Sulfur and selenium derivatives of quinazoline and pyrido[2,3-d]pyrimidine: Synthesis and study of their potential cytotoxic activity in vitro

DOI: 10.1016/j.ejmech.2011.10.056

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European Journal of Medicinal Chemistry p. 283 - 298 (2012)

Update date:2022-08-02

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Authors:

Moreno, Esther

Plano, Daniel

Lamberto, Iranzu

Font, María

Encío, Ignacio

Palop, Juan Antonio

Sanmartín, Carmen

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Article abstract of DOI:10.1016/j.ejmech.2011.10.056

The synthesis, cytotoxic activities and selectivities of 35 derivatives related to quinazoline and pyrido[2,3-d]pyrimidine are described. The synthesized compounds were screened in vitro against four tumoral cell lines - leukemia (CCRF-CEM), colon (HT-29), lung (HTB-54) and breast (MCF-7) - and two cell lines derived from non-malignant cell lines, one mammary (184B5) and one from bronchial epithelium (BEAS-2B). MCF-7 and HTB-54 were the most sensitive cell lines with GI₅₀ values below 10 μM for eleven and ten compounds, respectively. Two compounds (2o and 3a) were identified that evoked a marked cytotoxic effect in all cell lines tested and one compound, 7h, was potent and selective against MCF-7. A preliminary study into the mechanism of the potent derivatives 2o, 3a and 7h indicated that the cytotoxic activities of these compounds might be mediated by inducing cell death without affecting cell cycle phases.

Full text of DOI:10.1016/j.ejmech.2011.10.056

European Journal of Medicinal Chemistry 47 (2012) 283e298

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Original article

Sulfur and selenium derivatives of quinazoline and pyrido[2,3-d]pyrimidine:

Synthesis and study of their potential cytotoxic activity in vitro

Esther Moreno ^a, Daniel Plano ^a, Iranzu Lamberto ^a, María Font ^b, Ignacio Encío ^c, Juan Antonio Palop ^a

Carmen Sanmartín ^a

^aSección de síntesis, Departamento de Química Orgánica y Farmacéutica, University of Navarra, Irunlarrea, 1, E-31008 Pamplona, Spain

^bSección de modelización molecular, Departamento de Química Orgánica y Farmacéutica, University of Navarra, Irunlarrea, 1, E-31008 Pamplona, Spain

^cDepartamento de Ciencias de la Salud, Universidad Pública de Navarra, Avda. Barañain s/n, E-31008 Pamplona, Spain

a r t i c l e i n f o

a b s t r a c t

Article history:

Received 5 July 2011

Received in revised form

25 October 2011

Accepted 28 October 2011

Available online 6 November 2011

The synthesis, cytotoxic activities and selectivities of 35 derivatives related to quinazoline and pyrido

[2,3-d]pyrimidine are described. The synthesized compounds were screened in vitro against four tumoral

cell lines e leukemia (CCRF-CEM), colon (HT-29), lung (HTB-54) and breast (MCF-7) e and two cell lines

derived from non-malignant cell lines, one mammary (184B5) and one from bronchial epithelium (BEAS-

2B). MCF-7 and HTB-54 were the most sensitive cell lines with GI₅₀values below 10 mM for eleven and

ten compounds, respectively. Two compounds (2o and 3a) were identiﬁed that evoked a marked cyto-

toxic effect in all cell lines tested and one compound, 7h, was potent and selective against MCF-7. A

preliminary study into the mechanism of the potent derivatives 2o, 3a and 7h indicated that the cyto-

toxic activities of these compounds might be mediated by inducing cell death without affecting cell cycle

phases.

Keywords:

Cytotoxics

Antiproliferatives

Quinazolines

Pyridopyrimidines

1. Introduction

compounds are remarkable dihydrofolate reductase [18] and

tyrosine kinase [19e21] inhibitors such as geﬁtinib, lapatinib and

The disease of cancer has been ranked as a major health burden.

At present, a wide range of cytotoxic drugs with different mecha-

nisms of action are used to treat human cancer, either alone or in

combination. Numerous compounds are also in different phases of

clinical trials. The main drawback of these cytotoxic drugs is that

they do not discriminate between cancerous and normal cell types

and are accompanied by toxic side effects that are often cumulative

and dose limiting [1].

