pyrido[3⁰,2⁰:4,5]furo[3,2-d]pyrimidin-

4(3H)-one 14b. The reaction was carried out according to general

procedure D using formimidate 15b (0.10 g, 0.30 mmol) to give the

title product 18b (0.02 g, 20%) as a green solid. m.p. 260e263 ^ꢀC; ¹H

NMR (400 MHz; CDCl₃) 3.89 (3H, s, OMe), 7.07 (2H, d, J ¼ 8.8 Hz, H-

3⁰and H-5⁰), 7.37 (2H, d, J ¼ 8.8 Hz, H-2⁰and H-6⁰), 7.50 (1H, dd,

J ¼ 4.9, 7.7 Hz, H-8), 8.24 (1H, s, H-2), 8.47 (1H, dd, J ¼ 1.6, 7.7 Hz, H-

9), 8.67 (1H, dd, J ¼ 1.6, 4.9 Hz, H-7); IR: y_max(ﬁlm)/cm^ꢂ1: 3064,

3008, 2919, 2847, 1703, 1606, 1564, 1507, 1398, 1302, 1234, 1110; m/z

(ESIþ): 316 (MNa^þ, 100%); HRMS (ESI^þ) found (MNa^þ): 316.0687,

100%);

C₁₄H₇N₄OS³⁷ClNa requires 338.9892. Found (³⁵Cl MNa^þ): 336.9921,

₁₄H₇N₄OS³⁵ClNa requires 336.9921.

HRMS

(ESI^þ)

found

(

³⁷Cl

MNa^þ):

338.9891,

C

4.5.1.5. 8-Chloro-3-(4⁰-methoxyphenyl)pyrido[3⁰,2⁰:4,5]thieno[3,2-d]

[1,2,3]triazin-4(3H)-one 9e. The reaction was carried out according

to general procedure E using carboxamide 4e (0.15 g, 0.45 mmol) to

give the title product 9e (0.16 g, 99%) as a green solid. m.p.

289e290 ^ꢀC; ¹H NMR (400 MHz; CDCl₃) 3.90 (3H, s, OMe) 7.09 (2H,

d, J ¼ 8.9 Hz, H-3⁰and H-5⁰), 7.61 (2H, d, J ¼ 8.9 Hz, H-2⁰and H-6⁰),

8.80 (1H, d, J ¼ 2.4 Hz, H-9), 8.83 (1H, d, J ¼ 2.4 Hz, H-7); IR: y_max

(ﬁlm)/cm^ꢂ1: 3396, 3072, 2991, 2837, 1682, 1606, 1505, 1252, 1176,

1108, 1033; m/z (ESIþ): 369 (³⁷ClMNa^þ, 37%), 367 (³⁵ClMNa^þ, 100);

HRMS (ESI^þ) found (³⁷Cl MNa^þ): 369.0000, C₁₅H₉N₄O₂S³⁷ClNa re-

quires 368.9998. Found (³⁵Cl MNa^þ): 367.0026, C₁₅H₉N₄O₂S³⁵ClNa^þ

requires 367.0027.

C

₁₆H₁₁N₃O₃Na requires 316.0693.

4.5. Synthesis of the triazinones 9

4.5.1. General procedure E: synthesis of the triazinones 9 from

thienopyridine-2-carboxamides 4

To a solution of thienopyridine-2-carboxamides 4 (1.0 mmol) in

acetic acid (2 mL) at 0 ^ꢀC, was added a solution of sodium nitrite

(10 mmol) in water (3 mL) dropwise over 15 min. After 2 h the

resultant solid was collected by ﬁltration and recrystallized from

ethanol to give triazinones 9.

4.5.1.6. 8-Chloro-3-(3⁰-chlorophenyl)pyrido[3⁰,2⁰:4,5]thieno[3,2-d]

[1,2,3]triazin-4(3H)-one 9f. The reaction was carried out according

to general procedure E using carboxamide 4f (0.10 g, 0.29 mmol) to

give the title product 9f (0.10 g, 99%) as a white solid. m.p.

258e260 ^ꢀC; ¹H NMR (400 MHz; CDCl₃) 7.52e7.54 (2H, m, ArH),

4.5.1.1. 3-Phenylpyrido[3⁰,2⁰:4,5]thieno[3,2-d][1,2,3]triazin-4(3H)-

one 9a. The reaction was carried out according to general

432

J.M. Hung et al. / European Journal of Medicinal Chemistry 86 (2014) 420e437

7.61e7.64 (1H, m, ArH), 7.74e7.75 (1H, m, ArH), 8.81 (1H, d,

white solid. m.p. 135e137 ^ꢀC. lit. m.p. 130e131 [34]. ¹H NMR

(400 MHz; CDCl₃) 4.03 (2H, s, H-2), 7.15e7.19 (1H, m, AreH), 7.36

(2H, d, J ¼ 8.0 Hz, AreH), 7.53 (2H, d, J ¼ 8.0 Hz, AreH), 8.10 (1H, s,

NH). The ¹H NMR data was in agreement with the literature values

[35].

J ¼ 2.4 Hz, H-9), 8.85 (1H, d, J ¼ 2.4 Hz, H-7); IR: y_max(ﬁlm)/cm^ꢂ1

:

3335, 3053,1689, 1582,1505,1331,1253, 1107,1044; m/z (ESIþ): 374

(

³⁷Cl₂MNa^þ, 18%), 372 (³⁵Cl³⁷Cl MNa^þ, 65%), 370 (³⁵Cl₂MNa^þ,

100%); HRMS (ESI^þ) found ³⁷Cl₂MNa^þ): 374.9446,

₁₄H₆N₄OS³⁷Cl₂Na requires 374.9473. Found ³⁵Cl₂MNa^þ):

370.9522, C₁₄H₆N₄OS³⁵Cl₂Na requires 370.9532.

(

C

(

4.6.1.2. 2-Bromo-N-(4⁰-methoxyphenyl)acetamide 21b. The reaction

was carried out in accordance with general procedure F using p-

anisidine 22b (0.09 mL, 0.81 mmol) with the brown crude product

being further puriﬁed by trituration (DCM) to give the title product

21b (0.12 g, 52%) as a white solid. m.p. 129e132 ^ꢀC. lit. m.p.

131e132 ^ꢀC [36], ¹H NMR (400 MHz; CDCl₃) 3.80 (3H, s, OCH₃), 4.02

(2H, s, H-2), 6.89 (2H, d, J ¼ 9.0 Hz, H-3⁰and H-5⁰), 7.42 (2H, d,

J ¼ 9.0 Hz, H-2⁰and H-6⁰), 8.05 (1H, s, NH). The ¹H NMR data was in

agreement with the literature values [36].

4.5.1.7. 8-Bromo-3-phenylpyrido[3⁰,2⁰:4,5]thieno[3,2-d][1,2,3]triazin-

4(3H)-one 9g. The reaction was carried out according to general

procedure E using carboxamide 4g (0.06 g, 0.17 mmol) to give the

title product 9g (0.03 g, 50%) as a white solid. m.p. 218e220 ^ꢀC; ¹H

NMR (400 MHz; CDCl₃) 7.53e7.62 (3H, m, AreH), 7.67e7.70 (2H, m,

AreH), 8.93 (1H, d, J ¼ 2.2 Hz, H-9), 8.97 (1H, d, J ¼ 2.2 Hz, H-7); IR:

y_max(ﬁlm)/cm^ꢂ1: 3048, 2029, 2850, 1679, 1502, 1486, 1391, 1327,

1250,1097,1040; m/z (ESIþ): 382 (⁸¹Br MNa^þ, 96%), 380 (⁷⁹BrMNa^þ,

100%);

C

HRMS

₁₄H₇N₄OS⁸¹BrNa requires 382.9396. Found (⁷⁹Br MNa^þ): 380.9410,

₁₄H₇N₄OS⁷⁹BrNa requires 380.9416.

(ESI^þ)

found

(

⁸¹Br

MNa^þ)

382.9389,

4.6.1.3. 2-Bromo-N-(3⁰-chlorophenyl)acetamide 21c. The reaction

was carried out in accordance with general procedure F using 3-

chloroaniline 22c (0.10 g, 0.78 mmol) to give the title product 21c

(0.20 g, 100%) as a light orange solid. m.p. 80e83 ^ꢀC. lit. m.p.

80e81 ^ꢀC [36]. ¹H NMR (400 MHz; CDCl₃) 4.02 (2H, s, H-2), 7.15 (1H,

d, J ¼ 8.0 Hz, H-4⁰), 7.26e7.30 (1H, m, H-5⁰), 7.39 (1H, d, J ¼ 8.0 Hz,

H-6⁰), 7.65 (1H, s, H-2⁰), 8.12 (1H, s, NH). The ¹H NMR data was in

agreement with the literature values [36].

