Modulation of cAMP-specific PDE without emetogenic activity: New sulfide-like PDE7 inhibitors

Article

pubs.acs.org/jmc

Modulation of cAMP-Speciﬁc PDE without Emetogenic Activity: New

Sulﬁde-Like PDE7 Inhibitors

Ana M. García,^†Jose Brea,^‡Jose A. Morales-García,^§,∥Daniel I. Perez,^†Alejandro Gonzalez,^‡

́

Sandra Alonso-Gil,^§,∥Irene Gracia-Rubio,^⊥Clara Ros-Simo,^⊥Santiago Conde,^#María Isabel Cadavid,^‡

́

María Isabel Loza,^‡Ana Perez-Castillo,^§,∥Olga Valverde,^⊥Ana Martinez,*^,†and Carmen Gil*^,†

^†Centro de Investigaciones Biolog

́

icas (CSIC), Ramiro de Maeztu 9, 28040 Madrid, Spain

^‡Instituto de Farmacia Industrial, Facultad de Farmacia, Universidad de Santiago de Compostela, Campus Universitario Sur s/n,

15782 Santiago de Compostela, Spain

^§Instituto de Investigaciones Biomed

^∥Centro de Investigacion

Biomedica en Red sobre Enfermedades Neurodegenerativas (CIBERNED)

^⊥Departamento de Ciencias Experimentales y de la Salud. Universitat Pompeu Fabra, IMIM, Instituto Hospital del Mar de

Investigaciones Medicas. Dr. Aiguader 88, 08003 Barcelona, Spain

^#Instituto de Química Med

ica (CSIC), Juan de la Cierva 3, 28006 Madrid, Spain

́

icas (CSIC-UAM), Arturo Duperier 4, 28029 Madrid, Spain

́

S

* Supporting Information

ABSTRACT: A forward chemical genetic approach was

followed to discover new targets and lead compounds for

Parkinson’s disease (PD) treatment. By analysis of the cell

protection produced by some small molecules, a diphenyl

sulﬁde compound was revealed to be a new phosphodiesterase

7 (PDE7) inhibitor and identiﬁed as a new hit. This result

allows us to conﬁrm the utility of PDE7 inhibitors as a

potential pharmacological treatment of PD. On the basis of

these data, a diverse family of diphenyl sulﬁdes has been

developed and pharmacologically evaluated in the present

work. Moreover, to gain insight into the safety of PDE7

inhibitors for human chronic treatment, we evaluated the new compounds in a surrogate emesis model, showing nonemetic

eﬀects.

eﬀective treatment. Currently, the etiology of PD is not well

understood but is likely to involve both genetic and

environmental factors.⁵Neuropathologically, PD is characte-

rized by the loss of dopamine-producing neurons in the

substantia nigra pars compacta of the midbrain, followed by

striatal dopamine depletion and indirectly by cortical

dysfunction.⁶

Among the therapeutic agents for the palliative treatment of

PD, ^L-3,4-dihydroxyphenylalanine (L-dopa) is the drug most

frequently used. It ameliorates parkinsonian-associated motor

impediments but is relatively ineﬃcient in alleviating the

aﬀective and cognitive symptoms of the disease.⁷However,

long-term administration of ^L-dopa causes important side

eﬀects, such as dyskinesia, that contribute to lower quality of

life of patients.⁸Therefore, there is an urgent need for the

development of new therapeutic agents for PD or new

concomitant agents to avoid the high-dose administration of

L-dopa.⁹

INTRODUCTION

■

Parkinson’s disease (PD) is a progressive neurodegenerative

disorder that is characterized by the inability to initiate, execute,

and control movements. Common symptoms of PD patients

are tremor, rigidity, bradykinesia, akinesia, postural reﬂex

abnormalities, gait disturbance, etc.¹In addition to motor

diﬃculties, parkinsonian patients experience bouts of mild

depression and irritability as well as cognitive disturbances

deﬁned by memory and attention perturbations.²PD is a

disease of high incidence, especially in high-income countries,

where progressive aging of the population is one of the main

risk factors. With the partial exception of juvenile parkinsonism,

development of PD emerges during the ﬁfth and sixth decade,

aﬀecting 1 in 2000 people, and the prevalence is consistently

higher in men than in women. This incidence increases to 1 in

500 people older than 65 years.³Today, there are more than

one million PD patients in the USA and double this ﬁgure

worldwide, thus representing high social and healthcare costs.⁴

As a neurodegenerative disease, PD shares with these

devastating pathologies an unknown etiology, the progressive

destruction of speciﬁc areas of the brain, and the lack of an

Received: July 18, 2014

A

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Figure 1. Phenotypic screening of our in-house small molecule chemical library. Typical plot obtained in our chemical genetic approach to discover

new pharmacological targets for PD. Results for 27 diﬀerent compounds among a data set of 450 small molecules tested, including compound 2

(named as SC072), are depicted. SH-SY5Y cells were exposed to 6-OHDA [35 μM] during 24 h in the presence or absence of the small heterocyclic

compounds [10 μM]. The number of viable cells was measured by MTT assay. Each data point represents the mean SD of four replications in

three diﬀerent experiments. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001 versus 6-OHDA treated cultures (from ref 14).

In this context, we have reported the development of PDE7

inhibitors as a new approach to be explored for the treatment of

neurological disorders due to increasing levels of cAMP¹⁰and

speciﬁcally PDE7 inhibition by hindering dopaminergic cell

death and glial activation in a toxicity-based animal model of

PD. A heterocyclic small-molecule inhibitor of PDE7 that was

discovered in our research group, the 4-oxo-3-phenyl-2-thioxo-

1,2,3,4-tetrahydroquinazoline (1),¹¹conferred signiﬁcant neu-

ronal protection against diﬀerent insults both in the human

dopaminergic cell line SH-SY5Y and in primary rat

mesencephalic cell cultures. Quinazoline 1 treatment also

reduced microglial activation, protected dopaminergic neurons,

and improved motor function in the lipopolysaccharide rat

model of Parkinson’s disease. Moreover, quinazoline 1

neuroprotective eﬀects were abolished by blocking the cAMP

signaling pathways that operate through cAMP-dependent

PKA. These results showed for the ﬁrst time that inhibition of

the PDE7 enzyme leads to dopaminergic neuronal protection

and, therefore, its inhibitors may exert useful therapeutic

actions in patients with PD.^12,13Furthermore, the PDE7

inhibitor 1 (also known as S14) is currently in regulatory

toxicological studies to enter clinical trials for PD.

pharmacological analysis of the hits, which allowed us to

determine new targets for PD treatment (Figure 1). Thus, by

analyzing the cell protection produced by the 5-(3-chloro-

phenyl)-1,3-diphenyl-1,2,4-benzotriazepine (SC001), we could

demonstrate that GSK-3 is a good target for PD treatment.¹⁴

Here, we describe data obtained for the N-[2-(5-chloro-2-

nitrophenylthio)phenyl]acetamide (2), which was revealed to

be a new PDE7 inhibitor. The study here reported allows us to

conﬁrm the utility of PDE7 inhibitors as a potential

pharmacological treatment of PD.¹²Moreover, to gain insight

into the safety of PDE7 inhibitors, we here evaluate the new

compounds in a well-established surrogate emesis model,¹⁵

showing that modulation of PDE7 avoids gastrointestinal side

eﬀects such as those produced by PDE4 inhibitors. Human

emesis, which was found in clinical development of several

PDE4 inhibitors, has jeopardized their development and has

ruled out some promising candidates from reaching the

pharmaceutical market.

RESULTS AND DISCUSSION

■

Discovery of Sulﬁde-Like PDE7A Inhibitors. Our initial

screening was based on chemical diversity. From the chemical

structures collected in our in-house chemical library, we

selected 450 diverse small heterocyclic compounds for the

ﬁrst cell-based phenotypic screening. We analyzed all the

compounds that resulted in more than 65% cell survival in our

assay conditions (6-hydroxydopamine (6-OHDA)-induced

With the aim of discovering new targets for PD, a chemical

genetic approach involving the use of small molecules as

pharmacological tools was implemented in our group. First, a

screening of our in-house chemical library on a well-established

cellular model of PD was done, followed by a detailed

B

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a

Table 1. PDEs Panel from Sulﬁde 2

PDE7A

PDE3A

PDE4D

PDE4B

11 7% @ 10 μM

2

IC₅₀= 2.1 0.2 μM

10 3% @ 10 μM

10 2% @ 10 μM

a

Values represent the mean standard deviation of two independent experiments (n = 2) with duplicate measurements.

human neuroblastoma cell death, see Experimental Section).

Here, we report results derived from the analyses of the hit

compound 2. In the forward chemical genetics approach, one of

the key steps is the identiﬁcation of the target that produced the

phenotypic change. For that reason, we evaluated the diphenyl

sulﬁde 2 in our internal biological assays to determine its

potential target. Thus, we run ORAC experiments to look for

antioxidant activity, or potential inhibition of some protein

kinases, including GSK-3β, CK1δ, CK1ε, LRRK2, and ROCK,

and PDEs inhibition, such as PDE7A, PDE3A, PDE4B, and

PDE4D. In all cases, we used a ﬁxed concentration of

compound 2 of 10 μM, and we evaluated the percentage of

inhibition. When more than 50% inhibition was obtained, the

dose−response curve was determined and the IC₅₀value was

calculated. For sulﬁde 2, no antioxidant activity or protein

kinase inhibition was found in these experimental conditions.

However, a consistent and rather selective PDE7A inhibition

was found in human isoforms (Table 1), and thus, we can asses

the neuroprotection in our PD cell-based model due to PDE7

inhibition, conﬁrming our previous data with quinazoline-like

inhibitors.¹²

diﬀerent heterocycles, such as pyridine or thiophene, have been

assayed (Schemes 1−6).

In general, almost all the new synthesized compounds were

obtained by nucleophilic substitution of aryl halides and the

corresponding thiol or alkoxy derivatives. These reactions were

carried out using the base and solvent that was most convenient

in each case (Schemes 1, 2, and 5−6). As indicated in Scheme

1, optimization of the synthetic procedure of compounds 14

and 28 was carried out and microwave irradiation was used to

increase yields and reduce reaction times. In fact, sulﬁdes 3−14

were synthesized in dichloromethane and in the presence of

potassium carbonate as previously described.¹⁶The use of

microwave irradiation brought a signiﬁcant decrease of the

reaction time with respect to the reported procedure by

conventional heating (21 h). However, due to the frequent

dimerization of the thiol reagent, we tried other reaction

conditions such as ethanol/sodium acetate.¹⁷In this case, we

could observe a signiﬁcant reduction in the dimerization

product, although no signiﬁcant improvement in yields

(optimization of synthesis of compound 14). Moreover, for

further optimization, we decided to try other solvents with

higher boiling points such as acetonitrile or N,N-dimethylfor-

mamide. As a result of the increasing temperature, we were able

to signiﬁcantly reduce the reaction time as well as to increase

the yields (optimization of synthesis of compound 28).

To introduce a new point of diversity, compounds 28 and 8

were N-acetylated (Scheme 3) and the phenyl nitro groups

were converted into amino groups by treatment with a reducing

agent such as tin(II) chloride (Scheme 4).

In a second step, a small, focused subset of molecules from

our chemical library was selected to conﬁrm this speciﬁc

biological activity. Thus, seven more diphenyl sulﬁde-like

compounds similar to the hit were evaluated against PDE7A

(Figure 2), showing that almost all the evaluated compounds

inhibited PDE7A in the same range as the hit although with

lower potency.

In Vitro Evaluation of PDE7 Inhibition and Binding

Mode Studies. The new derivatives (3−58) were tested for

their inhibitory potencies against PDE7A1 using the recombi-

nant human isoenzyme as described in the Experimental

Section. All the compounds were tested at a ﬁxed concentration

of 10 μM, and the experiments were performed in duplicate.

The percentage of inhibition of PDE7A1 for all the compounds

at this concentration is shown in Table 2. When the percentage

of PDE7A1 inhibition was greater than 50%, the dose−

response curve was determined and the IC₅₀values were

calculated (Table 2). Some of these new compounds presented

IC₅₀values in the low micromolar range and thus are good hits

to be further explored as pharmacological agents.

For better characterization of the enzymatic inhibition of this

new family of PDE7 inhibitors, a kinetic study in which cAMP

concentration was varied was performed with two of the most

active compounds (28 and 42). Double-reciprocal plotting of

the data is depicted in Figure 3. The intercept of the plot in the

horizontal axis (1/[cAMP]) changes when the compound

concentrations increase, whereas the intercept in the vertical

axis (1/V) does not change. These results suggest that

diphenyl-sulﬁde derivatives act as competitive inhibitors of

cAMP in their binding to PDE7, targeting the catalytic binding

site.

Figure 2. Small focused subset of sulﬁde 2-related compounds selected

from our chemical library and evaluation as PDE7 inhibitors.

Optimisation of PDE7 Enzyme Potency. Given its

inhibition against PDE7A, its promising behavior in vitro, and

its chemical diversity from previously described inhibitors of

PDE7, compound 2 was selected for further biological activity

optimization. 2 has two phenyl rings connected by a sulﬁde

linker. We carried out a medicinal chemistry program to

increase the potency of the hit, consisting of the structural

modiﬁcation of the substituents attached to both phenyl rings

and changes in the nature of the linker and the substituents of

the phenyl rings. Thus, other linkers, an ether moiety and

To gain insight into the binding mode of the sulﬁde-like

inhibitors, docking experiments were carried out with the

docking program Glide (Schrodinger Inc.) according to the

procedure described in the Experimental Section for com-

C

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Scheme 1. General Procedure for the Preparation of Compounds 3−36

pounds 28 and 42. Considering the kinetic studies results, the

docking studies were focused on the cAMP binding site.

Regarding the sulﬁde 28, the binding mode shows a weak

aromatic π−π interaction of the poor-electron ring with Phe416

(Figure 4A). Moreover, the amine group present in the second

phenyl moiety may play a key role for the activity because it is

able to establish hydrogen bond interactions with oxygens from

Thr321 and Asp362, which could also explain the loss of

D

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Scheme 2. General Procedure for the Preparation of

Compounds 37−45

Scheme 5. General Procedure for the Preparation of

Compounds 50−54

Scheme 6. General Procedure for the Preparation of

Compounds 55−58

Scheme 3. General Procedure for the Preparation of

Compounds 46 and 47

in comparison with the 2-pyridine ones. Both PDE7 inhibitors,

28 and 42, are able to interact with the conserved Gln413.

When comparing the binding modes of 28 and 42, it is

important to mention that the π−π interaction between

Phe416 and one aromatic ring of the sulﬁde derivative is

conserved in both cases. However, the location of the second

aromatic ring is completely diﬀerent, probably due to the

butterﬂy shaped sulfur bridge, which allows the movement of

the ring looking for suitable interactions while maintaining the

π−π stacking with Phe416.

Scheme 4. General Procedure for the Preparation of

Compounds 48 and 49

Moreover, PDE3 and PDE4 inhibition of compounds with an

IC₅₀(PDE7A) ≤1.5 μM was calculated in order to assess safety

of these potential drug candidates in further development.

PDE3 and PDE4 inhibition in humans has been previously

related with cardiotoxic¹⁸and emetogenic¹⁹side eﬀects,

respectively (Table 3), and they should be avoided in future

chronic human treatment. The results show that diphenyl-

sulﬁde derivatives are inhibitors of the two PDE7 isoforms,

PDE7A and PDE7B, both of which are expressed in the brain,

while in general these compounds are rather selective regarding

PDE3 and PDE4. They do not present PDE3A inhibition at the

concentration assayed, while the inhibition of some PDE4

isoforms is at least 1 order of magnitude greater than that of

PDE7, if any. The most active PDE7A sulﬁde-type inhibitors,

compounds 28 and 42, were also evaluated for inhibition of

PDE1A, PDE5, PDE8A, PDE9A, and PDE10A at 10 μM.

Compound 28 inhibited these enzymes with a percentage of

inhibition of 36.0 1.0% (PDE1A), 28.0 3.0% (PDE5), 1.2

activity of compounds 22 and 27, which have a bromine atom

and an isopropyl group, respectively, in this position.

