Synthesis and biological evaluation of novel 2-amino-3-aroyl-4-neopentyl-5- substituted thiophene derivatives as allosteric enhancers of the A1 adenosine receptor

Bioorganic & Medicinal Chemistry 22 (2014) 148–166

Contents lists available at ScienceDirect

Bioorganic & Medicinal Chemistry

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

Synthesis and biological evaluation of novel 2-amino-3-aroyl-4-

neopentyl-5-substituted thiophene derivatives

as allosteric enhancers of the A₁adenosine receptor

a,

a

Romeo Romagnoli ^a,, Pier Giovanni Baraldi , Maria Dora Carrion , Olga Cruz-Lopez ,

Carlota Lopez Cara ^a, Giulia Saponaro ^a, Delia Preti ^a, Mojgan Aghazadeh Tabrizi ^a, Stefania Baraldi ^a,

Allan R. Moorman ^b, Fabrizio Vincenzi ^c, Pier Andrea Borea ^c, Katia Varani ^c

⇑

^aDipartimento di Scienze Chimiche e Farmaceutiche, Via Fossato di Mortara 17-19, Università di Ferrara, 44121 Ferrara, Italy

^bKing Pharmaceuticals Inc., Research and Development, 4000 Centre Green Way, Suite 300, Cary, NC 27513, United States

^cDipartimento di Scienze Mediche, Sezione di Farmacologia, Università di Ferrara, Ferrara, Italy

a r t i c l e i n f o

a b s t r a c t

Article history:

2-Amino-3-benzoyl thiophenes have been widely reported to act as allosteric enhancers at the A₁aden-

osine receptor. Their activity can be increased considerably by appropriate substitutions at the 4- and

5-positions of the thiophene ring. Substituent size at the thiophene C-4 position seemed to be a factor

closely related to activity, with the 4-neopentyl (2,2-dimethylpropyl) substitution showing the greatest

enhanced activity. A wide series of 2-amino-3-aroyl-4-neopentylthiophene derivatives with general

structure 3, characterized by the presence of different substituents (bromine, aryl and heteroaryl) at

the 5-position of the thiophene ring, have been identiﬁed as potent AEs at the A₁AR. With only one excep-

tion, all of the synthesized compounds proved to be superior to the reference compound PD 81,723 in a

functional assay. Derivatives 3p, 3u, 3am, 3ap and 3ar were the most active compounds in binding

(saturation and competition) and functional cAMP studies, being able to potentiate agonist [³H]CCPA

binding to the A₁receptor.

Received 20 September 2013

Accepted 21 November 2013

Available online 1 December 2013

Keywords:

A₁adenosine receptor

Allosteric modulation

G protein-coupled receptors

2-Amino-3-benzoylthiophene

1. Introduction

hypotension, reduction of neuropathic pain and inhibition of

lipolysis) occurs by the selective activation of the A₁AR,³ex-

Adenosine is a physiological extracellular modulator acting

pressed in high density in the CNS (cortex, hippocampus, cere-

bellum and thalamus) and fat cells, and in moderate to low

levels in many other tissues, such as bladder, lung, kidney and

heart.⁴Efforts to selectively target the A₁AR with modiﬁed

adenosine analogues or selective orthosteric agonists have been

limited by side effects due to the activation of the A₁AR in tis-

sues other than the therapeutic target, poor receptor subtype

via four distinct G protein-coupled receptors, named A₁, A_2A

,

A_2Band A₃, that are widely distributed throughout the body.¹

The A₁adenosine receptor (A₁AR) is coupled to a Gi-protein

signal transduction pathway to inhibit adenylate cyclase and

its activation reduces intracellular levels of cAMP.²A variety

of adenosine mediated effects (neuro- and cardio-protection,

selectivity and

a tendency to cause receptor desensitization

upon prolonged use.⁵

Abbreviations: GPCRs, G protein-coupled receptors; [³H]DPCPX, [³H]1,3-dipro-

pyl-8-cyclopentyl-xanthine; [³H]MRE-3008F20, [³H]5-N-(4-methoxyphenylcarba-

moyl)amino-8-propyl-2-(2-furyl)pyrazolo[4,3-e]-1,2,4-triazolo[1,5-c]pyrimidine;

[³H]CCPA, [³H]2-chloro-N⁶-cyclopentyladenosine; [³H]ZM 241385, [³H](4-(2-[7-

amino-2-(2-furil)[1,2.4]triazolo[2,3-a][1,3,5]triazin-5-ylamino]ethyl)phenol); CCPA,

2-chloro-N⁶-cyclopentyladenosine; CHO, chinese hamster ovary; cAMP, cyclic aden-

osine monophosphate; AE(s), allosteric enhancer(s); hA₁AR, human A₁adenosine

receptor; NBS, N-bromosuccinimide; PdCl₂(DPPF), [1,1⁰-bis(diphenylphosphino)

ferrocene] dichloropalladium (II) complex with dichloromethane; CsF, cesium

ﬂuoride; EWG, electron-withdrawing group; ERG, electron-releasing group; CNS,

central nervous system.

An opportunity for therapeutic intervention is provided by

targeting an allosteric site on the A₁AR with an allosteric enhan-

cer (AE).⁶The binding of an allosteric modulator to the allosteric

site of the A₁AR, structurally distinct from the orthosteric bind-

ing site, induces a reversible change of the A₁AR conformation

that ampliﬁes the potency and efﬁcacy of endogenous adeno-

sine, an important tissue protective agent released during ische-

mia, hypoxia or inﬂammation.⁷This approach can generate

selectivity in action as a consequence of both tissue-speciﬁc

and receptor-speciﬁc modulation.⁸Allosteric modulation of the

A₁AR may have potential therapeutic applications in the

⇑

Corresponding authors. Tel.: +39 (0)532455303; fax: +39 (0)532455953 (R.R.);

tel.: +39 (0)532455293; fax: +39 (0)532455953 (P.G.B.).

E-mail addresses: rmr@unife.it (R. Romagnoli), baraldi@unife.it (P.G. Baraldi).

http://dx.doi.org/10.1016/j.bmc.2013.11.043

R. Romagnoli et al. / Bioorg. Med. Chem. 22 (2014) 148–166

149

treatment of neuropathic pain, hypoxia and ischaemia-induced

injury, to mitigate allodynia and as cardioprotective agents.

Therefore, signiﬁcant research efforts have been directed to

the discovery of new small molecules acting as allosteric modu-

lators for the A₁AR.⁹

the compounds at a concentration of 10

l

M to reduce the cAMP

content of CHO cells expressing human A₁receptor. In this preli-

minary pharmacological evaluation, the neopentyl derivative 2d,

with a 35% reduction of cAMP production, was 2- to 3-fold more ac-

tive than the other compounds of this series (Table 1). Compound

2d was also more active than a small series of 4,5-disubstituted

2-amino-3-(4-chlorobenzoyl)thiophene analogues, corresponding

to the 4-CH₃, 5-C₆H₅(2e); 4-CH₃, 5-(4⁰-MeO-C₆H₄) (2f),

4-CH₃, 5-Br (2g), 4-C₆H₅, 5-CH₃(2h) and 4,5-di-C₆H₅(2i)^13aderiva-

tives, which caused a reduction of cAMP content ranging from 16%

to 24%. The results obtained with compound 2d make it possible to

identify in a neopentyl moiety at the 4-position of the 2-amino-

3-(4-chlorobenzoyl)thiophene scaffold an optimal substituent that

is critical for interaction with the allosteric site of the A₁AR. We

have therefore synthesized a ﬁrst series of compounds with general

formula 3, based on the 2-amino-4-neopentyl-thiophene skeleton,

containing different aroyl groups at the 3-position. Based upon pre-

vious studies, appropriate substituents on the benzoyl group were

selected from among those that improved activity, such as 4-chloro

(3e, 3j and 3q), 3-chloro (3a, 3f, 3k and 3r), 3,4-dichloro (3b, 3g, 3m

and 3s), 3-triﬂuoromethyl (3c, 3h, 3o and 3t) and 4-methyl (3d, 3i,

3p and 3u). By the synthesis of compounds 3l and 3n, we investi-

gated the inﬂuence on AE activity of further substitution (2-chloro

and 2,4-dichloro, respectively), on the 3-benzoyl group. These

molecules were also characterized by the presence of a hydrogen

(2d and 3a–d), bromine (3e–i), phenyl (3j–p) and 4-methoxyphenyl

(3q–u) at the 5-position of the 2-amino-3-aroyl-4-neopentyl-thio-

phene system.

The allosteric modulation of the A₁AR by 2-amino-3-aroyl thio-

phenes is well documented in several review articles.^6,8–10The 2-

amino and 3-benzoyl groups were found to be crucial for the AE

activity. Lipophilic substituents on the phenyl of the benzoyl moiety

impart a favourable ratio of allosteric enhancement to antagonism,

and include the 3-triﬂuoromethyl present in PD 81,723 (1a),^11a,11b

the 3,4-dichloro in LUF5484 (1b),^11cthe 4-chloro in T62 (1c), the

3-chloro in PD 71,605 (1d)^11a,11bas well as the

4-methyl (1e)^11c(Chart 1). A range of alkyl and aryl substituents

in the 4- and 5-positions of the 2-amino-3-aroyl thiophene system

have also been found to promote AE activity.¹²Large hydrophobic

alkyl or aryl groups at the 4-position have a beneﬁcial effect on AE

activity, which increased in the order: H < Me < Phenyl, while bulky

5-alkyl or aryl substituents favoured increased competitive antago-

nistic properties with the resulting compounds.¹³

To further study the role of various alkyl substitutions at the

4-position of the 2-amino-3-(4-chlorobenzoyl)thiophene nucleus,

the allosteric enhancement activity of compounds 2a (methyl),¹⁴

2b [isobutyl, CH₂CH(CH₃)₂], 2c (tert-butyl, C(CH₃)₃) and 2d [neo-

pentyl, CH₂C(CH₃)₃] was determined by measuring the ability of

Cl

R₀

The structural reﬁnement of the 5-phenyl and 5-(4⁰-methoxy-

phenyl)-thiophene derivatives 3j and 3q, respectively, led to the

synthesis of analogues 3v–au, based on the systematic modiﬁca-

tion of the 5-position of the 2-amino-3-(4-chlorobenzoyl)-4-neo-

pentyl-thiophene ring, with the goal of evaluating the effects on

AE activity due to insertion of substituents which included 3,4-

dimethyl-isoxazol-4-yl (3v), 1H-pyrazol-4-yl (3w), isomeric thio-

phene (3x and 3y), furan (3z and 3aa) and pyridine (3ab, 3ac

and 3ad), to end with the phenyl ring with electron-releasing

(alkyl, alkoxy, OCF₃) and electron-withdrawing (F and Cl) groups

(3ae–3au). By the preparation of compound 3ak, we have also

investigated the effect on AE activity due to the presence of a

vinyl spacer between a 4-chlorophenyl ring and the 5-position

of the thiophene ring.

R₄

O

R₄

O

NH₂

R₅

S

NH₂

R₅

S

1a (PD 81,723); R₀=3-CF_3,R₄=R₅=CH₃

1b (LUF5484); R₀=3,4-Cl₂, R₄,R₅=(CH₂)₄

1c (T62); R₀=4-Cl, R₄,R₅=(CH₂)₄

2a; R₄=CH₃, R₅=H

2b; R₄=CH₂CH(CH₃)₂, R₅=H

2c; R₄=C(CH₃)_3,R₅=H

2d; R₄=CH₂C(CH₃)₃, R₅=H

2e; R₄=CH₃, R₅=C₆H₅

2f; R₄=CH₃, R₅=4'-OMeC₆H₄

2g; R₄=CH₃, R₅=Br

1d (PD 71,605); R₀=3-Cl, R₄,R₅=(CH₂)₄

1e; R₀=4-CH_3,R₄,R₅=(CH₂)₄

2h; R₄=C₅H₅, R₅=CH₃

2i; R₄=R₅=C₆H₅

Cl

R₀

O

NH₂

2. Chemistry

NH₂

R₅

S

The target compounds 2d and 3a–au were synthesized as shown

in the reaction sequence outlined in Scheme 1. The

5-unsubstituted thiophene derivatives 2d, 3a–d and 5a–b were pre-

pared by a two-step procedure consisting of a Knoevenagel reaction

of 4,4-dimethylpentan-2-one with the appropriate benzoylaceto-

nitrile in toluene in the presence of b-alanine and acetic acid, fol-

lowed by isolation and puriﬁcation of the inseparable mixture of

the E- and Z-oleﬁn isomers 4a–g. Cyclization with sulfur in ethanol

in the presence of triethylamine (Gewald reaction)¹⁵provided the

target compounds. Subsequent reaction with phthalic anhydride

in acetic acid furnished, almost quantitatively, the corresponding

N-protected phthalimido intermediates 6a–g, which were trans-

formed to the 5-bromothiophene derivatives 7a–g by the chemose-

lective bromination with NBS in reﬂuxing acetonitrile. These latter

compounds were subjected to Suzuki cross-coupling conditions¹⁶

in the presence of the appropriate aryl/heteroarylboronic acid under

heterogeneous conditions [PdCl₂(dppf), CsF] in 1,4-dioxane under

heating to furnish derivatives 8a–al. For these latter compounds,

as well as for the 5-bromothiophene analogues 7a–g, the removal

of the N-protected phthaloyl group was accomplished by the use

of ethanolic hydrazine, to afford the derivatives 3e–au.

3v; R₅=3,4-(diCH₃)-isoxazol-4-yl

3w; R₅=1H-pyrazol-4-yl

3x; R₅=thiophen-2-yl

3a; R₀=3-Cl, R₅=H

3b; R₀=3,4-Cl_2,R₅=H

3c; R₀=3-CF_3,R₅=H

3y; R₅=thiophen-3-yl

3d; R₀=4-CH_3,R₅=H

3z; R₅=furan-2-yl

3e; R₀=4-Cl, R₅=Br

3aa; R₅=furan-3-yl

3f; R₀=3-Cl, R₅=Br

3ab; R₅=pyridin-4-yl

3g; R₀=3,4-Cl_2,R₅=Br

3h; R₀=3-CF_3,R₅=Br

3ac; R₅=pyridin-3-yl

3ad; R₅=pyridin-2-yl

3i; R₀=4-CH_3,R₅=Br

3ae; R₅=4'-F-C₆H₄

3j; R₀=4-Cl, R₅=C₆H₅

3af; R₅=2',3'-diF-C₆H₄

3ag; R₅=2',4'-diF-C₆H₄

3ah; R₅=2',5'-diF-C₆H₄

3ai; R₅=2',6'-diF-C₆H₄

3aj; R₅=4'-Cl-C₆H₄

3k; R₀=3-Cl, R₅=C₆H₅

3l; R₀=2-Cl, R₅=C₆H₅

3m; R₀=3,4-Cl_2,R₅=C₆H₅

3n; R₀=2,4-Cl_2,R₅=C₆H₅

3o; R₀=3-CF_3,R₅=C₆H₅

3p; R₀=4-CH_3,R₅=C₆H₅

3q; R₀=4-Cl, R₅=4'-OCH₃-C₆H₄

3r; R₀=3-Cl, R₅=4'-OCH₃-C₆H₄

3s; R₀=3,4-Cl_2,R₅=4'-OCH₃-C₆H₄

3t; R₀=3-CF_3,R₅=4'-OCH₃-C₆H₄

3u; R₀=4-CH_3,R₅=4'-OCH₃-C₆H₄

3ak; R₅=(E)-4'-Cl-C₆H₄CH=CH

3al; R₅=3',4'-diCl-C₆H₄

3am; R₅=3'-OCH₃-C₆H₄

3an; R₅=2'-OCH₃-C₆H₄

3ao; R₅=4'-OCH₃(CH₂)₂O-C₆H₄

3ap; R₅=3',4'-(diO-CH₂)-C₆H₃

3aq; R₅=4'-OCF₃-C₆H₄

3ar; R₅=4'-CH₃-C₆H₄

3as; R₅=3'-CH₃-C₆H₄

3at; R₅=2'-CH₃-C₆H₄

3au; R₅=4'-(CH₃)CH-C₆H₄

Chart 1. Chemical structures of 2-amino-3-aroyl thiophene derivatives 1a–e, 2a–i

and 3a–au, evaluated as allosteric modulators for the A₁adenosine receptor.

150

R. Romagnoli et al. / Bioorg. Med. Chem. 22 (2014) 148–166

Table 1

Effect of the novel allosteric enhancers 2a–i, 3a–au and of PD 81,723 in cAMP assay in hA₁CHO cells

Compound

% Inhibition of cAMP production^a

% Inhibition of cAMP production + CCPA^b

2a

2b

2c

2d

2e

2f

2g

2h

2i

3a

3b

3c

3d

3e

3f

3g

3h

3i

3j

3k

3l

3m

3n

3o

3p

3q

3r

3s

3t

3u

3v

3w

3x

3y

16

17

11

35

18

24

21

16

24

45

43

28

38

48

45

42

49

61

51

59

38

56

52

38

62

44

53

54

44

64

12

27

58

62

49

55

42

37

40

50

60

48

46

45

57

51

59

55

27

53

61

56

57

47

56

19

1

2

1

3

2

3

2

4

3

4

5

4

5

6

5

6

3

6

5

4

6

4

5

6

4

7

1

3

5

6

4

5

6

5

4

5

6

5

6

5

2

5

6

5

2

18

19

18

37

20

22

25

21

26

42

41

30

35

50

48

40

50

63

53

57

41

57

55

39

61

43

56

52

46

60

19

31

62

67

47

56

46

41

43

52

63

49

45

46

58

53

61

57

29

55

59

55

58

51

49

58

22

1

2

4

2

3

2

3

4

3

5

4

5

6

4

6

4

6

4

5

2

3

6

7

5

6

4

5

6

5

4

5

6

5

6

3

5

6

5

2

3z

3aa

3ab

3ac

3ad

3ae

3af

3ag

3ah

3ai

3aj

3ak

3al

3am

3an

3ao

3ap

3aq

3ar

3as

3at

3au

PD 81,723

a

Inhibition of the forskolin-stimulated cAMP production (in percentage) of the novel allosteric enhancers (10

Inhibition of the cAMP production (in percentage) of the novel allosteric enhancers (100 nM) in the presence of CCPA (1 pM). The values are expressed as the mean SEM,

l

M);

b

n = 3 independent experiments.

are thought to stabilize the active conformation of the A₁AR, leading

to a reduction in the cAMP content of the cells.¹⁷

3. Biological results and discussion

3.1. Functional assays

The reference compound PD 81,723 and the derivatives 2a–i

and 3a–au were tested alone at

a concentration of 10 lM

(Fig. 1A) or at a concentration of 100 nM in the presence of the

orthosteric agonist CCPA (1 pM) to assess enhancement of the

A₁AR agonist activity (Fig. 1B).