Growth factor signaling pathways have been a main focus of

research for novel targeted anticancer agents because of their

fundamental role in regulating key cellular functions, including

cell-proliferation, differentiation, metastasis and survival [2].

Quinazoline derivatives have attracted attention due to their

broad range of pharmacological activities, which include, among

others, antifungal [3], antimalarial [4], anti-inﬂammatory [5],

anticonvulsant [6], antibacterial [7] and antihypertensive [8], and

their anticancer activity [9e17] is one of the most promising

aspects as they act through multiple targets. Several of these

pazopanib. Some quinazolines interact with tubulin [22] and

interfere with its polymerization. Others act by modulating aurora

kinase activity [23] or have an effect in critical phases in the cell

cycle [11,24] or act as apoptosis inducers [16,17,25e27] (Fig. 1).

There are also numerous examples in the literature of

compounds with very diverse biological proﬁles shown by the

pyrido[2,3-d]pyrimidine nucleus. The family based on pyrido[2,3-d]

pyrimidines has been developed and these compounds show great

speciﬁcity for individual subgroups of receptor tyrosine kinases

[28,29]. Other members of this family inhibit non-receptor tyrosine

kinases such as Abl [30,31], Akt [32] or cyclin kinases [33]. Another

target for these derivatives is dihydrofolate reductase inhibition,

e.g. piritrexim [34], as well as cell cycle modulation [35] or as yet

undetermined mechanisms of action [36] (Fig. 1).

Based on the observations outlined above, these nuclei have

emerged as versatile templates for a diverse range of mechanisms

of anticancer activity and, considering our experience with these

heteroaromatic rings [26,37e41], we describe here the discovery of

a novel series of 2,4-disubstituted quinazoline and pyrido[2,3-d]

pyrimidine derivatives.

The different chemical groups selected for attachment to the

central nucleus in the new structures are based on the diverse

* Corresponding author. Tel.: þ34 948 425 600; fax: þ34 948 425 649.

E-mail address: jpalop@unav.es (J.A. Palop).

doi:10.1016/j.ejmech.2011.10.056

284

E. Moreno et al. / European Journal of Medicinal Chemistry 47 (2012) 283e298

Fig. 1. Chemical structures for some quinazolines and pyrido[2,3-d]pyrimidines with anticancer activity. (a) Trimetrexate; (b) Piritrexim; (c) Geﬁtinib; (d) PD166326.

reference literature, which was reviewed in our previous works in

order to optimize the substituents with respect to in vitro biological

activity. With this aim in mind, the following structural modiﬁca-

tions were considered (Fig. 2): quinazoline and pyridopyrimidine

were selected in order to explore the inﬂuence of the total number

of nitrogen atoms present in the central fragment [42], with the

subsequent variation of the number of centers potentially involved

in hydrogen bond formation; the inﬂuence of parameters like

electronic environment, which would affect the lipophilicity and

hence the activity of the target molecules [43], the length of the

chains and positions in the annular system [44]. Various linkers

were designed and incorporated between the central nucleus

scaffold and the ﬂexible aryl moiety in order to investigate their

effects on the biological activity. The linkages investigated were

(eNHe), which was inserted in position 4, and a sulfur [45] or

selenium atom, which were mainly introduced in the 2-position.

The choice of the latter linker was based on our research in recent

years, which has involved the design and synthesis of structurally

modiﬁed compounds containing selenium e many of which have

demonstrated potent antitumoral activity [46e50]. Recently, we

reported the positive effect on the cytotoxic properties achieved by

replacing sulfur with selenium [46,50] and it seemed reasonable to

assess the bioactivity of related compounds with this element

instead of sulfur.

2. Results and discussion

2.1. Chemistry

The synthesis of the 35 compounds described here was carried

out according to Schemes 1e3.

In order to prepare these molecules, the most appropriate

starting materials were the commercially available 2-mercapto-

4(3H)quinazolinone

2,4(1H,3H)-quinazolinedione B (Aldrich, 86-96-4) (Scheme 2),

pyrido[2,3-d]pyrimidine-2,4(1H,3H)-dione (Shanghai Richem

(Aldrich, 13906-09-7) (Scheme 1),

International Co., Ltd) and a compound that was prepared previ-

ously by us, 1c [51].