Regarding derivatives with 4-pyridine rings, such as

compound 42 (Figure 4B), docking studies revealed the

importance of the 4-pyridine ring for the activity, not only

because of the aromatic interaction with Phe384 but also

because the hydrogen bond formation with Asn260, which

could also explain the higher aﬃnity of 4-pyridine compounds

E

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Table 2. PDE7A1 Inhibition of Compounds (3−58) Together with References Compounds BRL50481, Quinazoline 1, and

a

Sulﬁde 2

compd

PDE7A % inh @ 10 μM

PDE7A IC₅₀(μM)

compd

PDE7A % inh @ 10 μM

PDE7A IC₅₀(μM)

BRL50481

99

76

58

74

42

60

38

63

83

71

79

36

69

59

2

4

6

3

4

1

2

1

4

1

0.09 0.02

4.68 0.23

2.10 0.32

6.48 1.06

30

31

32

33

34

35

36

37

38

39

40

41

42

43

44

45

46

47

48

49

50

51

52

53

54

55

56

57

58

1

87

74

70

77

4

2

1

2

1

2

1

2

1

2

1

3

1

4

1

3

2

1

3

4

1

1.50 0.02

2.20 0.13

3.30 0.71

2.20 0.10

2

3

4

5

8.77 1.34

6

17

69

81

56

69

85

80

91

56

7

9.27 0.68

3.97 0.35

2.20 0.10

1.47 0.22

5.20 1.10

1.60 0.36

7.98 1.23

3.60 0.11

1.29 0.64

0.18 0.09

1.88 0.21

0.72 0.06

3.80 0.63

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

28

29

1.01 0.10

2.14 0.63

5.45 1.17

5.75 1.36

5.69 0.41

3.20 0.83

6.18 0.21

2.88 0.17

0.99 0.05

66 13

69

68

78

64

49

79

23

11

33

31

17

15

10

81

6

1

2

1

6

1

2

4

2

3

1

2

26

13

46

27

12

2

6.50 1.03

32

3

44

18

36

17

0.37 0.07

-

a

Values represent the mean standard deviation of two independent experiments (n = 2) with duplicate measurements.

(PDE9A), and 29.4 5.6% (PDE10A). These results show that

these compounds can be considered fairly good hits as selective

PDE7 inhibitors. Finally, it is worth mentioning that cAMP

response element-binding protein (CREB) is a well-known

target of the cAMP signaling pathway, and the enhancement of

cAMP levels leads to an increase in its phosphorylation state.

Thus, as a surrogate marker of the cAMP increase induced by

our sulﬁde-like PDE7 inhibitors, we analyzed the phospho-

rylation levels of CREB in SH-SY5Y cultures after treatment

with the compounds 28 and 42. The use of an antiserum that

does not discriminate between CREB and p-CREB indicated

that the total levels of CREB were not aﬀected by the

treatments. Figure 5 clearly shows that the treatment with these

compounds resulted in an increase in the phosphorylation state

of CREB as a direct consequence of the up-regulation of cAMP

levels.

Eﬀects of New Sulﬁde-Like PDE7A Inhibitors on Cell

Viability in SH-SY5Y Cells. Our next step was to explore the

biological proﬁle of the new derivatives. First, we tested if the

new PDE7A inhibitors are able to protect the SH-SY5Y cell line

from cell death induced by the toxin 6-OHDA, a well-

established cell model for Parkinson’s disease. The ability of the

selected compounds (10, 12, 20, 28, 31, 41, 42, and 44, all with

an IC₅₀≤1.5 μM) to protect the human neuroblastoma cell line

from cell death was analyzed (Figure 6). As expected, treatment

of SH-SY5Y cells with 6-OHDA resulted in signiﬁcant cell

death, while compounds 20, 28, and 44 signiﬁcantly rescued

Figure 3. Studies of the PDE7A1 inhibition in the absence (control)

and in the presence of two diﬀerent concentrations of the sulﬁde

derivatives 28 and 42 incubating diﬀerent cAMP concentrations and

measuring AMP formation. Data represent the mean SEM (vertical

bars) of triplicate measurements.

0.8% (PDE8A), 7.0

(PDE10A), and compound 42 with 39.0

21.0 1.0% (PDE5), 2.4

1.0% (PDE9A), and 57.5

3.4%

4.0% (PDE1A),

1.1% (PDE8A), 1.0

1.0%

F

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Figure 5. Eﬀects of compounds 28 and 42 on phosphorylation state of

the cAMP response element-binding protein (CREB). Representative

Western blot showing the levels of p-CREB on SH-SY5Y after

treatment with compounds 28 and 42 (10 μM). Results are mean

values

SD from three independent experiments. Quantiﬁcation

analysis is shown. ***P ≤ 0.001 versus nontreated (basal) cultures.

Figure 6. SH-SY5Y cells were exposed to 6-OHDA [35 μM] during 16

h in the presence or absence of the PDE7A inhibitors at 10 μM. The

number of viable cell was measured by MTT assay. Values represent

the means SD from three diﬀerent experiments. ***p ≤ 0.001, *p ≤

0.05 versus 6-OHDA-treated cells.

Figure 4. Proposed binding mode for compound 28 (A) and 42 (B)

(PDB code 1ZKL) showing relevant interactions with nearby residues.

Distances are given in Å.

Anti-inﬂammatory Eﬀects of New Sulﬁde-Like PDE7A

Inhibitors. To further evaluate the role of PDE7A inhibitors in

inﬂammatory reactions of neuronal cells, we performed

experiments in diﬀerent cell-based assays. We used primary

cultures of astrocytes and microglia treated with lipopolysac-

charide (LPS), a potent inﬂammatory agent. The potential anti-

inﬂammatory activity of the selected PDE7A inhibitors was

tested by evaluating the production of nitrites from primary

human dopaminergic cells from death. Of note, derivative 28

increased cell survival with the same eﬃciency as two references

compounds: the standard PDE7 inhibitor BRL50481 and the

quinazoline-type PDE7 inhibitor 1, currently in pharmaceutical

development for PD.^12,20

a

b

Table 3. PDEs Proﬁling from Those Phenylsulﬁdes with an IC₅₀(PDE7A) ≤ 1.5μM (BRL50481 and Rolipram Are Included

c

As Standard References)

PDE7A IC₅₀

(μM)

PDE7B IC₅₀

(μM)

PDE4D

PDE4B

compd

PDE7B %inh @10 μM

PDE3A %inh @10 μM PDE4D %inh @10 μM IC₅₀(μM) PDE4B %inh @10 μM IC₅₀(μM)

10

12

20

28

31

41

42

44

1.47 0.17

1.01 0.23

0.99 0.19

0.37 0.06

1.50 0.13

1.29 0.17

0.18 0.08

0.72 0.06

42

71

92

82

74

39

94

76

2

1

2

1

22

16

60

37

46

44

27

33

2

4

2

1

4

3

2

1

2

1

2

6

1

2

3

2

4

1

2.20 0.23

1.41 0.07

3.00 0.18

1.50 0.07

44

7

30

4

13

24

65

74

28

35

69

77

0.6 0.02

13.9 1.6

7.3 1.3

3.0 0.4

6.6 1.2

4.4 0.7

a

b

BRL50481: IC₅₀(PDE7A) = 0.09 μM; 0.43% inh @10 μM (PDE3A); IC₅₀(PDE4B2) = 1.80 μM; IC₅₀(PDE4D3) = 1.28 μM. Rolipram: 3.10%

c

inh @10 μM (PDE7A); 9.42% inh @10 μM (PDE3A); IC₅₀(PDE4B2 ) = 97.9 nM; IC₅₀(PDE4D3) = 82.5 nM. Values represent the mean

standard deviation of two independent experiments (n = 2) with duplicate measurements.

G

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cultured glial cells (astrocytes and microglia). Cultures were

incubated with 10 μM of compounds 10, 12, 20, 28, 31, 41, 42,

and 44 for 1 h, and then cells were treated with LPS for a

further 24 h. When primary astrocytes and microglial cells were

stimulated with LPS (Figure 7), we observed a signiﬁcant

induction of nitrite production (approximately 5-fold) in the

culture medium, which was signiﬁcantly decreased after the

treatment with most of the selected PDE7A inhibitors.

correlation between the experimental and described perme-

ability values in the literature (Pe (exp) = 1.8979 (lit) − 1.667

(R²= 0.9506)) was obtained (see Figure S1 in Supporting

Information). Following the pattern established in the literature

for BBB permeation prediction²³that classify compounds as

CNS+ when they present a permeability >5.92 × 10⁻⁶cm s⁻¹,

we can consider that all the tested sulﬁde-like PDE7A inhibitors

(10, 12, 20, 28, 41, 42, and 44) are able to cross the BBB by

passive permeation, and thus they may be used as drug

candidates for CNS diseases (Table S2 in Supporting

Information).

On the basis of the above-reported results, we chose sulﬁde

28 for further in vivo studies because of its potency and

selective PDE proﬁle and its signiﬁcant eﬃcacy in both cellular

models and its ability to penetrate the brain.

Emetogenic Study of PDE7 Inhibitors. The results found

in cell cultures for the sulﬁde-like PDE7A inhibitors conﬁrm the

biological relevance of PDE7 as a valid target for PD.

Considering the great potential therapeutic use of this class

of compounds, it is necessary to pharmaceutically assess their

safety.

Until now, the severe gastrointestinal side eﬀects found in

the human clinical development of many diﬀerent PDE4

inhibitors have ruled out the compounds from entering the

market. Only roﬂumilast has been approved for the treatment

of EPOC, both by the FDA and EMA, with a narrow

therapeutic window. Emesis is a mechanism-based toxicity

associated with PDE4D inhibition in humans,²⁴which is not

easily reproduced in preclinical models. The most common and

expensive model is the use of ferrets. However, recently, some

surrogate models have been developed to determine human

emetogenic eﬀects in early development stages. In fact, it was

previously demonstrated that assessing the anesthesia-reversing

eﬀects of PDE4 inhibitors is a novel surrogate to evaluate the

emetic potential of these compounds in animal species that do

not have a vomiting reﬂex, e.g., mice.¹⁹In the presence of

PDE4 inhibitors, the percentage of mice exhibiting loss of the

righting reﬂex was reduced and the duration of anesthesia was

shortened.¹⁵

Three diﬀerent compounds were used as respective controls:

(R)-rolipram (1 mg/kg, sc), roﬂumilast (1 mg/kg, sc), and

BRL50481 (5 mg/kg, sc). Rolipram and roﬂumilast are

reference inhibitors of PDE4, exhibiting emetic-related

eﬀects,^19,25whereas BRL50481²⁰is a known inhibitor of

PDE7 in which the emetic eﬀects have not been shown. The

doses of each reference compound were selected in preliminary

experiments performed in our laboratory (detailed in the

Experimental Section). The return of the righting reﬂex (when

the mouse that was on its back turned itself spontaneously to a

prone position) was used as an end point to measure the

duration of anesthesia.

In our experimental conditions, the combined administration

of xylacine (10 mg/kg) and ketamine (100 mg/kg) induced

loss of the righting reﬂex within 15 min following injection.

Individual group comparisons showed that the administration

of rolipram (1 mg/kg) and roﬂumilast (1 mg/kg) induced a

decrease in the duration of anesthesia (both p < 0.05), but the

administration of the PDE7 inhibitor BRL50481 (5 mg/kg),

using a 5-fold greater dose, did not induce any eﬀect. Moreover,

the sulﬁde-like PDE7A inhibitor 28 at the doses evaluated (2.5,

5, and 10 mg/kg) was not able to modify the duration of

anesthesia induced by the combination of ketamine and

xylacine (Figure 8), supporting the absence of emetic-related

Figure 7. Eﬀect of PDE7 inhibitors on the inﬂammatory response of

astrocyte cultures (A) and microglial cells (B). Rat primary astrocyte

or microglial cultures were treated for 24 h with LPS (10 μg/mL) in

the absence or presence of PDE7 inhibitors (10 μM) and the

production of nitrite was evaluated by the Griess reaction. Values

represent the means SD from three diﬀerent experiments. ***p ≤

0.001, **p ≤ 0.01 versus LPS-treated cells.

Prediction of Blood−Brain Barrier Permeation. The

development of a new drug candidate for a neurological disease

is based not only on a good activity on the selected target but

also on the drug-like properties of the candidate. To target the

CNS system, the assessment of their ability to penetrate into

the brain is of utmost importance.

To determine whether these compounds have a suitable drug

proﬁle to be administered in vivo, we determined their ability to

cross the blood−brain barrier (BBB). The majority of

compounds enter the brain by transcellular passive diﬀusion,

which is driven by a concentration gradient between the blood

and the brain.²¹The parallel artiﬁcial membrane permeability

assay (PAMPA) is a high-throughput technique developed to

predict passive permeability through biological membranes.

Here, we used the PAMPA-BBB method described by Di et

al.²²that employs a brain lipid porcine membrane to determine

the ability of new inhibitors to penetrate into the brain. The in

vitro permeability (Pe) of commercial drugs through lipid

membrane extract together with the new PDE7A inhibitors was

determined (Table S2 in Supporting Information). A good

H

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eﬀects in PDE7 inhibitors in general and in the sulﬁde

compound 28 in particular.

none of them induced any eﬀect in the animals assayed,

conﬁrming the lack of emetogenic activity after the use of

cAMP-speciﬁc PDE7 inhibitors (Figures 8 and 9).

CONCLUSIONS

■

We corroborated the great potential of PDE7A inhibitors as a

new therapeutic option for neurodegenerative diseases,

speciﬁcally Parkinson’s disease. Moreover, we have shown

that PDE7 inhibitors may be a real therapeutic option for

human pharmacological treatments, as they do not have the

emetogenic activity found in clinical trials of PDE4 inhibitors.

PDE7 inhibitors are promising drug candidates for pathologies

in which cAMP signaling is involved such as the inﬂammatory

process of the central nervous system.

Here, we have shown that chemical genetic approaches

provide innovative hits that can be ﬁne-tuned by medicinal

chemistry programs to obtain valuable drug candidates. We

have shown here that sulﬁde-like PDE7A inhibitors are eﬀective

compounds in cellular models for Parkinson’s disease, they are

brain-penetrating drugs and these PDE7A inhibitors have no

emetic eﬀects in a surrogate animal model. Overall, PDE7A

inhibitors represent good drug candidates for CNS diseases,

especially Parkinson’s disease, as they are free of potential

emetic side eﬀects.

Figure 8. Eﬀects of compound 28 at diﬀerent concentrations (2.5, 5,

and 10 mg/kg) on the duration of anesthesia induced by the

combination of xylazine (10 mg/kg) and ketamine (100 mg/kg) in

CD1 mice. F(6, 107) = 4.617, p < 0.01. *p < 0.05 versus vehicle. The

evaluated compounds were injected subcutaneously (sc).

To conﬁrm the lack of emetic eﬀects when PDE7 is

inhibited, we evaluated the anesthesia-reversing behavior of

diﬀerent groups of mice exposed to the presence of three

chemically diverse PDE7 inhibitors previously synthesized in

our group. Currently, there are two approaches for studying the

function of a speciﬁc protein: classical genetics (using

antibodies, mutations, siRNAs, etc.) or chemical genetics

(using small chemical probes that target this protein). This is

the approach used here to show the lack of emetogenic activity

when PDE7 is down-regulated. Thus, a quinazoline derivative

(3-phenyl-2,4-dithioxo-1,2,3,4-tetrahydroquinazoline (TC3.6),

IC₅₀= 1.04 μM),²⁶a furan derivative (3,4,5-trimethoxybenzyl

5-phenyl-2-furoate (MR1.51), IC₅₀= 5.17 μM),²⁷and an

iminothiadiazole (5-(2-hydroxyethylimino)-2,3-diphenyl-2,5-di-

hydro-1,2,4-thiadiazole hydrobromide (VP1.15), IC₅₀= 1.11

μM)²⁸were used in this surrogate in vivo model. As expected,

EXPERIMENTAL SECTION

■

Chemical Procedures. Substrates were purchased from commer-

cial sources and used without further puriﬁcation. Melting points were

determined with a Mettler Toledo MP70 apparatus. Flash column

chromatography was carried out at medium pressure using silica gel

(E. Merck, grade 60, particle size 0.040−0.063 mm, 230−240 mesh

ASTM) with the indicated solvent as eluent. Compounds were

detected with UV light (254 nm). ¹H NMR spectra were obtained on

the Bruker AVANCE-300 spectrometer working at 300 MHz or on a

Varian INOVA 400 spectrometer working at 400 MHz. Typical

spectral parameters: spectral width 10 ppm, pulse width 9 μs (57°),

data size 32 K. ¹³C NMR experiments were carried out on the Bruker

AVANCE-300 spectrometer operating at 75 MHz or on a Varian

INOVA 400 spectrometer working at 100 MHz. The acquisition

parameters: spectral width 16 kHz, acquisition time 0.99 s, pulse width

Figure 9. Eﬀects of chemically diverse PDE7 inhibitors on the duration of anesthesia induced by the combination of xylazine (10 mg/kg) and

ketamine (80 mg/kg) in CD1 mice. (A) Compound tested: TC3.6, F(5, 45) = 12.003, p < 0.01. *p < 0.05 versus vehicle. (B) Compound tested

MR1.51, F(5, 58) = 9.776, p < 0.001. *p < 0.05 versus vehicle. (C) Compound tested: VP1.15, F(5, 56) = 5.508, p < 0.01. **p < 0.01 versus vehicle.