A reduction in cAMP content is indicated in Table 1 as a per-

centage inhibition of cAMP production relative to control (absence

of the test compound), in the absence or presence of the orthosteric

To assess the biological activity of the synthesized compounds

2a–i and 3a–au, we initially screened all molecules using a

functional assay, evaluating their ability to inhibit forskolin-stimu-

lated cAMP accumulation in intact CHO cells expressing the cloned

hA₁AR. When this receptor is in an active conformation in CHO cells,

it causes a measurable inhibition of adenylate cyclase activity. AEs

R. Romagnoli et al. / Bioorg. Med. Chem. 22 (2014) 148–166

151

R₀

O

CN

O

a

b

c

O

N

S

CN

NH₂

S

Ro

6a-g

O

2d, 3a-d and 5a-b

d

Ro

4a-g

R₀

O

4a, 6-7a; R₀=4-Cl

4b, 6-7b; R₀=3-Cl

e

O

NH₂

4c, 5a, 6-7c; R₀=2-Cl

4d, 6-7d; R₀=3,4-Cl₂

Br

N

S

Br

4e, 5b, 6-7e; R₀=2,4-Cl₂

4f, 6-7f; R₀=3-CF₃

7a-g

S

3e-i

O

4g, 6-7g; R₀=4-CH₃

f

R₀

O

8a; R₀=4-Cl, R₅=C₆H₅

8t; R₀=4-Cl, R₅=pyridin-3-yl

8u; R₀=4-Cl, R₅=pyridin-2-yl

8v; R₀=4-Cl, R₅=4'-F-C₆H₄

8w; R₀=4-Cl, R₅=2',3'-diF-C₆H₄

8x; R₀=4-Cl, R₅=2',4'-diF-C₆H₄

8y; R₀=4-Cl, R₅=2',5'-diF-C₆H₄

8z; R₀=4-Cl, R₅=2',6'-diF-C₆H₄

8aa; R₀=4-Cl, R₅=4'-Cl-C₆H₄

O

8b; R₀=3-Cl, R₅=C₆H₅

8c; R₀=2-Cl, R₅=C₆H₅

R₅

8a-al

N

S

8d; R₀=3,4-Cl_2,R₅=C₆H₅

8e; R₀=2,4-Cl_2,R₅=C₆H₅

8f; R₀=3-CF_3,R₅=C₆H₅

O

8g; R₀=4-CH_3,R₅=C₆H₅

e

8h; R₀=4-Cl, R₅=4'-OCH₃-C₆H₄

8i; R₀=3-Cl, R₅=4'-OCH₃-C₆H₄

8j; R₀=3,4-Cl_2,R₅=4'-OCH₃-C₆H₄

8k; R₀=3-CF_3,R₅=4'-OCH₃-C₆H₄

8l; R₀=4-CH_3,R₅=4'-OCH₃-C₆H₄

8m; R₀=4-Cl,

8ab; R₀=4-Cl,R₅=(E)-4'-Cl-C₆H₄CH=CH

8ac; R₀=4-Cl, R₅=3',4'-diCl-C₆H₄

8ad; R₀=4-Cl, R₅=3'-OCH₃-C₆H₄

8ae; R₀=4-Cl, R₅=2'-OCH₃-C₆H₄

8af; R₀=4-Cl, R₅=4'-OCH₃(CH₂)₂O-C₆H₄

8ag; R₀=4-Cl, R₅=3',4'-(diO-CH₂)-C₆H₃

8ah; R₀=4-Cl, R₅=4'-OCF₃-C₆H₄

8ai; R₀=4-Cl, R₅=4'-CH₃-C₆H₄

R₀

O

NH₂

R₅=3,4-(diCH₃)-isoxazol-4-yl

8n; R₀=4-Cl, R₅=1H-pyrazol-4-yl

8o; R₀=4-Cl, R₅=thiophen-2-yl

8p; R₀=4-Cl, R₅=thiophen-3-yl

8q; R₀=4-Cl, R₅=furan-2-yl

8r; R₀=4-Cl, R₅=furan-3-yl

8s; R₀=4-Cl, R₅=pyridin-4-yl

R₅

S

8aj; R₀=4-Cl, R₅=3'-CH₃-C₆H₄

3j-au

8ak; R₀=4-Cl, R₅=2'-CH₃-C₆H₄

8al; R₀=4-Cl, R₅=4'-(CH₃)CH-C₆H₄

Reagents. a: 4,4-dimethylpentan-2-one, AcOH, β-alanine, toluene, reflux ; b: S₈, TEA, EtOH,

reflux; c: phthalic anidride, AcOH, reflux; d: NBS, CH₃CN, reflux, e: NH₂NH_2,EtOH, reflux; f:

PdCl₂(DPPF), ArB(OH)_2,CsF, 1,4-dioxane, 65 °C.

Scheme 1.

agonist. The degree of inhibition of cAMP production was similar

under the two conditions tested.

while the presence of a bromine (3f) or the absence of a substituent

(3a) decreased the allosteric enhancement. For compound 3k,

moving the chlorine from the 3- to the 2-position of the benzoyl

moiety (compound 3l) led to a 1.5-fold reduction in activity. The

3,4-dichlorobenzoyl derivatives 3b, 3g, 3m and 3s showed the

same trend observed for the 3-chlorobenzoyl analogues 3a, 3f, 3k

and 3r, with the C-5 phenyl and 4-methoxyphenyl derivatives 3m

and 3s, respectively more active than the C-5 unsubstituted (3b)

and bromo- (3g) analogues. The 3-(3,4-dichlorobenzoyl)-5-phenyl

thiophene derivative 3m showed activity comparable to that of the

isomeric 2,4-dichlorobenzoyl counterpart, 3n.

Among the 2-amino-3-aroyl-4-neopentylthiophene derivatives

2d and 3a–au, only one compound (3v) was less active than PD

81,723 at the concentration tested. This suggests that the presence

of a 3,4-dimethyl-isoxazol-4-yl moiety at the 5-position of 2-ami-

no-3-(4-chlorobenzoyl)-5-neopentyl scaffold is detrimental to the

interaction of the ligand with the allosteric binding site of the

A₁AR. Three of the compounds (3c, 3w and 3an) were comparable

in activity to PD 81,723. All of the remaining new compounds were

more active at 10 lM than PD 81,723 and decreased the percent-

age of cAMP production from 37% to 62% (Table 1). With only a

few exceptions, the best results for inhibition of cAMP production

(>50%) were observed with the presence of an aryl or heteroaryl

(thiophene or furan) at the 5-position of the 2-amino-3-aroyl-4-

neopentyl-thiophene.

For the derivatives 3a, 3f, 3k and 3r, replacing the chlorine of

the 3-chlorobenzoyl moiety with a more lipophilic and isoelec-

tronic triﬂuoromethyl, to furnish compounds 3c, 3h, 3o and 3t,

respectively, led to a signiﬁcant reduction in activity, with the

exception of the 5-bromothiophene 3h.

In the series of 4-neopentyl-5-unsubstituted-thiophenes 2d and

3a–d, the 3-chloro (3a) and 3,4-dichloro (3b) derivatives tended to

be more potent than the 4-chloro (2d), 3-triﬂuoromethyl (3c) and

4-methyl (3d) analogues, with 3c being the least active compound

in the series.

In the series of four derivatives (3a, 3f, 3k and 3r) characterized

by the presence of a common 3-chlorobenzoyl moiety at the 3-po-

sition of 2-amino-4-neopentyl-thiophene skeleton and that differ

in the 5-substituent, it appeared that the most active compounds

had a phenyl (3k) or a 4-methoxyphenyl (3r) at the C-5 position,

For the 4-methylbenzoyl derivatives 3d, 3i, 3p and 3u, the

replacement of the hydrogen (3d) at the 5-position of the thio-

phene ring with a bromine (3i), phenyl (3p) or a 4⁰-methoxyphenyl

(3u) group led to a 1.5-fold reduction of cAMP production. A sim-

ilar effect was observed in the series of 4-chlorobenzoyl derivatives

2d, 3e, 3j and 3q, with the C-5 substituted analogues 3e (bromine),

3j (phenyl) and 3q (4⁰-methoxyphenyl) being more active than the

C-5 unsubstituted analogue 2d. Comparing the activities of deriva-

tives bearing the same substituent at the 5-position of the

thiophene ring (2d vs 3d, 3e vs 3i, 3j vs 3p, 3q vs 3u), the

152

R. Romagnoli et al. / Bioorg. Med. Chem. 22 (2014) 148–166

signiﬁcant difference in activity between 3m and 3p and the re-

100

80

60

40

20

0

A

lated C-5 4⁰-methoxyphenyl analogues 3s and 3u. Interestingly,

when a 4-methoxy group was introduced onto the 5-phenyl of

compound 3o, increased activity was observed at the tested

concentration.

Molecules with the same substituent at different positions and

therefore characterized by the same lipophilicity were also studied.

This is the case for a group of isomeric derivatives constituted by

the analogues substituted in the 3-position of the thiophene with

4-chlorobenzoyl (2d, 3e, 3j and 3q) and 3-chlorobenzoyl (3a, 3f,

3k and 3r) moieties. Comparing the activities of the molecules with

the same substituent on the 5-position of the thiophene ring (2d vs

3a, 3e vs 3f, 3j vs 3k, 3q vs 3r), the derivatives with the 3-chloro-

benzoyl group are generally more potent than the corresponding

analogues with the 4-chlorobenzoyl substituent. The exception to

this trend is with the 5-bromothiophenes 3e and 3f, where the

two compounds are effectively equal in activity.

The bioisosteric replacement of the phenyl in the 5-position of

the 2-amino-3-(4-chlorobenzoyl)-4-neopentyl-thiophene scaffold

(compound 3j) by a thiophene in either regioisomeric orientation

(to furnish the 2⁰-thienyl and 3⁰-thienyl derivatives 3x and 3y,

respectively), has a beneﬁcal effect on AE activity, with no differ-

ence in the reduction of cAMP production between the two iso-

mers. In contrast, the 3⁰,4⁰-dimethyl isoxazol-4-yl (3v) was not

tolerated at the C-5 position of the thiophene ring, while replace-

ment of 5-phenyl ring with an isosteric 1H-pyrazol-4-yl (3w)

reduced activity by approximately one-half.

100

80

60

40

20

0

B

With compounds 3z–3ad, the phenyl ring at C-5 was replaced

with heterocycles that possessed heteroatoms able to form hydro-

gen bonds, such as the isomeric furans (3z and 3aa) or pyridines

(3ab–3ad). In addition, especially with the pyridines, we could also

increase the hydrophilic properties of the molecules, since low

water solubility is one of the major limitations of 2-amino-3-ben-

zoyl thiophene derivatives. The three pyridine isomers 3ab–3ad

exhibited reduced AE activity when compared to the phenyl coun-

terpart 3j, while replacement of phenyl with furan-2-yl or furan-3-

yl rings (3z and 3aa, respectively), maintained the activity. Thus,

results of functional assay of both the more hydrophilic com-

pounds 3ab–3ad and hydrophobic compounds 3j, 3x and 3y, seem

to conﬁrm that the receptor domain of the allosteric site of the

A₁AR surrounding the 5-position of thiophene ring is principally

hydrophobic in nature.

Encouraged by the increased AE activity obtained with the

2-amino-4-(4-chlorobenzoyl)-4-neopentyl-5-phenylthiophene 3j,

we synthesized compounds 3q and 3ae–3au, to determine

whether various electron-donating (alkyl, alkoxy or OCF₃) or elec-

tron-withdrawing (F and Cl) substituents on the different positions

of the C-5 phenyl ring would lead to further increases in activity.

Turning speciﬁcally to the 4-mono-substituted phenyl derivatives

3q, 3ae, 3aj, 3ak, 3ao, 3aq, 3ar, and 3au, they showed highly var-

iable activity. The introduction of the weakly electron withdrawing

ﬂuorine group (compound 3e) had little overall effect on AE activ-

ity and increasing the size of the halogen from ﬂuorine to chlorine,

to furnish the derivative 3aj, led to a slight increase of the activity.

With the aim of determining if the presence of a second ﬂuorine

atom on the phenyl ring would lead to an increase of activity, the

diﬂuoro derivatives 3af–3ai were prepared. The 2⁰,4⁰-diﬂuoro-

(3ag), 2⁰,5⁰-diﬂuoro-(3ah) and 2⁰,6⁰-diﬂuoro-(3ai) derivatives main-

tain an AE activity comparable to that of the mono-substituted 4⁰-F

analogue 3ae, while the activity was superior for the 2⁰,3⁰-diﬂuoro

derivative 3af, in which the ﬂuorines are adjacent to each other.

Because the electronic properties of the di-ﬂuorophenyl substitu-

ents in compounds 3af–3ai are similar, the superior activity of

the 2⁰,3⁰-diﬂuoro derivative 3af may be due to steric or

hydrogen-bonding factors caused by the relative position of the

two ﬂuorine atoms on the phenyl ring.

Figure 1. Histograms showing the cAMP inhibition, expressed in pmol/10⁶cells,

mediated by novel allosteric enhancers at 10 M concentration (A). The effect of the

examined compounds (100 nM) was also studied in the presence of 1 pM CCPA (B).

Values are expressed as mean SEM of three separate experiments, as described in

Section 5.

l

substitution of the chloro at the 4-position of the benzoyl moiety

with a methyl, which has similar lipophilic character but an

opposite electronic effect, resulted in improved activity.

Starting from the 5-unsubstituted 4-chlorobenzoyl (2d), 3-trif-

luorobenzoyl (3c) and 4-methylbenzoyl (3d) thiophene analogues,

the insertion of a bromine at the C-5 position of thiophene ring, to

furnish compounds 3e, 3h and 3i, respectively, caused a 1.5-fold

increase in the percent inhibition of cAMP production, while there

was no difference in activity between the 3-chlorobenzoyl and

3,4-dichlorobenzoyl derivatives 3a and 3b vs the corresponding

5-bromo analogues 3f and 3g, respectively.

In comparing the 5-bromothiophene derivatives 3e–i, the

4-methylbenzoyl derivative 3i was more active than the other com-

pounds, which each showed a similar level of enhancement.

In contrast to the situation with the 4-chlorobenzoyl derivatives

3e and 3j and 4-methylbenzoyl analogues 3i and 3p, where

replacement of bromine (3e and 3i) with a phenyl (3j and 3p) in

the 5-position of the thiophene ring led to little change in activity,

with the isomeric 3-chlorobenzoyl and 3,4-dichlorobenzoyl

analogues 3f and 3g, respectively, replacement of bromine with

phenyl group, to furnish the corresponding derivatives 3k and

3m, caused a substantial increase of activity. In contrast, for the

3-triﬂuoromethylbenzoyl compounds 3h and 3o, the activity of the

5-phenyl derivative 3o was inferior to that the 5-bromo analogue

3h.

The introduction of an electron-releasing methoxy group at the

4-position of the C-5 phenyl ring of compounds 3j, 3k, 3m and 3o–

p has variable effects. Comparing 3j and 3k to the corresponding

C-5 4⁰-methoxyphenyl analogues 3q and 3r, the substituted

compounds showed slightly reduced activity, while there was no

R. Romagnoli et al. / Bioorg. Med. Chem. 22 (2014) 148–166

153

Relative to the activity of the 4-chloro derivative 3aj, the inser-

tion of an additional chlorine atom to the 3-position, affording the

3⁰,4⁰-dichloro analogue 3al, retained the activity.

Replacing chlorine with the electron-donating methyl group

(3ar) also maintained the activity, which was considerably reduced

by the substitution of the methyl with a less lipophilic and more

electron-releasing methoxy group (3q). For this latter compound,

the reduction in activity may be attributed to electronic factors.

We can exclude steric factors, due to the good activity shown by

lengthening the 4⁰-alkoxy moiety from methoxy to methoxyethy-

lenoxy (CH₃OCH₂CH₂O, derivative 3ao), characterized by the pres-

ence of an angularity component which extends signiﬁcantly

above or below the plane of the C-5 phenyl ring. The replacement

of the methoxyethylenoxy group with a 4⁰-OCF₃(compound 3aq)

maintained the level of cAMP reduction, which was comparable

to that of the 4⁰-Cl derivative 3aj. Starting from this latter

Table 2

A₁AR density expressed as B_maxvalues (A) obtained by [³H]CCPA binding assays in hA₁CHO membranes in the presence of 2a–i, 3a–au and of PD 81,723 (10

lM). Modulation by

the novel allosteric enhancers (10

l

M) on the CCPA afﬁnity (CCPA K_ishift) in [³H]DPCPX competition binding experiments (B)^a

Compound

(A)

(B)

B_max(fmol/mg protein)

B_maxshift (fold of increase)

CCPA K_i(nM)

CCPA K_ishift (fold of increase)

2a

2b

2c

2d

2e

2f

2g

2h

2i

3a

3b

3c

3d

3e

3f

3g

3h

3i

3j

3k

3l

3m

3n

3o

3p

3q

3r

3s

3t

3u

3v

3w

3x

3y

798 72

833 68

679 62

2038 186

771 64

1094 102

932 82

1.5 0.1

1.6 0.1

1.3 0.1

3.9 0.3

1.5 0.1

2.1 0.2

1.8 0.1

1.4 0.1

2.1 0.2

4.3 0.4

4.1 0.4

2.6 0.2

3.3 0.3

6.8 0.5

3.6 0.3

4.5 0.4

4.4 0.4

6.4 0.6

6.9 0.7

6.3 0.6

5.4 0.5

5.9 0.5

6.8 0.7

4.0 0.4

6.7 0.6

6.1 0.6

6.0 0.6

6.0 0.5

4.0 0.4

7.4 0.7

1.4 0.1

2.5 0.3

6.2 0.6

6.4 0.7

5.1 0.5

5.6 0.6

4.0 0.4

3.7 0.4

3.5 0.3

5.5 0.5

6.1 0.6

5.3 0.5

5.3 0.6

4.2 0.4

5.8 0.6

4.8 0.5

6.0 0.6

6.3 0.6

2.5 0.2

5.5 0.5

6.6 0.6

6.4 0.7

4.7 0.4

4.1 0.5

5.6 0.6

1.3 0.1

9.8 0.7

4.5 0.5

9.2 0.9

9.5 0.8

10.6 0.9

8.5 0.8

8.1 0.7

9.7 0.8

9.3 0.8

9.8 0.9

7.5 0.7

8.8 0.9

7.7 0.8

3.5 0.3

6.2 0.6

3.8 0.3

5.1 0.6

3.5 0.4

3.3 0.3

4.8 0.5

4.0 0.4

3.2 0.3

3.5 0.4

4.2 0.4

3.0 0.3

3.6 0.4

4.1 0.4

3.5 0.4

8.4 0.8

2.8 0.3

13.3 1.2

8.7 0.9

3.9 0.4

3.2 0.3

4.3 0.4

4.8 0.5

5.4 0.5

6.9 0.7

6.8 0.7

4.1 0.4

3.1 0.3

4.2 0.4

4.4 0.4

6.3 0.6

3.8 0.4

5.3 0.5

3.5 0.3

2.6 0.3

8.2 0.8

4.6 0.5

2.9 0.3

3.3 0.3

2.5 0.3

3.7 0.3

5.5 0.5

3.7 0.4

10.4 0.9

1.6 0.1

3.4 0.3

1.6 0.2

1.6 0.1

1.5 0.1

1.8 0.2

1.9 0.2

1.6 0.2

1.7 0.1

1.5 0.1

2.0 0.2

1.7 0.2

2.0 0.2

4.5 0.4

2.4 0.2

4.0 0.4

3.0 0.3

4.3 0.4

4.8 0.5

3.1 0.3

3.9 0.3

4.8 0.5

4.4 0.4

3.6 0.4

5.0 0.5

4.3 0.4

3.7 0.4

4.3 0.4

1.8 0.2

5.4 0.5

1.1 0.1

1.7 0.2

3.9 0.4

4.7 0.5

3.5 0.4

3.1 0.3

2.8 0.3

2.2 0.2

3.7 0.3

4.9 0.5

3.6 0.4

3.4 0.3

2.4 0.2

4.0 0.4

2.8 0.3

4.3 0.4

5.8 0.6

1.9 0.2

3.3 0.3

5.2 0.5

4.6 0.5

6.0 0.6

4.1 0.4

2.7 0.3

4.1 0.4

1.5 0.1

731 66

1126 107

2241 214

2148 206

1359 125

1726 163

3591 316

1877 169

2346 224

2286 218

3351 312

3649 324

3286 318

2863 256

3072 279

3603 277

2087 211

3493 322

3198 284

3139 314

3138 302

2084 198

3861 376

735 65

1308 138

3237 318

3332 341

2668 262

2922 297

2086 204

1928 194

1831 176

2876 277

3176 311

2758 261

2768 284

2194 208

3022 309

2509 252

3143 313

3292 317

1322 113

2879 270

3449 338

3337 346

3345 346

2449 234

2138 218

2932 296

685 62

3z

3aa

3ab

3ac

3ad

3ae

3af

3ag

3ah

3ai

3aj

3ak

3al

3am

3an

3ao

3ap

3aq

3ar

3as

3at

3au

PD 81,723

(A) = B_max(fmol/mg protein) and B_maxshift obtained in [³H]CCPA saturation binding experiments performed in the absence (B_max= 522 46 fmol/mg protein) or in the

presence of 10 M enhancers.

(B) = K_ivalues of CCPA in the presence of 10

l

M test compounds and CCPA shift = K_i(CCPA)/K_i(CCPA + 10

lM enhancers) where the K_iof CCPA was 15.1 1.6 nM.

a

The values are expressed as the mean SEM, n = 3 independent experiments.

154

R. Romagnoli et al. / Bioorg. Med. Chem. 22 (2014) 148–166

compound, the insertion of a vinyl (CH@CH) spacer between the

5-position of thiophene ring and the 4⁰-chlorophenyl moiety, to

afford the (E)-4⁰-chlorocinnamic derivative 3ak, resulted in a slight

reduction in activity relative to 3aj.

the most active compounds, each causing a B_maxshift of more than

6-fold. Figure 2 shows the effect of the allosteric modulators PD

81,723, 3p, 3u and 3ar at 10 l

M concentration in [³H]CCPA satura-

tion binding experiments on A₁AR binding parameters such as

afﬁnity and density. Interestingly, no differences were found in

afﬁnity values, suggesting that the enhancers were not able to

modify the K_Dvalues of the high afﬁnity binding sites labeled by

[³H]CCPA (K_Dranged from 1.0 0.1 to 1.2 0.1).