Compounds 1a and 1b (Scheme 1) were synthesized from A by

reaction with methyl or pentyl iodide in basic media. The reaction

of A, 1a, 1b or 1c with POCl₃and DMF (added as a catalyst) gave the

corresponding halide intermediates (DeG), which were used

without puriﬁcation. The derivatives containing

a chloro-

substituent in the 4-position proved to be very versatile interme-

diates, since the chlorine could be displaced with a variety of

nucleophiles. The nucleophilic displacement of the chloro-

substituent in the key intermediates with the appropriate amine

was achieved by heating an alcoholic solution under reﬂux to

generate the desired ﬁnal products 2-mercapto/2-alkylthio-4-

substituted quinazolines and the corresponding pyridopyr-

imidines (2aep) (Scheme 1), with yields ranging from 4% to 94%.

Chlorination of B or C with phosphoryl chloride, according to the

method previously reported by us [51] (Scheme 2), afforded the

corresponding dihalides. Nucleophilic displacement of the chloro

group(s) with selenourea or ammonia gave the target compounds

3a, 3b and 4-amino-2-chloroquinazoline or pyrido[2,3-d]pyrimi-

dine, respectively. Different methods were used to effect these

displacements depending on the type of nucleophile. For example,

3a and 3b were obtained using ethanol as the solvent and the other

derivatives were obtained with aqueous ammonia. The resulting

compounds also served as intermediates to prepare other members

of the series. Thus, 3a and 3b were converted to 3c, 3d, 3e and 3f by

reaction with the appropriate alkyl iodide in ethanol under reﬂux.

Treatment of 4-amino-2-chloroquinazoline or pyrido[2,3-d]

pyrimidine with selenourea in ethanol provided 4a and 4b and

these were alkylated to obtain the desired compounds 4c, 4d, 4e

and 4f.

We report here the synthesis of 35 2-thio-, 2-seleno-4-amino-

and 2,4-diseleno-derivatives of quinazoline and pyrido[2,3-d]

pyrimidines and the evaluation of their biological activity as

potential antitumoral agents.

The key isoselenocyanates 6a, 6b and 6c were synthesized in

two steps [52,53] (Scheme 3). The ﬁrst step involved formylation of

phenylalkylamines with ethyl formate (compounds 5a, 5b and 5c)

followed by treatment with triphosgene and selenium powder in

Fig. 2. Structural modiﬁcations carried out for the synthesized compounds.

E. Moreno et al. / European Journal of Medicinal Chemistry 47 (2012) 283e298

285

Scheme 1. Synthesis route of the compounds of series 1 and 2.

the presence of triethylamine to furnish the desired phenylalkyl

isoselenocyanates in moderate yields. Compounds were puriﬁed by

silica gel column chromatography using n-hexane/ethyl acetate as

the eluent and they were characterized by ¹H NMR spectroscopy.

Treatment of isoselenocyanates with o-aminobenzonitrile or 2-

amino-3-cyanopyridine in the presence of dry pyridine under

The purity of all products was determined by thin layer chro-

matography using several solvent systems of different polarity. All

compounds were pure and stable. The compounds were charac-

terized by infrared, ¹H and ¹³C nuclear magnetic resonance, mass

spectrometry and CHN microanalysis.

reﬂux

gave

the

corresponding

4-phenylalkylamino-2-

2.2. Biological evaluation

selenoquinazoline or 4-phenylalkylamino-2-selenopyrido[2,3-d]

pyrimidine, which tautomerize to give 7a, 7b, 7c and 7d (Scheme

3). The latter compound was obtained in very modest yield.

Alkylation of compounds 7a, 7b and 7c with methyl iodide or

pentyl iodide was performed in ethanol and compounds 7e, 7f, 7g,

7h and 7i (Scheme 3) were obtained with variable yields. It is

important to emphasize that under the same conditions the anal-

ogous reaction utilizing the pyridopyrimidine 7d failed to give any

alkylated product. Moreover, subsequent modiﬁcation of the

conditions (temperature, solvents) resulted in decomposition and

complex reaction mixtures were obtained. On the whole, hydro-

seleno group is alkylated with alkyl iodides due to the nucleophilic

character of selenium, nevertheless, this character is very sensitive

to electronic effects of neighboring groups. For example, rings

2.2.1. Cytotoxicity

All of the synthesized compounds were screened for their

cytotoxic and antiproliferative activities against a panel of four

human tumor cell lines: lung carcinoma (HTB-54), colon carcinoma

(HT-29), lymphocytic leukemia (CCRF-CEM) and breast adenocar-

cinoma (MCF-7) as well as two non-malignant cell lines e

mammary gland (184B5) and bronchial epithelium cell (BEAS-2B).