Rolipram and roﬂumilast, both PDE4 inhibitors, have been used as positive controls. The evaluated compounds were injected subcutaneously (sc).

I

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9 μs (57°), data size 32 K. Chemical shifts are reported in values

(ppm) relative to internal Me₄Si, and J values are reported in Hz.

HPLC analyses were performed on Alliance Waters 2690 equipment,

with a UV detector photodiode array Waters 2996 with MS detector

MicromassZQ (Waters), using an Sunﬁre column C18, 3.5 μm (50

mm × 4.6 mm) and acetonitrile and Milli-Q water (with 0.1% formic

acid) as mobile phase. The standard gradient consisted of a 5 min run

from 15% to 95% of acetonitrile at a ﬂow rate of 1 mL/min. Elemental

analysis results of all the new compounds were recorded on Heraeus

CHN-O rapid analyzer performed by the analytical department at

CENQUIOR (CSIC) and values were within 0.4% of the theoretical

values for all compounds; therefore, these compounds meet the

criteria of ≥95%. Additionally, purity of all ﬁnal compounds was found

to be ≥95% by LC/MS analysis. The microwave assisted syntheses

were carried out using a Biotage Initiator 8 single-mode cavity

instrument from Biotage. Experiments were performed with temper-

ature control mode in sealed microwave process vials. The

temperature was measured with an IR sensor on the outside of the

reaction vessel. Stirring was provided by an in situ magnetic stirrer.

General Synthetic Procedure for Compounds 3−45 and 55−

58. A solution of aryl thiol and aryl halide in the corresponding solvent

was prepared. The base was added, and the mixture was heated under

microwave irradiation during the time and temperature indicated in

each case. After cooling down, the solvent was evaporated and the

crude was washed with a saturated NaCl solution. The organic phase

was dried over magnesium sulfate and chromatographed on silica gel

column using as eluents mixtures of solvents in the portions indicated.

2-(4,5-Dichloro-2-nitrophenylthio)aniline (3). Reagents: 2-amino-

thiophenol (1.2 mmol, 1 equiv, 150.0 mg), 1,2-dichloro-4-ﬂuoro-5-

nitrobenzene (1.4 mmol, 1.2 equiv, 300.4 mg), and potassium

carbonate (6.0 mmol, 5 equiv, 826.7 mg). Solvent: anhydrous

dichloromethane. Microwave irradiation (80 °C, 1 h 30 min).

Puriﬁcation: ethyl acetate:hexane (1:4). Yield: orange solid (196.0

mg, 52%); mp 158−159 °C. ¹H NMR (300 MHz, DMSO-d₆): δ 8.51

(s, 1H), 7.35 (dd, J = 7.7, 1.6 Hz, 1H), 7.29 (ddd, J = 8.1, 7.2, 1.6 Hz,

1H), 6.84 (dd, J = 8.1, 1.2 Hz, 1H), 6.74 (s, 1H), 6.65 (ddd, J = 7.7,

7.2, 1.2 Hz, 1H), 5.65 (s, 2H). ¹³C NMR (75 MHz, DMSO-d₆): δ

151.6, 144.8, 136.7, 138.2, 138.1, 133.5, 128.5, 128.3, 128.2, 117.7,

116.1, 109.0. HPLC: purity 97%. MS (IE) (m/z): 315 [M], 317 [M +

2]. Anal. (C₁₂H₈Cl₂N₂O₂S) C, H, N, S.

2-[2-(Triﬂuoromethyl)-4-nitrophenylthio]aniline (4). Reagents: 2-

aminothiophenol (1.2 mmol, 1 equiv, 150.0 mg), 1-chloro-4-nitro-2-

(triﬂuoromethyl)benzene (1.4 mmol, 1.2 equiv, 324.3 mg), and

potassium carbonate (6.0 mmol, 5 equiv, 826.7 mg). Solvent:

anhydrous dichloromethane. Microwave irradiation (80 °C, 1h 30

min). Puriﬁcation: ethyl acetate:hexane (1:4). Yield: orange solid

(243.8 mg, 65%); mp 105−106 °C. ¹H NMR (300 MHz, DMSO-d₆):

δ 8.41 (d, J = 2.5 Hz, 1H), 8.33 (dd, J = 8.8, 2.5, Hz, 1H), 7.33 (dd, J =

7.4, 1.5 Hz, 1H), 7.30 (ddd, J = 8.0, 7.4, 1.5 Hz, 1H), 6.94 (dd, J = 8.8

Hz, 1H), 6.90 (dd, J = 8.0, 1.3 Hz, 1H), 6.65 (td, J = 7.4, 1.3 Hz, 1H),

5.57 (s, 2H). ¹³C NMR (75 MHz, DMSO-d₆): δ 152.0, 147.3, 145.2,

138.3, 133.6, 128.4, 128.1, 126.1 (q, J = 32.4 Hz), 123.8 (q, J = 274.3

Hz, CF₃), 123.0 (q, J = 5.9 Hz), 118.2, 116.5, 107.9. HPLC: purity

>99%. MS (ESI+) (m/z): 315 [M + 1]. Anal. (C₁₃H₉F₃N₂O₂S) C, H,

N, S.

2-(2-Chloro-4-nitrophenylthio)aniline (5). Reagents: 2-aminothio-

phenol (1.2 mmol, 1 equiv, 150.0 mg), 2-chloro-1-ﬂuoro-4-nitro-

benzene (1.4 mmol, 1.2 equiv, 252.4 mg), and potassium carbonate

(6.0 mmol, 5 equiv, 826.7 mg). Solvent: anhydrous dichloromethane.

Microwave irradiation (80 °C, 1h 30 min). Puriﬁcation: ethyl

acetate:hexane (1:4). Yield: orange solid (133.7 mg, 40%); mp 128−

129 °C. ¹H NMR (300 MHz, CDCl₃): δ 8.24 (d, J = 2.3 Hz, 1H), 7.90

(dd, J = 8.9, 2.3 Hz, 1H), 7.44 (ddd, J = 7.8, 1.6, 0.5 Hz, 1H), 7.36

(ddd, J = 8.2, 7.3, 1.6 Hz, 1H), 6.94−6.77 (m, 2H), 6.71 (d, J = 8.9 Hz,

1H). ¹³C NMR (75 MHz, CDCl₃): δ 149.7, 146.5, 145.7, 138.1, 133.1,

131.2, 126.1, 125.0, 122.4, 119.8, 116.2, 110.7. HPLC: purity 97%. MS

(ESI+) (m/z): 281 [M + 1], 283 [M + 3]. Anal. (C₁₂H₉ClN₂O₂S) C,

H, N, S.

(1.4 mmol, 1.2 equiv, 228.8 mg), and potassium carbonate (6.0 mmol,

5 equiv, 826.7 mg). Microwave irradiation (80 °C, 1 h 30 min).

Solvent: anhydrous dichloromethane. Microwave irradiation (80 °C, 1

h 30 min). Puriﬁcation: ethyl acetate:hexane (1:9). Yield: orange solid

(162.1 mg, 51%); mp 71−72 °C (lit.²⁹73−74 °C). H NMR (300

1

MHz, DMSO-d₆): δ 8.14 (dd, J = 8.8, 2.8 Hz, 1H), 7.53 (ddd, J = 9.1,

7.8, 2.9 Hz, 1H), 7.34 (dd, J = 7.7, 1.6 Hz, 1H), 7.25 (ddd, J = 8.2, 7.2,

1.6 Hz, 1H), 6.81 (ddd, J = 8.1, 1.4, 0.4 Hz, 1H), 6.76 (dd, J = 9.1, 5.3

Hz, 1H), 6.63 (ddd, J = 7.7, 7.2, 1.4 Hz, 1H), 5.56 (s, 2H). ¹³C NMR

(75 MHz, DMSO-d₆): δ 159.5 (d, J = 248.8 Hz), 148.6, 145.3 (d, J =

7.9 Hz), 137.9, 132.8, 132.5, 128.9 (d, J = 7.3 Hz), 121.7 (d, J = 21.9

Hz), 119.9, 116.2, 113.1 (d, J = 26.7 Hz), 112.7. HPLC: purity 97%.

MS (ESI+) (m/z): 265 [M +1]. Anal. (C₁₂H₉FN₂O₂S) C, H, N, S.

4-[2-(Triﬂuoromethyl)-4-nitrophenylthio]aniline (7). Reagents: 4-

aminothiophenol (1.2 mmol, 1 equiv, 150.0 mg), 1-chloro-4-nitro-2-

(triﬂuoromethyl)benzene (1.4 mmol, 1.2 equiv, 324.3 mg), and

potassium carbonate (6.0 mmol, 5 equiv, 826.7 mg). Solvent:

anhydrous dichloromethane. Microwave irradiation (100 °C, 45

min). Puriﬁcation: ethyl acetate:hexane (1:9). Yield: orange solid

1

(197.0 mg, 53%); mp 95−96 °C. H NMR (300 MHz, DMSO-d₆): δ

8.37 (d, J = 2.5 Hz, 1H), 8.30 (dd, J = 8.8, 2.5 Hz, 1H), 7.23 (d, J = 8.5

Hz, 2H), 7.01 (d, J = 8.8 Hz, 1H), 6.70 (d, J = 8.5 Hz, 2H), 5.83 (s,

2H). ¹³C NMR (75 MHz, DMSO-d₆): δ 150.8, 148.6, 144.0, 137.7,

127.7, 126.3 (q, J = 32.4 Hz), 122.7 (q, J = 274.6 Hz), 122.2 (q, J = 6.0

Hz), 116.4, 115.1. HPLC: purity 97%. MS (ESI+) (m/z): 315 [M +

1]. Anal. (C₁₃H₉F₃N₂O₂S) C, H, N, S.

4-(2-Chloro-4-nitrophenylthio)aniline (8). Reagents: 4-aminothio-

phenol (0.8 mmol, 1 equiv, 100.0 mg), 2-chloro-1-ﬂuoro-4-nitro-

benzene (1.0 mmol, 1.2 equiv, 168.3 mg), and potassium carbonate

(4.0 mmol, 5 equiv, 551.3 mg). Solvent: anhydrous dichloromethane.

Microwave irradiation (100 °C, 45 min). Puriﬁcation: ethyl

acetate:hexane (1:4). Yield: orange solid (202.0 mg, 45%); mp 138−

139 °C. ¹H NMR (300 MHz, DMSO-d₆): δ 8.26 (d, J = 2.6 Hz), 8.05

(dd, J = 8.9, 2.6 Hz, 1H), 7.22 (d, J = 8.5 Hz, 2H), 6.75 (d, J = 8.9 Hz,

1H), 6.70 (d, J = 8.5 Hz, 2H), 5.79 (s, 2H). ¹³C NMR (75 MHz,

DMSO-d₆): δ 151.0, 149.7, 144.24, 136.8, 127.9, 125.4, 123.8, 122.3,

115.0, 109.4. HPLC: purity >99%. MS (ESI+) (m/z): 281 [M + 1],

283 [M + 3]. Anal. (C₁₃H₉ClN₂O₂S) C, H, N, S.

4-(5-Chloro-2-nitrophenylthio)aniline (9). Reagents: 4-aminothio-

phenol (1.6 mmol, 1 equiv, 200.0 mg), 2,4-dichloro-1-nitrobenzene

(1.9 mmol, 1.2 equiv, 368.1 mg), and potassium carbonate (8.0 mmol,

5 equiv, 1.1 g). Solvent: anhydrous dichloromethane. Microwave

irradiation (100 °C, 45 min). Puriﬁcation: ethyl acetate:hexane (1:4).

Isomers mixture were obtained and separated by semipreparative

HPLC using as eluents methanol:acetonitrile 3:4. Yield: yellow solid

(34.1 mg, 77%); mp 113−114 °C (lit³⁰109−111 °C). ¹H NMR (400

MHz, DMSO-d₆) δ 8.22 (d, J = 8.8 Hz, 1H), 7.36 (dd, J = 8.8, 2.2 Hz,

1H), 7.21 (d, J = 8.5 Hz, 2H), 6.69 (d, J = 8.5 Hz, 2H), 6.66 (d, J = 2.2

Hz, 1H), 5.76 (s, 2H). ¹³C NMR (100 MHz, DMSO-d₆) δ 152.0,

144.2, 143.2, 140.2, 137.9, 128.5, 126.8, 125.9, 116.0, 112.1. HPLC:

purity >99%. MS (ESI+) (m/z): 281 [M + 1], 283 [M + 3]. Anal.

(C₁₃H₉ClN₂O₂S) C, H, N, S.

4-(3-Chloro-4-nitrophenylthio)aniline (10). Reagents: 4-amino-

thiophenol (1.6 mmol, 1 equiv, 200.0 mg), 2,4-dichloro-1-nitro-

benzene (1.9 mmol, 1.2 equiv, 368.1 mg), and potassium carbonate

(8.0 mmol, 5 equiv, 1103.0 mg). Solvent: anhydrous dichloromethane.

Microwave irradiation (100 °C, 45 min). Puriﬁcation: ethyl

acetate:hexane (1:4). Isomers mixture were obtained. These isomers

were separated by semipreparative HPLC using as eluents

methanol:acetonitrile 3:4. Yield: yellow solid (15.8 mg, 61%); mp

129−130 °C. ¹H NMR (400 MHz, DMSO-d₆): δ 7.94 (d, J = 8.7 Hz,

1H), 7.19 (d, J = 8.5 Hz, 2H), 7.12 (d, J = 2.0 Hz, 1H), 7.03 (dd, J =

8.7, 2.0 Hz, 1H), 6.65 (d, J = 8.5 Hz, 2H), 5.72 (s, 2H). ¹³C NMR

(100 MHz, DMSO-d₆): δ 151.8, 151.0, 144.0, 137.7, 127.5, 127.2,

126.9, 124.5, 115.8, 111.3. HPLC: purity >99%. MS (ESI+) (m/z):

281 [M + 1], 283 [M + 3]. Anal. (C₁₃H₉ClN₂O₂S) C, H, N, S.

4-Bromophenyl 5-Chloro-2-nitrophenyl sulﬁde (11). Reagents: 4-

bromothiophenol (1.1 mmol, 1 equiv, 200.0 mg), 2,4-dichoro-1-

nitrobenzene (1.3 mmol, 1.2 equiv, 243.7 mg), and potassium

carbonate (5.3 mmol, 5 equiv, 730.0 mg). Solvent: anhydrous

2-(4-Fluoro-2-nitrophenylthio)aniline (6). Reagents: 2-aminothio-

phenol (1.19 mmol, 1 equiv, 150.0 mg), 1,4-diﬂuoro-2-nitrobenzene

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dichloromethane. Microwave irradiation (80 °C, 1 h 30 min).

Puriﬁcation: ethyl acetate:hexane (1:4). Yield: yellow solid (114.4

d₆): δ 151.2, 145.6, 137.9, 134.8, 131.6, 130.4, 130.4, 130.1, 125.8,

122.4, 120.1, 116.4. HPLC: purity >99%. MS (ESI+) (m/z): 281 [M +

1], 283 [M + 3]. Anal. (C₁₂H₉ClN₂O₂S) C, H, N, S.

mg, 32%); mp 131−132 °C (lit³¹127.7−128.6 °C). H NMR (300

1

MHz, DMSO-d₆): δ 8.27 (d, J = 8.9 Hz, 1H), 7.78 (d, J = 8.5 Hz, 2H),

7.59 (d, J = 8.5 Hz, 2H), 7.49 (dd, J = 8.9, 2.2 Hz, 1H), 6.74 (d, J = 2.2

Hz, 1H). ¹³C NMR (75 MHz, DMSO-d₆): δ 143.5, 139.5, 139.4,

137.4, 133.6, 129.1, 127.9, 127.1, 126.3, 124.6. HPLC: purity 97%. MS

(IE) (m/z): 344 [M], 346 [M + 2]. Anal. (C₁₂H₇BrClNO₂S) C, H, N,

S.