Table 2 also reports the derived apparent afﬁnity (K_i) values for

CCPA, based on a one-state model of analysis, in the absence and in

the presence of the tested enhancers. This table also shows the

CCPA shift representing the ratio of apparent K_ivalues in the ab-

For compounds 3q, 3am and 3an, the position of the methoxy

substituent on the C-5 phenyl ring had inﬂuence on AE activity,

which increased in the order: ortho < < para < meta. Placing the

methoxy group on the 2-position of the phenyl ring reduced the

activity by approximately one-half relative to that of 3j. An in-

crease of activity was observed when the methoxy substituent

was moved from the 2⁰-position to either the 3⁰-(3am) or the

4⁰-(3q) position, which was more evident for the 3⁰-derivative

3am. Also the presence of a 3⁰,4⁰-methylenedioxy moiety on the

phenyl ring (3ap) led to an increase in the AE activity relative to 3j.

Among the three isomeric C-5 tolyl-thiophene derivatives 3ar-

3at, the 4⁰-methyl derivative 3ar was more active than the

3⁰-methyl and 2⁰-methyl counterparts (3as and 3at, respectively),

with these two latter compounds being equiactive. By replacing

the 4⁰-methyl group (3ar) with the more lipophilic and sterically

demanding isopropyl group (3au) activity was maintained.

By comparing the effects of ERG’s and EWG’s on the phenyl at

the C-5 position of the thiophene ring, no clear inﬂuence on allo-

steric enhancement was observed. In fact, several compounds

characterized by the presence of substituents with opposite elec-

tronic effects showed the same activity. For example, compound

3aj containing the electron-withdrawing chloro group showed

the same activity as compound 3ar containing the electron-donat-

ing methyl group.

sence and in the presence of the tested compounds at 10 lM con-

centration. In the hA₁CHO membranes, by using [³H]DPCPX as

radioligand, the K_ivalue of CCPA was 15.2 1.3 nM. Interestingly,

a signiﬁcant decrease in the apparent K_ivalue was due to the pres-

ence of the putative allosteric enhancers, suggesting an increase in

the high-afﬁnity binding sites. In the presence of PD 81,723, the

afﬁnity of CCPA increased by 1.5-fold. The CCPA afﬁnity data in

the presence of the derivatives 2b, 2f–g, 2i, 3b–u and 3w–au reveal

that the displacement curves are shifted left, suggesting even low-

er K_ivalues for CCPA. In particular, the largest afﬁnity shift has

been observed for compounds 3p, 3u, 3am, 3ap and 3ar. These

molecules enhanced the apparent afﬁnity of CCPA approximately

5.0, 5.4-, 5.8-, 5.2- and 6.0-fold, respectively, being twice as active

as the 5-unsubstituted derivatives 2b and 3a–d (Table 2) in this as-

say. Thus, the enhancers were able to mediate a shift of the A₁

receptors towards the high afﬁnity state as suggested from the in-

crease of the CCPA afﬁnity expressed as K_ivalues (Table 2). In Fig-

ure 3, representative binding curves for the displacement of

[³H]DPCPX by different concentrations of CCPA alone and in the

3.2. Antagonistic activity

Many of the currently available allosteric enhancers of agonist

binding to the hA₁AR have several non-speciﬁc actions. These

non-speciﬁc actions include antagonism at the A₁adenosine recep-

tor, especially at higher concentrations. The ability of compounds

2d and 3a–aq to displace the binding of [³H]DPCPX,

[³H]ZM241385 and [³H]MRE-3008-F20 at human A₁, A_2Aand A₃

presence of PD 81,723, 3p, 3u and 3ar at 10 lM concentration

are shown, demonstrating the apparent increase of the CCPA afﬁn-

ity in the presence of novel enhancers.

4000

A

Control

+ PD 81,723

+ 3ar

ARs were evaluated in CHO cells at a concentration of 10

The prototype enhancer PD 81,723 did not inhibit the binding of

the radiolabeled antagonists to A₁and A_2AARs, but at 10 M, it re-

lM.

3000

l

+ 3p

+ 3u

duced by 21% the binding of [³H]MRE-3008-F20 to A₃ARs.¹⁸None

of the examined derivatives signiﬁcantly inhibited the speciﬁc

binding of the radioligands to A₁, A_2Aand A₃ARs, causing inhibition

of radioligand binding of 11% or less (Table S1). For the most active

compounds in functional assays, such as 3i, 3k, 3p, 3u, 3x–y, 3af,

3aj, 3al and 3ap–ar it was possible to achieve a good separation

between high efﬁcacy in the inhibition of cAMP production and

binding to the orthosteric site.

2000

1000

0

5

10

15

20

[³H]CCPA free (nM)

4000

3000

2000

1000

0

B

3.3. Effect of enhancers on A₁AR binding parameters

Control

+ PD 81,723

+ 3ar

+ 3p

+ 3u

Saturation and competition experiments of the selective adeno-

sine A₁agonist [³H]CCPA to A₁receptors were performed to deter-

mine if the novel compounds modiﬁed the agonist binding

parameters. From these experiments, A₁receptor afﬁnity (K_D)

and density (B_max) were evaluated in the presence and in the ab-

sence of the examined compounds (PD 81,723, 2a–i and 3a–au

at a concentration of 10 lM) and were used to calculate the

increase of A₁density (B_maxshift) (Table 2).

0

1000

2000

Bound

3000

4000

The reference compound PD 81,723 induced a B_maxshift to hu-

man A₁adenosine receptors of 1.3-fold. Under the same experi-

mental conditions, with the exception of compounds 2a, 2c, 2e–i

and 3v, all of the new tested compounds were signiﬁcantly more

potent than PD 81,723. From the receptor density calculated in

the presence and in the absence of the novel enhancers, the deriv-

atives 3e, 3i–k, 3n, 3p, 3q–s, 3x–y, 3af, 3al–3am and 3ap–3ar were

Figure 2. [³H]-CCPA saturation binding curves at human A₁adenosine receptors

(A). Under control conditions, K_Dvalue was 1.1 0.1 nM and the B_maxwas

522 46 fmol/mg protein. In the presence of novel enhancers (10 lM), K_Dvalues

were similar to those obtained in controls and B_maxvalues were as reported in

Table 2. Values are the means and vertical lines are the SEM of three separate

experiments, as described in Section 5. Scatchard plots of the same experimental

data (B).

R. Romagnoli et al. / Bioorg. Med. Chem. 22 (2014) 148–166

155

100

80

60

40

20

0

2-amino-3-aroyl-4-neopentylthiophene derivatives (2d and 3a–

au) signiﬁcantly inhibited antagonist binding at the hA₁AR, hA₂AR,

or hA₃AR. Among these, derivatives 3p, 3u, 3am, 3ap and 3ar were

the most active compounds in binding (saturation and displace-

ment) experiments and functional cAMP assays.

Control

+ PD 81,723

+ 3ar

+ 3p

+ 3u

5. Experimental section

5.1. Chemistry

-12 -11 -10 -9

-8

-7

-6

-5

-4

log [CCPA] (M)

5.1.1. Materials and methods

Figure 3. Inhibition curves of speciﬁc [³H]-DPCPX binding to human A₁ARs of CCPA

in the absence and in the presence of novel enhancers (10 M). Afﬁnity values were

¹H and ¹³C NMR spectra were recorded on a Bruker AC 200 and

Varian 400 Mercury Plus spectrometer, respectively. Chemical

shifts (d) are given in parts per million (ppm) downﬁeld and J val-

ues are given in hertz. All products reported showed ¹H NMR spec-

tra in agreement with the assigned structures. Positive-ion

electrospray ionization (ESI) mass spectra were recorded on a dou-

ble-focusing ESI Micromass ZMD 2000 mass spectrometer. Melting

points (mp) were determined on a Buchi–Tottoli apparatus and are

uncorrected. Elemental analyses were conducted by the Microana-

lytical Laboratory of the Chemistry Department of the University of

Ferrara and were performed on a Yanagimoto MT-5 CHN recorder

analyzer. All tested compounds yielded data consistent with a pur-

ity of at least 95% as compared with the theoretical values. All reac-

tions were performed under an inert atmosphere of dry nitrogen,

unless otherwise described. Standard syringe techniques were ap-

plied for transferring dry solvents. Reaction courses and product

mixtures were routinely monitored by TLC on silica gel (precoated

F254 Merck plates) and visualized with aqueous KMnO₄. Flash

chromatography was performed using 230–400 mesh silica gel

and the solvent system indicated in the procedure. All commer-

cially available compounds were used without further puriﬁcation.

Organic solutions were dried over anhydrous Na₂SO₄. Dichloro-

methane (DCM) was distilled from calcium chloride and stored

over molecular sieves (3 Å). Petroleum ether refers to the fraction

boiling at 40–60 °C. Compounds 2a and 2i were synthesized fol-

lowing the synthetic procedures reported in the references 14

and 13a, respectively.

l

calculated by using a one-state model of analysis. Values are the means and vertical

lines are the SEM of three separate experiments as described in Section 5.

The results obtained from the competition and saturation

experiments conﬁrmed the good enhancer activity of most of the

synthesized compounds, in agreement with the cAMP functional

assay.

4. Conclusions

The current study describes the synthesis and biological evalu-

ation of a novel series of 2-amino-3-benzoyl-4-neopentyl thio-

phene derivatives, with variable modiﬁcations at the 5-position

of the thiophene as well as in the benzoyl system. The presence

of a neopentyl at the 4-position and a heteroaryl or variably substi-

tuted phenyl at the 5-position of 2-amino-3-aroyl-thiophene skel-

eton represented the best combination to afford a series of

compounds with improved AE activity. Among the 4-neopentyl

derivatives 2d and 3a–au, the 5-(3⁰,4⁰-dimethyl-isoxazol-4⁰yl) thi-

ophene derivative 3v was the least active compound in the series.

Replacement of a phenyl ring in the 5-position of the thiophene

ring with heterocycles that potentially could form a hydrogen bond

with the allosteric site of the A₁AR allowed the activity to be main-

tained with the furan derivatives 3z and 3aa. The activity was

slightly reduced with the more hydrophilic pyridine isomers

3ab–3ad. On the contrary, replacement of the phenyl ring with

the two isosteric/isoelectronic thienyl moieties (3x and 3y) was

well tolerated and increased the AE activity, with minimal differ-

ence in the reduction of cAMP production between 3x and 3y. This

similarity also occurred with the other isomeric pair tested, the

furanyl derivatives 3z and 3aa. In examining the effect of ERGs

and EWGs on the phenyl at the C-5 position of the thiophene, no

consistent pattern of effects on AE activity was observed. Staring

from the mono-ﬂuoro derivative 3ae, the introduction of an addi-

tional ﬂuorine atom retained [2⁰,4⁰-diF (3ag), 2⁰,5⁰-diF (3ah) and

2⁰,6⁰-diF (3ai) derivatives] or increased (2⁰,3⁰-diF, compound 3af)

the activity. There was little difference in activity between the

two 4⁰-alkylphenyl derivatives 3ar and 3au. In an effort to more

fully examine the steric effects of the alkoxy substituent at the

4⁰-position of the C-5 phenyl, the OCF₃and the more bulky

MeO(CH₂)₂O group (compounds 3aq and 3ao, respectively), were

prepared, both being more active relative to the 4⁰-OMe derivative

3q. The contribution of methyl and methoxy moieties to activity

was position-dependent. The 3⁰-methoxy derivative 3am had a

greater AE activity than the corresponding 4⁰- and 2⁰-methoxy

analogues (3q and 3am, respectively). Turning to the effects of

an electron-releasing group on the C5-phenyl moiety, we found

that a 4⁰-methyl group (3ar) caused a slight increase on AE activity

relative to the unsubstituted derivative 3j, which was reduced

moving the methyl group from the 4⁰- to the 3⁰- and 2⁰-positions.

A characteristic feature of AEs at the A₁AR is the propensity to

also cause antagonism at higher concentrations. None of the

5.2. General procedure (A) for the synthesis of compounds 4a–

g^12b

A mixture of 4,4-dimethylpentan-2-one (2.8 mL, 20 mmol) and

the appropriate aroylacetonitrile (20 mmol), acetic acid (2.6 mL,

43.3 mmol), b-alanine (180 mg, 2 mmol) and benzene (70 mL)

was heated to reﬂux in a Dean–Stark system. After 10 h, a second

addition of acetic acid (2.6 mL) and b-alanine (180 mg, 2 mmol)

was made. After 24 h, the reaction mixture was cooled to room

temperature, diluted with ethyl acetate (50 mL), washed with

water (30 mL), brine (30 mL), dried (Na₂SO₄), and ﬁnally concen-

trated in vacuo. The crude residue, constituting a mixture of

E- and Z-isomers, was puriﬁed by column chromatography on

silica gel to furnish the derivatives 4a–g.

5.2.1. (E/Z)-2-(4-Chlorobenzoyl)-3,5,5,-trimethylhex-2-

enenitrile (4a)

Following general procedure A, using 3-(4-chlorophenyl)-3-

oxo-propionitrile as b-ketonitrile, the crude was residue puriﬁed

by column chromatography on silica gel, eluting with petroleum

ether/ethyl acetate (19:1). Fractions containing the Knoevenagel

adduct 4a, as a mixture of geometric isomers, were combined

and concentrated to afford the desired product as a yellow oil

(yield 64%). ¹H NMR (CDCl₃) d: 0.93 (s, 9H), 1.13 (s, 9H), 2.11 (s,

3H), 2.38 (s, 3H), 2.54 (s, 2H), 2.60 (s, 2H), 7.47 (d, J = 6.4 Hz,

2 ꢀ 2H), 7.86 (d, J = 6.4 Hz, 2 ꢀ 2H). MS (ESI): [M+1]⁺= 276.2.

156

R. Romagnoli et al. / Bioorg. Med. Chem. 22 (2014) 148–166

5.2.2. (E/Z)-2-(3-Chlorobenzoyl)-3,5,5,-trimethylhex-2-

enenitrile (4b)

(s, 3H), 2.50 (s, 2H), 2.58 (s, 3H), 7.28 (d, J = 8.2 Hz, 2 ꢀ 2H),

7.84 (d, J = 8.2 Hz, 2 ꢀ 2H). MS (ESI): [M+1]⁺= 256.2.

Following general procedure A, using 3-(3-chlorophenyl)-3-

oxo-propionitrile as b-ketonitrile, the crude residue puriﬁed by

column chromatography on silica gel, eluting with petroleum

ether/ethyl acetate (9:1), afforded the desired product 4b as a yel-

low oil (yield: 53%). ¹H NMR (CDCl₃) d: 0.93 (s, 9H), 1.13 (s, 9H),

2.11 (s, 3H), 2.38 (s, 3H), 2.54 (s, 2H), 2.60 (s, 2H), 7.44 (t,

J = 7.6 Hz, 2 ꢀ 1H), 7.58 (d, J = 7.4 Hz, 2 ꢀ 1H), 7.78 (d, J = 7.4 Hz,

2 ꢀ 1H), 7.89 (s, 2 ꢀ 1H). MS (ESI): [M+1]⁺= 276.1.

5.3. General procedure (B) for the synthesis of compounds 2d,

3a–d and 5a–b^12b

A mixture of the Knoevenagel’s adduct (6.3 mmol), triethyl-

amine (1.1 mL, 7.65 mmol), and sulfur (243 mg, 7.6 mmol) in eth-

anol (20 mL) was heated to reﬂux for 2 h. After cooling to room

temperature, the mixture was concentrated and the residue dis-

solved in ethyl acetate (20 mL). The organic solution was washed

with 0.5 N HCl (5 mL), saturated aqueous NaHCO₃(5 mL), water

(5 mL), brine (5 mL), dried (Na₂SO₄), ﬁltered, and concentrated

in vacuo. The crude residue was puriﬁed by column chromatogra-

phy on silica gel using petroleum ether/ethyl acetate as eluent.

Fractions containing the desired product were combined and

concentrated to afford the desired product.

5.2.3. (E/Z)-2-(2-Chlorobenzoyl)-3,5,5,-trimethylhex-2-

enenitrile (4c)

Following general procedure A, using 3-(2-chlorophenyl)-3-

oxo-propionitrile as b-ketonitrile, the crude residue puriﬁed by

column chromatography on silica gel, eluting with petroleum

ether/ethyl acetate (19:1), afforded the desired product 4c as a col-

ourless oil (yield: 77%). ¹H NMR (CDCl₃) d: 0.91 (s, 9H), 1.14 (s, 9H),

2.12 (s, 3H), 2.34 (s, 3H), 2.52 (s, 2H), 2.62 (s, 2H), 7.32 (m, 4H), 7.74

(m, 4H). MS (ESI): [M+1]⁺= 276.2.

5.3.1. 2-Amino-5-(2,2-dimethylpropyl)thiophen-3-yl]

(4-chlorophenyl)methanone (2d)

Following general procedure B, derivative 2d was puriﬁed by

column chromatography using petroleum ether/ethyl acetate 9:1

as eluent. Yellow oil. Yield: 78%. ¹H NMR (CDCl₃) d: 0.64 (s, 9H),

2.12 (s, 2H), 5.92 (br s, 2H), 7.38 (s, 1H), 7.39 (d, J = 6.4 Hz, 2H),

7.57 (d, J = 6.4 Hz, 2H). MS (ESI): [M+1]⁺= 308.1. Anal. (C₁₆H_18-

ClNOS) C, H, N.

5.2.4. (E/Z)-2-(3,4-Dichlorobenzoyl)-3,5,5,-trimethylhex-2-

enenitrile (4d)

Following general procedure A, using 3-(3,4-dichlorophenyl)-3-

oxo-propionitrile (20 mmol) as b-ketonitrile, the crude residue,

constituting a mixture of E- and Z-isomers, was puriﬁed by column

chromatography on silica gel, eluting with petroleum ether:ethyl

acetate (19:1) to afford the desired product 4d as a yellow oil

(yield: 55%). ¹H NMR (CDCl₃) d: 0.93 (s, 9H), 1.13 (s, 9H), 2.13 (s,

3H), 2.39 (s, 3H), 2.55 (s, 2H), 2.61 (s, 2H), 7.62 (d, J = 6.4 Hz,

2 ꢀ 1H), 7.80 (m, 2 ꢀ 1H), 7.99 (s, 1H) 8.00 (s, 1H). MS (ESI):

[M+1]⁺= 310.1.

5.3.2. 2-Amino-5-(2,2-dimethylpropyl)thiophen-3-yl]

(3-chlorophenyl)methanone (3a)

Following general procedure B, derivative 3a was puriﬁed by

column chromatography using petroleum ether/ethyl acetate 9:1

as eluent. Yellow oil. Yield: 76%. ¹H NMR (CDCl₃) d: 0.64 (s, 9H),

2.10 (s, 2H), 5.92 (s, 1H), 6.04 (s, 2H), 7.36 (t, J = 6.8 Hz, 1H), 7.39

(d, J = 6.8 Hz, 1H), 7.56 (d, J = 6.8 Hz, 1H), 7.58 (s, 1H). MS (ESI):

[M+1]⁺= 308.2. Anal. (C₁₆H₁₈ClNOS) C, H, N.

5.2.5. (E/Z)-2-(2,4-Dichlorobenzoyl)-3,5,5,-trimethylhex-2-

enenitrile (4e)

Following general procedure A, using 3-(2,4-dichlorophenyl)-3-

oxo-propionitrile (20 mmol) as b-ketonitrile, the crude residue was

puriﬁed by column chromatography on silica gel, eluting with

petroleum ether/ethyl acetate (19:1), to afford the desired product

4e as a colourless oil (yield: 78%) d: ¹H NMR (CDCl₃) d: 1.03 (s, 9H),

1.09 (s, 9H), 2.29 (s, 3H), 2.31 (s, 3H), 2.62 (s, 2H), 2.83 (s, 2H), 6.95

(s, 1H), 7.01 (d, J = 6.4 Hz, 1H), 7.35 (d, J = 6.4 Hz, 1H), 7.36 (s, 1H),

7.54 (m, 2H). MS (ESI): [M+1]⁺= 310.1.

5.3.3. 2-Amino-5-(2,2-dimethylpropyl)thiophen-3-yl]

(3,4-dichlorophenyl)methanone (3b)

Following general procedure B, derivative 3b was puriﬁed by

column chromatography using petroleum ether/ethyl acetate 9:1

as eluent. Yellow oil. Yield: 78%. ¹H NMR (CDCl₃) d: 0.65 (s, 9H),

2.12 (s, 2H), 5.93 (s, 1H), 6.00 (br s, 2H), 7.41 (d, J = 7.2 Hz, 1H),

7.49 (d, J = 7.2 Hz, 1H), 7.69 (s, 1H). MS (ESI): [M+1]⁺= 342.3. Anal.

(C₁₆H₁₇Cl₂NOS) C, H, N.