4.5.1.8. 8-Bromo-3-(4⁰-methoxyphenyl)pyrido[3⁰,2⁰:4,5]thieno[3,2-d]

[1,2,3]triazin-4(3H)-one 9h. The reaction was carried out according

to general procedure E using carboxamide 4h (0.10 g, 0.26 mmol),

acetic acid (0.52 mL, 9.10 mmol) and sodium nitrite (0.50 g,

7.25 mmol) in water (2 mL) to give the title product 9h (0.10 g, 99%)

as a green solid. m.p. 281e282 ^ꢀC; ¹H NMR (400 MHz; CDCl₃) 3.90

(3H, s, OMe), 7.09 (2H, d, J ¼ 7.8 Hz, H-3⁰and H-5⁰), 7.61 (2H, d,

J ¼ 7.8 Hz, H-2⁰and H-6⁰), 8.92 (1H, d, J ¼ 2.4 Hz, H-9), 8.96 (1H, d,

J ¼ 2.4 Hz, H-7); IR: y_max(ﬁlm)/cm^ꢂ1: 3670, 3073, 2979, 2906, 2835,

1682, 1606, 1505, 1254, 1177, 1035; m/z (ESIþ): 412 (⁸¹Br MNa^þ,

93%), 410 (⁷⁹Br MNa^þ, 100%); HRMS (ESI^þ) found (⁸¹Br MNa^þ):

4.6.1.4. 2-Bromo-N-(3⁰-methoxyphenyl)acetamide 21d. The reaction

was carried out in accordance with general procedure F using 3-

methoxyaniline 22d (0.09 mL) to give the title product 21d

(0.15 g, 78%) as a brown solid. m.p. 87e89 ^ꢀC. lit. m.p. 98.5e99.5 ^ꢀC

[37]. ¹H NMR (400 MHz; CDCl₃) 3.81 (3H, s, OCH₃), 4.01 (2H, s, H-2),

6.72 (1H, d, J ¼ 8.1 Hz, H-4⁰), 7.01 (1H, d, J ¼ 8.1 Hz, H-6⁰), 7.22e7.26

(2H, m, H-2⁰and H-5⁰), 8.12 (1H, s, NH). ¹³C NMR (100 MHz; CDCl₃)

29.5 (C-2), 55.4 (OCH₃), 105.8 (C-2⁰), 111.0 (C-4⁰), 112.2 (C-6⁰), 129.8

(C-5⁰), 138.1 (C-1⁰), 160.2 (C-3⁰), 163.4 (C]O). The ¹H NMR data was

in agreement with the literature values [37].

412.9500,

C (

₁₅H₉N₄O₂S⁸¹BrNa^þrequires 412.9502. Found ⁷⁹Br

MNa^þ): 410.9515, C₁₅H₉N₄O₂S⁷⁹BrNa requires 410.9522.

4.5.1.9. 8-Bromo-3-(3⁰-chlorophenyl)pyrido[3⁰,2⁰:4,5]thieno[3,2-d]

[1,2,3]triazin-4(3H)-one 9i. The reaction was carried out according

to general procedure E using carboxamide 4i (0.10 g, 0.26 mmol),

acetic acid (0.52 mL, 9.14 mmol) and sodium nitrite (0.50 g,

7.25 mmol) in water (2 mL) to give the title product 9i (0.08 g, 82%)

as a white solid. m.p. 292e293 ^ꢀC; ¹H NMR (400 MHz; CDCl₃)

7.53e7.63 (3H, m, H-4⁰, H-5⁰and H-6⁰), 7.74 (1H, s, H-2⁰), 8.94 (1H, d,

4.6.1.5. 2-Bromo-N-(3⁰-bromophenyl)acetamide 21e. The reaction

was carried out in accordance with general procedure F using 3-

bromoaniline 4e (0.40 g, 2.33 mmol) to give the title product 21e

(0.68 g, 99%) as a yellow solid. m.p. 97e100 ^ꢀC. ¹H NMR (400 MHz;

CDCl₃) 4.02 (2H, s, H-2), 7.22 (1H, d, J ¼ 8.0 Hz, H-4⁰), 7.29e7.31 (1H,

m, H-5⁰), 7.45 (1H, d, J ¼ 8.0 Hz, H-6⁰), 7.78 (1H, s, H-2⁰), 8.13 (1H, s,

J ¼ 2.2 Hz, H-9), 8.97 (1H, d, J ¼ 2.2 Hz, H-7); IR: y_max(ﬁlm)/cm^ꢂ1

:

13

3671, 3099, 3054, 2978, 2902, 1689, 1579, 1503, 1412, 1252, 1097,

1048; m/z (ESIþ): 418 (⁸¹Br³⁷Cl MNa^þ, 29%), 416 (⁸¹Br³⁵Cl MNa^þ

and ⁷⁹Br³⁷ClMNa^þ, 100%), 414 (⁷⁹Br³⁵Cl MNa^þ, 78); HRMS (ESI^þ)

found (⁸¹Br³⁷Cl MNa^þ): 418.8983, C₁₄H₆N₄OS⁸¹Br³⁷ClNa requires

418.8988. Found (⁷⁹Br³⁵Cl MNa^þ): 414.9037, C₁₄H₆N₄OS⁷⁹Br³⁵ClNa

requires 414.9037.

NH); C NMR (100 MHz; CDCl₃) 29.3 (C-2), 118.5 (C-6⁰), 122.8 (C-

3⁰), 123.0 (C-2⁰), 128.3 (C-4⁰), 130.4 (C-5⁰), 138.2 (C-1⁰), 163.4 (C-1);

IR: y_max(ﬁlm)/cm^ꢂ1: 3249, 3182, 3107, 3070, 1649, 1606, 1589, 1537,

1471, 1421, 1235; m/z (ESIþ): 317 (⁸¹Br₂MNa^þ, 50%), 315 (⁷⁹Br⁸¹Br

MNa^þ, 100%), 313 (⁷⁹Br₂MNa^þ, 55%); HRMS (ESI^þ) found (⁸¹Br₂

MNa^þ): 317.8751, C₈H₇NO⁸¹Br₂Na requires 317.8746. Found (⁷⁹Br₂

MNa^þ) 313.8794, C₈H₇NO⁷⁹Br₂Na requires 313.8787.

4.6. Synthesis of the tetrahydrothieno[2,3-b]quinoline-2-

carboxamides 17and 23

4.6.1.6. 2-Bromo-N-(3⁰-nitrophenyl)acetamide 21f. The reaction was

carried out in accordance with general procedure F using 3-

nitroaniline 4f (0.10 g, 0.72 mmol) to give the title product 21f

(0.18 g, 93%) as a brown solid. m.p. 112e114 ^ꢀC. lit. m.p. 110e112

[38]. ¹H NMR (400 MHz; (CD₃)₂CO) 4.11 (2H, s, H-2), 7.62e7.67 (1H,

m, H-5⁰), 7.98e8.01 (2H, m, H-4⁰and H-6⁰), 8.72 (1H, s, H-2⁰), 10.02

(1H, s, NH). The ¹H NMR data was in agreement with the literature

values [38].

4.6.1. General procedure F: formation of the N-aryl

bromoacetamides 21aei

To a solution of substituted aniline 22aei (0.81 mmol) and

triethylamine (0.89 mmol) in DCM (2 mL) at 0 ^ꢀC was added bro-

moacetyl bromide (0.81 mmol) dropwise over 15 min, and stirring

was continued for an additional 1 h at 0 ^ꢀC. The mixture was diluted

with DCM (10 mL), washed with HCl (2 ꢁ 10 mL), H₂O (1 ꢁ 10 mL),

sat. aqueous NaHCO₃(1 ꢁ10 mL), brine (1 ꢁ10 mL), dried (Na₂SO₄),

and the solvent removed in vacuo to give bromoacetamides 21aei.

4.6.1.7. 2-Bromo-N-(3⁰-(triﬂuoromethyl)phenyl)acetamide

21g.