2-Bromophenyl 5-Chloro-2-nitrophenyl Sulﬁde (12). Reagents: 2-

bromothiophenol (0.8 mmol, 1 equiv, 150.0 mg), 2,5-dichloroni-

trobenzene (1.0 mmol, 1.2 equiv, 182.8 mg), and potassium carbonate

(4.0 mmol, 5 equiv, 547.2 mg). Solvent: anhydrous dichloromethane.

Microwave irradiation (80 °C, 1 h 30 min). Puriﬁcation: ethyl

acetate:hexane (1:9). Yield: yellow solid (43.0 mg, 16%); mp 100−101

°C. ¹H NMR (300 MHz, DMSO-d₆): δ 8.32 (d, J = 8.9 Hz, 1H), 7.93

(dd, J = 7.6, 1.8 Hz, 1H), 7.85 (dd, J = 7.6, 1.9 Hz, 1H), 7.58 (dd, J =

7.6, 1.8 Hz, 1H), 7.55 (dd, J = 7.6, 1.9 Hz, 1H), 6.57 (d, J = 2.0 Hz,

1H). ¹³C NMR (75 MHz, DMSO-d₆): δ 143.5, 139.7, 138.2, 138.1,

134.5, 133.1, 130.4, 129.9, 128.2, 128.1, 126.5 (2C). HPLC: purity

97%. MS (EI) (m/z): 344 [M], 346 [M + 2]. Anal. (C₁₂H₇BrClNO₂S)

C, H, N, S.

2-Bromophenyl 2-Chloro-4-nitrophenyl sulﬁde (13). Reagents: 2-

bromothiophenol (0.8 mmol, 1 equiv, 150.0 mg), 3-chloro-4-

ﬂuoronitrobenzene (1.0 mmol, 1.2 equiv, 166.8 mg), and potassium

carbonate (4.0 mmol, 5 equiv, 547.2 mg). Solvent: anhydrous

dichloromethane. Microwave irradiation (80 °C, 1 h 30 min).

Puriﬁcation: ethyl acetate:hexane (1:9). Yield: yellow solid (120.0

mg, 40%); mp 91−92 °C. ¹H NMR (300 MHz, DMSO-d₆): δ 8.36 (d,

J = 2.5 Hz, 1H), 8.09 (dd, J = 8.8, 2.5 Hz, 1H), 7.93 (dd, J = 7.0, 2.3

Hz, 1H), 7.76 (dd, J = 7.0, 2.3 Hz, 1H), 7.61−7.49 (m, 2H), 6.79 (d, J

= 8.8 Hz, 1H). ¹³C NMR (75 MHz, DMSO-d₆): δ 145.6, 144.5, 137.4,

134.4, 132.7, 130.0, 129.7, 129.4, 129.3, 127.2, 124.6, 123.0. HPLC:

purity 97%. MS (IE) (m/z): 344 [M], 346 [M + 2]. Anal.

(C₁₂H₇BrClNO₂S) C, H, N, S.

2-(5-Chloro-2-nitrophenylthio)aniline (14). Reagents: 2-amino-

thiophenol (1.6 mmol, 1 equiv, 200.0 mg), 2,4-dichloro-1-nitro-

benzene (1.6 mmol, 1 equiv, 306.8 mg), and sodium acetate (2.4

mmol, 1 equiv, 232.5 mg). Solvent: ethanol. Microwave irradiation (85

°C, 2 h). Puriﬁcation: ethyl acetate:hexane (1:9). Yield: orange solid

(224.0 mg, 50%); mp 134−135 °C (lit.³²132.0−133.5 °C). ¹H NMR

(300 MHz, DMSO-d₆): δ 8.28 (d, J = 8.8 Hz, 1H), 7.42 (dd, J = 8.8,

2.2 Hz, 1H), 7.34 (dd, J = 7.5, 1.6 Hz, 1H), 7.28 (ddd, J = 8.2, 7.5, 1.6

Hz, 1H), 6.84 (dd, J = 8.2, 1.3 Hz, 1H), 6.67 (dd, J = 7.5, 1.3 Hz, 1H),

6.63 (d, J = 2.2 Hz, 1H), 5.63 (s, 2H). ¹³C NMR (75 MHz, DMSO-

d₆): δ 150.8, 143.7, 139.5, 139.2, 137.3, 132.5, 128.0, 125.6, 125.3,

116.9, 115.2, 108.6. HPLC: purity >99%. MS (ESI+) (m/z): 281 [M +

1], 283 [M + 3]. Anal. (C₁₂H₉ClN₂O₂S) C, H, N, S.

3-(2-Chloro-4-nitrophenylthio)aniline (17). Reagents: 3-amino-

thiophenol (1.6 mmol, 1 equiv, 200.0 mg), 3-chloro-4-ﬂuoronitro-

benzene (1.6 mmol, 1 equiv, 280.8 mg), and sodium acetate (2.4

mmol, 1.5 equiv, 232.8 mg). Solvent: ethanol. Microwave irradiation

(85 °C, 2 h). Puriﬁcation: ethyl acetate:hexane (1:4). Yield: orange

solid (344.3 mg, 77%); mp 123−124 °C. ¹H NMR (300 MHz,

DMSO-d₆): δ 8.29 (d, J = 2.4 Hz, 1H), 8.08 (dd, J = 8.9, 2.4 Hz, 1H),

7.20 (t, J = 7.7 Hz, 1H), 6.90 (d, J = 8.9 Hz, 1H), 6.76 (t, J = 1.9 Hz,

1H), 6.75−6.69 (m, 2H), 5.49 (s, 2H). ¹³C NMR (75 MHz, DMSO-

d₆): δ 150.6, 147.8, 144.9, 131.0, 129.0, 127.7, 126.5, 124.2, 122.8,

121.7, 119.5, 115.9. HPLC: purity >99%. MS (ESI+) (m/z): 281 [M +

1], 283 [M + 3]. Anal. (C₁₂H₉ClN₂O₂S) C, H, N, S.

4-(4-Fluoro-2-nitrophenylthio)aniline (18). Reagents: 4-amino-

thiophenol (2.0 mmol, 1 equiv, 250.0 mg), 1,4-diﬂuoro-2-nitrobenzene

(2.0 mmol, 1 equiv, 317.7 mg), and sodium acetate (1.2 mmol, 1.5

equiv, 115.4 mg). Solvent: ethanol. Microwave irradiation (85 °C, 1h

30 min). Puriﬁcation: ethyl acetate:hexane (1:4). Yield: orange solid

(283.6 mg, 54%); mp 124−125 °C. ¹H NMR (300 MHz, DMSO-d₆):

δ 8.11 (dd, J = 8.7, 2.9 Hz, 1H), 7.54 (ddd, J = 9.0, 7.8, 2.9 Hz, 1H),

7.22 (d, J = 8.5 Hz, 2H), 6.87 (dd, J = 9.0, 5.3 Hz, 1H), 6.69 (d, J = 8.5

Hz, 2H), 5.74 (s, 2H). ¹³C NMR (75 MHz, DMSO-d₆): δ 151.5, 141.8

(d, J = 376 Hz), 137.6, 136.9 (d, J = 11.7 Hz), 129.8, 129.7 (d, J = 7.8

Hz), 122.5 (d, J = 22.4 Hz), 115.6, 112.9, 112.5. HPLC: purity 97%.

MS (ESI+) (m/z): 265 [M + 1]. Anal. (C₁₂H₉FN₂O₂S) C, H, N, S.

2-Bromophenyl 4,5-Dichloro-2-nitrophenyl Sulﬁde (19). Re-

agents: 2-bromothiophenol (0.8 mmol, 1 equiv, 150.0 mg), 1,2-

dichloro-4-ﬂuoro-5-nitrobenzene (0.8 mmol, 1 equiv, 166.5 mg), and

sodium acetate (1.2 mmol, 1.5 equiv, 115.4 mg). Solvent: ethanol.

Microwave irradiation (85 °C, 1h 30 min). Puriﬁcation: ethyl

acetate:hexane (1:10). Yield: yellow solid (49.2 mg, 17%); mp 156−

157 °C. ¹H NMR (300 MHz, DMSO-d₆): δ 8.57 (s, 1H), 7.93 (dd, J =

7.2, 1.2 Hz, 1H), 7.83 (dd, J = 7.2, 1.6 Hz, 1H), 7.68−7.42 (m, 2H),

6.78 (s, 1H). ¹³C NMR (75 MHz, DMSO-d₆): δ 143.9, 137.8, 137.7,

136.0, 134.4, 133.0, 130.2, 129.8, 129.4, 128.8, 128.5, 127.8. HPLC:

purity 97%. MS (IE) (m/z): 379 [M], 381 [M + 2]. Anal.

(C₁₂H₆BrCl₂NO₂S) C, H, N, S.

2-Bromophenyl 4-chloro-2-nitrophenyl sulﬁde (20). Reagents: 2-

bromothiophenol (0.8 mmol, 1 equiv, 150.0 mg), 2,5-dichloroni-

trobenzene (0.8 mmol, 1 equiv, 152.3 mg), and sodium acetate (1.2

mmol, 1.5 equiv, 115.4 mg). Solvent: ethanol. Microwave irradiation

(85 °C, 1 h 30 min). Puriﬁcation: ethyl acetate:hexane (1:9). Yield:

1

yellow solid (199.4 mg, 70%); mp 85−86 °C. H NMR (300 MHz,

DMSO-d₆): δ 8.33 (d, J = 2.4 Hz, 1H), 7.89 (dd, J = 7.4, 1.8 Hz, 1H),

7.80 (dd, J = 7.4, 2.0 Hz, 1H), 7.70 (dd, J = 8.8, 2.4 Hz, 1H), 7.56 (dd,

J = 7.4, 1.8 Hz, 1H), 7.51 (dd, J = 7.4, 2.0 Hz, 1H), 6.78 (d, J = 8.8 Hz,

1H). ¹³C NMR (75 MHz, DMSO-d₆): δ 145.5, 137.7, 134.4, 134.3,

134.2, 132.6, 130.8, 130.6, 129.7, 129.6, 129.5, 125.5. HPLC: purity

97%. MS (IE) (m/z): 344 [M], 346 [M + 2]. Anal. (C₁₂H₇BrClNO₂S)

C, H, N, S.

2-(4-Chloro-2-nitrophenylthio)aniline (15). Reagents: 2-amino-

thiophenol (1.6 mmol, 1 equiv, 200.0 mg), 2,5-dichloronitrobenzene

(1.6 mmol, 1 equiv, 306.8 mg), and sodium acetate (2.4 mmol, 1.5

equiv, 232.5 mg). Solvent: ethanol. Microwave irradiation (85 °C, 2

h). Puriﬁcation: ethyl acetate:hexane (3:17). Yield: yellow solid (268.1

4-Bromophenyl 2-Chloro-4-nitrophenyl Sulﬁde (21). Reagents: 4-

bromothiophenol (1.1 mmol, 1 equiv, 200.0 mg), 3-chloro-4-

ﬂuoronitrobenzene (1.1 mmol, 1 equiv, 185.7 mg), and sodium

acetate (1.6 mmol, 1.5 equiv, 153.9 mg). Solvent: ethanol. Microwave

irradiation (85 °C, 1h 30 min). Puriﬁcation: ethyl acetate:hexane

(1:9). Yield: yellow solid (216.4 mg, 57%); mp 127−128 °C. ¹H NMR

(300 MHz, DMSO-d₆): δ 8.34 (d, J = 2.5 Hz, 1H), 8.06 (dd, J = 8.9,

2.5 Hz, 1H), 7.78 (d, J = 8.5 Hz, 2H), 7.58 (d, J = 8.5 Hz, 2H), 6.90

(d, J = 8.9 Hz, 1H). ¹³C NMR (75 MHz, DMSO-d₆): δ 146.1, 145.3,

137.1, 133.6, 129.6, 127.7, 127.2, 124.4, 124.4, 122.9. HPLC: purity

>99%. MS (IE) (m/z): 344 [M], 346 [M + 2]. Anal.

(C₁₂H₇BrClNO₂S) C, H, N, S.

4-Bromophenyl 4-Chloro-2-nitrophenyl Sulﬁde (22). Reagents: 4-

bromothiophenol (1.1 mmol, 1 equiv, 200.0 mg), 3-chloro-4-

ﬂuoronitrobenzene (1.1 mmol, 1 equiv, 203.1 mg), and sodium

acetate (1.6 mmol, 1.5 equiv, 153.9 mg). Solvent: ethanol. Microwave

irradiation (85 °C, 1h 30 min). Puriﬁcation: ethyl acetate:hexane

(1:9). Yield: yellow solid (310.0 mg, 85%); mp 155−156 °C (lit.³⁵155

mg, 60%); mp 121−122 °C (lit.³³122.0 °C). H NMR (300 MHz,

1

DMSO-d₆): δ 8.28 (d, J = 2.1 Hz, 1H), 7.65 (dd, J = 8.8, 2.1 Hz, 1H),

7.34 (dd, J = 7.7, 1.6 Hz, 1H), 7.25 (ddd, J = 8.2, 7.2, 1.6 Hz, 1H), 6.81

(dd, J = 8.2, 1.3 Hz, 1H), 6.74 (d, J = 8.8 Hz, 1H), 6.63 (ddd, J = 7.7,

7.2, 1.3 Hz, 1H), 5.57 (s, 2H). ¹³C NMR (75 MHz, DMSO-d₆): δ

150.8, 145.4, 137.3, 136.1, 133.8, 132.3, 129.3, 128.4, 125.4, 116.7,

115.1, 108.8. HPLC: purity >99%. MS (ESI+) (m/z): 281 [M + 1],

283 [M + 3]. Anal. (C₁₂H₉ClN₂O₂S) C, H, N, S.

3-(4-Chloro-2-nitrophenylthio)aniline (16). Reagents: 3-amino-

thiophenol (1.2 mmol, 1 equiv, 150.0 mg), 2,5-dichloronitrobenzene

(1.2 mmol, 1 equiv, 230.4 mg), and sodium acetate (1.8 mmol, 1.5

equiv, 174.6 mg). Solvent: ethanol. Microwave irradiation (85 °C, 2

h). Puriﬁcation: ethyl acetate:hexane (1:9). Yield: orange solid (124.8

mg, 32%); mp 96−97 °C (lit.³⁴94.0 °C). ¹H NMR (400 MHz,

DMSO-d₆): δ 8.24 (d, J = 2.4 Hz, 1H), 7.66 (dd, J = 8.8, 2.4 Hz, 1H),

7.15 (t, J = 7.8 Hz, 1H), 6.92 (d, J = 8.8 Hz, 1H), 6.72 (t, J = 2.0 Hz,

1H), 6.73−6.65 (m, 2H), 5.44 (s, 2H). ¹³C NMR (100 MHz, DMSO-

K

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Journal of Medicinal Chemistry

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°C). ¹H NMR (300 MHz, DMSO-d₆): δ 8.29 (d, J = 2.4 Hz, 1H), 7.74

(d, J = 8.7 Hz, 2H), 7.67 (dd, J = 8.8, 2.4 Hz, 1H), 7.55 (d, J = 8.7 Hz,

2H), 6.91 (d, J = 8.8 Hz, 1H). ¹³C NMR (75 MHz, DMSO-d₆): δ

145.9, 137.5, 135.9, 134.6, 133.8, 130.8, 130.6, 129.9, 125.7, 124.6.

HPLC: purity >99%. MS (IE) (m/z): 344 [M], 346 [M + 2]. Anal.

(C₁₂H₇BrClNO₂S) C, H, N, S.

2-Bromobenzyl 2-Chloro-4-nitrophenyl Sulﬁde (23). Reagents: 2-

bromobenzyl mercaptan (0.5 mmol, 1 equiv, 100.8 mg), 3-chloro-4-

ﬂuoronitrobenzene (0.5 mmol, 1 equiv, 87.1 mg), and sodium acetate

(0.7 mmol, 1.5 equiv, 72.2 mg). Solvent: ethanol. Microwave

irradiation (85 °C, 1h 30 min). Puriﬁcation: ethyl acetate:hexane

(1:9). Yield: yellow solid (57.0 mg, 32%); mp 107−108 °C. ¹H NMR

(300 MHz, DMSO-d₆): δ 8.30 (d, J = 2.4 Hz, 1H), 8.18 (dd, J = 8.8,

2.4 Hz, 1H), 7.73 (d, J = 8.8 Hz, 1H), 7.68 (dd, J = 7.9, 1.3 Hz, 1H),

7.61 (dd, J = 7.6, 1.7 Hz, 1H), 7.39 (td, J = 7.6, 1.3 Hz, 1H), 7.28 (td, J

= 7.9, 1.7 Hz, 1H), 4.52 (s, 2H). ¹³C NMR (75 MHz, DMSO-d₆): δ

146.3, 145.1, 134.6, 133.5, 131.9, 130.5, 130.2, 128.6, 126.8, 124.6,

124.4, 123.0, 36.6. HPLC: purity >99%. MS (IE) (m/z): 358 [M], 360

[M + 2]. Anal. (C₁₃H₉BrClNO₂S) C, H, N, S.