5.2.6. (E/Z)-2-(3-Triﬂuoromethylbenzoyl)-3,5,5,-trimethylhex-2-

enenitrile (4f)

Following general procedure A, using 3-(3-triﬂuoromethyl-

phenyl)-3-oxo-propionitrile as b-ketonitrile, the crude residue,

constituting a mixture of E- and Z-isomers, was puriﬁed by column

chromatography on silica gel, eluting with petroleum ether/ethyl

acetate (19:1) to afford the desired product 4f as a yellow oil

(Yield: 44%). ¹H NMR (CDCl₃) d: 1.14 (s, 9H), 1.16 (s, 9H), 2.15 (s,

2H), 2.42 (s, 2H), 2.58 (s, 3H), 2.63 (s, 3H), 7.63 (m, 2 ꢀ 1H), 7.83

5.3.4. 2-Amino-5-(2,2-dimethylpropyl)thiophen-3-yl]

(3-triﬂuoromethylphenyl)methanone (3c)

Following general procedure B, derivative 3c was puriﬁed by

column chromatography on silica gel using petroleum ether/ethyl

acetate 19:1 as eluent. Yellow oil. Yield: 84%. ¹H NMR (CDCl₃) d:

0.60 (s, 9H), 2.03 (s, 2H), 5.93 (s, 1H), 6.14 (br s, 2H), 7.56 (t,

J = 7.8 Hz, 1H), 7.75 (m, 2H), 7.84 (s, 1H). MS (ESI):

[M+1]⁺= 342.4. Anal. (C₁₇H₁₈F₃NOS) C, H, N.

(m, 2 ꢀ 1H), 8.03 (m,

2

ꢀ 1H), 8.18 (s, 2 ꢀ 1H). MS (ESI):

[M+1]⁺= 310.3.

5.3.5. 2-Amino-5-(2,2-dimethylpropyl)thiophen-3-yl]

(4-methylphenyl)methanone (3d)

5.2.7. (E/Z)-2-(4-Methylbenzoyl)-3,5,5,-trimethylhex-2-

enenitrile (4g)

Following general procedure B, derivative 3d, the crude residue

was puriﬁed by column chromatography on silica gel using petro-

leum ether/ethyl acetate 7:3 as eluent. Yellow solid. Yield: 68%,

mp: 112–114 °C. ¹H NMR (CDCl₃) d: 0.64 (s, 9H), 2.17 (s, 2H),

2.41 (s, 3H), 5.69 (br s, 2H), 5.92 (s, 1H), 7.21 (d, J = 7.8 Hz, 2H),

7.51 (d, J = 7.8 Hz, 2H). ¹³C NMR (100 MHz, DMSO-d₆) d: 20.97,

Following general procedure A, using 3-(4-methylphenyl)-3-

oxo-propionitrile as b-ketonitrile, the crude residue constituting

a mixture of E- and Z-isomers, was puriﬁed by column chroma-

tography on silica gel, eluting with petroleum ether/ethyl acetate

(9:1) to afford the desired product 4g as a yellow oil (yield: 50%).

¹H NMR (CDCl₃) d: 0.92 (s, 9H), 1.13 (s, 9H), 2.36 (s, 3H), 2.43

R. Romagnoli et al. / Bioorg. Med. Chem. 22 (2014) 148–166

157

29.17 (3C), 31.34, 42.89, 105.45, 115.56, 128.67 (4C), 136.12,

138.05, 141.2, 162.81, 191.83. MS (ESI): [M+1]⁺= 288.2. Anal. (C_17-

H₂₁NOS) C, H, N.

yellow solid. Yield: 83%, mp 131–133 °C. ¹H NMR (CDCl₃) d: 0.82

(s, 9H), 2.69 (s, 2H), 7.10 (s, 1H), 7.27 (t, J = 8.2 Hz, 2H), 7.46 (d,

J = 8.2 Hz, 2H), 7.72 (m, 5H). MS (ESI): [M+1]⁺= 472.1.

5.3.6. 2-Amino-5-(2,2-dimethylpropyl)thiophen-3-yl]

(2-chlorophenyl)methanone (5a)

5.4.5. 2-[3-(2,4-Dichlorobenzoyl)-4-(2,2-dimethylpropyl)

thiophen-2-yl]isoindoline-1,3-dione (6e)

Following general procedure B, derivative 5a was puriﬁed by

column chromatography using petroleum ether/ethyl acetate 9:1

as eluent. Yield: 72%. Yellow solid, mp: 130–131 °C. ¹H NMR

(CDCl₃) d: 0.65 (s, 9H), 1.91 (s, 2H), 5.83 (s, 1H), 6.76 (br s, 1H),

7.39 (m, 4H). MS (ESI): [M+1]⁺= 308.1.

Following general procedure C, the residue was triturated with

petroleum ether (20 mL), furnishing the desired product 6e as a yel-

low solid. Yield: 68%; mp: 134–136 °C. ¹H NMR (CDCl₃) d: 0.68 (s,

9H), 2.69 (s, 2H), 7.07 (d, J = 6.4 Hz, 1H), 7.15 (s, 1H), 7.16 (s, 1H),

7.36 (d, J = 6.4 Hz, 1H), 7.75 (m, 4H). MS (ESI): [M+1]⁺= 472.2.

5.3.7. 2-Amino-5-(2,2-dimethylpropyl)thiophen-3-yl]

(2,4-dichlorophenyl)methanone (5b)

5.4.6. 2-[4-(2,2-Dimethylpropyl)-3-(3-triﬂuoromethylbenzoyl)-

thiophen-2-yl]isoindoline-1,3-dione (6f)

Following general procedure B, derivative 5b was puriﬁed by

column chromatography using petroleum ether/ethyl acetate 9:1

as eluent. Yellow solid. Yield: 74%, mp: 138–139 °C. ¹H NMR

(CDCl₃) d: 0.67 (s, 9H), 1.93 (s, 2H), 5.84 (br s, 2H), 6.02 (s, 1H),

7.31 (s, 1H), 7.39 (d, J = 6.4 Hz, 1H), 7.44 (d, J = 6.4 Hz, 1H). MS

(ESI): [M+1]⁺= 342.2.

Following general procedure C, the residue was puriﬁed by col-

umn chromatography on silica gel eluting with petroleum ether/

ethyl acetate 4:1, to furnish the compound 6f as a brown oil. Yield:

83%. ¹H NMR (CDCl₃) d: 0.68 (s, 9H), 2.74 (s, 2H), 7.11 (s, 1H), 7.34

(t, J = 8.4 Hz, 1H), 7.42 (d, J = 8.4 Hz, 1H), 7.70 (m, 4H), 7.80 (d,

J = 8.4 Hz, 1H), 7.93 (s, 1H). MS (ESI): [M+1]⁺= 472.1.

5.4. General procedure (C) for the synthesis of compounds 6a–g

5.4.7. 2-[4-(2,2-Dimethylpropyl)-3-(4-methylbenzoyl)-

thiophen-2-yl]isoindoline-1,3-dione (6g)

To a solution of 2-amino-3-aroyl-4-(2,2-dimethylpropyl)thio-

phene (5 mmol) in acetic acid (20 mL), was added phthalic anhy-

dride (0.88 g, 5.9 mmol) and the mixture heated to reﬂux for 6 h.

The solvent was removed in vacuo and the residue dissolved in

ethyl acetate (20 mL). The organic solution was washed with a sat-

urated aqueous solution of NaHCO₃(5 mL), water (5 mL), brine

(5 mL), dried (Na₂SO₄), ﬁltered, and concentrated in vacuo. The

crude product was then stirred for 1 h with petroleum ether

(20 mL), furnishing the desired product by ﬁltration. Alternatively,

the residue was puriﬁed by column chromatography (eluent

EtOAc-petroleum ether) on silica gel.

Following general procedure C, the residue was puriﬁed by col-

umn chromatography on silica gel eluting with petroleum ether/

ethyl acetate 4:1, to furnish the compound 6g as a pink solid. Yield:

73%; mp 169–170 °C. ¹H NMR (CDCl₃) d: 0.82 (s, 9H), 2.14 (s, 3H),

2.71 (s, 2H), 6.98 (s, 1H), 7.04 (d, J = 8.8 Hz, 2H), 7.55 (d, J = 8.8 Hz,

2H), 7.72 (m, 4H). MS (ESI): [M+1]⁺= 418.2.

5.5. General procedure (D) for the synthesis of compounds 7a–g

To a solution of thiophene derivative 6a–g (0.5 mmol) in CH₃CN

(5 mL) was added NBS (180 mg, 2 mmol). The mixture was heated

to reﬂux for 2 h, then cooled to room temperature. The solvent was

removed under reduced pressure, and the residue, dissolved in

EtOAc (10 mL), was sequentially washed with a saturated aqueous

solution of NaHCO₃(2 mL), water (1 mL), brine (1 mL), dried (Na_2-

SO₄), ﬁltered, and concentrated to give a residue puriﬁed by col-

umn chromatography on silica gel or triturated with petroleum

ether.

5.4.1. 2-[3-(4-Chlorobenzoyl)-4-(2,2-dimethylpropyl)thiophen-

2-yl]isoindoline-1,3-dione (6a)

Following general procedure C, the crude product was stirred

for 1 h with petroleum ether (20 mL) furnishing the desired prod-

uct 6a by ﬁltration as a yellow solid. Yield: 77%, mp 179–181 °C. ¹H

NMR (CDCl₃) d: 0.62 (s, 9H), 2.68 (s, 2H), 7.09 (s, 1H), 7.18 (d,

J = 6.4 Hz, 2H), 7.65 (d, J = 6.4 Hz, 2H), 7.72 (m, 4H). MS (ESI):

[M+1]⁺= 438.1.

5.5.1. 2-[5-Bromo-3-(4-chlorobenzoyl)-4-(2,2-dimethylpropyl)

thiophen-2-yl]isoindoline-1,3-dione (7a)

5.4.2. 2-[3-(3-Chlorobenzoyl)-4-(2,2-dimethylpropyl)thiophen-

2-yl]isoindoline-1,3-dione (6b)

Following general procedure D, after workup as described

previously, the residue was triturated with petroleum ether for

10 min, to furnish 7a as a white solid. Yield: 96%, mp 180–

183 °C. ¹H NMR (CDCl₃) d: 0.85 (s, 9H), 2.79 (s, 2H), 7.18 (d,

J = 6.4 Hz, 2H), 7.62 (d, J = 6.4 Hz, 2H), 7.74 (m, 4H). MS (ESI):

[M+1]⁺= 516.1, [M+3]⁺= 518.2.

Following general procedure C, the residue was puriﬁed by col-

umn chromatography on silica gel using petroleum ether and ethyl

acetate (8.5:1.5) as eluent, to furnish the compound 6b as a brown

solid. Yield: 78%, mp 142–144 °C. ¹H NMR (CDCl₃) d: 0.69 (s, 9H),

2.72 (s, 2H), 7.08 (s, 1H), 7.30 (t, J = 8.2 Hz, 1H), 7.44 (d,

J = 8.2 Hz, 1H), 7.73 (m, 4H), 7.82 (d, J = 8.4 Hz, 1H), 7.90 (s, 1H).

MS (ESI): [M+1]⁺= 438.2.

5.5.2. 2-[5-Bromo-3-(3-chlorobenzoyl)-4-(2,2-dimethylpropyl)

thiophen-2-yl]isoindoline-1,3-dione (7b)

Following general procedure D, after workup as described pre-

viously, the residue was puriﬁed by column chromatography on

silica gel using petroleum ether/ethyl acetate 17:3 as eluent, to

give the desired compound 7b as a yellow solid. Yield: 87%. mp:

143–144 °C. ¹H NMR (CDCl₃) d: 0.86 (s, 9H), 2.63 (s, 2H), 7.16 (m,

2H), 7.53 (d, J = 7.6 Hz, 1H), 7.67 (s, 1H), 7.74 (m, 4H). MS (ESI):

[M+1]⁺= 516.0, [M+3]⁺= 518.1.

5.4.3. 2-[3-(2-Chlorobenzoyl)-4-(2,2-dimethylpropyl)thiophen-

2-yl]isoindoline-1,3-dione (6c)

Following general procedure C, the residue was triturated with

petroleum ether (20 mL), furnishing the desired product 6c as a

yellow solid. Yield: 73%; mp: 157–158 °C. ¹H NMR (CDCl₃) d:

0.89 (s, 9H), 2.72 (s, 2H), 7.07 (m, 4H), 7.44 (d, J = 6.4 Hz, 1H),

7.70 (m, 4H). MS (ESI): [M+1]⁺= 438.1.

5.5.3. 2-[5-Bromo-3-(2-chlorobenzoyl)-4-(2,2-dimethylpropyl)

thiophen-2-yl]isoindoline-1,3-dione (7c)

Following general procedure D, after workup as described

previously, the residue was triturated with petroleum ether for

5.4.4. 2-[3-(3,4-Dichlorobenzoyl)-4-(2,2-dimethylpropyl)

thiophen-2-yl]isoindoline-1,3-dione (6d)

Following general procedure C, the residue was triturated with

petroleum ether (20 mL), furnishing the desired product 6d as a

10 min, to furnish 7c as

a yellow solid. Yield: 96%, mp

158

R. Romagnoli et al. / Bioorg. Med. Chem. 22 (2014) 148–166

135–136 °C. ¹H NMR (CDCl₃) d: 0.91 (s, 9H), 2.76 (s, 2H), 7.12 (m,

3H), 7.42 (d, J = 6.4 Hz, 1H), 7.71 (m, 4H). MS (ESI):

[M+1]⁺= 516.1, [M+3]⁺= 518.1.

affording compound 8a as a white solid. Yield: 90%, mp 115–

117 °C.¹H NMR (CDCl₃) d: 0.56 (s, 9H), 2.94 (s, 2H) 7.26 (d, 2H,

J = 6.4 Hz), 7.42 (d, 2H, J = 6.4 Hz), 7.52 (m, 2H), 7.74 (m, 7H). MS

(ESI): [M+1]⁺= 514.1.

5.5.4. 2-[5-Bromo-3-(3,4-dichlorobenzoyl)-4-(2,2-

dimethylpropyl)thiophen-2-yl]isoindol-ine-1,3-dione (7d)

Following general procedure D, after workup as described previ-

ously, the residue was puriﬁed by column chromatography on silica

gel eluting with a mixture of petroleum ether/ethyl acetate 9:1 as

eluent, to give the desired compound 7d as a yellow solid. Yield:

78%. mp 158–160 °C. ¹H NMR (CDCl₃) d: 0.85 (s, 9H), 2.80 (s, 2H),

7.27 (d, J = 8.2 Hz, 1H), 7.48 (d, J = 8.2 Hz, 1H), 7.76 (m, 5H). MS

(ESI): [M+1]⁺= 550.8, [M+3]⁺= 552.8.

5.6.2. 2-[3-(3-Chlorobenzoyl)-4-(2,2-dimethylpropyl)-5-

(phenyl)thiophen-2-yl]isoindoline-1,3-dione (8b)

Following general procedure E, after workup as described previ-

ously, the residue was puriﬁed by column chromatography on sil-

ica gel using petroleum ether/ethyl acetate 9:1 as eluent. affording

the desired intermediate 8b as a white solid. Yield: 62%, mp 91–

93 °C. ¹H NMR (CDCl₃) d: 0.58 (s, 9H), 2.97 (s, 2H), 7.17 (d, 1H,

J = 7.6 Hz), 7.49 (m, 6H), 7.78 (m, 6H). MS (ESI): [M+1]⁺= 514.1.

5.5.5. 2-[5-Bromo-3-(2,4-dichlorobenzoyl)-4-(2,2-

dimethylpropyl)thiophen-2-yl]isoindol-ine-1,3-dione (7e)

Following general procedure D, after workup as described pre-

viously, the residue was triturated with a mixture of diethyl

ether/petroleum ether for 30 min to give the desired product 7e

as a yellow solid. Yield: 84%, mp 165–166 °C. ¹H NMR (CDCl₃) d:

0.91 (s, 9H), 2.77 (s, 2H), 7.09 (d, J = 8.4 Hz, 1H), 7.16 (s, 1H), 7.43

(d, J = 8.4 Hz, 1H), 7.78 (m, 4H). MS (ESI): [M+1]⁺= 550.6,

[M+3]⁺= 552.7.

5.6.3. 2-[3-(2-Chlorobenzoyl)-4-(2,2-dimethylpropyl)-5-

(phenyl)thiophen-2-yl]isoindoline-1,3-dione (8c)

Following general procedure E, after workup as described previ-

ously, the residue was puriﬁed by column chromatography on sil-

ica gel using petroleum ether/ethyl acetate 4:1 as eluent affording

the desired intermediate 8c as a cream colored solid. Yield: 77%,

mp 87–90 °C. ¹H NMR (CDCl₃) d: 0.90 (s, 9H), 2.82 (s, 2H), 7.22

(m, 3H), 7.54 (m, 5H), 7.62 (d, J = 6.4 Hz, 1H), 7.81 (m, 4H). MS

(ESI): [M+1]⁺= 514.2.

5.5.6. 2-[5-Bromo-4-(2,2-dimethylpropyl)-3-(3-

triﬂuoromethylbenzoyl)thiophen-2-yl]isoindoline-1,3-dione

(7f)

5.6.4. 2-[3-(3,4-Dichlorobenzoyl)-4-(2,2-dimethylpropyl)-5-

phenylthiophen-2-yl]isoindoline-1,3-dione (8d)

Following general procedure D, after workup as described pre-

viously, the residue was puriﬁed by column chromatography on

silica gel, eluting with a mixture of petroleum ether/ethyl acetate

9:1 to give the desired product 7f as a yellow oil. Yield: 87%. ¹H

NMR (CDCl₃) d: 0.88 (s, 9H), 2.86 (s, 2H), 7.34 (t, J = 7.6 Hz, 1H),

7.42 (d, J = 7.6 Hz, 1H), 7.70 (m, 4H), 7.83 (d, J = 7.6 Hz, 1H), 7.94

(s, 1H). MS (ESI): [M+1]⁺= 550.1, [M+3]⁺= 552.1.

Following general procedure E, after workup as described previ-

ously, the residue was puriﬁed by column chromatography on sil-

ica gel using petroleum ether/ethyl acetate 19:1 as eluent,

affording the desired intermediate 8d as a yellow solid. Yield:

55%, mp 171–173 °C. ¹H NMR (CDCl₃) d: 0.56 (s, 9H), 2.79 (s, 2H),

7.32 (s, 1H), 7.44 (d, J = 7.8 Hz, 1H), 7.46 (d, J = 7.8 Hz, 1H), 7.52

(m, 5H), 7.74 (m, 4H). MS (ESI): [M+1]⁺= 548.1.

5.5.7. 2-[5-Bromo-4-(2,2-dimethylpropyl)-3-(4-methylbenzoyl)

thiophen-2-yl]isoindoline-1,3-dione (7g)

5.6.5. 2-[3-(2,4-Dichlorobenzoyl)-4-(2,2-dimethylpropyl)-5-

(phenyl)thiophen-2-yl]iso-indoline-1,3-dione (8e)

Following general procedure D, after workup as described pre-

viously, the residue was puriﬁed by column chromatography on

silica gel, eluting with a mixture of petroleum ether/ethyl acetate

4:1 to give the desired product 7g as a white solid. Yield: 80%,

mp 169–170 °C. ¹H NMR (CDCl₃) d: 0.86 (s, 9H), 2.13 (s, 2H), 2.82

(s, 3H), 6.97 (d, J = 7.8 Hz, 2H), 7.56 (d, J = 7.8 Hz, 2H), 7.68 (m,

4H). MS (ESI): [M+1]⁺= 495.9, [M+3]⁺= 497.9.

Following general procedure E, after workup as described previ-

ously, the residue was puriﬁed by column chromatography on sil-

ica gel using petroleum ether/ethyl acetate 9:1 as eluent, affording

the desired intermediate 8e as a yellow oil. Yield: 89%. ¹H NMR

(CDCl₃): d 0.88 (s, 9H), 2.74 (s, 2H), 7.11 (d, J = 6.4 Hz, 1H), 7.18

(s, 1H), 7.45 (m, 6H), 7.78 (m, 4H). MS (ESI): [M+1]⁺= 548.2.

5.6. General procedure (E) for the synthesis of compounds 8a–al

5.6.6. 2-[4-(2,2-Dimethylpropyl)-5-(phenyl)-3-(3-

triﬂuoromethylbenzoyl)thiophen-2-yl]isoindoline-1,3-dione

(8f)

Following general procedure E, after workup as described previ-

ously, the residue was puriﬁed by column chromatography on silica

gel using petroleum ether/ethyl acetate (4:1) as eluent, affording

the desired intermediate 8f as a yellow solid. Yield: 63%, mp 170–

172 °C. ¹H NMR (CDCl₃) d: 0.57 (s, 9H), 2.99 (s, 2H), 7.39 (d,

J = 7.6 Hz, 1H), 7.43 (m, 6H), 7.71 (m, 4H), 7.86 (d, J = 7.6 Hz, 1H),

8.08 (s, 1H). MS (ESI): [M+1]⁺= 548.2.