The reaction was carried out in accordance with general procedure

F using 3-(triﬂuoromethyl)aniline 4g (0.40 g, 2.48 mmol) to give the

title product 21g (0.70 g, 100%) as a brown solid. m.p. 78e81 ^ꢀC. lit.

m.p. 82 [39]. ¹H NMR (400 MHz; CDCl₃) 4.04 (2H, s, H-2), 7.41e7.50

(2H, m, H-4⁰and H-5⁰), 7.75 (1H, d, J ¼ 8.0 Hz, H-6⁰), 7.83 (1H, s, H-

4.6.1.1. 2-Bromo-N-phenylacetamide 21a. The reaction was carried

out in accordance with general procedure F using aniline 22a

(0.10 mL, 1.07 mmol) to give the title product 21a (0.12 g, 51%) as a

J.M. Hung et al. / European Journal of Medicinal Chemistry 86 (2014) 420e437

433

2⁰), 8.28 (1H, s, NH). The ¹H data was in agreement with the liter-

ature values [40].

4.6.2.1. 3-Amino-5-oxo-N-phenyl-5,6,7,8-tetrahydrothieno[2,3-b]

quinoline-2-carboxamide 17a. The reaction was carried out in

accordance with general procedure G using 2-bromo-N-phenyl-

acetamide 21a (0.11 g, 0.49 mmol) to give the title product 17a

(0.07 g, 41%) as a yellow solid. m.p. 280e282 ^ꢀC. lit. m.p. 291e292 ^ꢀC

[13]. ¹H NMR (400 MHz; d₆-DMSO) 2.13e2.19 (2H, m, H-7), 2.73

(2H, t, J ¼ 6.2 Hz, H-6), 3.20 (2H, t, J ¼ 6.2 Hz, H-8), 7.07e7.12 (1H, m,

H-4⁰), 7.34 (2H, dd, J ¼ 1.0, 8.3 Hz, H-3⁰and H-5⁰), 7.59 (2H, s, NH₂),

7.69 (2H, dd, J ¼ 1.0, 8.3 Hz, H-2⁰and H-6⁰), 9.05 (1H, s, H-4), 9.50

(1H, s, NH); ¹³C NMR (100 MHz; d₆-DMSO) 21.2 (C-7), 32.3 (C-8),

38.1 (C-6), 96.1 (C-2) 121.2 (C-2⁰and C-6⁰), 123.5 (C-4⁰), 124.4 (C-4a),

125.2 (C-3a),128.4 (C-3⁰and C-5⁰), 130.0 (C-4), 138.8 (C-1⁰), 147.4 (C-

3), 162.1 (C-9a), 163.7 (NC]O), 164.0 (C-8a), 197.0 (C-5); IR: y_max

(ﬁlm)/cm^ꢂ1: 3408, 3348, 3292, 3048, 2954, 2884, 1676, 1627, 1584,

1528, 1433, 1315, 1232; m/z (ESIþ): 360 (MNa^þ, 100%); HRMS (ESI^þ)

found (MNa^þ) 360.0777, C₁₈H₁₅N₃O₂SNa requires 360.0777.

4.6.1.8. 2-Bromo-N-(3⁰,5⁰-dimethoxyphenyl)acetamide 21h. The re-

action was carried out in accordance with general procedure F us-

ing 3,5-dimeoxyaniline 4h (0.40 g, 2.61 mmol) to give the title

product 21h (0.70 g, 100%) as an orange solid. m.p. 90e93 ^ꢀC. ¹H

NMR (400 MHz; CDCl₃) 3.78 (6H, s, OCH₃), 4.00 (2H, s, H-2), 6.28

(1H, t, J ¼ 2.2 Hz, H-4⁰), 6.76 (2H, d, J ¼ 2.2 Hz, H-2⁰, and H-6⁰), 8.08

(1H, s, NH); ¹³C NMR (100 MHz; CDCl₃) 29.5 (C-2), 55.4 (OCH₃), 97.5

(C-4⁰), 98.4 (C-2⁰and C-6⁰), 138.7 (C-1⁰), 161.1 (C-3⁰and C-5⁰), 163.7

(C-1); IR: y_max(ﬁlm)/cm^ꢂ1: 3267, 3222, 3161, 3099, 2957, 2840,

1670, 1598, 1555, 1418, 1393, 1299, 1200, 1151; m/z (ESIþ): 297 (⁸¹Br

MNa^þ, 91%), 295

(

⁷⁹Br MNa^þ, 100%); HRMS (ESI^þ) found

⁸¹BrMNa^þ): 297.9883, C₁₀H₁₂NO⁸₃¹BrNa requires 297.9876. Found

⁷⁹Br MNa^þ) 295.9900, C₁₀H₁₂NO⁷₃⁹BrNa^þrequires 295.9893.

4.6.2.2. 3-Amino-N-(4⁰-methoxyphenyl)-5-oxo-5,6,7,8-

tetrahydrothieno[2,3-b]quinoline-2-carboxamide 17b. The reaction

was carried out in accordance with general procedure G using 2-

bromo-N-(4-methoxyphenyl)acetamide 21b (0.12 g, 0.49 mmol)

to give the title product 17b (0.03 g, 14%) as a yellow solid. m.p.

321e324 ^ꢀC. ¹H NMR (400 MHz; d₆-DMSO) 2.12e2.19 (2H, m, H-7),

2.73 (2H, t, J ¼ 6.4 Hz, H-6), 3.20 (2H, t, J ¼ 6.4 Hz, H-8), 3.75 (3H, s,

OCH₃), 6.91 (2H, d, J ¼ 9.0 Hz, H-3⁰and H-5⁰), 7.51 (2H, s, NH₂), 7.56

(2H, d, J ¼ 9.0 Hz, H-2⁰and H-6⁰), 9.02 (1H, s, H-4), 9.41 (1H, s, NH);

¹³C NMR (100 MHz; DMSO-d6) 21.2 (C-7), 32.3 (C-8), 38.1 (C-6), 55.1

(OCH₃) 96.3 (C-2) 113.6 (C-3⁰and C-5⁰), 123.0 (C-2⁰and C-6⁰), 124.4

(C-4a), 125.2 (C-3a), 129.9 (C-4) 131.6 (C-1⁰), 147.1 (C-3), 155.6 (C-4⁰)

162.1 (C-9a) 163.5 (NC]O), 164.0 (C-8a), 197.1 (C-5); IR: y_max(ﬁlm)/

cm^ꢂ1: 3428, 3332, 2952, 1674, 1593, 1507, 1462, 1408, 1231, 1033; m/

z (ESIþ): 390 (MNa^þ, 100%); HRMS (ESI^þ) found (MNa^þ): 390.0874,

4.6.1.9. 2-Bromo-N-(3⁰,5⁰-dichlorophenyl)acetamide 21i. The reac-

tion was carried out in accordance with general procedure F using

3,5-dichloroaniline 4i (0.50 g, 3.09 mmol) to give the title product

21i (0.70 g, 100%) as a brown solid. m.p. 90e95 ^ꢀC. ¹H NMR

(400 MHz; CDCl₃) 3.78 (6H, s, OCH₃), 4.00 (2H, s, H-2), 6.28 (1H, t,

J ¼ 2.2 Hz, H-4⁰), 6.76 (2H, d, J ¼ 2.2 Hz, H-2⁰, and H-6⁰), 8.08 (1H, s,

13

NH). C NMR (100 MHz; CDCl₃) 29.1 (C-2), 118.2 (C-2⁰and C-6⁰),

125.2 (C-4⁰), 135.4 (C-3⁰and C-5⁰), 138.7 (C-1⁰), 163.6 (C-1); IR: y_max

(ﬁlm)/cm^ꢂ1: 3297, 3260, 3183, 3121, 3081, 1666, 1586, 1536, 1437,

1409, 1313, 1113; m/z (ESIþ): 309 (³⁷Cl₂⁸¹Br MNa^þ, 5%), 307

(

³⁵Cl³⁷Cl⁸¹Br MNa^þand ³⁷Cl⁷₂⁹Br MNa^þ, 42%), 305 (³⁵Cl₂⁸¹Br MNa^þ

and ³⁵Cl³⁷Cl⁷⁹Br MNa^þ, 100%), 303 (³⁵Cl⁷₂⁹Br MNa^þ, 62%); HRMS

(ESI^þ) found (³⁷Cl⁸₂¹Br MNa^þ): 309.8831, C₈H₆³⁷Cl₂⁸¹BrNONa requires

309.8823. Found (³⁵Cl⁷₂⁹Br MNa^þ): 303.8910, C₈H₆³⁵Cl⁷₂⁹BrNONa re-

quires 303.8902.

C

₁₉H₁₇N₃O₃SNa^þrequires 390.0883.