Cytotoxicity assays were based on the reactivity of MTT [3-(4,5-

dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium

bromide]

described by Mosmann [54]. Results are expressed as GI₅₀, i.e. the

concentration that reduces by 50% the growth of treated cells with

respect to untreated controls, TGI, the concentration that

completely inhibits cell growth, and LC₅₀, the concentration that

kills 50% of the cells. The cytotoxic effect of each substance was

without electron-donating groups or p-deﬁcient rings cannot react.

In light of these ﬁndings, the corresponding alkylated pyridopyr-

imidines were not obtained and the importance of this structural

change on the biological activity could not be evaluated.

tested at ﬁve different concentrations between 0.01 and 100 mM.

The GI₅₀, TGI and LC₅₀results are reported in Table 1. As a positive

control we used cisplatin and etoposide at the same concentration.

286

E. Moreno et al. / European Journal of Medicinal Chemistry 47 (2012) 283e298

So, compounds 3a, 4a and 7h displayed a very interesting pattern of

selective cytostatic effect against these cell lines. On the other hand,

compounds 2o and 3a can be classiﬁed as cytotoxic agents owing to

their narrow difference between cytostatic and cytotoxic parame-

ters, with LC₅₀values below 10 mM.

In this primary screening we included isoselenocyanate deriva-

tives (6aec), used assyntheticintermediates, becausethey havebeen

described as effective antitumor agents for many types of cancer [53].

The following preliminary structureeactivity relationships can

be drawn for the screened compounds:

a) As a general trend, the pyridopyrimidines and quinazolines

with a SeH group in the positions 2 and 4 usually have better

antiproliferative activity than the corresponding analogs with

selenoalkyl functionality (e.g. 3a versus 3c and 3d; 3b versus 3e

and 3f).

b) In order to evaluate the importance of the long linear hydro-

phobic chain between the central nucleus and the phenyl ring,

we synthesized compounds 2c (n ¼ 0), 2d (n ¼ 1), 2h (n ¼ 2), 2i

(n ¼ 3) and 2j (n ¼ 4). The results did not show any correlation

between the chain length and the activity of compounds,

except for HT-29 e for which an increase in antiproliferative

effect was observed from n ¼ 0 (GI₅₀¼ 65.84

M) to n ¼ 3

(GI₅₀¼ 19.60 M).

c) In some cases, replacement of the sulfur unit in derivatives 2 by

a selenium fragment in derivatives 7 did not signiﬁcantly affect

the biological activity. Surprisingly, compounds 2aeb showed

better activities than their isosteric selenium analogs in several

cancer cell lines.

Compared to the activities of cisplatin and etoposide, the anti-

proliferative effects of our derivatives are cell-line dependent.

Among the thirty eight analogs synthesized and examined, thirteen

compounds showed GI₅₀values below that of etoposide

(GI₅₀¼ 19.95

MCF-7. If we consider the HT-29 cell line, twelve were more active

than etoposide (GI₅₀¼ 31.62 M) and ﬁve had values below that of

M). Finally, compounds 2b, 2o and 3a

mM) and six below that of cisplatin (GI₅₀¼ 3.16

mM) in

cisplatin (GI₅₀¼ 7.94

exhibited better activity than etoposide (GI₅₀¼ 12.59

M) in CCRF-

CEM cell line.