A stirred suspension of bromothiophene 7a–g (0.5 mmol) and

the appropriate aryl/heteroarylboronic acid (0.75 mmol) in diox-

ane (6 mL containing 2 drops of water) was degassed under a

stream of nitrogen over 10 min, then treated with PdCl₂(DPPF)

(41 mg, 0.05 mmol) and CsF (190 mg, 1.25 mmol). The reaction

mixture was heated under nitrogen at 45 °C for 30 min, then at

65 °C for 6 h (or 95 °C for 18 h for compounds 8s–u). The reaction

mixture was cooled to ambient temperature, diluted with CH₂Cl₂

(10 mL), ﬁltered on a pad of celite and evaporated in vacuo. The

residue was dissolved with CH₂Cl₂(15 mL), and the resultant solu-

tion was washed sequentially with water (5 mL) and brine (5 mL).

The organic layer was dried and evaporated, and the residue was

puriﬁed by column chromatography on silica gel.

5.6.7. 2-[4-(2,2-Dimethylpropyl)-3-(4-methylbenzoyl)-5-

(phenyl)thiophen-2-yl]isoindoline-1,3-dione (8g)

Following general procedure E, after workup as described previ-

ously, the residue was puriﬁed by column chromatography on sil-

ica gel using petroleum ether/ethyl acetate 17:3 as eluent,

affording the desired intermediate 8g as a yellow solid. Yield:

61%, mp 95–96 °C. ¹H NMR (CDCl₃) d: 0.57 (s, 9H), 2.13 (s, 3H),

2.96 (s, 2H), 6.98 (d, J = 7.8 Hz, 2H), 7.52 (m, 5H), 7.64 (d,

J = 7.8 Hz, 2H), 7.72 (m, 4H). MS (ESI): [M+1]⁺= 494.0.

5.6.1. 2-[3-(4-Chlorobenzoyl)-4-(2,2-dimethylpropyl)-5-

phenylthiophen-2-yl]isoindoline-1,3-dione (8a)

Following general procedure E, after workup as described previ-

ously, the residue was puriﬁed by ﬂash chromatography on silica

gel using light petroleum ether/ethyl acetate 9:1 as eluent,

R. Romagnoli et al. / Bioorg. Med. Chem. 22 (2014) 148–166

159

5.6.8. 2-[3-(4-Chlorobenzoyl)-4-(2,2-dimethylpropyl)-5-(4-

5.6.14. 2-[3-(4-Chlorobenzoyl)-4-(2,2-dimethylpropyl)-5-

(pyrazol-4-yl)thiophen-2-yl]isoindoline-1,3-dione (8n)

methoxyphenyl)-thiophen-2-yl]isoindoline-1,3-dione (8h)

Following general procedure E, after workup as described previ-

ously, the crude material was puriﬁed by column chromatography

on silica gel, eluting with ethyl acetate/petroleum ether 3:17, to

furnish the desired intermediate 8h as a yellow solid. Yield: 55%,

mp 170–172 °C. ¹H NMR (CDCl₃) d: 0.57 (s, 9H), 2.90 (s, 2H), 3.86

(s, 3H), 6.95 (d, J = 8.8 Hz, 2H), 7.20 (d, J = 9.0 Hz, 2H), 7.43 (d,

J = 9.0 Hz, 2H), 7.72 (m, 6H). MS (ESI): [M+1]⁺= 544.0.

A solution of 7a and 1-(tert-butyloxycarbonyl)pyrazole-4-boro-

nic acid pinacol ester (1.7 mmol, 500 mg) in anhydrous dioxane

(12 mL) was degassed under a stream of nitrogen for approximately

10 min. To the solution was added [1,1⁰bis(diphenylphosphino)fer-

rocene]-dichloropalladium (II) methylene chloride complex

(0.10 mmol, 82 mg) and CsF (2.75 mmol, 420 mg). The mixture

was heated under nitrogen to 40 °C for 1 h, then to 60 °C for three

days. After cooling to room temperature, the reaction mixture

was diluted with methylene chloride (35 mL), ﬁltered through a

pad of Celite, and the combined ﬁltrates concentrated. The residue

was dissolved in 1:1 v/v mixture of triﬂuoroacetic acid and toluene

(20 mL), stirred at ambient temperature for one hour, concentrated,

dissolved in 1:1 v/v mixture of acetic acid and toluene, heated to re-

ﬂux for two hours, and cooled to room temperature. The mixture

was diluted with 2-propanol (6 mL), washed with a saturated aque-

ous solution of sodium bicarbonate (10 mL), and the aqueous layer

back-extracted with methylene chloride containing a small amount

of 2-propanol (2 ꢀ 15 mL). The combined organic phases were

dried (MgSO4), ﬁltered, and concentrated, then puriﬁed by column

chromatography on silica gel, eluting with a gradient of 2% ? 20%

ethyl acetate in methylene chloride to furnish the desired interme-

diate 8n as a pale tan solid. Yield: 37%, mp 297–299 °C. ¹H NMR

(CDCl₃) d: 0.66 (s, 9H), 2.89 (s, 2H), 7.21 (d, J = 8.5 Hz, 2H), 7.69

(d, J = 8.5 Hz, 2H), 7.70 (m, 2H), 7.78 (m, 2H), 7.79 (s, 2H). MS

(ESI): [M+1]⁺= 504.0.

5.6.9. 2-[3-(3-Chlorobenzoyl)-4-(2,2-dimethylpropyl)-5-(4-

methoxyphenyl)thiophen-2-yl]isoindoline-1,3-dione (8i)

Following general procedure E, after workup as described previ-

ously, the residue was puriﬁed by column chromatography on sil-

ica gel using petroleum ether/ethyl acetate 9:1 as eluent affording

the desired intermediate 8i as a yellow solid. Yield: 58%, mp 110–

112 °C. ¹H NMR (CDCl₃) d: 0.58 (s, 9H), 2.93 (s, 2H), 3.87 (s, 3H),

6.96 (d, J = 8.8 Hz, 2H), 7.13 (m, 2H), 7.46 (d, J = 8.8 Hz, 2H), 7.74

(m, 6H). MS (ESI): [M+1]⁺= 544.2.

5.6.10. 2-[3-(3,4-Dichlorobenzoyl)-4-(2,2-dimethylpropyl)-5-(4-

methoxyphenyl)thiophen-2-yl]isoindoline-1,3-dione (8j)

Following general procedure E, after workup as described previ-

ously, the residue was puriﬁed by column chromatography on sil-

ica gel using petroleum ether/ethyl acetate 19:1 as eluent,

affording the desired intermediate 8j as a yellow solid. Yield:

63%, mp 81–83 °C. ¹H NMR (CDCl₃) d: 0.58 (s, 9H), 2.91 (s, 2H),

3.87 (s, 3H), 6.96 (d, J = 8.6 Hz, 2H), 7.24 (d, J = 8.6 Hz, 2H), 7.42

(d, J = 8.8 Hz, 1H), 7.54 (d, J = 8.8 Hz, 1H), 7.77 (m, 4H), 7.84 (s,

1H). MS (ESI): [M+1]⁺= 578.1.

5.6.15. 2-[3-(4-Chlorobenzoyl)-4-(2,2-dimethylpropyl)-5-

(thiophen-2-yl)thiophen-2-yl]isoindoline-1,3-dione (8o)

Following general procedure E, after workup as described previ-

ously, the residue was puriﬁed by column chromatography on sil-

ica gel, eluting with methylene chloride to furnish the desired

5.6.11. 2-[4-(2,2-Dimethylpropyl)-5-(4-methoxyphenyl)-3-(3-

triﬂuoromethylbenzoyl)-thiophen-2-yl]isoindoline-1,3-dione

(8k)

intermediate 8o as

a pale yellow solid. Yield: 77%, mp

160–163 °C. ¹H NMR (CDCl₃) d: 0.66 (s, 9H), 3.03 (s, 2H), 7.09

(dd, J = 5.0 and 3.5 Hz, 1H), 7.28 (m, 3H), 7.41 (dd, J = 5.0 and

1.0 Hz, 1H), 7.74 (m, 4H), 7.80 (m, 2H). MS (ESI): [M+1]⁺= 520.0.

Following general procedure E, after workup as described previ-

ously, the residue was puriﬁed by column chromatography on sil-

ica gel using petroleum ether/ethyl acetate 19:1 as eluent,

affording the desired intermediate 8k as a yellow solid. Yield:

54%, mp 156–158 °C. ¹H NMR (CDCl₃) d: 0.59 (s, 9H), 2.95 (s, 2H),

3.87 (s, 3H), 7.00 (d, J = 8.8 Hz, 2H), 7.39 (d, J = 7.6 Hz, 1H), 7.47

(d, J = 8.8 Hz, 2H), 7.71 (m, 5H), 7.88 (d, J = 7.6 Hz, 1H), 8.03 (s,

1H). MS (ESI): [M+1]⁺= 578.2.

5.6.16. 2-[3-(4-Chlorobenzoyl)-4-(2,2-dimethylpropyl)-5-

(thiophen-3-yl)thiophen-2-yl]isoindoline-1,3-dione (8p)

Following general procedure E, after workup as described previ-

ously, the residue was puriﬁed by column chromatography on sil-

ica gel, eluting with a mixture of ethyl acetate/heptane 3:7 to

furnish the desired intermediate 8p as a very pale yellow solid.

Yield: 72%, mp 175–178 °C. ¹H NMR (CDCl₃) d: 0.61 (s, 9H), 2.94

(s, 2H), 7.20 (d, J = 8.5 Hz, 2H), 7.28 (m, 1H), 7.43 (m, 2H), 7.73

(m, 4H), 7.79 (m, 2H). MS (ESI): [M+1]⁺= 520.0.

5.6.12. 2-[4-(2,2-Dimethylpropyl)-5-(4-methoxyphenyl)-3-(4-

methylbenzoyl)-thiophen-2-yl]isoindoline-1,3-dione (8l)

Following general procedure E, after workup as described previ-

ously, the residue was puriﬁed by column chromatography on sil-

ica gel using petroleum ether/ethyl acetate 19:1 as eluent,

affording the desired intermediate 8l as a yellow oil. Yield: 63%.

¹H NMR (CDCl₃) d: 0.58 (s, 9H), 2.12 (s, 3H), 2.86 (s, 2H), 3.86 (s,

3H), 6.75 (d, J = 8.8 Hz, 2H), 6.95 (d, J = 6.2 Hz, 2H), 7.02 (d,

J = 6.2 Hz, 2H), 7.46 (d, J = 8.8 Hz, 2H), 7.69 (m, 4H). MS (ESI):

[M+1]⁺= 524.3.

5.6.17. 2-[3-(4-Chlorobenzoyl)-4-(2,2-dimethylpropyl)-5-

(furan-2-yl)thiophen-2-yl]isoindoline-1,3-dione (8q)

Following general procedure E, after workup as described previ-

ously, the residue was puriﬁed by column chromatography on sil-

ica gel, eluting with a mixture of methylene chloride/heptane 2:1

to furnish the desired intermediate 8q as a pale yellow solid. Yield:

77%, mp 184–186 °C. ¹H NMR (CDCl₃) d: 0.71 (s, 9H), 3.01 (s, 2H),

6.49 (m, 1H), 6.60 (m, 1H), 7.22 (d, J = 8.5 Hz, 2H), 7.50 (m, 1H),

7.69 (d, J = 8.5 Hz, 2H), 7.74 (m, 2H), 7.80 (m, 2H). MS (ESI):

[M+23]⁺= 526.0.

5.6.13. 2-[3-(4-Chlorobenzoyl)-5-(3,5-dimethylisoxazol-4-yl)-4-

(2,2-dimethylpropyl)-thiophen-2-yl]isoindoline-1,3-dione (8m)

Following general procedure E, after workup as described previ-

ously, the residue was puriﬁed by column chromatography on sil-

ica gel, eluting with methylene chloride followed by 2% ethyl

acetate in methylene chloride to afford the desired product 8m

as a pale tan solid. Yield: 65%, mp 212–214 °C. ¹H NMR (CDCl₃)

d: 0.66 (s, 9H), 2.31 (s, 3H), 2.44 (s, 3H), 2.64 (s, 2H), 7.21 (d,

J = 8.5 Hz, 2H), 7.67 (d, J = 8.5 Hz, 2H), 7.74 (m, 2H), 7.79 (m, 2H).

MS (ESI): [M+1]⁺= 533.0.

5.6.18. 2-[3-(4-Chlorobenzoyl)-4-(2,2-dimethylpropyl)-5-

(furan-3-yl)-thiophen-2-yl]isoindoline-1,3-dione (8r)

Following general procedure E, after workup as described previ-

ously, the residue was puriﬁed by column chromatography on sil-

ica gel, eluting with a gradient of 50–60% methylene chloride in

heptane to furnish the desired intermediate 8r as a pale yellow so-

lid. Yield: 55%, mp 214-217 °C. ¹H NMR (CDCl₃) d: 0.68 (s, 9H), 2.89

160

R. Romagnoli et al. / Bioorg. Med. Chem. 22 (2014) 148–166

(s, 2H), 6.63 (m, 1H), 7.20 (d, J = 8.5 Hz, 2H), 7.52 (m, 1H), 7.64 (m,

1H), 7.68 (d, J = 8.5 Hz, 2H), 7.73 (m, 2H), 7.78 (m, 2H). MS (ESI):

[M+1]⁺= 504.0.

5.6.25. 2-[3-(4-Chlorobenzoyl)-5-(2,5-diﬂuorophenyl)-4-(2,2-

dimethylpropyl)thiophen-2-yl]isoindoline-1,3-dione (8y)

Following general procedure E, after workup as described previ-

ously, the residue was puriﬁed by column chromatography on sil-

ica gel, eluting with a mixture of methylene chloride/heptane 3:2,

to furnish the desired intermediate 8y as a pale yellow solid. Yield:

95%, mp 165–167 °C. ¹H NMR (CDCl₃) d: 0.60 (s, 9H), 2.78 (s, 2H),

7.07 (m, 3H), 7.23 (d, J = 8.5 Hz, 2H), 7.68 (m, 4H), 7.75 (m, 2H).

MS (ESI): [M+1]⁺= 550.0.

5.6.19. 2-[3-(4-Chlorobenzoyl)-4-(2,2-dimethylpropyl)-5-

(pyridin-4-yl)thiophen-2-yl]isoindoline-1,3-dione (8s)

Following general procedure E, after workup as described previ-

ously, the residue was puriﬁed by column chromatography on

silica gel, eluting with a mixture of ethyl acetate/heptane 1:3 to

furnish the desired intermediate 8s as a very pale tan solid. Yield:

54%, mp 227–230 °C.¹H NMR (CDCl₃) d: 0.58 (s, 9H), 2.97 (s, 2H),

7.22 (d, J = 8.5 Hz, 2H), 7.47 (dd, J = 4.5 and 1.5 Hz, 2H), 7.70 (d,

J = 8.5 Hz, 2H), 7.74 (m, 2H), 7.80 (m, 2H), 8.71 (dd, J = 4.5 and

1.5 Hz, 2H). MS (ESI): [M+1]⁺= 515.0.

5.6.26. 2-[3-(4-Chlorobenzoyl)-5-(2,6-diﬂuorophenyl)-4-(2,2-

dimethylpropyl)-thiophen-2-yl]isoindoline-1,3-dione (8z)

Following general procedure E, after workup as described

previously, the residue was puriﬁed by column chromatography

on silica gel, eluting with a mixture of methylene chloride/hep-

tane 2:1, to afford the desired product 8z as a pale beige solid.

Yield: 48%, mp 114–116 °C. ¹H NMR (CDCl₃) d: 0.61 (s, 9H), 2.66

(s, 2H), 7.03 (t, J = 8.0 Hz, 2H), 7.24 (d, J = 8.5 Hz, 2H), 7.37 (m,

2H), 7.71 (d, J = 8.5 Hz, 2H), 7.75 (m, 2H), 7.81 (m, 2H). MS

(ESI): [M+1]⁺= 550.0.

5.6.20. 2-[3-(4-Chlorobenzoyl)-4-(2,2-dimethylpropyl)-5-

(pyridin-3-yl)thiophen-2-yl]isoindoline-1,3-dione (8t)

Following general procedure E, after workup as described previ-

ously, the residue was puriﬁed by column chromatography on sil-

ica gel, eluting with a mixture of ethyl acetate/heptane 2:3 to

furnish the desired intermediate 8t as a very pale tan solid. Yield:

24%, mp 217–220 °C. ¹H NMR (CDCl₃) d: 0.57 (s, 9H), 2.93 (s, 2H),

7.22 (d, J = 8.5 Hz, 2H), 7.41 (m, 1H), 7.68 (m, 4H), 7.80 (m, 2H),

7.85 (m, 1H), 8.65 (m, 1H), 8.80 (m, 1H). MS (ESI): [M+1]⁺= 515.0.

5.6.27. 2-[3-(4-Chlorobenzoyl)-5-(4-chlorophenyl)-4-(2,2-

dimethylpropyl)thiophen-2-yl]isoindoline-1,3-dione (8aa)

Following general procedure E, after workup as described previ-

ously, the residue was puriﬁed by column chromatography on sil-

ica gel, eluting with a mixture of heptane/ethyl acetate 4:1, to

furnish the desired intermediate 8aa as a pale yellow solid. Yield:

97%, mp 110–112 °C. ¹H NMR (CDCl₃) d: 0.59 (s, 9H), 2.90 (s, 2H),

7.21 (d, J = 8.5 Hz, 2H), 7.37 (dd, J = 2.0 and 8.0 Hz, 1H), 7.54 (d,

J = 8.0 Hz, 1H), 7.64 (d, J = 2.0 Hz, 1H), 7.69 (d, J = 8.5 Hz, 2H),

7.72 (m, 2H), 7.78 (m, 2H). MS (ESI): [M+1]⁺= 548.5.

5.6.21. 2-[3-(4-Chlorobenzoyl)-4-(2,2-dimethylpropyl)-5-

(pyridin-2-yl)thiophen-2-yl]isoindoline-1,3-dione (8u)

Following general procedure E, after workup as described previ-

ously, the residue was puriﬁed by column chromatography on sil-

ica gel, eluting with 5% ethyl acetate in heptane to furnish the

desired intermediate 8u as a very pale tan solid. Yield: 24%, mp

187–190 °C. ¹H NMR (CDCl₃) d: 0.60 (s, 9H), 3.16 (s, 2H), 7.23 (d,

J = 8.5 Hz, 2H), 7.30 (m, 1H), 7.68 (m, 1H), 7.74 (m, 4H), 7.81 (m,

3H), 8.69 (m, 1H). MS (ESI): [M+1]⁺= 515.0.

5.6.28. (E)-2-[3-(4-Chlorobenzoyl)-5-(2-(4-chlorophenyl)ethen-

1-yl)-4-(2,2-dimethyl-propyl)thiophen-2-yl]isoindoline-1,3-

dione (8ab)

Following general procedure E, after workup as described previ-

ously, the residue was puriﬁed by column chromatography on sil-

ica gel, eluting with methylene chloride/heptane 3:1, to afford the

desired product 8ab as a pale yellow foam. Yield: 29%. ¹H NMR

(CDCl₃) d: 0.84 (s, 9H), 2.87 (s, 2H), 6.90 (d, J = 16.0 Hz, 1H), 7.20

(d, J = 8.5 Hz, 2H), 7.26 (d, J = 16.0 Hz, 1H), 7.34 (d, J = 8.5 Hz, 2H),

7.40 (d, J = 8.5 Hz, 2H), 7.66 (d, J = 8.5 Hz, 2H), 7.74 (m, 2H), 7.80

(m, 2H). MS (ESI): [M+1]⁺= 574.1.

5.6.22. 2-[3-(4-Chlorobenzoyl)-4-(2,2-dimethylpropyl)-5-(4-

ﬂuorophenyl)-thiophen-2-yl]isoindoline-1,3-dione (8v)

Following general procedure E, after workup as described previ-

ously, the residue was puriﬁed by column chromatography on sil-

ica gel, eluting with a mixture of methylene chloride/heptane 2:1,

to furnish the desired intermediate 8v as a very pale yellow solid.

Yield: 71%, mp 171–173 °C. ¹H NMR (CDCl₃) d: 0.57 (s, 9H), 2.89 (s,

2H), 7.15 (m, 2H), 7.21 (d, J = 8.5 Hz, 2H), 7.50 (m, 2H), 7.71 (m,

4H), 7.78 (m, 2H). MS (ESI): [M+1]⁺= 532.0.