4. 6. 2. 3. 3-Amin o-N -(3⁰-chlorophenyl)-5-o xo-5 , 6 , 7 , 8 -

tetrahydrothieno[2,3-b]quinoline-2-carboxamide 17c. The reaction

was carried out in accordance with general procedure G using 2-

bromo-N-(3-chlorophenyl)acetamide 21c (0.12 g, 0.49 mmol) to

give the title product 17c (0.14 g, 77%) as a yellow solid. m.p.

276e278 ^ꢀC. ¹H NMR (400 MHz; d₆-DMSO) 2.12e2.19 (2H, m, H-7),

2.73 (2H, t, J ¼ 6.2 Hz, H-6), 3.20 (2H, t, J ¼ 6.2 Hz, H-8), 7.13 (1H, d,

J ¼ 8.0 Hz, H-4⁰), 7.33e7.37 (1H, m, H-5⁰), 7.64 (3H, d, J ¼ 8.0 Hz, H6⁰

and NH₂), 7.90 (1H, s, H-2⁰), 9.04 (1H, s, H-4), 9.65 (1H, s, NH). The

¹H NMR data was in agreement with the literature values [11].

4.6.1.10. 5-Oxo-2-thioxo-1,2,5,6,7,8-hexahydroquinoline-3-

carbonitrile 18. A mixture of 1,3-cyclohexanedione 19 (0.50 g,

4.46 mmol) and dimethyl formamide dimethyl acetyl (0.59 mL,

4.46 mmol) in DMF (15 mL) was stirred, under an atmosphere of

nitrogen, for 24 h at room temperature to form enamine 20.

Separately, a solution of sodium hydride (0.21 g, 8.92 mmol) and

cyanothioacetamide (0.45 g, 4.46 mmol) in DMF (15 mL) was stirred

for 10 min under an atmosphere of nitrogen, at room temperature,

and then transferred into the enamine 20 mixture and stirred for a

further 24 h. The mixture was then acidiﬁed to pH 4 using conc. HCl

and stirred further for 24 h. The resultant solid was ﬁltered and

recrystallized from ethanol to give the title product 18 (0.73 g, 82%)

as a brown solid. m.p. >350 ^ꢀC. [lit. [15] m.p. >300 ^ꢀC]; ¹H NMR

(400 MHz; d₆-DMSO) 2.02e2.08 (2H, m, H-7), 2.51e2.54 (2H, m, H-

6), 2.99e3.02 (2H, m, H-8), 8.24 (1H, s, H-4), 14.4 (1H, s, NH); ¹³C

NMR (100 MHz; DMSO-d₆) 19.9 (C-8), 26.7 (C-9), 36.5 (C-7), 114.8

(CN) 116.2 and 118.2 (C-5 and C-3), 140.1 (C-4), 161.4 (C-2), 180.6 (C-

10), 192.5 (C]O). The ¹H NMR data was in agreement with the

literature values [15].

4.6.2.4. 3-Amino-N-(3⁰-methoxyphenyl)-5-oxo-5,6,7,8-

tetrahydrothieno[2,3-b]quinoline-2-carboxamide 17d. The reaction

was carried out in accordance with general procedure G using 2-

bromo-N-(3-methoxyphenyl)acetamide 21d (0.12 g, 0.49 mmol)

to give the title product 17d (0.14 g, 77%) as a yellow solid. m.p.

248e250 ^ꢀC. ¹H NMR (400 MHz; d₆-DMSO) 2.13e2.19 (2H, m, H-7),

2.73 (2H, t, J ¼ 6.4 Hz, H-6), 3.20 (2H, t, J ¼ 6.4 Hz, H-8), 3.76 (3H, s,

OCH₃), 6.67 (1H, d, J ¼ 8.1 Hz, H-4⁰), 7.21e7.25 (1H, m, H-5⁰), 7.32

(1H, d, J ¼ 8.1 Hz, H-6⁰), 7.38 (1H, s, H-2⁰), 7.59 (2H, s, NH₂), 9.04 (1H,

s, H-4), 9.46 (1H, s, NH); ¹³C NMR (100 MHz; d₆-DMSO) 21.2 (C-7),

32.4 (C-8), 38.1 (C-6), 55.0 (OCH₃) 96.0 (C-2), 106.6 (C-2⁰), 109.1 (C-

4⁰), 113.2 (C-6⁰), 124.4 (C-4a), 125.2 (C-3a), 129.1 (C-5⁰), 130.0 (C-4),

140.1 (C-1⁰), 147.6 (C-3), 159.3 (C-3⁰) 162.1 (C-9a), 163.7 (NC]O),

164.1 (C-8a), 197.1 (C-5); IR: y_max(ﬁlm)/cm^ꢂ1: 3416, 3325, 3046,

2945, 2842, 1673, 1631, 1582, 1519, 1428, 1243, 1158, 1044; m/z

(ESIþ): 390 (MNa^þ, 100%); HRMS (ESI^þ) found (MNa^þ): 390.0881,

4.6.2. General procedure G: synthesis of 5-oxo-tetrahydrothieno

[2,3-b]quinoline-2-carboxamides analogues 17aei

A mixture of 2-bromoacetamides 21aei (0.49 mmol), 5-oxo-2-

thioxo-1,2,5,6,7,8-hexahydroquinoline-3-carbonitrile

18

(0.49 mmol) and anhydrous sodium carbonate (0.52 mmol) in ab-

solute ethanol (2 mL) was stirred at reﬂux for 18 h. The mixture was

cooled to room temperature, and the solvent was removed in vacuo.

The crude product was washed using small amounts of water and

the remaining solid, ﬁltered and recrystallized from methanol to

give the 5-oxo-tetrahydrothieno[2,3-b]quinolines 17aei.

C₁₉H₁₇N₃O₃SNa requires 390.0883.

4. 6. 2. 5 . 3 -Amin o-N -(3⁰- b rom op h enyl) - 5 - oxo - 5, 6, 7 , 8 -

tetrahydrothieno[2,3-b]quinoline-2-carboxamide 17e. The reaction

434

J.M. Hung et al. / European Journal of Medicinal Chemistry 86 (2014) 420e437

was carried out in accordance with general procedure G using 2-

bromo-N-(3-bromophenyl)acetamide 21e (0.14 g, 0.49 mmol) to

give the title product 17e (0.18 g, 87%) as a yellow solid. m.p.

301e303 ^ꢀC. ¹H NMR (400 MHz; d₆-DMSO) 2.11e2.18 (2H, m, H-7),

2.72 (2H, t, J ¼ 6.2 Hz, H-6), 3.19 (2H, t, J ¼ 6.2 Hz, H-8), 7.23 (1H, d,

J ¼ 8.0 Hz, H-4⁰), 7.26e7.30 (1H, m, H-5⁰), 7.63 (2H, s, NH₂), 7.67 (1H,

d, J ¼ 8.0 Hz, H-6⁰), 8.04 (1H, s, H-2⁰), 9.03 (1H, s, H-4), 9.61 (1H, s,

NH); ¹³C NMR (100 MHz; d₆-DMSO) 21.2 (C-7), 32.4 (C-8), 38.1 (C-

6), 96.4 (C-2) 119.7 (C-6⁰), 121.2 (C-3⁰), 123.2 (C-2⁰), 124.4 (C-4a)

125.2 (C-3a) 125.6 (C-4⁰), 130.1 (C-4), 130.3 (C-5⁰), 141.3 (C-1⁰), 147.6

(C-3), 162.2 (C-9a) 163.9 (NC]O), 164.1 (C-8a), 197.0 (C-5); IR: y_max

(ﬁlm)/cm^ꢂ1: 3432, 3352, 3312, 1677, 1634, 1582, 1521, 1417, 1245,

1054; m/z (ESIþ): 440 (⁸¹Br MNa^þ, 46%), 438 (⁷⁹Br MNa^þ, 45%), 360

([MNa-Br]^þ, 100%). HRMS (ESI^þ) found (⁸¹Br MNa^þ): 439.9865,

130.1 (C-4), 140.7 (C-1⁰), 147.6 (C-3), 160.2 (C-3⁰and C-5⁰), 162.1 (C-

9a), 163.7 (NC]O), 164.1 (C-8a), 197.0 (C-5); IR: y_max(ﬁlm)/cm^ꢂ1

3467, 3348, 3279, 2941, 2838, 1686, 1633, 1580, 1540, 1476, 1450,

1424, 1267, 1156, 1066; m/z (ESIþ): 420 (MNa^þ, 100%); HRMS (ESI^þ)

found (MNa^þ): 420.0997, C₂₀H₁₉N₃O₄SNa requires 420.0988.