2-Bromobenzyl 4-Chloro-2-nitrophenyl Sulﬁde (24). Reagents: 2-

bromobenzyl mercaptan (0.5 mmol, 1 equiv, 100.8 mg), 2,5-

dichloronitrobenzene (0.5 mmol, 1 equiv, 95.2 mg), and sodium

acetate (0.7 mmol, 1.5 equiv, 72.2 mg). Solvent: ethanol. Microwave

irradiation (85 °C, 1h 30 min). Puriﬁcation: ethyl acetate:hexane

(1:10). Yield: yellow solid (80.0 mg, 45%); mp 157−158 °C. ¹H NMR

(300 MHz, DMSO-d₆): δ 8.27 (d, J = 2.3 Hz, 1H), 7.83 (dd, J = 8.7,

2.3 Hz, 1H), 7.76 (d, J = 8.7 Hz, 1H), 7.67 (dd, J = 7.7, 1.3 Hz, 1H),

7.56 (dd, J = 7.7, 1.7 Hz, 1H), 7.38 (td, J = 7.7, 1.3 Hz, 1H), 7.27 (td, J

= 7.7, 1.7 Hz, 1H), 4.44 (s, 2H). ¹³C NMR (75 MHz, DMSO-d₆): δ

146.0, 134.9, 134.4, 134.0, 133.0, 131.7, 130.0, 129.8, 129.6, 128.2,

125.4, 124.2, 37.2. HPLC: purity >99%. MS (IE) (m/z): 358 [M], 360

[M + 2]. Anal. (C₁₃H₉BrClNO₂S) C, H, N, S.

(IE) (m/z): 307 [M], 309 [M + 2]. Anal. (C₁₅H₁₄ClNO₂S) C, H, N,

S.

4-(4-Chloro-2-nitrophenylthio)aniline (28). Reagents: 4-amino-

thiophenol (1.2 mmol, 1 equiv, 150.0 mg), 2,5-dichloronitrobenzene

(1.2 mmol, 1 equiv, 230.1 mg), and cesium carbonate (1.8 mmol, 1.5

equiv, 585.5 mg). Solvent: acetonitrile. Microwave irradiation (130 °C,

8 min). Puriﬁcation: ethyl acetate:hexane (1:4). Yield: yellow solid

(241.5 mg, 58%); mp 130−131 °C (lit.³⁶127−129 °C). H NMR

1

(300 MHz, DMSO-d₆): δ 8.24 (d, J = 2.4 Hz, 1H), 7.65 (dd, J = 8.8,

2.4 Hz, 1H), 7.20 (d, J = 8.6 Hz, 2H), 6.83 (d, J = 8.8 Hz, 1H), 6.67

(d, J = 8.6 Hz, 2H), 5.74 (s, 2H). ¹³C NMR (75 MHz, DMSO-d₆): δ

151.6, 144.2, 139.9, 137.2, 134.0, 129.1, 129.0, 125.1, 115.2, 111.7.

HPLC: purity >99%. MS (ESI+) (m/z): 281 [M + 1], 283 [M + 3].

Anal. (C₁₂H₉ClN₂O₂S) C, H, N, S.

4-Chloro-2-nitrophenyl 4-nitrophenyl Sulﬁde (29). Reagents: 4-

nitrothiophenol (1.0 mmol, 1 equiv, 150.0 mg), 2,5-dichloronitro-

benzene (1.0 mmol, 1 equiv, 185.6 mg), and cesium carbonate (1.5

mmol, 1.5 equiv, 472.6 mg). Solvent: acetonitrile. Microwave

irradiation (130 °C, 8 min). Puriﬁcation: ethyl acetate:hexane (1:9).

Yield: yellow solid (147.6 mg, 49%); mp 134−135 °C (lit.³⁷135−137

°C). ¹H NMR (300 MHz, DMSO-d₆): δ 8.35 (d, J = 2.4 Hz, 1H), 8.29

(d, J = 8.8 Hz, 2H), 7.77 (d, J = 8.8 Hz, 2H), 7.74 (d, J = 8.7, 2.4 Hz,

1H) 7.25 (d, J = 8.7 Hz, 1H). ¹³C NMR (75 MHz, DMSO-d₆): δ

148.2, 147.9, 141.1, 134.6, 134.0, 133.2, 132.5, 131.7, 125.7, 125.2.

HPLC: purity >99%. MS (IE) (m/z): 309 [M], 311 [M + 2]. Anal.

(C₁₂H₇ClN₂O₄S) C, H, N, S.

2-Chloro-4-nitrophenyl 4-Nitrophenyl Sulﬁde (30). Reagents: 4-

nitrothiophenol (1.0 mmol, 1 equiv, 150.0 mg), 3-chloro-4-

ﬂuoronitrobenzene (1.0 mmol, 1 equiv, 169.7 mg), and cesium

carbonate (1.5 mmol, 1.5 equiv, 472.6 mg). Solvent: acetonitrile.

Microwave irradiation (130 °C, 8 min). Puriﬁcation: ethyl

acetate:hexane (1:9). Yield: yellow solid (234.0 mg, 78%); mp 146−

147 °C (lit.³⁸144 °C). ¹H NMR (300 MHz, DMSO-d₆): δ 8.44 (d, J =

2.4 Hz, 1H), 8.29 (d, J = 9.0 Hz, 2H), 8.13 (dd, J = 8.8, 2.4 Hz, 1H),

7.73 (d, J = 9.0 Hz, 2H), 7.36 (d, J = 8.8 Hz, 1H). ¹³C NMR (75 MHz,

DMSO-d₆): δ 147.5, 146.8, 142.2, 139.4, 133.1, 132.9, 131.2, 125.0,

124.8, 123.1. HPLC: purity >99%. MS (IE) (m/z): 309 [M], 311 [M

+ 2]. Anal. (C₁₂H₇ClN₂O₄S) C, H, N, S.

2-Chloro-4-nitrophenyl 2-isopropylphenyl Sulﬁde (25). Reagents:

2-isopropyl isopropylthiophenol (0.7 mmol, 1 equiv, 100.0 mg), 3-

chloro-4-ﬂuoronitrobenzene (0.7 mmol, 1 equiv, 115.3 mg), and

sodium acetate (1.0 mmol, 1.5 equiv, 93.1 mg). Solvent: ethanol.

Microwave irradiation (85 °C, 1h 30 min). Puriﬁcation: ethyl

1

acetate:hexane (1:40). Yield: brown oil (143.6 mg, 48%). H NMR

(300 MHz, DMSO-d₆): δ 8.34 (d, J = 2.5 Hz, 1H), 8.07 (dd, J = 8.9,

2.5 Hz, 1H), 7.66−7.61 (m, 2H), 7.59 (dd, J = 7.7, 1.0 Hz, 1H), 7.43−

7.39 (m, 1H), 6.64 (d, J = 8.9 Hz, 1H), 3.34 (m, 1H), 1.16 (d, J = 6.9

Hz, 6H). ¹³C NMR (75 MHz, DMSO-d₆): δ 153.2, 147.7, 145.3,

137.4, 132.2, 129.5, 128.4, 127.9, 126.5, 126.3, 124.7, 123.2, 31.1, 23.9.

HPLC: purity 97%. MS (IE) (m/z): 307 [M], 309 [M + 2]. Anal.

(C₁₅H₁₄ClNO₂S) C, H, N, S.

4-Chloro-2-nitrophenyl Phenyl Sulﬁde (31). Reagents: thiophenol

(1.8 mmol, 1 equiv, 200.0 mg), 2,5-dichloronitrobenzene (1.8 mmol, 1

equiv, 348.5 mg), and cesium carbonate (2.7 mmol, 1.5 equiv, 887.2

mg). Solvent: acetonitrile. Microwave irradiation (130 °C, 8 min).

Puriﬁcation: ethyl acetate:hexane (5:95). Yield: yellow solid (319.1

mg, 66%); mp 81−82 °C (lit.³⁹83−84 °C). H NMR (400 MHz,

1

DMSO-d₆): δ 8.27 (d, J = 2.4 Hz, 1H), 7.64 (dd, J = 8.8, 2.4 Hz, 1H),

7.62−7.57 (m, 2H), 7.57−7.51 (m, 3H), 6.82 (d, J = 8.8 Hz, 2H). ¹³C

NMR (100 MHz, DMSO-d₆): δ 145.6, 136.8, 135.7, 134.5, 130.9,

130.8, 130.5, 130.2, 130.1, 125.6. HPLC: purity >99%. MS (IE) (m/z):

265 [M], 267 [M + 2]. Anal. (C₁₂H₈ClNO₂S) C, H, N, S.

2-Chloro-4-nitrophenyl 4-isopropylphenyl Sulﬁde (26). Reagents:

4-isopropylthiophenol (0.7 mmol, 1 equiv, 100.0 mg), 3-chloro-4-

ﬂuoronitrobenzene (0.7 mmol, 1 equiv, 115.3 mg), and sodium acetate

(1.0 mmol, 1.5 equiv, 93.1 mg). Solvent: ethanol. Microwave

irradiation (85 °C, 1h 30 min). Puriﬁcation: ethyl acetate:hexane

2-Chloro-4-nitrophenyl phenyl Sulﬁde (32). Reagents: thiophenol

(1.4 mmol, 1 equiv, 150.0 mg), 3-chloro-4-ﬂuoronitrobenzene (1.4

mmol, 1 equiv, 238.7 mg), and cesium carbonate (2.0 mmol, 1.5 equiv,

665.4 mg). Solvent: acetonitrile. Microwave irradiation (130 °C, 8

min). Yield: gray solid (163.0 mg, 45%); mp 109−110 °C (lit.⁴⁰117−

1

(1:60). Yield: pale-yellow solid (57.4 mg, 30%); mp 71−72 °C. H

NMR (300 MHz, DMSO-d₆): δ 8.32 (d, J = 2.4 Hz, 1H), 8.07 (dd, J =

8.9, 2.4 Hz, 1H), 7.56 (d, J = 8.2 Hz, 2H), 7.46 (d, J = 8.2 Hz, 2H),

6.80 (d, J = 8.9 Hz, 1H), 2.98 (hep, J = 6.9 Hz, 1H), 1.24 (d, J = 6.9

Hz, 6H). ¹³C NMR (75 MHz, DMSO-d₆): δ 151.3, 147.5, 145.0,

135.5, 129.0, 128.7, 126.3, 124.6, 124.3, 122.9, 33.20, 23.54. HPLC:

purity 97%. MS (IE) (m/z): 307 [M], 309 [M + 2]. Anal.

(C₁₅H₁₄ClNO₂S) C, H, N, S.

1

118.5 °C). H NMR (300 MHz, DMSO-d₆): δ 8.35 (d, J = 2.4 Hz,

1H), 8.08 (dd, J = 8.9, 2.4 Hz, 1H), 7.71−7.56 (m, 5H), 6.84 (d, J =

8.9 Hz, 1H). ¹³C NMR (75 MHz, DMSO-d₆): δ 147.4, 145.5, 135.8,

131.1, 129.7, 128.3, 127.1, 124.7, 123.2. HPLC: purity >99%. MS (IE)

(m/z): 265 [M], 267 [M + 2]. Anal. (C₁₂H₈ClNO₂S) C, H, N, S.

4-(4-Chloro-2-nitrophenoxy)aniline (33). Reagents: 4-aminophe-

nol (1.6 mmol, 1.5 equiv, 170.5 mg), 2,5-dichloronitrobenzene (1.0

mmol, 1 equiv, 200.0 mg), and potassium carbonate (2.1 mmol, 2

equiv, 287.5 mg). Solvent: N,N-dimethylformamide. Microwave

irradiation (150 °C, 10 min). Puriﬁcation: ethyl acetate:hexane

(1:6). Yield: yellow solid (177.0 mg, 40%); mp 91−92 °C. ¹H

NMR (300 MHz, DMSO-d₆): δ 8.11 (d, J = 2.7 Hz, 1H), 7.64 (dd, J =

9.1, 2.7 Hz, 1H), 6.90 (d, J = 9.1 Hz, 1H), 6.83 (d, J = 8.8 Hz, 2H),

6.59 (d, J = 8.8 Hz, 2H), 5.15 (s, 2H). ¹³C NMR (75 MHz, DMSO-d₆)

δ 150.5, 146.6, 143.9, 140.1, 134.2, 125.3, 124.8, 120.9, 119.5, 114.8.

4-Chloro-2-nitrophenyl 4-isopropylphenyl Sulﬁde (27). Reagents:

4-isopropyl isopropylthiophenol (0.7 mmol, 1 equiv, 100.0 mg), 2,5-

dichloronitrobenzene (0.7 mmol, 1 equiv, 125.9 mg), and sodium

acetate (1.0 mmol, 1.5 equiv, 95.5 mg). Solvent: ethanol. Microwave

irradiation (85 °C, 1h 30 min). Puriﬁcation: ethyl acetate:hexane

1

(1:60). Yield: yellow solid (117.1 mg, 60%); mp 108−109 °C. H

NMR (300 MHz, DMSO-d₆): δ 8.29 (d, J = 2.4 Hz, 1H), 7.68 (dd, J =

8.8, 2.4 Hz, 1H), 7.53 (d, J = 8.2 Hz, 2H), 7.43 (d, J = 8.2 Hz, 2H),

6.84 (d, J = 8.8 Hz, 1H), 2.97 (hep, J = 6.9 Hz, 1H), 1.23 (d, J = 6.9

Hz, 1H). ¹³C NMR (75 MHz, DMSO-d₆): δ 151.0, 145.0, 136.9,

135.4, 134.1, 129.9, 128.5, 125.2, 33.2, 23.5. HPLC: purity 97%. MS

L

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HPLC: purity >99%. MS (ESI+) (m/z): 265 [M + 1], 267 [M + 3].

Anal. (C₁₂H₉ClN₂O₃) C, H, N.

7.7, 1.1 Hz, 1H), 7.63 (dd, J = 8.8, 2.3 Hz, 1H), 7.43 (ddd, J = 7.7, 4.8,

1.1 Hz, 1H), 7.38 (d, J = 2.3 Hz, 1H). ¹³C NMR (75 MHz, DMSO-

d₆): δ 154.3, 151.2, 146.9, 138.9, 138.8, 133.8, 131.6, 128.5, 127.7,

127.4, 123.9. HPLC: purity 97%. MS (ESI+) (m/z): 267 [M + 1], 269

[M + 3]. Anal. (C₁₁H₇ClN₂O₂S) C, H, N, S.

4-(2-Chloro-4-nitrophenoxy)aniline (34). Reagents: 4-aminophe-

nol (1.7 mmol, 1.5 equiv, 186.5 mg), 3-chloro-4-ﬂuoronitrobenzene

(1.1 mmol, 1 equiv, 200.0 mg), and potassium carbonate (2.3 mmol, 2

equiv, 314.6 mg). Solvent: N,N-dimethylformamide. Microwave

irradiation (150 °C, 10 min). Puriﬁcation: ethyl acetate:hexane

(1:4). Yield: yellow solid (163.3 mg, 54%); mp 92−93 °C. ¹H

NMR (300 MHz, DMSO-d₆): δ 8.38 (d, J = 2.3 Hz, 1H), 8.13 (dd, J =

8.8, 2.3 Hz, 1H), 6.87 (d, J = 8.8 Hz, 1H), 6.83 (d, J = 8.8 Hz, 1H),

6.63 (d, J = 8.8 Hz, 1H), 5.20 (s, 2H). ¹³C NMR (75 MHz, DMSO-

d₆): δ 160.3, 147.4, 143.7, 141.8, 126.3, 124.9, 122.3, 121.6, 115.9,

115.3. HPLC: purity >99%. MS (ESI+) (m/z): 265 [M + 1], 267 [M

+ 3]. Anal. (C₁₂H₉ClN₂O₃) C, H, N.