5.6.29. 2-[3-(4-Chlorobenzoyl)-5-(3,4-dichlorophenyl)-4-(2,2-

dimethylpropyl)thiophen-2-yl]isoindoline-1,3-dione (8ac)

Following general procedure E, after workup as described previ-

ously, the residue was puriﬁed by column chromatography on sil-

ica gel, eluting with a mixture of heptane/ethyl acetate 4:1, to

furnish the desired intermediate 8ac as a pale tan solid. Yield:

95%. mp 210–213 °C. ¹H NMR (CDCl₃) d: 0.59 (s, 9H), 2.90 (s, 2H),

7.21 (d, J = 8.5 Hz, 2H), 7.37 (dd, J = 2.0 and 8.0 Hz, 1H), 7.54 (d,

J = 8.0 Hz, 1H), 7.64 (d, J = 2.0 Hz, 1H), 7.69 (d, J = 8.5 Hz, 2H),

7.72 (m, 2H), 7.78 (m, 2H). MS (ESI): [M+23]⁺= 604.0.

5.6.23. 2-[3-(4-Chlorobenzoyl)-5-(2,3-diﬂuorophenyl)-4-(2,2-

dimethylpropyl)thiophen-2-yl]isoindoline-1,3-dione (8w)

Following general procedure E, after workup as described previ-

ously, the residue was puriﬁed by column chromatography on sil-

ica gel, eluting with methylene chloride to furnish the desired

intermediate 8w as a white solid. Yield: 80%, mp 162–164 °C. ¹H

NMR (CDCl₃) d: 0.59 (s, 9H), 2.77 (s, 2H), 7.15 (m,1H), 7.27 (m,

4H), 7.75 (m, 4H), 7.81 (m, 2H), 7.69 (d, J = 8.5 Hz, 2H), 7.72 (m,

2H), 7.78 (m, 2H). MS (ESI): [M+1]⁺= 550.0.

5.6.30. 2-[3-(4-Chlorobenzoyl)-4-(2,2-dimethylpropyl)-5-(3-

methoxyphenyl)-thiophen-2-yl]isoindoline-1,3-dione (8ad)

Following general procedure E, after workup as described

previously, the crude material was puriﬁed by column chroma-

tography on silica gel, eluting with ethyl acetate/petroleum

ether 3:17, to furnish the desired intermediate 8ad as a yellow

solid. Yield: 55%, mp 153–156 °C. ¹H NMR (CDCl₃) d: 0.62 (s,

9H), 2.94 (s, 2H), 3.86 (s, 3H), 6.96 (m, 1H), 7.03 (m, 2H),

7.22 (d, J = 8.8 Hz, 2H), 7.38 (t, J = 7.6 Hz, 1H), 7.72 (d,

J = 8.8 Hz, 2H), 7.75 (m, 4H). MS (ESI): [M+1]⁺= 544.0.

5.6.24. 2-[3-(4-Chlorobenzoyl)-5-(2,4-diﬂuorophenyl)-4-(2,2-

dimethylpropyl)thiophen-2-yl]isoindoline-1,3-dione (8x)

Following general procedure E, after workup as described previ-

ously, the residue was puriﬁed by column chromatography on sil-

ica gel, eluting with a mixture of methylene chloride/heptane 3:2,

to furnish the desired intermediate 8x as a pale yellow solid. Yield:

68%, mp 150–152 °C. ¹H NMR (CDCl₃) d: 0.59 (s, 9H), 2.74 (s, 2H),

7.03 (m, 2H), 7.22 (d, J = 8.5 Hz, 2H), 7.50 (m, 1H), 7.68 (m, 4H),

7.81 (m, 2H). MS (ESI): [M+1]⁺= 550.0.

R. Romagnoli et al. / Bioorg. Med. Chem. 22 (2014) 148–166

161

5.6.31. 2-[3-(4-Chlorobenzoyl)-4-(2,2-dimethylpropyl)-5-(2-

5.6.37. 2-[3-(4-Chlorobenzoyl)-4-(2,2-dimethylpropyl)-5-(2-

methylphenyl)-thiophen-2-yl]isoindoline-1,3-dione (8ak)

methoxyphenyl)-thiophen-2-yl]isoindoline-1,3-dione (8ae)

Following general procedure E, after workup as described pre-

viously, the crude material was puriﬁed by column chromatogra-

phy on silica gel, eluting with ethyl acetate/petroleum ether 3:17,

to furnish the desired intermediate 8ae as a yellow solid. Yield:

55%, mp 115–117 °C. ¹H NMR (CDCl₃) d: 0.56 (s, 9H), 2.73 (s,

2H), 3.93 (s, 3H), 6.94 (d, J = 8.8 Hz, 2H), 7.04 (m, 2H), 7.230 (d,

J = 8.8 Hz, 2H), 7.41 (m, 2H), 7.75 (m, 4H). MS (ESI):

[M+1]⁺= 544.1.

Following general procedure E, after workup as described

previously, the residue was puriﬁed by column chromatography

on silica gel, eluting with a gradient of methylene chloride/hep-

tane (from 2:1 to 4:1), to furnish the desired intermediate 8ak

as a yellow solid. Yield: 72%, mp 154–156 °C. ¹H NMR (CDCl₃)

d: 0.57 (s, 9H), 2.41 (s, 3H), 2.92 (s, 2H), 7.21 (d, J = 8.0 Hz,

2H), 7.25 (d, J = 8.0 Hz, 2H), 7.41 (d, J = 8.5 Hz, 2H), 7.71 (m,

4H), 7.77 (m, 2H). MS (ESI): m/z 528.0 (M+H). MS (ESI):

[M+1]⁺= 528.1.

5.6.32. 2-[3-(4-Chlorobenzoyl)-4-(2,2-dimethylpropyl)-5-(4-(2-

(methoxy)ethoxy)phenyl)-thiophen-2-yl]isoindoline-1,3-dione

(8af)

5.6.38. 2-[3-(4-Chlorobenzoyl)-5-(4-(1-(methyl)ethyl)phenyl)-4-

(2,2-dimethylpropyl)-thiophen-2-yl]isoindoline-1,3-dione (8al)

Following general procedure E, after workup as described previ-

ously, the residue was puriﬁed by column chromatography on sil-

ica gel, eluting with a mixture of ethyl acetate/heptane 3:7, to

furnish the desired intermediate 8al as a very pale yellow solid.

Yield: 74%, mp 120–122 °C. ¹H NMR (CDCl₃) d: 0.56 (s, 9H), 1.30

(d, J = 7.0 Hz, 6H), 2.93 (s, 2H), 2.94 (m, 1H), 7.20 (d, J = 8.5 Hz,

2H), 7.30 (d, J = 8.5 Hz, 2H), 7.44 (d, J = 8.5 Hz, 2H), 7.73 (m, 4H),

7.79 (m, 2H). MS (ESI): [M+1]⁺= 556.2.

Following general procedure E, after workup as described previ-

ously, the residue was puriﬁed by column chromatography on sil-

ica gel, eluting with a mixture of ethyl acetate/heptane 1:3, to

furnish the desired intermediate 8af as a very pale tan solid. Yield:

54%, mp 227–230 °C. ¹H NMR (CDCl₃) d: 0.58 (s, 9H), 2.97 (s, 2H),

7.22 (d, J = 8.5 Hz, 2H), 7.47 (dd, J = 4.5 and 1.5 Hz, 2H), 7.70 (d,

J = 8.5 Hz, 2H), 7.74 (m, 2H), 7.80 (m, 2H), 8.71 (dd, J = 4.5 and

1.5 Hz, 2H). MS (ESI): [M+1]⁺= 588.2.

5.7. General procedure (F) for the synthesis of compounds 3e-

au¹⁴

5.6.33. 2-[5-(Benzo[d][1,3]dioxol-5-yl)-3-(4-chlorobenzoyl)-4-

(2,2-dimethylpropyl)thio-phen-2-yl]isoindoline-1,3-dione (8ag)

Following general procedure E, after workup as described previ-

ously, the residue was puriﬁed by column chromatography on sil-

ica gel, eluting with methylene chloride to furnish the desired

intermediate 8ag as a pale yellow solid. Yield: 95%, mp 213–

215 °C. ¹H NMR (CDCl₃) d: 0.61 (s, 9H), 2.89 (s, 2H), 6.03 (s, 2H),

6.88 (d, J = 8.5 Hz, 1H), 6.98 (m, 2H), 7.20 (d, J = 8.5 Hz, 2H), 7.68

(m, 4H), 7.75 (m, 2H). MS (ESI): [M+1]⁺= 558.0.

A stirred suspension of thiophene derivative 7a–g or 8a–al

(0.5 mmol) and hydrazine monohydrate (29 lL, 0.6 mmol,

1.2 equiv) in absolute EtOH (10 mL) was reﬂuxed for 1 h. The sol-

vent was evaporated, and the residue partitioned between CH₂Cl₂

(10 mL) and water (5 mL). The organic phase was washed with

brine (2 mL), dried (Na₂SO₄) and concentrated in vacuo to obtain

a residue that was puriﬁed by column chromatography on silica

gel.

5.6.34. 2-[3-(4-Chlorobenzoyl)-4-(2,2-dimethylpropyl)-5-(4-

(triﬂuoromethoxy)phenyl)-thiophen-2-yl]isoindoline-1,3-

dione (8ah)

5.7.1. (2-Amino-4-(2,2-dimethylpropyl)-5-bromothiophen-3-yl)

(4-chlorophenyl)-methanone (3e)

Following general procedure E, after workup as described pre-

viously, the residue was puriﬁed by column chromatography on

silica gel, eluting with methylene chloride/heptane 2:1, to afford

the desired product 8ah as a white solid. Yield: 84%, mp 99–

102 °C. ¹H NMR (CDCl₃): d 0.57 (s, 9H), 2.91 (s, 2H), 7.21 (d,

J = 8.5 Hz, 2H), 7.31 (d, J = 8.5 Hz, 2H), 7.56 (d, J = 8.5 Hz, 2H),

7.70 (d, J = 8.5 Hz, 2H), 7.74 (m, 2H), 7.79 (m, 2H). MS (ESI):

[M+1]⁺= 598.2.

Following the general procedure (F), the residue was puriﬁed

by column chromatography on silica gel, eluting with petroleum

ether/ethyl acetate 9:1, to afford the desired product 3e as a yel-

low oil. Yield 90%. ¹H NMR (CDCl₃) d: 0.69 (s, 9H), 2.25 (s, 2H),

5.88 (br s, 2H), 7.46 (d, J = 8.6 Hz, 2H), 7.53 (d, J = 8.6 Hz, 2H).

¹³C NMR (100 MHz, DMSO-d₆) d: 29.46 (4C), 33.44, 93.45,

114.19, 128.51 (2C), 130.82 (2C), 135.24, 136.45, 138.42,

161.88, 189.76. MS (ESI): [M]⁺= 386.0, [M+2]⁺= 388.2. Anal. (C_16-

H₁₇ClBrClNOS) C, H, N.

5.6.35. 2-[3-(4-Chlorobenzoyl)-4-(2,2-dimethylpropyl)-5-(4-

methylphenyl)-thiophen-2-yl]isoindoline-1,3-dione (8ai)

Following general procedure E, after workup as described

previously, the crude material was puriﬁed by column chroma-

tography on silica gel, eluting with ethyl acetate/petroleum

ether 2:8, to furnish the desired intermediate 8ai as a yellow

solid. Yield: 63%, mp 162–165 °C. ¹H NMR (CDCl₃) d: 0.84 (s,

9H), 1.57 (s, 3H), 2.79 (s, 2H), 7.18 (d, J = 8.8 Hz, 2H), 7.66 (d,

J = 8.8 Hz, 2H), 7.74 (m, 8H). MS (ESI): [M+1]⁺= 528.2.

5.7.2. (2-Amino-4-(2,2-dimethylpropyl)-5-bromothiophen-3-yl)

(3-chlorophenyl)methanone (3f)

Following the general procedure (F), the residue was puriﬁed by

column chromatography on silica gel, eluting with petroleum

ether/ethyl acetate 9:1, to afford the desired product 3f as a yellow

oil. Yield 95%. ¹H NMR (CDCl₃) d: 0.70 (s, 9H), 2.24 (s, 2H), 5.93 (br

s, 2H), 7.42 (d, J = 8.2 Hz, 1H), 7.49 (m, 2H), 7.57 (s, 1H). MS (ESI):

[M]⁺= 386.0, [M+2]⁺= 388.1. Anal. (C₁₆H₁₇ClBrClNOS) C, H, N.

5.6.36. 2-[3-(4-Chlorobenzoyl)-4-(2,2-dimethylpropyl)-5-(3-

methylphenyl)-thiophen-2-yl]isoindoline-1,3-dione (8aj)

Following general procedure E, after workup as described

previously, the crude material was puriﬁed by column chroma-

tography on silica gel, eluting with ethyl acetate/petroleum

ether1:4, to furnish the desired intermediate 8aj as a yellow so-

lid. Yield: 58%. mp 149–151 °C. ¹H NMR (CDCl₃) d: 0.85 (s, 9H),

1.56 (s, 3H), 2.79 (s, 2H), 7.18 (d, J = 8.6 Hz, 2H), 7.24 (s, 1H),

7.34 (d, J = 4.8 Hz, 1H), 7.63 (d, J = 8.6 Hz, 2H), 7.74 (m, 6H).

MS (ESI): [M+1]⁺= 528.2.

5.7.3. (2-Amino-4-(2,2-dimethylpropyl)-5-bromothiophen-3-yl)

(3,4-dichloro-phenyl)methanone (3g)

Following the general procedure (F), the residue was puriﬁed by

column chromatography on silica gel, eluting with petroleum

ether/ethyl acetate 9:1, to afford the desired product 3g as a yellow

solid. Yield: 78%, mp 134–136 °C. ¹H NMR (CDCl₃) d: 0.71 (s, 9H),

2.26 (s, 2H), 5.92 (br s, 2H), 7.42 (d, J = 7.6 Hz, 1H), 7.49 (s, 1H),

7.72 (d. J = 7.8 Hz, 1H). MS (ESI): [M]⁺= 419.1, [M+2]⁺= 421.1. Anal.

(C₁₆H₁₆ClBrCl₂NOS) C, H, N.

162

R. Romagnoli et al. / Bioorg. Med. Chem. 22 (2014) 148–166

5.7.4. (2-Amino-4-(2,2-dimethylpropyl)-5-bromothiophen-3-yl)

(3-triﬂuoromethylphenyl)methanone (3h)

121.79, 127.01, 128.60 (2C), 129.23, 129.88 (2C), 130.50, 130.70,

130.99, 131.27, 134.03, 134.72, 140.54, 162.28, 188.62. MS (ESI):

[M+1]⁺= 418.1. Anal. (C₂₂H₂₁Cl₂NOS) C, H, N.

Following the general procedure (F), the residue was puriﬁed by

column chromatography on silica gel, eluting with petroleum

ether/ethyl acetate 9:1, to afford the desired product 3h as a yellow

oil. Yield 93%. ¹H NMR (CDCl₃) d: 0.68 (s, 9H), 2.18 (s, 2H), 6.03 (br

s, 2H), 7.56 (t, J = 7.6 Hz, 1H), 7.73 (m, 2H), 7.85 (s, 1H). ¹³C NMR

(100 MHz, DMSO-d₆) d: 29.43 (4C), 33.43, 93.64, 113.88, 122.21,

125.15, 128.04, 129.42, 129.69, 133.01, 135.04, 140.52, 162.76,

189.19. MS (ESI): [M]⁺= 419.8, [M+2]⁺= 421.9. Anal. (C₁₇H₁₇BrF_3-

NOS) C, H, N.

5.7.10. (2-Amino-4-(2,2-dimethylpropyl)-5-phenylthiophen-3-yl)

(2,4-dichlorophenyl)-methanone (3n)

Following the general procedure (F), the residue was crystal-

lized from petroleum ether to give the desired product 3n as a yel-

low solid. Yield: 81%, mp 166–167 °C. ¹H NMR (CDCl₃) d: 0.54 (s,

9H), 2.28 (s, 2H), 6.68 (br s, 2H), 7.32 (d, J = 6.4 Hz, 1H), 7.38 (m,

6H), 7.46 (d, J = 6.4 Hz, 1H). MS (ESI): [M+1]⁺= 418.1. Anal. (C₂₂H_21-

Cl₂NOS) C, H, N.

5.7.5. (2-Amino-4-(2,2-dimethylpropyl)-5-bromothiophen-3-yl)

(4-methylphenyl)methanone (3i)

5.7.11. (2-Amino-4-(2,2-dimethylpropyl)-5-phenylthiophen-3-yl)

(3-triﬂuoromethylphenyl)-methanone (3o)

Following the general procedure (F), the residue was puriﬁed by

column chromatography on silica gel, eluting with petroleum

ether/ethyl acetate (9:1) to afford the desired product 3i as a

brown solid. Yield 75%, mp 123–124 °C. ¹H NMR (CDCl₃) d: 0.69

(s, 9H), 2.24 (s, 2H), 2.43 (s, 3H), 5.88 (br s, 2H), 7.41 (d,

J = 8.6 Hz, 2H), 7.53 (d, J = 8.6 Hz, 2H). ¹³C NMR (100 MHz, DMSO-

d₆) d: 21.03, 29.44 (4C), 33.36, 93.03, 114.79, 128.89 (2C), 129.00

(2C), 135.56, 137.10, 141.88, 160.96, 191.07. MS (ESI):

[M+1]⁺= 365.7, [M+3] ⁺= 367.9. Anal. (C₁₇H₂₀BrNOS) C, H, N.

Following the general procedure (F), the residue was puriﬁed by

column chromatography on silica, eluting with petroleum ether/

ethyl acetate 9:1, to give the desired product 3o as a yellow solid.

Yield: 83%, mp 116–118 °C. ¹H NMR (CDCl₃) d: 0.42 (s, 9H), 2.26 (s,

2H), 6.12 (br s, 2H), 7.32 (m, 5H), 7.59 (t, J = 7.6 Hz, 1H), 7.76 (d,

J = 7.8 Hz, 1H), 7.87 (d, J = 7.8 Hz, 1H), 7.95 (s, 1H). ¹³C NMR

(100 MHz, DMSO-d₆) d: 29.28 (4C), 33.41, 115.94, 121.75, 125.18,

122.12, 125.18, 127.04, 127.70, 128.62 (2C), 129.64 (2C), 129.90,

130.86, 132.95, 134.71, 141.25, 162.68, 189.73. MS (ESI):

[M+1]⁺= 418.2. Anal. (C₂₃H₂₂F₃NOS) C, H, N.

5.7.6. (2-Amino-4-(2,2-dimethylpropyl)-5-phenylthiophen-3-yl)

(4-chlorophenyl)-methanone (3j)

Following the general procedure (F), the residue was puriﬁed by

column chromatography on silica gel, eluting with petroleum

ether/ethyl acetate 3:17, to afford the desired product 3j as a yellow

solid. Yield: 89%, mp 153–155 °C. ¹H NMR (CDCl₃) d: 0.44 (s, 9H),

2.35 (s, 2H), 6.33 (br s, 2H), 7.37 (m, 5H), 7.46 (d, J = 8.4 Hz, 2H),

7.68 (d, J = 8.4 Hz, 2H). ¹³C NMR (100 MHz, DMSO-d₆) d: 29.34

(4C), 33.42, 116.29, 121.49, 126.94, 128.42 (2C), 128.57 (2C),

129.93 (2C), 130.82 (2C), 131.13, 134.82, 136.07, 139.16, 161.81,

190.34. MS (ESI): [M+1]⁺= 384.1. Anal. (C₂₂H₂₂ClNOS) C, H, N.

5.7.12. (2-Amino-4-(2,2-dimethylpropyl)-5-phenylthiophen-3-yl)

(4-methylphenyl)-methanone (3p)

Following the general procedure (F), the residue was puriﬁed

by column chromatography on silica, eluting with petroleum

ether/ethyl acetate 17:3, to give the desired product 3p as a yellow

solid. Yield: 81%, mp 78–80 °C. ¹H NMR (CDCl₃) d: 0.45 (s, 9H), 2.42

(s, 3H), 2.78 (s, 2H), 5.77 (br s, 2H), 7.23 (m, 5H), 7.36 (d, J = 7.2 Hz,

2H), 7.64 (d, J = 7.2 Hz, 2H). ¹³C NMR (100 MHz, DMSO-d₆) d:

21.00, 29.33 (4C), 33.35, 126.00, 128.35, 128.54 (2C), 128.82

(2C), 129.00, 129.18 (2C), 129.94 (2C), 131.53, 134.95, 137.79,

144.78, 160.85, 191.67. MS (ESI): [M+1]⁺= 364.2. Anal. (C₂₃H_25-

NOS) C, H, N.