Compounds 2o and 3a were the most active, with LC₅₀<10

mM in

all the cell lines tested, while the rest of them only exhibited these

values in one or twocell lines (Table 1). Withregard toselectivity, the

cytotoxicity of all compounds described in this work was assayed

against two nontumoral lines, one mammary gland cell line (184B5)

and one bronchial epithelium line (BEAS-2B). The therapeutic index

was calculated from the ratio of GI₅₀on normal cells and tumoral

cells. Ratios between 3 and 6 denote moderate selectivity, ratios

greaterthan 6indicatehigh selectivity, while compoundsthatdo not

fulﬁll either of these criteria are rated as non-selective [55]. It is

important to point out that derivatives 1b, 3c and 7h were highly

selective in MCF-7 cells, as evidenced by the ratios of their GI₅₀

values (GI_{50 184B5}/GI_{50 MCF-7}ranged from >12 for 1b, 12 for 3c and

1418 for 7h). The rest of the derivatives showed similar parameters

in tumoral and normal cells. On the basis of these results we can

conclude that 7h, due to its potency and selectivity, and 2o and 3a,

due to their potent cytotoxic effect in all the cell lines, are promising

candidates to provide some insight into the molecular mechanisms

involved in the antiproliferative activity.

Scheme 2. Synthesis route of the compounds of series 3 and 4.

As guidance with regard to selectivity, all of the compounds were

further examined for toxicity in a mammary gland cell line derived

from non-malignant cells (184B5) and another derived from non-

malignant bronchial epithelium cells (BEAS-2B) (Table 2). The

analyses were all carried out with a minimum of three independent

experiments and values were calculated after 72 h exposure.

In terms of cell line sensitivity, the highest activities were

observed in MCF-7 and HTB-54 cell lines with GI₅₀values below

2.2.2. Cell death and effects on cell cycle progression

In the searchfora possible mechanism ofaction for the antitumor

activity, we investigated the ability of selected compounds (2o, 3a

and 7h) to induce internucleosomal degradation of genomic DNA (a

hallmark of apoptosis) and their effects on the cell cycle. In this

10 mM for eleven and ten compounds, respectively. Different

proﬁles were observed for the compounds attending to cytostatic

(mean GI₅₀and mean TGI) and cytotoxic parameters (mean LC₅₀).

E. Moreno et al. / European Journal of Medicinal Chemistry 47 (2012) 283e298

287

Scheme 3. Synthesis route of the compounds of series 5, 6 and 7.

investigation the MCF-7 cell line, i.e. the most sensitive to the tested

compounds, was used. The internucleosomal status of the cells was

investigated after 24 h of treatment with 1, 5,10,15 and 20 mM of the

corresponding compound using the Apo-Direct kit (BD Pharmingen)

[56] based on the TUNEL technique. Camptothecin was used as

a positivecontrol. The results obtained are shown in Fig. 3. Regarding

cell death, all of the compounds studied, and 3a in particular, dis-

played an increase in their capacity to induce DNA degradation and

the formation of oligonucleosomal fragments relative to the control

In addition, it is well known that numerous drug candidates have

failed during clinical tests because of problems related to ADME

(absorption, distribution, metabolism and excretion) properties. A

very preliminary computational study designed to predict the

absorption properties of our active and selective compounds found

for each speciﬁc bioassay was performed (PreADMET program). The

results are presented in Table 4. Human Intestinal Absorption (HIA)

and Caco-2 permeability are good indicators of drug absorbance in

the intestine and Caco-2 monolayer penetration, respectively.

Human Intestinal Absorption data are the sum of bioavailability

and absorption evaluated from the ratio of excretion or cumulative

excretion in urine, bile and feces [59]. The predicted percentages of

intestinal absorption are excellent for all of the compounds tested,

with values above 98% in all cases. The compounds present good

permeability values in Caco-2 cells, ranging from 44 to 56 [60].

Hence, theoretically, all of these compounds should present good

passive oral absorption and differences in their bioactivity cannot

be attributed to this property.

(DMSO control) at 5, 10, 15 and 20

mM. These values for 3a were 1.7-

fold at 5 M to 9-fold at 20 M higher than those for the control. In

order to ascertainwhether this effect is time-dependent we checked

the induction of cell death for these derivatives at 4, 12, 24 and 48 h

with the drug concentration at 10 mM. Cells treated with the selected

compounds evidence cell death at 12 h. These compounds induced

a time-dependent cell death (Fig. 4). For 3a, the cell death increased

in all time tested although compounds 2o and 7h showed a slightly

decrease in cell death at 48 h.

Flow cytometry analysis of cell cycle distribution was carried out

after treatment of MCF-7 cells for 4, 12 and 24 h with the corre-

3. Conclusions

sponding compounds (2o, 3a and 7h) at 10 mM (Table 3). DNA ﬂow

cytometric analysis indicated that treatment of the cells with these

derivatives did not induce any speciﬁc phase arrest of the cell cycle

relative to the control.