N-[4-(2-Chloro-4-nitrophenoxy)phenyl]acetamide (35). Reagents:

4′-hydroxyacetophenone (1.3 mmol, 1.5 equiv, 194.2 mg), 3-chloro-4-

ﬂuoronitrobenzene (0.9 mmol, 1 equiv, 150.0 mg), and potassium

carbonate (1.7 mmol, 2 equiv, 235.7 mg). Solvent: N,N-dimethylfor-

mamide. Microwave irradiation (150 °C, 10 min). Puriﬁcation: ethyl

acetate:hexane (1:3). Yield: yellow solid (202.4 mg, 78%); mp 158−

159 °C. ¹H NMR (300 MHz, DMSO-d₆): δ 10.08 (s, 1H), 8.44 (d, J =

2.7 Hz, 1H), 8.15 (dd, J = 9.2, 2.7 Hz, 1H), 7.68 (d, J = 9.0 Hz, 1H),

7.15 (d, J = 9.0 Hz, 1H), 6.95 (d, J = 9.2 Hz, 1H), 2.05 (s, 3H). ¹³C

NMR (75 MHz, DMSO-d₆): δ 168.2, 158.7, 148.9, 142.1, 137.1, 130.0,

126.0, 124.5, 120.7, 120.6, 116.8, 23.9. HPLC: purity >99%. MS (ESI

+) (m/z): 307 [M + 1], 309 [M + 3]. Anal. (C₁₄H₁₁ClN₂O₄) C, H, N.

N-[4-(4-Chloro-2-nitrophenoxy)phenyl]acetamide (36). Reagents:

4′-hydroxyacetophenone (1.2 mmol, 1.5 equiv, 177.2 mg), 2,5-

dichloronitrobenzene (0.8 mmol, 1 equiv, 150.0 mg), and potassium

carbonate (1.6 mmol, 2 equiv, 215.6 mg). Solvent: N,N-dimethylfor-

mamide. Microwave irradiation (150 °C, 10 min). Yield: white solid

(145.9 mg, 72%); mp 156−157 °C. ¹H NMR (300 MHz, DMSO-d₆):

δ 10.04 (s, 1H), 8.18 (d, J = 2.6 Hz, 1H), 7.70 (dd, J = 9.1, 2.7 Hz,

1H), 7.63 (d, J = 9.0 Hz, 1H), 7.07 (d, J = 9.1 Hz, 1H), 7.06 (d, J = 9.0

Hz, 1H), 2.03 (s, 3H). ¹³C NMR (75 MHz, DMSO-d₆): δ 168.2,

149.96, 148.9, 140.9, 136.4, 134.5, 126.7, 125.1, 121.3, 120.7, 119.6,

23.9. HPLC: purity >99%. MS (ESI+) (m/z): 307 [M + 1], 309 [M +

3]. Anal. (C₁₄H₁₁ClN₂O₄) C, H, N.

2-[(4-Chloro-2-nitrophenyl)thio]-pyridine (40). Reagents: 2-mer-

captopyridine (1.3 mmol, 1 equiv, 150.0 mg), 2,5-dichloronitroben-

zene (1.3 mmol, 1 equiv, 259.1 mg), and potassium carbonate (1.3

mmol, 1 equiv, 186.2 mg). Solvent: N,N-dimethylformamide.

Microwave irradiation (150 °C, 10 min). Yield: yellow solid (332.4

mg, 92%); mp 79−80 °C (lit.³⁵82 °C). ¹H NMR (300 MHz, DMSO-

d₆): δ 8.54−8.46 (m, 1H), 8.28 (d, J = 2.3 Hz, 1H), 7.83 (td, J = 7.7,

1.9 Hz, 1H), 7.75 (dd, J = 8.6, 2.3 Hz, 1H), 7.52 (dd, J = 7.9, 1.9 Hz,

1H), 7.46 (d, J = 8.6 Hz, 1H), 7.35 (ddd, J = 7.5, 4.8, 1.9 Hz, 1H). ¹³C

NMR (75 MHz, DMSO-d₆) δ 155.1, 150.9, 149.8, 138.6, 135.3, 133.9,

133.1, 128.9, 126.3, 125.4, 123.4. HPLC: purity 97%. MS (ESI+) (m/

z): 267 [M + 1], 269 [M + 3]. Anal. (C₁₁H₇ClN₂O₂S) C, H, N, S.

2-[(2-Chloro-4-nitrophenyl)thio]-pyridine (41). Reagents: 2-mer-

captopyridine (1.3 mmol, 1 equiv, 150.0 mg), 3-chloro-4-ﬂuoroni-

trobenzene (1.3 mmol, 1 equiv, 236.9 mg), and potassium carbonate

(1.3 mmol, 1 equiv, 186.2 mg). Solvent: N,N-dimethylformamide.

Microwave irradiation (150 °C, 10 min). Yield: brown solid (275.7

mg, 76%); mp 104−105 °C. ¹H NMR (300 MHz, DMSO-d₆): δ 8.54

(ddd, J = 4.8, 1.9, 0.9 Hz, 1H), 8.40 (d, J = 2.5 Hz, 1H), 8.14 (dd, J =

8.7, 2.5 Hz, 1H), 7.85 (td, J = 7.7, 1.9 Hz, 1H), 7.60 (d, J = 8.7 Hz,

1H), 7.52 (dt, J = 7.7, 1.0 Hz, 1H), 7.38 (ddd, J = 7.7, 4.8, 1.0 Hz, 1H).

¹³C NMR (75 MHz, DMSO-d₆): δ 153.8, 150.7, 146.9, 141.2, 138.4,

134.2, 132.9, 125.9, 124.6, 123.0, 122.6. HPLC: purity 97%. MS (ESI

+) (m/z): 267 [M + 1], 269 [M + 3]. Anal. (C₁₁H₇ClN₂O₂S) C, H, N,

S.

4-[(2-Chloro-4-nitrophenyl)thio]-pyridine (42). Reagents: 4-mer-

captopyridine (0.9 mmol, 1 equiv, 100.0 mg), 3-chloro-4-ﬂuoroni-

trobenzene (0.9 mmol, 1 equiv, 157.9 mg), and potassium carbonate

(0.9 mmol, 1 equiv, 124.1 mg). Solvent: N,N-dimethylformamide.

Microwave irradiation (150 °C, 10 min). Puriﬁcation: ethyl

acetate:hexane (3:7). Yield: pale-yellow solid (62.0 mg, 52%); mp

133−134 °C. ¹H NMR (300 MHz, DMSO-d₆): δ 8.58 (d, J = 6.2 Hz,

2H), 8.45 (d, J = 2.4 Hz, 1H), 8.17 (dd, J = 8.7, 2.4 Hz, 1H), 7.53 (d, J

= 8.7 Hz, 1H), 7.39 (d, J = 6.2 Hz, 2H). ¹³C NMR (75 MHz, DMSO-

d₆): δ 150.6, 147.2, 142.3, 140.3, 134.2, 132.6, 125.0, 124.9, 123.1.

HPLC: purity 97%. MS (ESI+) (m/z): 267 [M + 1], 269 [M + 3].

Anal. (C₁₁H₇ClN₂O₂S) C, H, N, S.

4-[(5-Chloro-2-nitrophenyl)thio]-pyridine (43). Reagents: 4-mer-

captopyridine (0.9 mmol, 1 equiv, 100.0 mg), 2,4-dichloronitroben-

zene (0.9 mmol, 1 equiv, 172.6 mg), and potassium carbonate (0.9

mmol, 1 equiv, 124.1 mg). Solvent: N,N-dimethylformamide.

Microwave irradiation (150 °C, 10 min). Puriﬁcation: ethyl

acetate:hexane (3:7). Yield: yellow solid (43.0 mg, 45%); mp 149−

150 °C. ¹H NMR (300 MHz, DMSO-d₆): δ 8.64 (d, J = 6.0 Hz, 1H),

8.26 (d, J = 8.9 Hz, 1H), 7.64 (dd, J = 8.9, 2.1 Hz, 1H), 7.52 (d, J = 6.0

Hz, 1H), 7.23 (d, J = 2.1 Hz, 1H). ¹³C NMR (75 MHz, DMSO-d₆): δ

150.9, 146.3, 142.5, 139.2, 133.8, 130.4, 128.3, 127.7, 126.6. HPLC:

purity 97%. MS (ESI+) (m/z): 267 [M + 1], 269 [M + 3]. Anal.

(C₁₁H₇ClN₂O₂S) C, H, N, S.

4-[(4-Chloro-2-nitrophenyl)thio]-pyridine (44). Reagents: 4-mer-

captopyridine (0.9 mmol, 1 equiv, 100.0 mg), 2,5-dichloronitroben-

zene (0.9 mmol, 1 equiv, 172.6 mg), and potassium carbonate (0.9

mmol, 1 equiv, 124.1 mg). Solvent: N,N-dimethylformamide.

Microwave irradiation (150 °C, 10 min). Puriﬁcation: ethyl

acetate:hexane (3:7). Yield: yellow solid (213.0 mg, 88%); mp 106−

107 °C. ¹H NMR (300 MHz, DMSO-d₆): δ 8.58 (d, J = 6.1 Hz, 2H),

8.33 (d, J = 1.9 Hz, 1H), 7.76 (dd, J = 8.7, 1.9 Hz, 1H), 7.43 (d, J = 6.1

Hz, 2H), 7.37 (d, J = 8.7 Hz, 1H). ¹³C NMR (75 MHz, DMSO-d₆): δ

150.5, 148.9, 143.3, 134.1, 133.9, 132.8, 128.9, 125.6, 125.2. HPLC:

purity 97%. MS (ESI+) (m/z): 267 [M + 1], 269 [M + 3]. Anal.

(C₁₁H₇ClN₂O₂S) C, H, N, S.

2-Chloro-4-nitrophenyl 2-Thienyl Sulﬁde (37). Reagents: 2-

thiophenethiol (0.6 mmol, 1 equiv, 75.0 mg), 3-chloro-4-ﬂuoroni-

trobenzene (0.6 mmol, 1 equiv, 113.5 mg), and potassium carbonate

(1.9 mmol, 3 equiv, 267.8 mg). Solvent: tetrahydrofuran. Microwave

irradiation (40 °C, 30 min). Puriﬁcation: ethyl acetate:hexane (1:40).

1

Yield: yellow solid (139.8 mg, 80%); mp 79−80 °C. H NMR (300

MHz, DMSO-d₆): δ 8.34 (d, J = 2.4 Hz, 1H), 8.13 (dd, J = 8.9, 2.4 Hz,

1H), 8.07 (dd, J = 5.4, 1.3 Hz, 1H), 7.61 (dd, J = 3.6, 1.3 Hz, 1H), 7.34

(dd, J = 5.4, 3.6 Hz, 1H), 6.86 (d, J = 8.9 Hz, 1H). ¹³C NMR (75

MHz, DMSO-d₆): δ 147.4, 145.8, 139.8, 135.9, 129.9, 129.0, 126.4,

124.7, 124.4, 123.5. HPLC: purity >99%. MS (IE) (m/z): 271 [M],

273 [M + 2]. Anal. (C₁₀H₆ClN₂O₂S₂) C, H, N, S.

4-Chloro-2-nitrophenyl 2-Thienyl Sulﬁde (38). Reagents: 2-

thiophenethiol (0.9 mmol, 1 equiv, 125.0 mg), 2,5-dichloronitroben-

zene (1.1 mmol, 1 equiv, 206.8 mg), and potassium carbonate (3.2

mmol, 3 equiv, 446.5 mg). Solvent: tetrahydrofuran. Microwave

irradiation (40 °C, 30 min). Puriﬁcation: ethyl acetate:hexane (1:40).

Yield: yellow solid (269.0 mg, 92%); mp 124−125 °C. ¹H NMR (300

MHz, DMSO-d₆): δ 8.32 (d, J = 2.4 Hz, 1H), 8.04 (dd, J = 5.3, 1.3 Hz,

1H), 7.76 (dd, J = 8.8, 2.4 Hz, 1H), 7.59 (dd, J = 3.5, 1.3 Hz, 1H), 7.33

(dd, J = 5.3, 3.5 Hz, 1H), 6.91 (d, J = 8.8 Hz, 1H). ¹³C NMR (75

MHz, DMSO-d₆): δ 144.9, 139.7, 137.6, 135.6, 134.9, 130.8, 129.8,

129.1, 126.8, 125.7. HPLC: purity 98%. MS (IE) (m/z): 271 [M], 273

[M + 2]. Anal. (C₁₀H₆ClN₂O₂S₂) C, H, N, S.

2-[(5-Chloro-2-nitrophenyl)thio]-pyridine (39). Reagents: 2-mer-

captopyridine (0.9 mmol, 1 equiv, 100.0 mg), 2,4-dichloronitroben-

zene (0.9 mmol, 1 equiv, 172.6 mg), and potassium carbonate (0.9

mmol, 1 equiv, 124.1 mg). Solvent: N,N-dimethylformamide.

Microwave irradiation (150 °C, 10 min). Puriﬁcation: ethyl

acetate:hexane (3:7). Yield: yellow solid (37.0 mg, 15%); mp 95−96

°C. ¹H NMR (100 MHz, DMSO-d₆): δ 8.59 (dd, J = 4.8, 1.9 Hz, 1H),

8.23 (d, J = 8.8 Hz, 1H), 7.89 (td, J = 7.7, 1.9 Hz, 1H), 7.64 (dd, J =

4-(4-Acethyl-2-nitrophenylthio)pyridine (45). Reagents: 4-mercap-

topyridine (1.3 mmol, 1 equiv, 150.0 mg), 4-bromo-3-nitroacetophe-

none (1.3 mmol, 1 equiv, 329.3 mg), and potassium carbonate (1.3

mmol, 1 equiv, 186.2 mg). Solvent: N,N-dimethylformamide.

M

dx.doi.org/10.1021/jm501090m | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

1

Microwave irradiation (150 °C, 10 min). Puriﬁcation: methanol:di-

chloromethane (1:20). Yield: orange solid (146.9 mg, 40%); mp 134−

135 °C. ¹H NMR (300 MHz, DMSO-d₆): δ 8.68 (d, J = 6.0 Hz, 2H),

8.65 (d, J = 2.0 Hz, 1H), 8.12 (dd, J = 8.5, 2.0 Hz, 1H), 7.58 (d, J = 6.0

Hz, 2H), 7.30 (d, J = 8.5 Hz, 1H). ¹³C NMR (75 MHz, DMSO-d₆): δ

195.7, 162.3, 151.0, 146.5, 141.5, 138.1, 135.2, 133.0, 130.7, 127.6,

125.2, 30.8. HPLC: purity 97%. MS (ESI+) (m/z): 275 [M + 1]. Anal.

(C₁₃H₁₀N₂O₃S) C, H, N, S.

Yield: orange solid (50.2 mg, 30%); mp 82−83 °C. H NMR (300

MHz, DMSO-d₆): δ 8.67 (d, J = 4.9 Hz, 2H), 8.61 (d, J = 6.1 Hz, 2H),

7.65 (d, J = 6.1 Hz, 2H), 7.34 (t, J = 4.9 Hz, 1H). ¹³C NMR (75 MHz,

DMSO-d₆): δ 168.8, 158.4, 150.0, 140.2, 127.5, 118.8. HPLC: purity

97%. MS (ESI+) (m/z): 190 [M + 1]. Anal. (C₉H₇N₃S) C, H, N, S.

2-[(1,1′-Biphenyl)-4-ylthio]-5-methyl-3-nitropyridine (52). Re-

agents: biphenyl-4-thiol (0.8 mmol, 1 equiv, 150.0 mg) and 2-

chloro-5-methyl-3-nitropyridine (0.8 mmol, 1 equiv, 138.9 mg).

Puriﬁcation: dichloromethane. Yield: yellow solid (89.3 mg, 55%);

4-(Phenylthio)pyridine (55). Reagents: thiophenol (1.8 mmol, 1

equiv, 200.0 mg), 4-bromopyridine hydrochloride (1.8 mmol, 1 equiv,

353.0 mg), and cesium carbonate (3.6 mmol, 2 equiv, 1.18 g). Solvent:

acetonitrile. Microwave irradiation (130 °C, 35 min). Puriﬁcation:

1

mp 133−134 °C. H NMR (500 MHz, DMSO-d₆): δ 8.49 (s, 1H),

8.48 (s, 1H), 7.74 (d, J = 8.5 Hz, 2H), 7.72 (dd, J = 7.4, 1.2 Hz, 2H),

7.60 (d, J = 8.5 Hz, 2H), 7.49 (t, J = 7.4 Hz, 2H), 7.40 (t, J = 7.4 Hz,

1H), 2.33 (s, 3H). ¹³C NMR (125 MHz, DMSO-d₆): δ 154.9, 153.2,

141.6, 141.5, 139.6 136.6, 134.5, 131.5, 129.5, 128.9, 128.4, 128.0,

127.3, 17.1. HPLC: purity 97%. MS (ESI+) (m/z): 323 [M + 1]. Anal.