5.7.7. (2-Amino-4-(2,2-dimethylpropyl)-5-phenylthiophen-3-yl)

(3-chlorophenyl)-methanone (3k)

Following the general procedure (F), the residue was puriﬁed

by column chromatography on silica, eluting with petroleum

ether/ethyl acetate 9:1, to give the desired product 3k as a yel-

low solid. Yield: 95%, mp 158–160 °C. ¹H NMR (CDCl₃) d: 0.45

(s, 9H), 2.34 (s, 2H), 6.02 (br s, 2H), 7.39 (m, 5H), 7.48 (d,

J = 7.2 Hz, 2H), 7.59 (d, J = 7.4 Hz, 2H), 7.68 (s, 1H). ¹³C NMR

(100 MHz, DMSO-d₆) d: 29.35 (4C), 33.43, 116.07, 121.54,

126.99, 127.64, 128.26, 128.60 (2C), 129.93, 130.29 (2C),

130.99, 131.07, 133.06, 134.77, 142.46, 162.39, 189.81. MS

(ESI): [M+1]⁺= 384.0. Anal. (C₂₂H₂₂ClNOS) C, H, N.

5.7.13. (2-Amino-4-(2,2-dimethylpropyl)-5-(4-methoxyphenyl)

thiophen-3-yl)(4-chloro-phenyl)methanone (3q)

Following the general procedure (F), the residue was puriﬁed

by column chromatography on silica gel, eluting with petroleum

ether/ethyl acetate 17:3, to afford the desired product 3q as a

yellow solid. Yield: 90%, mp 150–152 °C. ¹H NMR (CDCl₃) d:

0.45 (s, 9H), 2.35 (s, 2H), 3.83 (s, 3H), 5.91 (br s, 2H), 6.88 (d,

J = 8.8 Hz, 2H), 7.28 (d, J = 8.8 Hz, 2H), 7.43 (d, J = 8.6 Hz, 2H),

7.64 (d, J = 8.4 Hz, 2H). ¹³C NMR (100 MHz, DMSO-d₆) d: 29.36

(4C), 33.32, 54.99, 113.97 (2C), 116.16, 121.42, 126.87, 128.38

(2C), 130.39, 130.80 (2C), 131.21 (2C), 135.99, 139.25, 158.20,

161.51, 190.25. MS (ESI): [M+1]⁺= 414.2. Anal. (C₂₃H₂₄ClNO₂S)

C, H, N.

5.7.8. (2-Amino-4-(2,2-dimethylpropyl)-5-phenylthiophen-3-yl)

(2-chlorophenyl)-methanone (3l)

Following the general procedure (F), the residue was puriﬁed by

column chromatography on silica gel, eluting with petroleum

ether/ethyl acetate 1:4, to give the desired product 3l as a yellow

oil. Yield: 90%. ¹H NMR (CDCl₃) d: 0.69 (s, 9H), 2.25 (s, 2H), 5.88

(br s, 2H), 7.41 (d, J = 8.6 Hz, 2H), 7.53 (d, J = 8.6 Hz, 2H), 7.68 (m,

5H). MS (ESI): [M+1]⁺= 384.2. Anal. (C₂₂H₂₂ClNOS) C, H, N.

5.7.14. (2-Amino-4-(2,2-dimethylpropyl)-5-(4-methoxyphenyl)

thiophen-3-yl)(3-chloro-phenyl)methanone (3r)

Following the general procedure (F), the residue was puriﬁed by

column chromatography on silica gel, eluting with petroleum

ether/ethyl acetate 17:3, to afford the desired product 3r as a yel-

low solid. Yield: 88%, mp 74–76 °C. ¹H NMR (CDCl₃) d: 0.45 (s, 9H),

2.30 (s, 2H), 3.83 (s, 3H), 6.02 (br s, 2H), 6.88 (d, J = 8.8 Hz, 2H), 7.29

(d, J = 8.8 Hz, 2H), 7.33 (m, 1H), 7.42 (d, J = 7.2 Hz, 1H), 7.52 (d,

J = 7.4 Hz, 1H), 7.67 (s, 1H). ¹³C NMR (100 MHz, DMSO-d₆) d:

29.36 (4C), 33.32, 54.98, 113.99 (2C), 115.94, 121.48, 126.80,

127.62, 128.26, 129.32, 130.26, 130.99, 131.21 (2C), 133.03,

142.55, 158.21, 162.12, 189.71. MS (ESI): [M+1]⁺= 414.1. Anal.

(C₂₃H₂₄ClNO₂S) C, H, N.

5.7.9. (2-Amino-4-(2,2-dimethylpropyl)-5-phenylthiophen-3-yl)

(3,4-dichlorophenyl)-methanone (3m)

Following the general procedure (F), the residue was puriﬁed by

column chromatography on silica gel, eluting with petroleum

ether/ethyl acetate 9:1, to give the desired product 3m as a yellow

solid. Yield: 95%, mp 194–196 °C. ¹H NMR (CDCl₃) d: 0.45 (s, 9H),

2.36 (s, 2H), 5.96 (br s, 2H), 7.37 (m, 2H), 7.54 (m, 5H), 7.79 (s,

1H). ¹³C NMR (100 MHz, DMSO-d₆) d: 29.37 (4C), 33.48, 115.82,

R. Romagnoli et al. / Bioorg. Med. Chem. 22 (2014) 148–166

163

5.7.15. (2-Amino-4-(2,2-dimethylpropyl)-5-(4-methoxyphenyl)

thiophen-3-yl)(3,4-dichloro-phenyl)methanone (3s)

acetate 17:3, to provide the desired product 3x as a yellow so-

lid. Yield: 94%, mp 164–166 °C. ¹H NMR (CDCl₃) d: 0.55 (s, 9H),

2.48 (s, 2H), 5.94 (br s, 2H), 7.01 (dd, J = 5.0 and 3.5 Hz, 1H),

7.04 (dd, J = 3.5 and 1.0 Hz, 1H), 7.27 (m, 1H), 7.43 (d,

J = 8.5 Hz, 2H), 7.62 (d, J = 8.5 Hz, 2H). MS (ESI): [M+1]⁺= 390.0.

Anal. (C₂₀H₂₀ClNOS₂) C, H, N.

Following the general procedure (F), the residue was puriﬁed by

column chromatography on silica gel, eluting with ethyl acetate/

petroleum ether 3:17, to furnish the desired product 3s as a yellow

solid. Yield 95%, mp 182–184 °C, ¹H NMR (CDCl₃) d: 0.46 (s, 9H), 2.31

(s, 2H), 3.84 (s, 3H), 6.00 (br s, 2H), 6.89 (d, J = 6.6 Hz, 1H), 7.29 (d,

J = 6.6 Hz, 1H), 7.33 (d, J = 7.4 Hz, 2H), 7.54 (d, J = 7.4 Hz, 2H), 7.79

(s, 1H). ¹³C NMR (100 MHz, DMSO-d₆) d: 29.40 (4C), 33.38, 54.99,

114.00 (2C), 115.70, 121.73, 126.76, 129.22, 130.23, 130.49,

130.55, 130.67, 131.17 (2C), 133.96, 140.64, 158.24, 162.04,

188.51. MS (ESI): [M+1]⁺= 448.2. Anal. (C₂₃H₂₃Cl₂NO₂S) C, H, N.

5.7.21. (2-Amino-4-(2,2-dimethylpropyl)-5-(thiophen-3-yl)

thiophen-3-yl)(4-chlorophenyl)-methanone (3y)

Following the general procedure (F), the residue was puriﬁed by

column chromatography on silica, eluting with heptane/ethyl ace-

tate 22:3, to provide the desired product 3y as a yellow solid. Yield:

93%, mp 171–173 °C. ¹H NMR (CDCl₃) d: 0.49 (s, 9H), 2.36 (s, 2H),

5.91 (br s, 2H), 7.14 (dd, J = 5.0 and 1.5 Hz, 1H), 7.23 (dd, J = 3.0

and 1.5 Hz, 1H), 7.34 (dd, J = 5.0 and 3.0 Hz, 1H), 7.43 (d,

J = 8.5 Hz, 2H), 7.63 (d, J = 8.5 Hz, 2H). MS (ESI): [M+1]⁺= 390.0.

Anal. (C₂₀H₂₀ClNOS₂) C, H, N.

5.7.16. (2-Amino-4-(2,2-dimethylpropyl)-5-(4-methoxyphenyl)

thiophen-3-yl)(3-triﬂuoro-methylphenyl)methanone (3t)

Following the general procedure (F), the residue was puriﬁed by

column chromatography on silica gel, eluting with petroleum ether/

ethyl acetate 9:1, to give the desired product 3t as a yellow solid.

Yield: 75%, mp 154–156 °C. ¹H NMR (CDCl₃) d: 0.43 (s, 9H), 2.22

(s, 2H), 3.83 (s, 3H), 6.11 (br s, 2H), 6.88 (d, J = 8.8 Hz, 2H), 7.23 (d,

J = 8.8 Hz, 2H), 7.61 (t, J = 7.6 Hz, 1H), 7.78 (d, J = 7.8 Hz, 1H), 7.86

(d, J = 7.8 Hz, 1H), 7.98 (s, 1H). ¹³C NMR (100 MHz, DMSO-d₆) d:

29.30 (4C), 33.31, 54.98, 114.03 (2C), 115.81, 121.69, 125.18,

126.74, 127.66, 128.46, 128.88, 129.61, 130.13, 131.18 (2C),

132.93, 141.34, 158.26, 162.42, 189.63. MS (ESI): [M+1]⁺= 448.0.

Anal. (C₂₄H₂₄F₃NO₂S) C, H, N.

5.7.22. (2-Amino-4-(2,2-dimethylpropyl)-5-(furan-2-yl)

thiophen-3-yl)(4-chlorophenyl)-methanone (3z)

Following the general procedure (F), the residue was puriﬁed by

column chromatography on silica, eluting with heptane/ethyl ace-

tate 22:3, to provide the desired product 3z as a yellow-orange so-

lid. Yield: 93%, mp 119–121 °C. ¹H NMR (CDCl₃) d: 0.57 (s, 9H), 2.49

(s, 2H), 5.99 (br s, 2H), 6.34 (m, 1H), 6.42 (m, 1H), 7.39 (m, 1H), 7.42

(d, J = 8.5 Hz, 2H), 7.60 (d, J = 8.5 Hz, 2H). MS (ESI): [M+1]⁺= 374.0.

Anal. (C₂₀H₂₀ClNO₂S) C, H, N.

5.7.17. (2-Amino-4-(2,2-dimethylpropyl)-5-(4-methoxyphenyl)

thiophen-3-yl)(4-methyl-phenyl)methanone (3u)

Following the general procedure (F), the residue was puriﬁed

by column chromatography on silica gel, eluting with petroleum

ether/ethyl acetate 17:3, to give the desired product 3u as a yel-

low solid. Yield: 81%, mp 78–80 °C. ¹H NMR (CDCl₃) d: 0.45 (s,

9H), 2.42 (s, 3H), 2.78 (s, 2H), 5.60 (s, 1H), 5.77 (br s, 2H),

7.23 (m, 6H), 7.36 (d, J = 7.2 Hz, 2H), 7.64 (d, J = 7.2 Hz, 2H).

¹³C NMR (100 MHz, DMSO-d₆) d: 21.00, 29.36, 33.26 (3C),

33.32, 54.98, 113.94 (2C), 121.15, 128.54 (2C), 129.12, 129.56

(2C), 130.81, 131.21 (2C), 135.47, 137.87, 141.36, 158.13,

160.52, 191.63. MS (ESI): [M+1]⁺= 393.8. Anal. (C₂₄H₂₇NO₂S) C,

H, N.

5.7.23. (2-Amino-4-(2,2-dimethylpropyl)-5-(furan-3-yl)

thiophen-3-yl)(4-chlorophenyl)-methanone (3aa)

Following the general procedure (F), the residue was puriﬁed by

column chromatography on silica gel, the residue was puriﬁed by

column chromatography on silica, eluting with methylene chlo-

ride, providing the desired product 3aa as a yellow solid. Yield:

85%, mp 98–100 °C. ¹H NMR (CDCl₃) d: 0.55 (s, 9H), 2.33 (s, 2H),

6.50 (br s, 2H), 7.42 (d, J = 8.5 Hz, 1H), 7.44 (m, 1H), 7.50 (m, 1H),

7.30 (d, J = 8.5 Hz, 2H), 7.61 (d, J = 8.5 Hz, 2H). MS (ESI):

[M+1]⁺= 374.0. Anal. (C₂₀H₂₀ClNO₂S) C, H, N.

5.7.18. (2-Amino-5-(3,5-dimethylisoxazol-4-yl)-4-(2,2-

dimethylpropyl)thiophen-3-yl)(4-chlorophenyl)methanone

(3v)

Following the general procedure (F), the residue was puriﬁed

by column chromatography on silica gel, eluting with heptane/

ethyl acetate 99:1, to provide the desired product 3v as a yel-

low solid. Yield: 83%, mp 247–250 °C. ¹H NMR (CDCl₃) d: 0.53

(s, 9H), 2.03 (br s, 2H), 2.23 (s, 3H), 2.36 (s, 3H), 5.90 (br s,

2H), 7.44 (d, J = 8.5 Hz, 2H), 7.59 (d, J = 8.5 Hz, 2H). MS (ESI):

[M+1]⁺= 403.0. Anal. (C₂₁H₂₃ClN₂O₂S) C, H, N.

5.7.24. (2-Amino-4-(2,2-dimethylpropyl)-5-(pyridine-4-yl)

thiophen-3-yl)(4-chlorophenyl)-methanone (3ab)

Following the general procedure (F), the residue was puriﬁed

by column chromatography on silica gel, eluting with heptane/

ethyl acetate 22:3, to provide the desired product 3ab as a yellow

solid. Yield: 87%, mp 180–182 °C. ¹H NMR (CDCl₃) d: 0.47 (s, 9H),

2.44 (s, 2H), 6.04 (br s, 2H), 7.32 (dd, J = 4.5 and 1.5 Hz, 2H), 7.45

(d, J = 8.5 Hz, 2H), 7.65 (d, J = 8.5 Hz, 2H), 8.59 (dd, J = 4.5 and 1.5

H, 2H). MS (ESI): [M+1]⁺= 385.0. Anal. (C₂₁H₂₁ClNO₂S) C, H, N.

5.7.25. (2-Amino-4-(2,2-dimethylpropyl)-5-(pyridin-3-yl)

thiophen-3-yl)(4-chlorophenyl)-methanone (3ac)

5.7.19. (2-Amino-4-(2,2-dimethylpropyl)-5-(pyrazol-4-yl)

thiophen-3-yl)(4-chlorophenyl)-methanone (3w)

Following the general procedure (F), the residue was puriﬁed by

column chromatography on silica gel, eluting with heptane/ethyl

acetate 2:1, to provide the desired product 3ac as a very pale tan

solid. Yield: 85%, mp 212–214 °C. ¹H NMR (CDCl₃) d: 0.46 (s, 9H),

2.35 (s, 2H), 5.97 (br s, 2H), 7.32 (m, 1H), 7.45 (d, J = 8.5 Hz, 2H),

7.66 (d, J = 8.5 Hz, 2H), 7.70 (m, 1H), 8.54 (m, 1H), 8.66 (m, 1H).

MS (ESI): [M+1]⁺= 385.0. Anal. (C₂₁H₂₁ClNO₂S) C, H, N.

Following the general procedure (F), the residue was puriﬁed

by column chromatography on silica, eluting with methylene

chloride/ethyl acetate 3:1, to provide the desired product 3w

as a yellow solid. Yield: 90%, mp 80–82 °C. ¹H NMR (CDCl₃) d:

0.54 (s, 9H), 2.33 (s, 2H), 5.89 (br s, 2H), 7.43 (d, J = 8.5 Hz,

2H), 7.62 (d, J = 8.5 Hz, 2H), 7.65 (m, 3H). MS (ESI):

[M+1]⁺= 374.0. Anal. (C₁₉H₂₀ClN₃OS) C, H, N.

5.7.26. (2-Amino-4-(2,2-dimethylpropyl)-5-(pyridin-2-yl)

thiophen-3-yl)(4-chlorophenyl)-methanone (3ad)

5.7.20. (2-Amino-4-(2,2-dimethylpropyl)-5-(thiophen-2-yl)

thiophen-3-yl)(4-chlorophenyl)-methanone (3x)

Following the general procedure (F), the residue was puriﬁed by

column chromatography on silica gel, eluting with heptane/ethyl

Following the general procedure (F), the residue was puriﬁed

by column chromatography on silica, eluting with heptane/ethyl

164

R. Romagnoli et al. / Bioorg. Med. Chem. 22 (2014) 148–166

acetate 3:1, to provide the desired product 3ad as a yellow solid.

Yield: 87%, mp 170–172 °C. ¹H NMR (CDCl₃) d: 0.51 (s, 9H), 2.75

(s, 2H), 6.09 (br s, 2H), 7.11 (m, 1H), 7.43 (d, J = 8.5 Hz, 2H), 7.51

(m, 1H), 7.62 (m, 3H), 8.58 (m, 1H). MS (ESI): [M+1]⁺= 385.0. Anal.

(C₂₁H₂₁ClNO₂S) C, H, N.

2.33 (s, 2H), 5.93 (br s, 2H), 7.37 (m, 4H), 7.44 (d, J = 8.5 Hz, 2H),

7.64 (d, J = 8.5 Hz, 2H). MS (ESI): [M+1]⁺= 418.0. Anal. (C₂₂H₂₁Cl_2-

NOS) C, H, N.

5.7.33. (E)-(2-Amino-5-(2-(4-chlorophenyl)ethen-1-yl)-4-(2,2-

dimethylpropyl)thiophen-3-yl)(4-chlorophenyl)methanone

(3ak)

5.7.27. (2-Amino-4-(2,2-dimethylpropyl)-5-(4-ﬂuorophenyl)

thiophen-3-yl)(4-chlorophenyl)-methanone (3ae)

Following the general procedure (F), the residue was puriﬁed by

column chromatography on silica, eluting with methylene

chloride/heptane 3:1, affording the desired product 3ak as a yel-

low-orange solid. Yield: 80%, mp 171–174 °C. ¹H NMR (CDCl₃) d:

0.69 (s, 9H), 2.32 (s, 2H), 6.11 (br s, 2H), 6.49 (d, J = 16 Hz, 1H),

7.12 (d, J = 16 Hz, 1H),7.28 (d, J = 8.5 Hz, 2H), 7.32 (d, J = 8.5 Hz,

2H), 7.41 (d, J = 8.5 Hz, 2H), 7.57 (d, J = 8.5 Hz, 2H). MS (ESI):

[M+1]⁺= 444.2. Anal. (C₂₄H₂₃Cl₂NOS) C, H, N.

Following the general procedure (F), the residue was puriﬁed by

column chromatography on silica gel, eluting with heptane/ethyl

acetate 4:1, providing the desired product 3ae as a yellow solid.

Yield: 95%, mp 169–171 °C. ¹H NMR (CDCl₃) d: 0.45 (s, 9H), 2.31

(s, 2H), 5.92 (br s, 2H), 7.07 (m, 2H), 7.35 (m, 2H), 7.44 (d,

J = 8.5 Hz, 2H), 7.65 (d, J = 8.5 Hz, 2H). MS (ESI): [M+1]⁺= 402.0.

Anal. (C₂₂H₂₁ClFNOS) C, H, N.

5.7.34. (2-Amino-5-(3,4-dichlorophenyl)-4-(2,2-

dimethylpropyl)thiophen-3-yl)(4-chlorophenyl)-methanone

(3al)

5.7.28. (2-Amino-5-(2,3-diﬂuorophenyl)-4-(2,2-dimethylpropyl)

thiophen-3-yl)(4-chlorophenyl)methanone (3af)

Following the general procedure (F), the residue was puriﬁed by

column chromatography on silica gel, eluting with heptane/ethyl

acetate 17:3, providing the desired product 3af as a yellow-orange

solid. Yield: 96%, mp 173–175 °C. ¹H NMR (CDCl₃) d: 0.48 (s, 9H),

2.21 (s, 2H), 5.95 (br s, 2H), 7.08 (m, 3H), 7.44 (d, J = 8.5 Hz, 2H),

7.64 (d, J = 8.5 Hz, 2H). MS (ESI): [M+1]⁺= 420.0. Anal. (C₂₂H₂₀ClF_2-

NOS) C, H, N.

Following the general procedure (F), the residue was puriﬁed by

column chromatography on silica gel, eluting with a gradient of

heptane/methylene chloride (from 1:2 to 1:3), providing the de-

sired product 3al as a yellow solid. Yield: 87%, mp 189–191 °C.

¹H NMR (CDCl₃) d: 0.48 (s, 9H), 2.33 (s, 2H), 5.95 (br s, 2H), 7.23

(dd, J = 8.5 Hz, 2H), 7.43 (m, 2H), 7.48 (d, J = 2.0 Hz, 1H), 7.63 (d,

J = 8.5 Hz, 2H). MS (ESI): [M+1]⁺= 452.0. Anal. (C₂₂H₂₀Cl₃NOS) C,

H, N.