In conclusion, we have synthesized a series of 35 sulfur and

selenium quinazoline and pyrido[2,3-d]pyrimidine compounds and

investigated their abilities to inhibit in vitro the proliferation of

leukemia (CCRF-CEM), colon (HT-29), lung (HTB-54) and breast

(MCF-7) cancer cell lines as well as their effect on two derived from

non-malignant cell lines, one mammary (184B5) and one from

bronchial epithelium (BEAS-2B). Some derivatives exhibited good

antiproliferative activity and in some cases this was higher than

that for reference drugs etoposide and cisplatin, mainly in MCF-7.

The most promising compounds in these series were 2o and 3a,

which showed stronger antiproliferative activities against all of cell

lines tested, and 7h, which exhibited good activity against MCF-7

and also displayed weak cytotoxicity to non-malignant cells. In

order to elucidate the mechanisms of action, the cytotoxicity cell

death status and cell cycle distribution were examined in MCF-7.

The three compounds all induced cell death in a time and dose-

dependent manner without involving cell cycle perturbation.

2.2.3. ADME prediction

As a part of our study, the compliance of compounds to Lip-

inski’s rule of ﬁve was evaluated [57]. Although the cytotoxic effects

of lead compounds are thought to be primarily due to their ability

to modulate cell death, other factors such as solubility, stability

and/or efﬂux properties within the cell may also contribute. The

freely accessible program OSIRIS Explorer Properties [58] was used

to analyze the results given in Table 4 and it was found that the

selected active compounds comply with these rules with the

exception of 7h, which did not comply for c Log P. However, the

reference drug for pharmacological testing (etoposide,

c Log P ¼ 0.53; MW ¼ 588; n-OHNH ¼ 3; n-ON ¼ 13) did not fulﬁll

two of Lipinski’s rules when analyzed with this program.

288

E. Moreno et al. / European Journal of Medicinal Chemistry 47 (2012) 283e298

Table 1

Average GI₅₀, TGI and LC₅₀values (mM) for compounds.

Ref.