(C₁₈H₁₄N₂O₂S) C, H, N, S.

1

ethyl acetate:hexane (1:2). Yield: pale-yellow oil (209.8 mg, 63%). H

NMR (300 MHz, DMSO-d₆): δ 8.35 (d, J = 6.2 Hz, 2H), 7.68−7.41

(m, 5H), 7.00 (d, J = 6.2 Hz, 2H). ¹³C NMR (75 MHz, DMSO-d₆): δ

149.6, 149.0, 134.9, 130.3, 129.9, 128.6, 120.5. HPLC: purity 97%. MS

(ESI+) (m/z): 188 [M + 1]. Anal. (C₁₁H₉NS) C, H, N, S.

2-(1,1′-Biphenyl)-4-ylthiopyridine (53). Reagents: biphenyl-4-thiol

(0.8 mmol, 1 equiv, 150.0 mg) and 2-bromopyridine (0.8 mmol, 1

equiv, 127.2 mg). Puriﬁcation: ethyl acetate:hexane (1:10). Yield: pale-

4-(4-Methoxyphenylthio)pyridine (56). Reagents: 4-methoxythio-

phenol (1.4 mmol, 1 equiv, 200.0 mg), 4-bromopyridine hydrochloride

(1.4 mmol, 1 equiv, 225.5 mg), and cesium carbonate (2.1 mmol, 2

equiv, 697.2 mg). Solvent: acetonitrile. Microwave irradiation (130 °C,

35 min). Puriﬁcation: ethyl acetate:hexane (1:9). Yield: pale-yellow

1

yellow solid (147.2 mg, 70%); mp 41−42 °C. H NMR (500 MHz,

DMSO-d₆): δ 8.40 (ddd, J = 4.8, 1.7, 0.8 Hz, 1H), 7.77 (d, J = 8.1 Hz,

2H), 7.71 (dd, J = 7.3, 1.8 Hz, 2H), 7.67 (m, 1H), 7.64 (d, J = 8.1 Hz,

2H), 7.48 (t, J = 7.8 Hz, 2H), 7.39 (t, J = 7.8 Hz, 1H), 7.15 (ddd, J =

7.4, 4.8, 0.8 Hz, 1H), 8.40 (dd, J = 7.4, 0.8 Hz, 1H). ¹³C NMR (125

MHz, DMSO-d₆): δ 160.1, 150.1, 141.3, 139.5, 137.9, 135.5, 129.8,

129.5, 128.4, 128.3, 127.2, 121.7, 121.1. HPLC: purity 97%. MS (ESI

+) (m/z): 264 [M + 1]. Anal. (C₁₇H₁₃NS) C, H, N, S.

1

solid (160.4 mg, 53%), mp 96−97 °C. H NMR (300 MHz, DMSO-

d₆): δ 8.32 (d, J = 6.4 Hz, 2H), 7.52 (d, J = 8.6 Hz, 2H), 7.09 (d, J =

8.6 Hz, 2H), 6.92 (d, J = 6.4 Hz, 2H). ¹³C NMR (75 MHz, DMSO-

d₆): δ 166.41, 156.04, 155.07, 155.03, 142.90, 142.86, 125.39, 123.94,

121.53, 121.49, 61.05. HPLC: purity 97%. MS (ESI+) (m/z): 218 [M

+ 1]. Anal. (C₁₂H₁₁NOS) C, H, N, S.

5-Methyl-3-nitro-2-(3-aminophenylthio)pyridine (54). Reagents:

3-aminothiophenol (1.2 mmol, 1 equiv, 150.0 mg) and 2-chloro-5-

methyl-3-nitropyridine (1.2 mmol, 1 equiv, 206.8 mg). Puriﬁcation:

methanol:dichloromethane (1:30). Yield: yellow solid (155.0 mg,

50%); mp 157−158 °C. ¹H NMR (300 MHz, DMSO-d₆): δ 8.31 (d, J

= 2.1 Hz, 1H), 8.21 (d, J = 2.1 Hz, 1H), 7.15 (t, J = 7.8 Hz, 1H), 6.86

(dt, J = 7.6, 1.4 Hz, 1H), 6.80 (m, 1H), 6.68 (ddd, J = 8.1, 2.3, 1.4 Hz,

1H), 3.66 (s, 2H), 2.29 (s, 3H). ¹³C NMR (75 MHz, DMSO-d₆): δ

154.6, 147.5, 133.9, 130.8, 130.3, 130.1, 126.2, 122.4, 116.7, 17.7.

HPLC: purity 98%. MS (ESI+) (m/z): 262 [M + 1]. Anal.

(C₁₂H₁₁N₃O₂S) C, H, N, S.

N-[4-(4-Chloro-2-nitrophenylthio)phenyl]acetamide (46). A

mixture of 100.0 mg of 4-(4-chloro-2-nitrophenylthio)aniline (28)

(0.4 mmol, 1 equiv), 0.025 mL of acethyl chloride (0.4 mmol, 1

equiv), and tetrahydrofuran (1 mL) was heated under microwave

irradiation (110 °C, 20 min). After cooling down, the solvent was

evaporated, and the crude was dissolved in ethyl acetate (100 mL) and

was washed with three aqueous solutions: HCl 0.1 M (100 mL),

saturated NaHCO₃(100 mL), and saturated NaCl (100 mL). The

organic phase was dried over magnesium sulfate and puriﬁed by silica

gel column chromatography, using ethyl acetate:hexane (1:5) as

4-(3-Aminophenylthio)pyridine (57). Reagents: 3-aminothiophenol

(1.6 mmol, 1 equiv, 200.0 mg), 4-bromopyridine hydrochloride (1.6

mmol, 1 equiv, 310.8 mg), and cesium carbonate (3.2 mmol, 2 equiv,

1040.0 mg). Solvent: acetonitrile. Microwave irradiation (130 °C, 35

min). Puriﬁcation: methanol:dichloromethane (1:9). Yield: yellow

1

solid (100.3 mg, 33%); mp 89−90 °C. H NMR (300 MHz, DMSO-

d₆): δ 8.34 (d, J = 6.3 Hz, 2H), 7.14 (t, J = 7.8 Hz, 1H), 7.02 (d, J = 6.3

Hz, 2H), 6.74 (t, J = 2.0 Hz, 1H), 6.69 (dd, J = 8.0, 1.5 Hz, 1H), 6.66

(dd, J = 7.3, 1.5 Hz, 1H), 2.78 (s, 2H). ¹³C NMR (75 MHz, DMSO-

d₆): δ 150.3, 149.8, 149.3, 130.5, 128.3, 121.4, 120.4, 119.2, 115.3.

HPLC: purity 97%. MS (ESI+) (m/z): 203 [M + 1]. Anal.

(C₁₁H₁₀N₂S) C, H, N, S.

3-(4-Methoxyphenylthio)pyridine (58). Reagents: 4-methoxythio-

phenol (1.4 mmol, 1 equiv, 200.0 mg), 3-bromopyridine (1.4 mmol, 1

equiv, 225.5 mg), and cesium carbonate (2.1 mmol, 1.5 equiv, 697.2

mg). Solvent: acetonitrile. Microwave irradiation (130 °C, 35 min).

Puriﬁcation: ethyl acetate:hexane (1:2). Yield: pale-yellow oil (180.5

1

mg, 58%). H NMR (300 MHz, DMSO-d₆): δ 8.35 (d, J = 2.4 Hz,

1H), 8.29 (dd, J = 4.8, 1.5 Hz, 1H), 7.36 (d, J = 8.8 Hz, 2H), 7.35 (m,

1H), 7.07 (dd, J = 8.0, 4.7 Hz, 1H), 6.84 (d, J = 8.8 Hz, 2H), 3.76 (s,

3H). ¹³C NMR (75 MHz, DMSO-d₆): δ 160.7, 149.3, 147.2, 136.4,

136.1, 135.8, 124.1, 123.0, 115.7, 55.8. HPLC: purity 97%. MS (ESI+)

(m/z): 218 [M + 1]. Anal. (C₁₂H₁₁NOS) C, H, N, S.

1

eluents. Yield: yellow solid (80.0 mg, 70%); mp 197−198 °C. H

NMR (300 MHz, DMSO-d₆): δ 10.24 (s, 1H), 8.28 (d, J = 2.4 Hz,

1H), 7.75 (d, J = 8.6 Hz, 1H), 7.66 (dd, J = 8.8, 2.4 Hz, 1H), 7.54 (d, J

= 8.6 Hz, 1H), 6.80 (d, J = 8.8 Hz, 1H), 2.08 (s, 3H). ¹³C NMR (75

MHz, DMSO-d₆): δ 169.2, 145.7, 145.0, 141.6, 136.9, 134.5, 130.1,

129.8, 125.6, 122.6, 120.8, 24.5. HPLC: purity >99%. MS (ESI+) (m/

z): 323 [M + 1], 325 [M + 3]. Anal. (C₁₄H₁₁ClN₂O₃S) C, H, N, S.

N-[4-(2-Chloro-4-nitrophenylthio)phenyl]acetamide (47). A

mixture of 100.0 mg of 4-(2-chloro-4-nitrophenylthio)aniline (8) (0.4

mmol, 1 equiv), 0.025 mL of acethyl chloride (0.4 mmol, 1 equiv), and

tetrahydrofuran (1 mL) were heated under microwave irradiation (110

°C, 20 min). After cooling down, the solvent was evaporated, and the

crude was dissolved in ethyl acetate (100 mL) and was washed with

three aquous solutions: HCl 0.1 M (100 mL), saturated NaHCO₃

(100 mL), and saturated NaCl (100 mL). The organic phase was dried

over magnesium sulfate, and the product was obtained as a yellow

solid (80.5 mg, 70%); mp 166−167 °C. ¹H NMR (300 MHz, DMSO-

d₆): δ 10.27 (s, NH, 1H), 8.31 (d, J = 2.6 Hz, 1H), 8.05 (dd, J = 8.9,

2.6 Hz, 1H), 7.79 (d, J = 8.7 Hz, 2H), 7.56 (d, J = 8.7 Hz, 2H), 6.75

(d, J = 8.9 Hz, 1H), 2.08 (s, CH₃, 3H). ¹³C NMR (75 MHz, DMSO-

d₆): δ 169.2, 148.4, 145.3, 142.0, 136.9, 129.1, 126.5, 124.6, 123.2,

General Synthetic Procedure for Compounds 50−54. A

mixture of aryl thiol, corresponding 2-halidearylpyridine, and water

was heated under microwave irradiation (100 °C, 30 min). After

cooling down, ethyl acetate was added (100 mL) and the crude was

washed with a saturated NaCl solution (100 mL). The organic phase

was dried over magnesium sulfate and chromatographed on silica gel

column using as eluents mixtures of solvents in the portions indicated.

5-Methyl-3-nitro-2-(4-pyridinylthio)pyridine (50). Reagents: 4-

mercaptopyridine (1.4 mmol, 1 equiv, 150.0 mg) and 2-chloro-5-

methyl-3-nitropyridine (1.4 mmol, 1 equiv, 234.9 mg). Puriﬁcation:

ethyl acetate:hexane (1:4). Yield: orange solid (255.2 mg, 76%); mp

128−129 °C. ¹H NMR (300 MHz, DMSO-d₆): δ 8.61 (d, J = 6.1 Hz,

2H), 8.52 (m, 2H), 7.54 (d, J = 6.1 Hz, 2H), 2.36 (s, 3H). ¹³C NMR

(75 MHz, DMSO-d₆): δ 154.4, 150.2, 150.0, 141.8, 140.5, 134.2,

132.0, 128.8, 16.7. HPLC: purity 97%. MS (ESI+) (m/z): 248 [M +

1]. Anal. (C₁₁H₉N₃O₂S) C, H, N, S.

2-(4-Pyridinylthio)pyrimidine (51). Reagents: 4-mercaptopyridine

(0.9 mmol, 1 equiv, 100.0 mg) and 2-chloropyrimidine (0.9 mmol, 1

equiv, 103.9 mg). Puriﬁcation: methanol:dichloromethane (1:20).

N

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120.9, 120.7, 24.5. HPLC: purity >99%. MS (ESI+) (m/z): 323 [M +

1], 325 [M + 3]. Anal. (C₁₄H₁₁ClN₂O₃S) C, H, N, S.

window, they were screened by using IMAP ﬂuorescence polarization

(FP) assay (Molecular Devices R8175).

First, 0.05U of PDE4B2 (Calbiochem no. 524736), 0.05 units of

PDE4D3 (Calbiochem no. 524733), 0.15 units of PDE5 (Calbiochem

no. 524738), 2 units of PDE1 (Abcam @ab125661), and 0.05 units of

PDE9 (Abcam no. ab54113) were incubated in a 96-well black half

area plate with 1 nM of either ﬂuorescein adenosine 3′,5′-cyclic

phosphate or ﬂuorescein guanosine 3′,5′-cyclic phosphate (PDE5)

contained in the kit and inhibitors in 40 μL of assay buﬀer. Then plates

were mixed on a shaker for 10 s and incubated at ambient temperature

for 60 min. IMAP binding reagent was added (60 μL of a 1 in 600

dilution in binding buﬀer of the kit stock solution) to terminate the

assay. Plates were allowed to stand at ambient temperature for 1 h.

The FP ratio was measured with a Tecan Ultra Evolution reader.

IC₅₀values were calculated by nonlinear regression ﬁtting using

GraphPad Prism. Data (ﬂuorescence polarization vs log concentration)

was ﬁtted to a sigmoidal dose−response equation: Y = Bottom + (Top

2-(4-Aminophenylthio)-5-chloroaniline (48). A mixture of 4-

(4-chloro-2-nitrophenylthio)aniline (28) (0.4 mmol, 1 equiv, 100.0

mg), tin chloride(II) dihydrate (2.0 mmol, 5.5 equiv, 442.1 mg), and 6

mL of ethanol were heated under microwave irradiation (110 °C, 5

min). After cooling down, the solvent was evaporated, and the crude

was dissolved in ethyl acetate (100 mL) and was washed with with

saturated NaHCO₃(100 mL) and saturated NaCl (100 mL) solutions.

The organic phase was dried over magnesium sulfate and puriﬁed by

silica gel column chromatography using ethyl acetate:hexane (1:3) as

1

eluents. Yield: dark-blue oil (70.0 mg, 78%). H NMR (300 MHz,

DMSO-d₆): δ 7.01 (d, J = 8.3 Hz, 1H), 7.00 (d, J = 8.5 Hz, 1H), 6.68

(d, J = 2.3 Hz, 1H), 6.48 (d, J = 8.5 Hz, 1H), 6.47 (d, J = 8.3, 2.3 Hz,

1H), 5.47 (s, 2H), 5.26 (s, 2H). ¹³C NMR (75 MHz, DMSO-d₆): δ

149.6, 149.1, 135.0, 133.4, 133.3, 117.9, 117.7, 116.3, 115.0, 113.7.

HPLC: purity >99%. MS (ESI+) (m/z): 251 [M + 1], 253 [M + 3].

Anal. (C₁₂H₁₁ClN₂S) C, H, N, S.

− Bottom)/(1 + 10^((logIC^−X)×n)), where Bottom and Top were the

50

minimum and maximal inhibition for PDE, respectively, IC₅₀was the

concentration of compound that inhibited the PDE activity in a 50%,

and n was the slope of the concentration−response curve.

4-(4-Aminophenylthio)-3-chloroaniline (49). A mixture of 4-

(2-chloro-4-nitrophenylthio)aniline (8) (0.4 mmol, 1 equiv, 100.0

mg), tin chloride(II) dihydrate (2.0 mmol, 5.5 equiv, 442.1 mg), and

ethanol (6 mL) were heated under microwave irradiation (110 °C, 5

min). After cooling down, the solvent was evaporated, and the crude

was dissolved in ethyl acetate (100 mL) and was washed with

saturated NaHCO₃(100 mL) and saturated NaCl (100 mL) solutions.

The organic phase was dried over magnesium sulfate and puriﬁed by

silica gel column chromatography using ethyl acetate:hexane (1:3) as

Docking Studies. To carry out the docking studies, the PDE7A

enzyme employed was based on the crystal structure reported in the

Protein Data Bank (PDB code 1ZKL). IBMX, a well-known inhibitor

of PDE, whose crystal structure in complex with the enzyme is

reported in the Protein Data Bank (PDB code 1ZKL), was docked as

reference compound. Protein was prepared for docking using Protein

Prepared Wizard (Schrodinger Inc.). Water molecules coordinated

with both magnesium and zinc atoms were conserved for the docking

development. Hydrogens were incorporated at neutral pH, and the

whole structure was minimized using force ﬁeld OPLS2005.