:

4.6.2.9. 3-Amino-N-(3⁰,5⁰-dichlorophenyl)-5-oxo-5,6,7,8-

tetrahydrothieno[2,3-b]quinoline-2-carboxamide 17i. The reaction

was carried out in accordance with general procedure G using 2-

bromo-N-(3,5-dichlorophenyl)acetamide 22i (0.50 g, 1.78 mmol)

to give the title product 17i (0.25 g, 35%) as a yellow solid. m.p.

269e270 ^ꢀC. ¹H NMR (400 MHz; d₆-DMSO) 2.12e2.15 (2H, m, H-7),

2.70 (2H, t, J ¼ 5.5 Hz, H-6), 3.17 (2H, t, J ¼ 5.5 Hz, H-8), 7.20 (1H, s,

H-4⁰), 7.65 (2H, s, NH₂), 7.82 (2H, s, H-2⁰and H-6⁰), 9.00 (1H, s, H-4);

¹³C NMR (100 MHz; d₆-DMSO) 21.2 (C-7), 32.3 (C-8), 38.1 (C-6), 96.3

(C-2) 118.8 (C-2⁰and C-6⁰), 121.8 (C-4⁰), 124.4 (C-4a), 125.1 (C-3a),

130.1 (C-4), 133.6 (C-3⁰and C-5⁰), 142.4 (C-1⁰), 147.9 (C-3), 162.3 (C-

C

₁₈H₁₄N₃O₂S⁸¹BrNa requires 439.9862. Found

437.9883, C₁₈H₁₄N₃O₂S⁷⁹BrNa requires 437.9882.

(

⁷⁹Br MNa^þ)

4.6.2.6. 3-Amino-N-(3⁰-nitrophenyl)-5-oxo-5,6,7,8-tetrahydrothieno

9a), 164.1 (NC]O), 164.2 (C-8a), 197.0 (C-5); IR: y_max(ﬁlm)/cm^ꢂ1

:

[2,3-b]quinoline-2-carboxamide 17f. The reaction was carried out in

3433, 3327, 2941,1675,1648,1578, 1527,1439,1399,1265,1230; m/z

accordance with general procedure

G using 2-bromo-N-(3-

(ESIþ): 431 (³⁷Cl₂MNa^þ, 16%), 429 (³⁵Cl³⁷Cl MNa^þ68%), 427 (³⁵Cl₂

nitrophenyl)acetamide 21f (0.13 g, 0.49 mmol) to give the title

product 17f (0.19 g, 100%) as a yellow solid. m.p. 290e292 ^ꢀC. ¹H

NMR (400 MHz; d₆-DMSO) 2.13e2.19 (2H, m, H-7), 2.74 (2H, t,

J ¼ 6.2 Hz, H-6), 3.21 (2H, t, J ¼ 6.2 Hz, H-8), 7.61e7.65 (1H, m, H-5⁰),

7.74 (2H, s, NH₂), 7.94 (1H, d, J ¼ 8.0 Hz, H-4⁰), 8.15 (1H, d, J ¼ 8.0 Hz,

H-6⁰), 8.77 (1H, s, H-2⁰), 9.08 (1H, s, H-4), 9.95 (1H, s, NH); ¹³C NMR

(100 MHz; d₆-DMSO) 21.2 (C-7), 32.4 (C-8), 38.1 (C-6), 95.1 (C-2)

114.8 (C-2⁰),117.7 (C-4⁰),124.5 (C-4a),125.0 (C-3a) 126.6 (C-6⁰) 129.8

(C-5⁰), 130.3 (C-4), 140.3 (C-1⁰), 147.8 (C-3), 148.5 (C-3⁰), 162.3 (C-9a)

164.0 (NC]O), 164.5 (C-8a), 197.0 (C-5); IR: y_max(ﬁlm)/cm^ꢂ1: 3444,

3322, 2960, 2871, 1672, 1643, 1583, 1525, 1320, 1296, 1238, 1096; m/

z (ESIþ): 405 (MNa^þ, 49%), 360 ([MNa-NO₂]^þ, 100%). HRMS (ESI^þ)

found (MNa^þ): 405.0632, C₁₈H₁₄N₄O₄SNa requires 405.0628.

MNa^þ, 100%); HRMS (ESI^þ) found ³⁷Cl₂MNa^þ): 431.9936,

(

C

₁₈H₁₃N₃O₂S³⁷Cl₂Na requires 431.9939. Found

(

³⁵Cl₂MNa^þ):

427.9998, C₁₈H₁₃N₃O₂S³⁵Cl₂Na requires 427.9998.

4.6.3. General procedure H: synthesis of 5-hydroxy-

tetrahydrothieno[2,3-b]quinoline-2-carboxamides 23aei

A solution of sodium borohydride (0.30 mmol) in methanol

(1 mL) was added dropwise to a solution of 5-oxo-tetrahydrothieno

[2,3-b]quinoline-2-carboxamide 17 (0.30 mmol) in THF (15 mL)

over 15 min. The mixture was stirred for 2 h at room temperature,

before H₂O (10 mL) was added and the stirring was continued for

further 5 min. The volatile solvents were removed in vacuo, and the

remaining mixture diluted with H₂O (90 mL). The aqueous mixture

was extracted with ethyl acetate (3 ꢁ 100 mL), the organic layers

were combined and further washed with H₂O (3 ꢁ 100 mL), dried

(Na₂SO₄) and concentrated in vacuo to give 5-hydroxy-tetrahy-

drothieno[2,3-b]quinolines 23aei.

4.6.2.7. 3-Amino-5-oxo-N-(3⁰-(triﬂuoromethyl)phenyl)-5,6,7,8-

tetrahydrothieno[2,3-b]quinoline-2-carboxamide 17g. The reaction

was carried out in accordance with general procedure G using 2-

bromo-N-(3-(triﬂuoromethyl)phenyl)acetamide 21g (0.14 g,

0.49 mmol) to give the title product 17g (0.16 g, 88%) as a yellow

solid. m.p. 289e292 ^ꢀC. ¹H NMR (400 MHz; d₆-DMSO) 2.12e2.18

(2H, m, H-7), 2.72 (2H, t, J ¼ 6.3 Hz, H-6), 3.19 (2H, t, J ¼ 6.3 Hz, H-8),

7.39 (1H, d, J ¼ 8.0 Hz, H-4⁰), 7.53e7.57 (1H, m, H-5⁰), 7.66 (2H, s,

NH₂), 7.97 (1H, d, J ¼ 8.0 Hz, H-6⁰), 8.18 (1H, s, H-2⁰), 9.04 (1H, s, H-

4), 9.77 (1H, s, NH); ¹³C NMR (100 MHz; d₆-DMSO) 21.2 (C-7), 32.4

(C-8), 38.1 (C-6), 96.2 (C-2) 117.0 (C-2⁰), 119.2 (C-4⁰), 122.9 (C-3⁰),

124.4 (C-4a and C-6⁰), 125.2 (C-3a), 128.8 (CF₃), 129.5 (C-5⁰), 130.1

(C-4), 140.5 (C-1⁰), 147.7 (C-3), 162.2 (C-9a), 164.1 (NC]O), 164.1 (C-

8a), 197.0 (C-5); IR: y_max(ﬁlm)/cm^ꢂ1: 3427, 3353, 3311, 3074, 2955,

2876, 1675, 1631, 1581, 1525, 1475, 1402, 1301, 1231, 1094; IR: y_max

(ﬁlm)/cm^ꢂ1: 3429, 3362, 3313, 2953, 1677, 1642, 1585, 1538, 1439,

1318, 1251, 1228, 1107; m/z (ESIþ): 428 (MNa^þ, 75%), 360 ([MNa-

CF₃]^þ, 100%). HRMS (ESI^þ) found (MNa^þ) 428.0657,

4.6.3.1. 3-Amino-5-hydroxy-N-phenyl-5,6,7,8-tetrahydrothieno[2,3-

b]quinoline-2-carboxamide 23a. The reaction was carried out in

accordance with general procedure

H using 5-oxo-tetrahy-

drothieno[2,3-b]quinoline 17a (0.10 g, 0.30 mmol) to give the title

product 23a (0.07 g, 66%) as a yellow solid. m.p. 225e228 ^ꢀC. ¹H

NMR (400 MHz; d₆-DMSO) 1.71e1.83 (2H, m, H-7), 1.99e2.03 (2H,

m, H-6), 2.91e2.97 (2H, m, H-8), 4.72e4.76 (1H, m, H-5), 5.40 (1H,

d, J ¼ 6.1 Hz, OH), 7.05e7.09 (1H, m, AreH), 7.32 (2H, d, J ¼ 8.4 Hz,

AreH), 7.39 (2H, s, NH₂), 7.69 (2H, d, J ¼ 8.4 Hz, AreH), 8.52 (1H, s,

H-4), 9.35 (1H, s, NH); ¹³C NMR (100 MHz; d₆-DMSO) 18.4 (C-7),

32.0 (C-8), 32.3 (C-6), 66.5 (C-5) 95.6 (C-2) 121.1 (AreCH), 123.3

(AreCH), 124.5 (C-3a), 128.3 (AreCH), 130.7 (C-4), 132.2 (C-4a)

139.0 (AreC), 147.2 (C-3), 157.0 (C-9a) 158.8 (C-8a), 164.1 (C]O); IR:

y_max(ﬁlm)/cm^ꢂ1: 3442, 3299, 3055, 2932, 1590, 1521, 1488, 1436,

1317, 1244, 1068; m/z (ESIþ): 362 (MNa^þ, 100%), 340 (MH^þ, 99%).