5.7.29. (2-Amino-5-(2,4-diﬂuorophenyl)-4-(2,2-dimethylpropyl)

thiophen-3-yl)(4-chlorophenyl)methanone (3ag)

5.7.35. (2-Amino-4-(2,2-dimethylpropyl)-5-(3-methoxyphenyl)

thiophen-3-yl)(4-chloro-phenyl)methanone (3am)

Following the general procedure (F), the residue was puriﬁed by

column chromatography on silica gel, eluting with heptane/ethyl

acetate 22:3, providing the desired product 3ag as a yellow solid.

Yield: 94%, mp 164–166 °C. ¹H NMR (CDCl₃) d: 0.48 (s, 9H), 2.17

(s, 2H), 5.91 (br s, 2H), 6.85 (m, 2H), 7.29 (m, 1H), 7.44 (d,

J = 8.5 Hz, 2H), 7.64 (d, J = 8.5 Hz, 2H). MS (ESI): [M+1]⁺= 420.0.

Anal. (C₂₂H_2oClF₂NOS) C, H, N.

Following the general procedure (F), the residue was puriﬁed by

column chromatography on silica gel, eluting with petroleum

ether/ethyl acetate 4:1, to afford the desired product 3am as a yel-

low solid. Yield: 64%, mp 168–170 °C. ¹H NMR (CDCl₃) d: 0.46 (s,

9H), 2.37 (s, 2H), 3.83 (s, 3H), 5.93 (br s, 2H), 6.86 (d, J = 8.2 Hz,

1H), 6.95 (s, 1H), 7.00 (m, 1H), 7.23 (m, 1H), 7.43 (d, J = 8.4 Hz,

2H), 7.67 (d, J = 8.4 Hz, 2H). MS (ESI): [M+1]⁺= 414.2. Anal. (C₂₃H_24-

ClNO₂S) C, H, N.

5.7.30. (2-Amino-5-(2,5-diﬂuorophenyl)-4-(2,2-dimethylpropyl)

thiophen-3-yl)(4-chlorophenyl)methanone (3ah)

5.7.36. (2-Amino-4-(2,2-dimethylpropyl)-5-(2-methoxyphenyl)

thiophen-3-yl)(4-chloro-phenyl)methanone (3an)

Following the general procedure (F), the residue was puriﬁed by

column chromatography on silica gel, eluting with heptane/ethyl

acetate 22:3, providing the desired product 3ah as a yellow solid.

Yield: 95%, mp 165–167 °C. ¹H NMR (CDCl₃) d: 0.48 (s, 9H), 2.21

(s, 2H), 5.95 (s, 2H), 6.97 (m, 3H), 7.44 (d, J = 8.5 Hz, 2H), 7.64 (d,

J = 8.5 Hz, 2H). MS (ESI): [M+1]⁺= 420.0. Anal. (C₂₂H₂₀ClF₂NOS) C,

H, N.

Following the general procedure (F), the residue was puriﬁed by

column chromatography on silica gel, eluting with petroleum

ether/ethyl acetate 17:3, to afford the desired product 3an as a yel-

low solid. Yield: 60%, mp 180–182 °C. ¹H NMR (CDCl₃) d: 0.54 (s,

9H), 2.16 (s, 2H), 3.83 (s, 3H), 5.85 (br s, 2H), 6.96 (m, 2H), 7.27

(m, 2H), 7.40 (d, J = 8.8 Hz, 2H), 7.64 (d, J = 8.8 Hz, 2H). ¹³C NMR

(100 MHz, DMSO-d₆) d: 29.21 (4C), 32.87, 55.42, 111.61, 115.73,

117.21, 120.26, 122.92, 128.35 (2C), 129.18, 130.78 (2C), 132.58,

132.62, 136.01, 139.22, 156.97, 162.01, 190.30. MS (ESI):

[M+1]⁺= 414.1. Anal. (C₂₃H₂₄ClNO₂S) C, H, N.

5.7.31. (2-Amino-4-(2,2-dimethylpropyl)-5-(2,6-diﬂuorophenyl)

thiophen-3-yl)(4-chloro-phenyl)methanone (3ai)

Following the general procedure (F), the residue was puriﬁed by

column chromatography on silica gel, eluting with heptane/ethyl

acetate 22:3, to provide the desired product 3ai as a yellow solid.

Yield: 87%, mp 175–177 °C. ¹H NMR (CDCl₃) d: 0.49 (s, 9H), 2.12

(s, 2H), 5.93 (br s, 2H), 6.96 (t, J = 8.0 Hz, 2H), 7.28 (m, 1H), 7.43

(d, J = 8.5 Hz, 2H), 7.63 (d, J = 8.5 Hz, 2H). MS (ESI):

[M+1]⁺= 420.0. Anal. (C₂₂H₂₀ClF₂NOS) C, H, N.

5.7.37. (2-Amino-4-(2,2-dimethylpropyl)-5-(4-(2-(methoxy)

ethoxy)phenyl)thiophen-3-yl)(4-chlorophenyl)-methanone

(3ao)

Following the general procedure (F), the residue was puriﬁed

by column chromatography on silica gel eluting with methylene

chloride/ethyl acetate 49:1, affording the desired product 3ao as

a

yellow-orange solid. Yield: 85%, mp 168–170 °C. ¹H NMR

5.7.32. (2-Amino-5-(4-chlorophenyl)-4-(2,2-dimethylpropyl)

thiophen-3-yl)(4-chlorophenyl)-methanone (3aj)

Following the general procedure (F), the residue was puriﬁed by

column chromatography on silica gel, eluting with heptane/meth-

ylene chloride 1:3, providing the desired product 3aj as a yellow

solid. Yield: 87%, mp 162–164 °C.¹H NMR (CDCl₃) d: 0.45 (s, 9H),

(CDCl₃) d: 0.44 (s, 9H), 2.31 (br s, 2H), 3.47 (s, 3H), 3.77 (t,

J = 5.0 Hz, 2H), 4.14 (t, J = 5.0 Hz, 2H), 5.91 (br s, 2H), 6.93 (d,

J = 8.5 Hz, 2H), 7.29 (d, J = 8.5 Hz, 2H), 7.43 (d, J = 8.5 Hz, 2H),

7.64 (d, J = 8.5 Hz, 2H). MS (ESI): [M+1]⁺= 458.0. Anal. (C₂₅H_28-

ClNO₃S) C, H, N.

R. Romagnoli et al. / Bioorg. Med. Chem. 22 (2014) 148–166

165

5.7.38. (2-Amino-4-(2,2-dimethylpropyl)-5-(3,4-

methylenedioxyphenyl)thiophen-3-yl)(4-chlorophenyl)

methanone (3ap)

5.8. Biology experiments

5.8.1. Materials

Following the general procedure (F), the residue was puriﬁed by

column chromatography on silica gel, eluting with heptane/ethyl

acetate 17:3, providing the desired product 3ap as a yellow-orange

solid. Yield: 91%, mp 190–192 °C. ¹H NMR (CDCl₃) d: 0.48 (s, 9H),

2.32 (s, 2H), 5.90 (br s, 2H), 6.00 (s, 2H), 6.81 (d, J = 8.5 Hz, 1H),

6.88 (m, 3H), 7.43 (d, J = 8.5 Hz, 2H), 7.64 (d, J = 8.5 Hz, 2H). MS

(ESI): [M+1]⁺= 428.0. Anal. (C₂₃H₂₃ClNO₃S) C, H, N.

[³H]DPCPX ([³H]1,3-dipropyl-8-cyclopentyl-xanthine; speciﬁc

activity, 120 Ci/mmol) and [³H]CCPA ([³H]2-chloro-N⁶-cyclopentyl-

adenosine; speciﬁc activity, 55 Ci/mmol) were obtained from

Perkin Elmer Research Products (Boston, MA); [³H]ZM 241385

([³H](4-(2-[7-amino-2-(2-furil)[1,2.4]triazolo[2,3-a][1,3,5]triazin-

5-ylamino]ethyl)phenol); speciﬁc activity, 17 Ci/mmol) was ob-

tained from Biotrend (Cologne, Germany); [³H]MRE-3008-F20

([³H]5-N-(4-methoxyphenylcarbamoyl)amino-8-propyl-2-

(2-furyl)pyrazolo[4,3-e]-1,2,4-triazolo[1,5-c]pyrimidine; speciﬁc

activity, 67 Ci/mmol) was obtained from Amersham International

(Buckinghamshire, UK). DPCPX (1,3-dipropyl-8-cyclopentyl-

xanthine), R-PIA ((R)-N⁶-(L-2-Phenylisopropyl)adenosine) and

CCPA (2-chloro-N⁶-cyclopentyladenosine) were obtained from

Sigma (St. Louis, MO, USA). All other reagents were of analytical

grade and obtained from commercial sources.

5.7.39. (2-Amino-4-(2,2-dimethylpropyl)-5-(4-

(triﬂuoromethoxy)phenyl)thiophen-3-yl)(4-chlorophenyl)

methanone (3aq)

Following the general procedure (F), the residue was puriﬁed by

column chromatography on silica, eluting with methylene chlo-

ride/ethyl acetate 49:1, affording the desired product 3aq as a yel-

low solid. Yield: 83%, mp 137–139 °C. ¹H NMR (CDCl₃) d: 0.45 (s,

9H), 2.34 (s, 2H), 5.92 (br s, 2H), 7.22 (d, J = 8.5 Hz, 2H), 7.41 (d,

J = 8.5 Hz, 2H), 7.44 (d, J = 8.5 Hz, 2H), 7.65 (d, J = 8.5 Hz, 2H). MS

(ESI): [M+1]⁺= 468.0. Anal. (C₂₃H₂₁ClF₃NO₂S) C, H, N.

5.8.2. Cell membrane preparation

The hA₁CHO, hA_2ACHO and hA₃CHO cells were grown adher-

ently and maintained in Dulbecco’s modiﬁed Eagle’s medium with

nutrient mixture F12, containing 10% fetal calf serum, penicillin

(100 U/mL), streptomycin (100 lg/mL), L-glutamine (2 mM), gene-

5.7.40. (2-Amino-4-(2,2-dimethylpropyl)-5-(4-methylphenyl)

thiophen-3-yl)(4-chloro-phenyl)methanone (3ar)

ticine (G418) 0.2 mg/mL at 37 °C in 5% CO₂/95% air. For membrane

preparation the culture medium was removed and the cells were

washed with phosphate-buffered saline and scraped off T75 ﬂasks

in ice-cold hypotonic buffer (5 mM Tris–HCl, 1 mM EDTA, pH 7.4).

The cell suspension was homogenized with a Polytron, the homog-

enate was spun for 10 min at 1000g and the supernatant was then

centrifuged for 30 min at 100,000g. The membrane pellet was sus-

pended in 50 mM Tris–HCl buffer (pH 7.4) for A₁ARs, in 50 mM

Tris–HCl, 10 mM MgCl₂(pH 7.4) for A_2AARs, in 50 mM Tris–HCl,

10 mM MgCl₂, 1 mM EDTA (pH 7.4) for A₃ARs. The membranes

were incubated with 2–3 IU/mL of adenosine deaminase to reduce

the endogenous adenosine. The protein concentration was deter-

mined according to a Bio-Rad method with bovine albumin as a

standard reference.¹⁹

Following the general procedure (F), the residue was puriﬁed by

column chromatography on silica gel, eluting with petroleum ether/

ethyl acetate 4:1 to afford the desired product 3ar as a yellow solid.

Yield 78%, mp 134–136 °C. ¹H NMR (CDCl₃) d: 0.44 (s, 9H), 2.22 (s,

3H), 2.38 (s, 2H), 5.93 (br s, 2H), 7.22 (d, J = 8.8 Hz, 2H), 7.45 (d,

J = 8.2 Hz, 2H), 7.56 (d, J = 8.2 Hz, 2H), 7.68 (d, J = 8.8 Hz, 2H). MS

(ESI): [M+1]⁺= 398.0. Anal. (C₂₃H₂₄ClNOS) C, H, N.

5.7.41. (2-Amino-4-(2,2-dimethylpropyl)-5-(3-methylphenyl)

thiophen-3-yl)(4-chloro-phenyl)methanone (3as)

Following the general procedure (F), the residue was puriﬁed

by column chromatography on silica gel, eluting with petroleum

ether/ethyl acetate 19:1, to afford the desired product 3as as a

yellow solid. Yield: 67%, mp 180–182 °C. ¹H NMR (CDCl₃) d: 0.48

(s, 9H), 2.24 (s, 3H), 2.37 (s, 2H), 5.83 (br s, 2H), 7.19 (d,

J = 8.4 Hz, 1H), 7.38 (d, J = 8.4 Hz, 2H), 7.42 (s, 1H); 7.56 (d,

J = 8.2 Hz, 2H), 7.63 (m, 2H). MS (ESI): [M+1]⁺= 398.1. Anal. (C_23-

H₂₄ClNOS) C, H, N.

5.8.3. Binding experiments in hA₁CHO membranes

5.8.3.1. [³H]CCPA binding experiments.

Saturation binding

experiments of [³H]CCPA (0.05–20 nM) to hA₁CHO membranes

were performed in triplicate at 25 °C for 90 min in 50 mM Tris–

HCl, pH 7.4, in the absence and presence of the tested compounds

at the ﬁnal concentration of 10

l

M.²⁰Non-speciﬁc binding was

M R-PIA.

5.7.42. (2-Amino-4-(2,2-dimethylpropyl)-5-(2-methylphenyl)

thiophen-3-yl)(4-chloro-phenyl)methanone (3at)

deﬁned as binding in the presence of 1

l

5.8.3.2. [³H]DPCPX competition binding experiments.

Com-

Following the general procedure (F), the residue was puriﬁed

by column chromatography on silica gel, eluting with 2% ethyl

acetate in methylene chloride to afford the desired product 3at

as a yellow solid. Yield 94%, mp 160–162 °C. ¹H NMR (CDCl₃) d:

0.45 (s, 9H), 2.34 (s, 2H), 2.37 (s, 3H), 5.91 (br s, 2H), 7.17 (d,

J = 8.0 Hz, 2H), 7.28 (d, J = 8.0 Hz, 2H), 7.43 (d, J = 8.5 Hz, 2H),

7.65 (d, J = 8.5 Hz, 2H). MS (ESI): [M+1]⁺= 398.0. Anal. (C₂₃H_24-

ClNOS) C, H, N.

petition binding experiments of 1 nM [³H]DPCPX were performed in

triplicate in 50 mM Tris–HCl, pH 7.4, for 90 min at 25 °C. The effect

of the different tested compounds at a concentration of 10

the CCPA curve (0.01 nM–1

M) was investigated.²¹Non-speciﬁc

binding was deﬁned as binding in the presence of 1 M DPCPX.

lM on

l

5.8.3.3. Assay of the antagonistaActivity versus A₁, A_2Aand A₃

ARs. A₁, A_2Aand A₃AR competition binding experiments were

performed using 1 nM [³H]DPCPX, 1 nM [³H]ZM 241385 and 2 nM

[³H]MRE-3008-F20 as radioligands, respectively.^21–23Membrane

suspensions were incubated in 50 mM Tris–HCl, pH 7.4, at 25 °C

for 120 min, in 50 mM Tris–HCl, 10 mM MgCl₂, pH 7.4, at 4 °C for

60 min, and in 50 mM Tris HCl, 10 mM MgCl₂, 1 mM EDTA, pH

7.4 at 4 °C for 120 min to study A_1,A_2Aand A₃ARs, respectively.

Non-speciﬁc binding was deﬁned as the binding in the presence

of 1 lM DPCPX or ZM 241385 or MRE-3008-F20 for A₁, A_2Aand

A₃ARs, respectively. Inhibition was expressed as percentage of con-

trol speciﬁc binding (100%). Test agents were dissolved in DMSO

5.7.43. (2-Amino-4-(2,2-dimethylpropyl)-5-(4-(1-(methyl)ethyl)

phenyl)thiophen-3-yl)(4-chlorophenyl)methanone (3au)

Following the general procedure (F), the residue was puriﬁed by

column chromatography on silica gel, eluting with heptane/ethyl

acetate 22:3, providing the desired product as a yellow solid. Yield:

87%, mp 155–157 °C. ¹H NMR (CDCl₃) d: 0.48 (s, 9H), 1.27 (d,

J = 7.0 Hz, 6H), 2.35 (s, 2H), 2.96 (m, 1H), 5.90 (br s, 2H), 7.22 (d,

J = 8.5 Hz, 2H), 7.30 (d, J = 8.5 Hz, 2H), 7.44 (d, J = 8.5 Hz, 2H),

7.65 (d, J = 8.5 Hz, 2H). MS (ESI): [M+1]⁺= 426.0. Anal. (C₂₅H₂₈

ClNOS) C, H, N.

166

R. Romagnoli et al. / Bioorg. Med. Chem. 22 (2014) 148–166

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and added to the assay from a 100-fold concentrated solution in

DMSO. Control incubations also contained 1% DMSO.

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assay mixture through Whatman GF/B glass ﬁber ﬁlters using a

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many). The ﬁlter bound radioactivity was counted by Packard Tri

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5.8.4. Effect of the novel compounds in cyclic AMP assays

Human A₁CHO cells (10⁶cells/mL) were prepared as described

above and were suspended in 0.5 mL incubation mixture phosphate

buffer, containing 1.0 IU adenosine deaminase/mL and 0.5 mM

4-(3-butoxy-4-methoxybenzyl)-2-imidazolidinone (Ro 20-1724) as

a phosphodiesterase inhibitor and preincubated for 10 min in a shak-

ing bath at 37 °C. The effect of allosteric enhancers were studied at

10 lM concentration that was added to the mixture for a further

10 min. The effect of allosteric enhancers (100 nM) was also studied

in the presence of a low concentration of CCPA (1 pM). Forskolin

1 lM was added for 5 min and was used to stimulate the activity of

adenylatecyclase. Thereactionwasterminatedbytheadditionofcold

6% trichloroacetic acid (TCA). The TCA suspension was centrifuged at

2000gfor 10 minat 4 °C and thesupernatantwasextractedfourtimes

with water saturated diethyl ether. The ﬁnal aqueous solution was

tested for cAMP levels by a competition protein binding assay.

Samples ofcAMPstandards (0–10 pmol) were added toeach test tube

containing trizma base 0.1 M, aminophylline 8.0 mM, mercap-

toethanol 6.0 mM, pH 7.4 and [³H]-cAMP (at the ﬁnal concentration

of1 nM).Thebindingprotein, previouslypreparedfrombeefadrenals,

was added to the samples and incubated at 4 °C for 150 min. At the

end of the incubation time and after the addition of charcoal, the

samples were centrifuged at 2000g for 10 min. The clear supernatant

was mixed with 4 mL of Ultima Gold (Perkin Elmer) and counted in a

Packard Tri Carb 2810 TR scintillation counter (Perkin Elmer).

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5.8.5. Data analysis

Saturation and competition binding experiments were analysed

with the program LIGAND, which performed weighted, non-linear,

least squares curve ﬁtting program.²⁴Inhibitory binding constants,

K_i, were also calculated from the IC₅₀values according to the Cheng

and Prusoff equation K_i¼ IC₅₀=ð1 þ ½C^ꢁꢂ=K^ꢁÞ, where [C⁄] is the con-

D

centration of the radioligand and K_D⁄ its dissociation constant.²⁵

All experimental data are expressed as mean standard error of

the mean (S.E.M.) of three or four independent experiments per-

formed in duplicate.

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Acknowledgement

18. In our experiments, the reference compound PD 81,723 (at a concentration

of 10

transfected in CHO cells. For the same reference compound, Bruns (Ref. 11a)

l

M) did not inhibit [³H]DPCPX binding to human A1 receptors

We wish to thank Dr. Alberto Casolari for technical assistance.

showed a Ki value of 11 lM obtained in competition binding experiments by

using [³H]DPCPX as radioligand on rat membranes. Furthermore, data

performed on CHO-K1cells stably expressing the human A₁receptors (Ref.

12c) reported an inhibition of [³H]DPCPX binding to human A₁receptors by

Supplementary data

PD 81,723 only of 42 7%, when tested at 100 lM. We speculate that

species differences in afﬁnity binding of PD 81,723 may explain the

discrepancy between the data.

Supplementary data associated (Supplementary data (Table S1)

on antagonist activity of compounds PD 81,723, 2a–i and 3a–au,

synthetic procedure for the preparation of compounds 2b–c and

2e–h. Elemental analyses of compounds 3a–au) with this article

can be found, in the online version, at http://dx.doi.org/10.1016/

j.bmc. 2013.11.043.

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References and notes

1. (a) Jacobson, K. A.; Gao, Z.-G. Nat. Rev. Drug Discov. 2006 , 5 , 247; (b) Fredholm, B. B.;

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Article Doi

Synthesis and biological evaluation of novel 2-amino-3-aroyl-4-neopentyl-5- substituted thiophene derivatives as allosteric enhancers of the A1 adenosine receptor

DOI: 10.1016/j.bmc.2013.11.043

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