1

eluents. Yield: dark-yellow oil (102.3 mg, 76%). H NMR (300 MHz,

DMSO-d₆): δ 7.00 (d, J = 8.5 Hz, 1H), 6.82 (d, J = 8.4 Hz, 1H), 6.63

(d, J = 2.2 Hz, 1H), 6.52 (d, J = 8.5 Hz, 1H), 6.42 (dd, J = 8.4, 2.2 Hz,

1H), 5.45 (s, 2H), 5.32 (s, 2H). ¹³C NMR (75 MHz, DMSO-d₆): δ

149.7, 149.2, 134.7, 134.0, 133.4, 120.4, 118.5, 115.0, 114.5, 113.9.

HPLC: purity >99%. MS (ESI+) (m/z): 251 [M + 1], 253 [M + 3].

Anal. (C₁₂H₁₁ClN₂S) C, H, N, S.

Radiometric Phosphodiesterase Inhibition Assay. The

methodology used for measuring human recombinant PDE7A1,

PDE7B, PDE8A, PDE10A2, and PDE3A activity was based in a

scintillation proximity assay (SPA) from PerkinElmer (TRKQ7090).

The activity of the phosphodiesterase was measured by coincubating

the enzyme with [³H]cAMP, and the hydrolysis of the nucleotide was

quantiﬁed by radioactivity measurement after binding of [³H]AMP to

scintillation binding bead.

Ligands were prepared using Ligprep (Schrodinger Inc.). The

ligands were minimized using OPLS2005 force ﬁeld, and all the

possible states at target pH 5−9 were generated. The lowest energy

conformer for every molecule were docked in the PDE7A protein

(PDB code1ZKL) by using the automatic docking program Glide

(Schrodinger Inc.). Glide was used to generate a grid based on the

centroid of select residues in the binding pocket (Phe384, Gln413, and

Phe416). The box size was set to the value of 20 × 20 × 20 Å³. This

box size encompasses the entire PDE7A binding pocket both in width

and depth. Extra precision (XP) mode and ﬂexible docking with ring

sampling were selected for the docking setup. After docking, the

receptor−ligand complexes were minimized using the OPLS2005 all

atom force ﬁeld.

SH-SY5Y Cell Culture. The human neuroblastoma SH-SY5Y cell

line (Sigma-Aldrich) was cultured in F12 medium/EMEM containing

2 mM glutamine, 1% nonessential amino acids, and 15% fetal bovine

serum (FBS) under standard culture conditions (37 °C and 5% CO₂).

On attaining semiconﬂuence, cells were pretreated for 1 h with the

diﬀerent compounds at several concentrations (30 μM for BRL50481

and 10 μM for the rest of tested compounds). After that, 6-OHDA (35

μM, Sigma) was added to the cultures and incubated for 16 h. Finally,

cultures were processed for cell viability assay. Some cultures were

treated for 16 h only with the compounds 28 and 42 for Western blot

analysis.

Primary Cell Cultures. Glial cells were prepared from neonatal rat

cerebral cortex, as previously described by Luna-Medina et al.⁴¹Brieﬂy,

after removal of the meninges, the cerebral cortex was dissected,

dissociated, and incubated with 0.25% trypsin/EDTA at 37 °C for 1 h.

After centrifugation, the pellet was washed 3 times with HBSS (Gibco)

and the cells were plated on noncoated ﬂasks and maintained in

HAMS/DMEM (1:1) medium containing 10% FBS. After 15 days, the

ﬂasks were agitated on an orbital shaker for 4 h at 240 rpm at 37 °C,

the supernatant was collected, centrifuged, and the cellular pellet

containing the microglial cells resuspended in complete medium

(HAMS/DMEM (1:1) containing 10% FBS) and seeded on uncoated

96-well plates. Cells were allowed to adhere for 2 h, and the medium

was removed to eliminate nonadherent oligodendrocytes. New fresh

medium containing 10 ng/mL of GM-CSF was added. The remaining

astroglial cells adhered on the ﬂasks were then trypsinized, collected,

Either 0.02 units of PDE7A1 (Calbiochem no. 524751), 0.02 units

of PDE3A (Calbiochem no. 524742), 0.02 units of PDE10A

(Calbiochem no. 524739), 0.5 units of PDE7B (Abcam no.

ab79800), or 0.5 units of PDE8A (Abcam no. ab125540) were

incubated in a 96-well ﬂexiplate with 5 nCi of [³H]cAMP and

inhibitors in 100 μL of assay buﬀer (contained in the kit) for 20 min at

30 °C. After the incubation time, 50 μL of a solution of SPA-beads

(approximately 1 mg per well) were added to each well, and the plate

was shaken for 1 h at room temperature. Finally, beads were settled for

30 min and radioactivity was detected in a Microbeta Trilux reader.

IC₅₀values were calculated by nonlinear regression ﬁtting using

GraphPad Prism. Data (radioactivity vs log concentration) was ﬁtted

to a sigmoidal dose−response equation: Y = Bottom + (Top-

Bottom)/(1 + 10^((logIC^−X)×n)), where Bottom and Top were the

50

minimum and maximal inhibition for PDE, respectively, IC₅₀was the

concentration of compound that inhibited the PDE activity in a 50%,

and n was the slope of the concentration−response curve.

The mode of inhibitory action for compound 28 and 42 was

determined by varying the concentration of unlabeled cAMP in the

reaction cocktail within the range of 5 nM to 2 μM in the presence of a

ﬁxed concentration of [³H]-cAMP tracer and inhibitor concentrations.

Enzyme activity data were analyzed using Lineweaver−Burk plots

using GraphPad Prism.

Fluorescence Polarization Phosphodiesterase Inhibition

Assay. PDE4B2, PDE4D3, PDE1A, PDE5, and PDE9 phosphodies-

terases showed a little signal/background ratio when they were

screened by using the radiometric assay. To increase this signal

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centrifuged, and plated onto 96-well plates with complete medium.

The purity of cultures obtained by this procedure was >98% as

determined by immunoﬂuorescence with the OX42 (microglial

marker) and the GFAP (astroglial marker) antibodies. After 2 days

in culture, cells were pretreated for 2 h with BRL50481 (30 μM), and

the diﬀerent compounds at 10 μM were also treated with

lipopolysaccharide (LPS; 10 μg/mL) for 24 h. Nitrite production on

cultures was then measured.

Cell Viability Assay. Cell viability was measured using the MTT

assay from Roche, based on the ability of viable cells to reduce yellow

MTT to blue formazan. Cells cultured for 16 h were incubated with

MTT (0.5 mg/mL, 4h) and subsequently solubilized in 10% SDS/0.01

M HCl for 1 h in the dark. The extent of reduction of MTT was

quantiﬁed by absorbance measurement at 595 nm according to the

manufacturer’s protocol.

Western Blot Analysis. Proteins were isolated from cell cultures

following standard methods. Brieﬂy, cells were resuspended in ice-cold

cell lysis buﬀer (Cell Signaling Technology) with protease inhibitor

cocktail (Roche) and incubated for 15−30 min on ice. A total amount

of 30 μg of protein was loaded on a 12% SDS-PAGE gel, and after

electrophoresis, proteins were transferred to nitrocellulose membranes

(Protran, Whatman) and blots probed with primary antibodies against

p-CREB (rabbit; Cell Signaling) and CREB (rabbit; Cell Signaling).

Secondary peroxidase-conjugated donkey antirabbit (Amersham

Biosciences, GE Healthcare) antibody was used. Values in the ﬁgure

are the average of the quantiﬁcation of at least three independent

experiments corresponding to three diﬀerent samples.

Nitrites Measurement. Accumulation of nitrites in media was

assayed by the standard Griess reaction. After stimulation of cells with

LPS during 24 h, supernatants were collected and mixed with an equal

volume of Griess reagent (Sigma). Samples were then incubated at

room temperature for 15 min and absorbance read using a plate reader

at 492/540 nm.

CNS Penetration: In Vitro Parallel Artiﬁcial Membrane

Permeability Assay (PAMPA) Blood−Brain Barrier (BBB).

Prediction of the brain penetration was evaluated using a parallel

artiﬁcial membrane permeability assay (PAMPA).²²Ten commercial

drugs, phosphate buﬀer saline solution at pH 7.4 (PBS), ethanol, and

dodecane were purchased from Sigma, Acros Organics, Merck,

Aldrich, and Fluka. The porcine polar brain lipid (PBL) (catalogue

no. 141101) was from Avanti Polar Lipids. The donor plate was a 96-

well ﬁltrate plate (Multiscreen IP sterile plate PDVF membrane, pore

size is 0.45 μM, catalogue no. MAIPS4510), and the acceptor plate was

an indented 96-well plate (Multiscreen, catalogue no. MAMCS9610)

both from Millipore. Filter PDVF membrane units (diameter 30 mm,

pore size 0.45 μm) from Symta were used to ﬁltered the samples. A

96-well plate UV reader (Thermoscientiﬁc, Multiskan spectrum) was

used for the UV measurements. Test compounds [(3−5 mg of

caﬀeine, enoxacine, hydrocortisone, desipramine, oﬂoxacine, pirox-

icam, and testosterone), (12 mg of promazine), and 25 mg of

verapamil and atenolol] were dissolved in EtOH (1000 μL). Then 100

μL of this compound stock solution was taken and 1400 μL of EtOH

and 3500 μL of PBS pH 7.4 buﬀer were added to reach 30% of EtOH

concentration in the experiment. These solutions were ﬁltered. The

acceptor 96-well microplate was ﬁlled with 180 μL of PBS/EtOH (70/

30). The donor 96-well plate was coated with 4 μL of porcine brain

lipid in dodecane (20 mg mL⁻¹), and after 5 min, 180 μL of each

compound solution was added. Then 1−2 mg of every compound to

have their ability to pass the brain barrier determined were dissolved in

1500 μL of EtOH and 3500 μL of PBS pH 7.4 buﬀer, ﬁltered, and then

added to the donor 96-well plate. Then the donor plate was carefully

put on the acceptor plate to form a “sandwich”, which was left

undisturbed for 2 h and 30 min at 25 °C. During this time, the

compounds diﬀused from the donor plate through the brain lipid

membrane into the acceptor plate. After incubation, the donor plate

was removed. A UV plate reader determined the concentration of

compounds and commercial drugs in the acceptor and the donor wells.

Every sample was analyzed at 3−5 wavelengths, in three wells, and in

two independent runs. Results are given as the mean [standard

deviation (SD)], and the average of the two runs were reported. Ten

quality control compounds (previously mentioned) of known BBB

permeability were included in each experiment to validate the analysis

set.

Emesis Surrogate Model. In vivo experiments were performed

using CD1 male mice (25−30 g) purchased in Charles Rivers

(France). Mice were hosed in a temperature (21

1 °C) and

humidity (60 20%) controlled environment and grouped four per

cage. Food and water were available ad libitum. A period of 1 week to

habituation to colony facilities was allowed prior to experiments.

Procedures were conducted according to standard ethical guidelines

(European Communities Council Directive 86/609 EEC) and

approved by the local ethical committee (CEEA−PRBB). All in vivo

experiments were performed in the colony facilities of the University

Pompeu Fabra (Ubiomex−PRBB). To select the doses of BRL50481

(and rolipram and roﬂumilast) for the in vivo study, a preliminary set

of experiments was performed on a surrogate model for emesis

potential in mice (data not shown). In the case of BRL50481, the

doses assayed were 0.1, 1, 5, and 10 mg/kg (sc) in mice. Any of the

doses used reversed the anesthetic eﬀect of the mixture of ketamine/

xylacine in mice. Because only one dose of BRL50481 was shown in

the manuscript, we selected for that, the dose of 5 mg/kg that is high

enough to discard an emetic eﬀect of this compound.

The selected compound 28 shows an inhibitory eﬀect on PDE7 of

81% at a concentration of 10 μM while producing a light eﬀect on

PDE4 (4% of inhibition). Considering this residual eﬀect on PDE4

activity, we selected a range of relative high doses of the compound 28

(2.5, 5, and 10 mg/kg) in order to completely discard a possible

emetic eﬀect and, in consequence, this undesirable side eﬀect of the

compound 28.

Experiments were performed by following procedures previously

reported¹⁹and adapted to our experimental conditions. Brieﬂy, mice

were anesthetized with the mixture solution of xylacine (10 mg/kg)

and ketamine (100 mg/kg) administered by intraperitoneal (ip) route.

Fifteen minutes later, the mice were injected subcutaneously (sc) with

the evaluated compound, sulﬁde 28, at three diﬀerent doses (2.5, 5,

and 10 mg/kg) or the vehicle, polyethylene glycol (PEG). All

compounds were freshly dissolved in 60% (v/v) PEG (in saline) and

administered in a volume of injection of 0.1 mL/10 g body weight.

Three diﬀerent compounds were used as respective controls: (R),

rolipram (1 mg/kg, sc); roﬂumilast (1 mg/kg, sc); and BRL50481 (5

mg/kg, sc). Rolipram and roﬂumilast were purchased in Sigma

Chemical Co (Madrid, Spain). BRL50481 was purchased in Tocris.

Rolipram and roﬂumilast are reference inhibitors of PDE4, exhibiting

emetic-related eﬀects, whereas BRL50481 is an inhibitor of PDE7

showing no emetic-related eﬀects. The return of the righting reﬂex

(when the mouse backed and turned itself spontaneously to prone

position) was used as an end point to measure the duration of

anesthesia. The duration of anesthesia is expressed as the mean

SEM. Statistically signiﬁcant diﬀerences among groups were analyzed

using ANOVA, and a subsequent posthoc analysis was applied when

required (Dunnett test). The level of signiﬁcance in all the studies was

p < 0.05.

In our experimental conditions, the combined administration of

xylacine (10 mg/kg) and ketamine (100 mg/kg) induced loss of

righting reﬂex within 15 min following injection. The overall duration

of anesthesia measured was 80 7.1 min (n = 10). One-way ANOVA

for the duration of the anesthesia calculated for the diﬀerent

compounds evaluated exhibited signiﬁcant eﬀects (F(6, 107) =

4.617; p < 0.01; n = 10−19 per group).

Three additional PDE7 inhibitors were evaluated in order to

support data obtained with the compound 28: the quinazoline

derivative TC3.6, the furan derivative MR1.51, and the iminothiadia-

zole VP1.15. In vivo experiments for evaluating these compounds were

performed in a diﬀerent set of experiments and conducted as above-

described for the compound 28. The only diﬀerence in the

methodology was that mice were anesthetized with the mixture

solution of xylacine (10 mg/kg) and ketamine (80 mg/kg). In this

conditions, the overall duration of anesthesia measured was 120 16.1

min (n = 10).

P

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For the in vivo experiments, data were analyzed using One-Way

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ASSOCIATED CONTENT

* Supporting Information

■

S

Elemental analyses of all synthesized compounds, Linear

correlation between experimental and reported permeability

of commercial drugs using the PAMPA-BBB assay, and

permeability eﬀective (Pe 10⁻⁶cm·s⁻¹) in the PAMPA-BBB

assay for 10 commercial drugs, used in the experiment

validation, and diﬀerent sulﬁde-like PDE7 inhibitors with

their predictive penetration in the CNS. This material is

available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION

Corresponding Authors

*For C.G.: phone, +34 91 8373112; fax, +34 91 5360432, E-

mail, carmen.gil@csic.es.

■

*For A.M.: phone, +34 91 8373112; fax, +34 91 5360432; E-

mail, ana.martinez@csic.es.

Notes

The authors declare no competing ﬁnancial interest.

ACKNOWLEDGMENTS

■

Financial support from MINECO and FEDER founds (UE

program) (project nos. IPT-2012-0762-300000 and SAF2012-

33600) and MICINN (SAF2010-15793 to O.V. and SAF2010-

16365 to A.P.-C.) are acknowledged. A.M.G. and D. I. P.

acknowledge a predoctoral and postdoctoral grants respectively

to the CSIC (JAE program). CIBERNED is funded by the

Instituto de Salud Carlos III. J.A.M-G. is a fellow from

CIBERNED. BRAINco Biopharma SL reviewed and funded the

pro-emetic studies of rolipram, roﬂumilast, BRL50481, VP1.15,

and TC3.6 compounds

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Modulation of cAMP-specific PDE without emetogenic activity: New sulfide-like PDE7 inhibitors

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