HRMS (ESI^þ) found (MNa^þ) 362.0935, C₁₈H₁₇N₃O₂SNa requires

362.0934.

C

₁₉H₁₄F₃N₃O₂SNa requires 428.0651.

4.6.2.8. 3-Amino-N-(3⁰,5⁰-dimethoxyphenyl)-5-oxo-5,6,7,8-

tetrahydrothieno[2,3-b]quinoline-2-carboxamide 17h. The reaction

was carried out in accordance with general procedure G using 2-

4.6.3.2. 3-Amino-5-hydroxy-N-(4⁰-methoxyphenyl)-5,6,7,8-

tetrahydrothieno[2,3-b]quinoline-2-carboxamide 23b. The reaction

was carried out in accordance with general procedure H using 5-

oxo-tetrahydrothieno[2,3-b]quinoline 17b (0.10 g, 0.27 mmol) to

give the title product 23b (0.10 g, 99%) as a yellow solid. m.p.

250e253 ^ꢀC. ¹H NMR (400 MHz; d₆-DMSO) 1.74e1.81 (2H, m, H-7),

2.00e2.03 (2H, m, H-6), 2.86e3.00 (2H, m, H-8), 3.75 (3H, s, OCH₃)

4.72e4.76 (1H, m, H-5), 5.40 (1H, d, J ¼ 6.3 Hz, OH), 6.90 (2H, d,

J ¼ 8.0 Hz, H-3⁰and H-5⁰), 7.33 (2H, s, NH₂), 7.56 (2H, d, J ¼ 8.0 Hz, H-

bromo-N-(3,5-dimethoxyphenyl)acetamide

21h

(0.40

g,

1.46 mmol) to give the title product 17h (0.17 g, 29%) as a yellow

solid. m.p. 279e280 ^ꢀC. ¹H NMR (400 MHz; d₆-DMSO) 2.13e2.16

(2H, m, H-7), 2.72 (2H, t, J ¼ 5.6 Hz, H-6), 3.19 (2H, t, J ¼ 5.6 Hz, H-8),

3.74 (6H, s, OCH₃), 6.23 (1H, s, H-4⁰), 7.03 (2H, s, H-2⁰and H-6⁰), 7.60

(2H, s, NH₂), 9.03 (1H, s, H-4), 9.38 (1H, s, NH); ¹³C NMR (100 MHz;

d₆-DMSO) 21.2 (C-7), 32.3 (C-8), 38.1 (C-6), 55.1 (OCH₃), 95.6 and

96.0 (C-2 and C-4⁰), 99.0 (C-2⁰and C-6⁰), 124.4 (C-4a), 125.1 (C-3a),

J.M. Hung et al. / European Journal of Medicinal Chemistry 86 (2014) 420e437

435

2⁰and H-6⁰), 8.49 (1H, s, H-4), 9.25 (1H, s, NH); ¹³C NMR (100 MHz;

d₆-DMSO) 18.4 (C-7), 32.0 (C-8), 32.3 (C-6), 55.1 (OCH₃), 66.5 (C-5),

95.8 (C-2), 133.5 (C-3⁰and C-5⁰), 122.9 (C-2⁰and C-6⁰), 124.6 (C-3a),

130.6 (C-4), 131.9 (C-1⁰), 132.2 (C-4a), 146.8 (C-3), 155.5 (C-4⁰) 156.9

(C-9a) 158.6 (C-8a), 163.9 (C]O); IR: y_max(ﬁlm)/cm^ꢂ1: 3672, 3420,

3295, 2924, 1590, 1503, 1298, 1228, 1172, 1032; m/z (ESIþ): 392

(MNa^þ, 100%). HRMS (ESI^þ): found (MNa^þ) 392.1038,

C

₁₈H₁₆N₃O₂S⁸¹BrNa requires 442.0019. Found (⁷⁹BrMNa) 440.0033,

₁₈H₁₆N₃O₂S⁷⁹BrNa requires 440.0039.

4.6.3.6. 3-Amino-5-hydroxy-N-(3⁰-nitrophenyl)-5,6,7,8-

tetrahydrothieno[2,3-b]quinoline-2-carboxamide 23f. The reaction

was carried out in accordance with general procedure H using 5-

oxo-tetrahydrothieno[2,3-b]quinoline 17f (0.10 g, 0.26 mmol) to

give the title product 23f (0.10 g, 98%) as a yellow solid. m.p.

259e260 ^ꢀC. ¹H NMR (400 MHz; d₆-DMSO) 1.71e1.84 (2H, m, H-7),

1.99e2.03 (2H, m, H-6), 2.92e2.98 (2H, m, H-8), 4.72e4.77 (1H, m,

H-5), 5.41 (1H, d, J ¼ 6.3 Hz, OH), 7.56 (2H, s, NH₂), 7.59e7.63 (1H, m,

H-5⁰), 7.92 (1H, d, J ¼ 8.3 Hz, H-4⁰), 8.15 (1H, d, J ¼ 8.3 Hz, H-6⁰), 8.56

(1H, s, H-4), 8.77 (1H, s, H-2⁰), 9.83 (1H, s, NH); ¹³C NMR (100 MHz;

d₆-DMSO) 18.4 (C-7), 31.9 (C-8), 32.3 (C-6), 66.5 (C-5), 95.6 (C-2),

114.8 (C-2⁰), 117.5 (C-4⁰), 124.3 (C-3a), 126.6 (C-6⁰) 129.7 (C-5⁰), 131.0

(C-4), 132.4 (C-4a), 140.4 (C-1⁰), 147.8 (C-3), 148.2 (C-3⁰), 157.1 (C-

9a), 159.2 (C-8a), 164.4(C]O); IR: y_max(ﬁlm)/cm^ꢂ1: 3422, 3304,

2948, 2922, 2852, 1585, 1527, 1320, 1297, 1243, 1053; m/z (ESIþ):

407 (MNa^þ, 86%), 360 (MNa^þ-NO₂, 100%). HRMS (ESI^þ): found

(MNa^þ) 407.0780, C₁₈H₁₆N₄O₄SNa requires 407.0784.

C

₁₉H₁₉N₃O₃SNa requires 392.1039.

4.6.3.3. 3-Amino-N-(3⁰-chlorophenyl)-5-hydroxy-5,6,7,8-

tetrahydrothieno[2,3-b]quinoline-2-carboxamide 23c. The reaction

was carried out in accordance with general procedure H using 5-

oxo-tetrahydrothieno[2,3-b]quinoline 17c (0.10 g, 0.27 mmol) to

give the title product 23c (0.10 g, 98%) as a yellow solid. m.p.

255e257 ^ꢀC. ¹H NMR (400 MHz; d₆-DMSO) 1.71e1.85 (2H, m, H-7),

1.99e2.03 (2H, m, H-6), 2.87e3.00 (2H, m, H-8), 4.72e4.76 (1H, m,

H-5), 5.41 (1H, d, J ¼ 6.2 Hz, OH), 7.11 (1H, d, J ¼ 8.0 Hz, H-4⁰),

7.32e7.36 (1H, m, H-5⁰), 7.47 (2H, s, NH₂), 7.70 (1H, d, J ¼ 8.0 Hz, H-

6⁰), 7.91 (1H, s, H-2⁰), 8.53 (1H,s H-4), 9.51 (1H, s, NH); ¹³C NMR

(100 MHz; d₆-DMSO) 18.4 (C-7), 31.9 (C-8), 32.3 (C-6), 66.5 (C-5),

95.0 (C-2) 119.1 (C-6⁰), 120.2 (C-2⁰), 122.8 (C-4⁰), 124.4 (C-3a), 130.0

(C-5⁰),130.8 (C-4),132.3 (C-3⁰),132.7 (C-4a),140.6 (C-1⁰),147.8 (C-3),

157.0 (C-9a), 159.0 (C-8a), 164.2 (C]O); IR: y_max(ﬁlm)/cm^ꢂ1: 3469,

3381, 3344, 3287, 2933, 2863,1583,1516,1414,1311,1240,1044; m/z

(ESIþ): 398 (³⁷ClMNa^þ, 18%), 396 (³⁵ClMNa^þ, 48%), 360 (MNa^þ-Cl,

4.6.3.7. 3-Amino-5-hydroxy-N-(3⁰-(triﬂuoromethyl)phenyl)-5,6,7,8-

tetrahydrothieno[2,3-b]quinoline-2-carboxamide 23g. The reaction

was carried out in accordance with general procedure H using 5-

oxo-tetrahydrothieno[2,3-b]quinoline 17g (0.10 g, 0.25 mmol) to

give the title product 23g (0.10 g, 99%) as a yellow solid. m.p.

258e260 ^ꢀC. ¹H NMR (400 MHz; d₆-DMSO) 1.71e1.85 (2H, m, H-7),

1.99e2.03 (2H, m, H-6), 2.87e3.00 (2H, m, H-8), 4.72e4.76 (1H, m,

H-5), 5.41 (1H, d, J ¼ 6.0 Hz, OH), 7.40 (1H, d, J ¼ 8.0 Hz, H-4⁰), 7.51

(2H, s, NH₂), 7.56 (1H, t, J ¼ 8.0 Hz, H-5⁰), 8.00 (1H, d, J ¼ 8.0 Hz, H-

6⁰), 8.20 (1H, s, H-2⁰), 8.55 (1H, s, H-4), 9.66 (1H, s, NH); ¹³C NMR

(100 MHz; d₆-DMSO) 18.4 (C-7), 31.9 (C-8), 32.3 (C-6), 66.5 (C-5),

94.8 (C-2), 116.8 (C-2⁰), 119.4 (C-4⁰), 122.9 (C-3⁰), 124.2 and 124.4 (C-

6⁰and C-3a), 129.2 (CF₃), 129.5 (C-5⁰) 130.9 (C-4), 132.3 (C-4a), 139.9

(C-1⁰), 147.9 (C-3), 157.1 (C-9a), 159.1 (C-8a), 164.3 (C]O); IR: y_max

(ﬁlm)/cm^ꢂ1: 3423, 3308, 2922, 2853, 1593, 1549, 1438, 1319, 1115,

1057; m/z (ESIþ): 430 (CF₃MNa^þ, 100%); HRMS (ESI^þ): found

(MNa^þ) 430.0809, C₁₉H₁₆F₃N₃O₂SNa requires 430.0808.

100%).

C

HRMS

₁₈H₁₆N₃O₂S³⁷ClNa requires 398.0515. Found (³⁵ClMNa^þ) 396.0541,

₁₈H₁₆N₃O₂S³⁵ClNa requires 396.0544.

(ESI^þ):

found

(

³⁷ClMNa^þ)

398.0513,

4.6.3.4. 3-Amino-5-hydroxy-N-(3⁰-methoxyphenyl)-5,6,7,8-

tetrahydrothieno[2,3-b]quinoline-2-carboxamide 23d. The reaction

was carried out in accordance with general procedure H using 5-

oxo-tetrahydrothieno[2,3-b]quinoline 17d (0.10 g, 0.27 mmol) to

give the title product 23d (0.10 g, 98%) as a yellow solid. m.p.

236e239 ^ꢀC. ¹H NMR (400 MHz; d₆-DMSO) 1.79e1.87 (2H, m, H-7),

2.04e2.08 (2H, m, H-6), 2.96e3.02 (2H, m, H-8), 3.80 (3H, s, OCH₃),

4.77e4.81 (1H, m, H-5), 5.45 (1H, d, J ¼ 6.1 Hz, OH), 6.70 (1H, d,

J ¼ 8.0 Hz, H-4⁰), 7.24e7.28 (1H, m, H-5⁰), 7.37 (1H, d, J ¼ 8.0 Hz, H-

6⁰), 7.43 (1H, s, H-2⁰), 7.46 (2H, s, NH₂), 8.57 (1H, s, H-4), 9.36 (1H, s,

NH); ¹³C NMR (100 MHz; d₆-DMSO) 18.4 (C-7), 32.0 (C-8), 32.3 (C-

6), 55.0 (OCH₃), 66.5 (C-5), 95.5 (C-2), 106.6 (C-2⁰), 108.9 (C-4⁰),

113.2 (C-6⁰), 124.5 (C-3a), 129.1 (C-5⁰), 130.7 (C-4), 132.3 (C-4a),

140.2 (C-1⁰), 147.3 (C-3), 157.0 (C-9a) 158.8 (C-8a), 159.3 (C-3⁰) 164.1

(C]O); IR: y_max(ﬁlm)/cm^ꢂ1: 3447, 3338, 3271, 2932, 2833, 1586,

1521, 1448, 1430, 1243, 1161, 1037; m/z (ESIþ): 392 (MNa^þ, 39%),

360 (MNa^þ-OCH₃, 100%); HRMS (ESI^þ): found (MNa^þ) 392.1035,

4.6.3.8. 3-Amino-N-(3⁰,5⁰-dimethoxyphenyl)-5-hydroxy-5,6,7,8-

tetrahydrothieno[2,3-b]quinoline-2 carboxamide 23h. The reaction

was carried out in accordance with general procedure H using 5-

oxo-tetrahydrothieno[2,3-b]quinoline 17h (0.10 g, 0.25 mmol) to

give the title product 23h (0.73 g, 73%) as a yellow solid. m.p.

199e201 ^ꢀC. ¹H NMR (400 MHz; d₆-DMSO) 1.71e1.85 (2H, m, H-7),

1.99e2.03 (2H, m, H-6), 2.87e3.00 (2H, m, H-8), 3.73 (6H, s, OCH₃)

4.72e4.76 (1H, m, H-5), 5.39 (1H, d, J ¼ 6.2 Hz, OH), 6.22 (1H, t,

J ¼ 2.1 Hz, H-4⁰), 7.04 (2H, d, J ¼ 2.1 Hz, H-2⁰and H-6⁰) 7.42 (2H, s,

NH₂), 8.53 (1H,s H-4), 9.25 (1H, s, NH); ¹³C NMR (100 MHz; d₆-

DMSO) 18.4 (C-7), 32.0 (C-8), 32.3 (C-6), 55.1 (OCH₃), 66.5 (C-5),

95.4 (C-2 and C-4⁰), 98.9 (C-2⁰and C-6⁰) 124.5 (C-3a), 130.8 (C-4),

132.3 (C-4a), 140.8 (C-1⁰), 147.4 (C-3), 156.9 (C-9a), 158.8 (C-8a),

160.2 (C-3⁰and C-5⁰), 164.1 (C]O); IR: y_max(ﬁlm)/cm^ꢂ1: 3671, 3411,

3297, 2928, 2851, 1594, 1529, 1475, 1452, 1416, 1258, 1237, 1155,

1050; m/z (ESIþ): 422 (MNa^þ, 100%); HRMS (ESI^þ): found (MNa^þ)

422.1143, C₂₀H₂₁N₃O₄SNa requires 422.1145.

C

₁₉H₁₉N₃O₃SNa requires 392.1039.

4.6.3.5. 3-Amino-N-(3⁰-bromophenyl)-5-hydroxy-5,6,7,8-

tetrahydrothieno[2,3-b]quinoline-2-carboxamide 23e. The reaction

was carried out in accordance with general procedure H using 5-

oxo-tetrahydrothieno[2,3-b]quinoline 17e (0.10 g, 0.24 mmol) to

give the title product 23e (0.10 g, 97%) as a yellow solid. m.p.

240e241 ^ꢀC. ¹H NMR (400 MHz; d₆-DMSO) 1.74e1.83 (2H, m, H-7),

1.97e2.05 (2H, m, H-6), 2.91e2.97 (2H, m, H-8), 4.72e4.76 (1H, m,

H-5), 5.40 (1H, d, J ¼ 6.2 Hz, OH), 7.24 (1H, d, J ¼ 8.0 Hz, H-4⁰),

7.26e7.30 (1H, m, H-5⁰) 7.48 (2H, s, NH₂), 7.70 (1H, d, J ¼ 8.0 Hz, H-

6⁰), 8.05 (1H, s, H-2⁰), 8.54 (1H, s, H-4), 9.49 (1H, s, NH); ¹³C NMR

(100 MHz; d₆-DMSO) 18.4 (C-7), 32.0 (C-8), 32.3 (C-6), 66.5 (C-5),

95.0 (C-2), 119.5 (C-6⁰), 121.2 (C-3⁰), 123.1 (C-2⁰), 124.4 (C-3a), 125.7

(C-4⁰), 130.3 (C-5⁰), 131.9 (C-4), 132.3 (C-4a), 140.8 (C-1⁰), 147.8 (C-3),

157.1 (C-9a), 159.0 (C-8a), 164.2 (C]O); IR: y_max(ﬁlm)/cm^ꢂ1: 3446,

3322, 2929, 2852, 1580, 1524, 1474, 1400, 1305, 1243, 1056; m/z

(ESIþ): 442 (⁸¹BrMNa^þ, 61%), 440 (⁷⁹BrMNa^þ, 57%), 360 (MNa^þ-Br,

4.6.3.9. 3-Amino-N-(3⁰,5⁰-dichlorophenyl)-5-hydroxy-5,6,7,8-

tetrahydrothieno[2,3-b]quinoline-2-carboxamide 23i. The reaction

was carried out in accordance with general procedure H using 5-

oxo-tetrahydrothieno[2,3-b]quinoline 17i (0.10 g, 0.25 mmol) to

give the title product 23i (0.10 g, 100%) as a yellow solid. m.p.

267e269 ^ꢀC. ¹H NMR (400 MHz; d₆-DMSO) 1.71e1.83 (2H, m, H-7),

1.99e2.03 (2H, m, H-6), 2.87e3.01 (2H, m, H-8), 4.73e4.76 (1H, m,

H-5), 5.42 (1H, d, J ¼ 6.1 Hz, OH), 7.26 (1H, t, J ¼ 2.0 Hz, H-4⁰), 7.55

100%).

HRMS

(ESI^þ):

found

(

⁸¹BrMNa^þ)

442.0012,

436

J.M. Hung et al. / European Journal of Medicinal Chemistry 86 (2014) 420e437

(2H, s, NH₂), 7.88 (2H, d, J ¼ 2.0 Hz, H-2⁰and H-6⁰), 8.56 (1H, s, H-4),

9.63 (1H, s, NH); ¹³C NMR (100 MHz; d₆-DMSO) 18.3 (C-7), 31.9 (C-

8), 32.3 (C-6), 66.5 (C-5), 94.5 (C-2), 118.6 (C-2⁰and C-6⁰), 122.1 (C-

4⁰),124.3 (C-3a),131.0 (C-4),132.4 (C-4a),133.7 (C-3⁰and C-5⁰),141.6

(C-1⁰), 148.3 (C-3), 157.1 (C-9a), 159.3 (C-8a), 164.2 (C]O); IR: y_max

(ﬁlm)/cm^ꢂ1: 3432, 3320, 2942, 2922, 2854, 1580, 1528, 1492, 1443,

1404, 1311, 1264, 1056; m/z (ESIþ): 434 (³⁷Cl₂MNa^þ, 15%), 432

[3] Early Breast Cancer Trialists' Collaborative Group (EBCTCG), Effects of

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(

³⁵Cl³⁷ClMNa^þ, 70%), 430 (³⁵Cl₂MNa^þ, 100%); HRMS (ESI^þ): found

³⁷Cl₂MNa) 434.0102, C₁₈H₁₅N₃O₂S³⁷Cl₂Na requires 434.0096.

Found (³⁵Cl³⁷ClMNa) 432.0130, C₁₈H₁₅N₃O₂S³⁵Cl³⁷ClNa requires

432.0125. Found (³⁵Cl₂lMNa) 430.0159, C₁₈H₁₅N₃O₂S³⁵Cl₂Na re-

quires 430.0154.

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growth of human tumour cells, Eur. J. Med. Chem. 54 (2012) 463e469.

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4.7. Biological testing

The compounds obtained were submitted to the National Can-

cer Institute's Developmental Therapeutic Program (DTP) where

they were screened against a panel of sixty human tumour cell lines

(NCI-60, for further information see Refs. [23,41,42] and references

therein). Furthermore, the protocol is given in the Supplementary

Information.

PLC-g inhibitor on the proliferation, morphology, migration and cell cycle of

breast cancer cells, Med. Chem. Commun. 5 (2014) 99e106.

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tion: an ef ﬁ cient approach for the synthesis of 2,5-disubstituted 1,3,4-

The compounds were docked to the crystal structure of PLC-d1

(PDB ID: 1DJX, resolution 2.3 Å), which was obtained from the

Protein Data Bank (PDB) [43,44]. The Scigress Ultra version 7.7.0.47

program [45] was used to prepare the crystal structure for docking,

i.e., hydrogen atoms were added, the co-crystallised ligand (D-

Myo-Inositol-1,4,5-Triphosphate, IP₃) was removed as well as

crystallographic water molecules. The Scigress software suite was

also used to build the inhibitors and the MM2 [46] force ﬁeld was

used to optimise the structures. The centre of the binding pocket

was deﬁned as the position of the Ca^2þion (x ¼ 126.257, y ¼ 38.394,

z ¼ 22.370) with 10 Å radius. Fifty docking runs were allowed for

each ligand with default search efﬁciency (100%). The basic amino

acids lysine and arginine were deﬁned as protonated. Furthermore,

aspartic and glutamic acids were assumed to be deprotonated. The

GoldScore (GS) [47] ChemScore (CS) [48,49], ChemPLP [50] and ASP

[51] scoring functions were implemented to validate the predicted

binding modes and relative energies of the ligands using the GOLD

v5.2 software suite.

oxadiazole derivatives by

a Ugi-4CR/aza-Wittig sequence, Org. Lett. 12

(2010) 2852e2855.

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reaction of a secondary amine and a 2-hydroxybenzaldehyde derivative with

an isocyanide in the presence of silica gel: an ef ﬁ cient one-pot synthesis of

benzo[b]furan derivatives, Tetrahedron Lett. 50 (2009) 5625e5627.

[19] E. Vessally, A. Ramazani, E. Yaaghubi, Green synthesis and characterization of

novel

a -acyloxycarboxamides through three-component reaction between

pyridine carbaldehydes, cyclohexyl isocyanide, and benzoic acid derivatives,

Monatsh. Chem. 142 (2011) 625e630.

[20] A. Ramazani, M. Rouhani, A. Rezaei, N. Shajari, A. Souldozi, The reaction of (N-

isocyanimino)triphenylphosphorane with biacetyl in the presence of aromatic

carboxylic acids: ef ﬁ cient one-pot three-component reaction for the synthesis

of 3-(5-aryl-1,3,4-oxadiazol-2-yl)-3-hydroxybutan-2-one derivatives, Helv.

Chim. Acta 94 (2011) 282e288.

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120e144.

Acknowledgements

Funding for this work was obtained from the University of

Auckland (9136/3706824), Maurice and Phyllis Paykel Trust (9136/

3700837), the McClelland Trust (9136/3704422) and Auckland

Medical Research Foundation (9136/3701442). This work is also

supported by facilities at the Auckland Cancer Society Research

Centre.

[23] M. Azizmohammadi, M. Khoobi, A. Ramazani, S. Emami, A. Zarrin, O. Firuzi,

R. Miri, A. Sha ﬁ ee, 2H-Chromene derivatives bearing thiazolidine-2,4-dione,

rhodanine or hydantoin moieties as potential anticancer agents, Eur. J. Med.

Chem. 59 (2013) 15e22.

Appendix A. Supplementary data

[24] A. Ramazani, M. Khoobi, A. Torkaman, F.Z. Nasrabadi, H. Forootanfar,

M. Shakibaie, M. Jafari, A. Ameri, S. Emami, M.A. Faramarzi, A. Foroumadi,

A. Sha ﬁ ee, One-pot, four-component synthesis of novel cytotoxic agents 1-(5-

aryl-1,3,4-oxadiazol-2-yl)-1-(1H-pyrrol-2-yl)methanamines, Eur. J. Med.

Chem. 78 (2014) 151e156.

Supplementary data related to this article can be found at http://

dx.doi.org/10.1016/j.ejmech.2014.09.001.

[25] G.S. Khan, B.D. Dickson, D. Barker, Synthesis of benzoic acids and poly-

benzamides containing tertiary alkylamino functionality, Tetrahedron 68

(2012) 1790e1801.

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Article Doi

Synthesis and cytotoxicity of thieno[2,3-b]pyridine and furo[2,3-b]pyridine derivatives

DOI: 10.1016/j.ejmech.2014.09.001

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