R⁰

CCRF-CEM^a

HT-29^b

GI₅₀

HTB-54^c

GI₅₀

MCF-7^d

GI₅₀

TGI^f

LC₅₀

TGI

LC₅₀

TGI

LC₅₀

TGI

LC₅₀

CH₃

C₆H₅

4-OCH₃eC₆H₄

C₆H₅

4-OCH₃eC₆H₄

4-SCH₃eC₆H₄

4-SeCH₃eC₆H₄

C₆H₅

4-SeCH₃eC₆H₄

C₆H₅

4-OCH₃eC₆H₄

4-SCH₃eC₆H₄

CH₃

52.13

82.41

10.38

74.60

87.90

93.68

>100

63.31

32.87

39.49

46.99

>100

56.27

>100

5.35

85.97

>100

41.01

>100

72.78

87.82

>100

98.55

>100

16.84

>100

6.01

>100

71.63

>100

59.03

>100

9.03

74.59

10.14

6.49

>100

63.50

28.63

>100

87.37

89.55

>100

7.03

>100

63.91

>100

8.75

30.40

15.23

13.49

55.80

37.87

93.68

7.22

57.01

50.55

50.64

>100

31.37

19.79

5.08

>100

45.53

>100

82.30

>100

44.49

>100

71.44

49.98

51.58

>100

14.81

>100

65.88

7.93

>100

77.57

>100

89.13

>100

80.16

>100

63.57

>100

68.35

>100

98.46

66.09

>100

76.26

>100

7.82

27.23

7.08

81.44

>100

36.47

>100

82.81

85.14

>100

63.96

>100

75.20

>100

4.37

>100

22.88

46.14

50.73

95.13

67.72

>100

59.47

>100

53.84

61.67

7.55

>100

78.39

>100

95.88

>100

9.13

CH₃

65.84

36.53

70.18

34.66

>100

29.17

19.60

>100

14.42

87.14

>100

4.32

63.87

8.98

7.46

>100

52.19

36.30

27.63

40.30

44.14

5.91

20.96

57.49

48.77

62.20

71.64

42.89

6.35

42.83

73.63

72.66

72.68

83.59

7.94

78.35

41.52

30.92

47.29

36.41

80.29

77.21

32.03

61.95

13.04

39.42

48.07

0.05

48.92

2.92

9.83

5.97

38.35

>100

3.28

>100

5.98

>100

2.99

>100

45.22

33.76

>100

87.94

76.78

>100

82.74

>100

ND^h

4.03

CH₃

31.22

65.70

54.85

>100

78.48

>100

26.55

>100

15.23

0.04

44.28

17.98

34.62

78.21

8.78

5.98

37.20

5.32

31.22

7.11

45.52

>100

74.58

33.04

37.88

37.73

40.35

84.24

>100

60.54

3.63

>100

25.66

30.89

65.13

34.77

68.44

12.93

28.72

39.30

25.53

>100

71.90

30.51

39.75

>100

48.71

34.48

35.02

1.00

>100

52.39

61.47

>100

67.97

>100

42.75

57.70

60.87

53.83

>100

68.93

61.63

>100

86.88

72.46

83.90

79.43

50.12

>100

79.11

92.06

>100

72.57

86.67

82.44

82.13

>100

83.50

>100

89.58

74.81

>100

77.28

19.98

44.54

>100

33.11

80.96

>100

62.08

68.13

>100

40.84

0.15

94.60

>100

90.38

>100

85.01

95.45

81.17

>100

>100C

>100

99.40

95.33

>100

96.56

88.69

84.04

>100

50.99

33.41

>100

9.98

>100

60.87

>100

72.44

81.11

81.28

37.27

46.48

28.57

42.99

22.67

27.89

0.62

CH₃

5.77

59.33

>100

94.00

>100

67.01

98.61

>100

47.13

65.03

>100

CH₃

C₆H₅

>100

42.73

48.12

0.03

15.80

42.24

54.05

0.05

4-OCH₃eC₆H₄

4-SCH₃eC₆H₄

C₆H₅

4-OCH₃eC₆H₄

4-SCH₃eC₆H₄

C₆H₅

Cisplatin

Etoposide

4-SCH₃eC₆H₄

34.73

3.16

19.95

12.59

31.62

Lymphocytic leukemia.

Colon carcinoma.

Lung carcinoma.

Breast adenocarcinoma.

Concentration that inhibits 50% of cell growth.

Concentration that inhibits 100% of cell growth.

Concentration that kills 50% of cells.

ND: No data.

4. Experimental protocols

spectrometer (Rheinstetten, Germany) using TMS as the internal

standard. The IR spectra were obtained on a Thermo Nicolet FTeIR

Nexus spectrophotometer with KBr pellets. Elemental microanal-

yses were carried out on vacuum-dried samples using a LECO CHN-

900 Elemental Analyzer. Silica gel 60 (0.040e0.063 mm)

1.09385.2500 (Merck KGaA, 64271 Darmstadt, Germany) was used

for Column Chromatography and Alugram^ÒSIL G/UV₂₅₄(Layer:

4.1. Chemistry

Melting points were determined with a Mettler FP82þFP80

apparatus (Greifense, Switzerland) and have not been corrected.

The ¹H NMR spectra were recorded on a Bruker 400 UltrashieldÔ

E. Moreno et al. / European Journal of Medicinal Chemistry 47 (2012) 283e298

289

Table 2

4.1.1. 4-Hydroxy-2-pentylthioquinazoline (1b)

Average GI₅₀, TGI and LC₅₀values (

lines.

M) for compounds in 184B5 and BEAS-2B cell

BEAS-2B^b

The synthetic route previously published by Kane et al. was

followed with a slight modiﬁcation. The corresponding 4-hydroxy-

2-mercaptoquinazoline (11.2 mmol) was dissolved in 0.4 N aqueous

NaOH (50 mL). A solution of C₅H₁₁I (13.4 mmol) in EtOH (25 mL)

was added. The mixture was stirred for 18 h at 70 ^ꢀC and then

neutralized with acetic acid. The resulting precipitate was collected

and was used without puriﬁcation. Yield: 89%; mp 161e162 ^ꢀC. IR

(KBr) cm^ꢁ1: 3159 (m, CeH); 2954e2862 (m, CeH); 1675 (m, C]O);

Compound 184B5^a

GI₅₀

(m (mM) LC₅₀(mM) GI₅₀(m (mM) LC₅₀(mM)

M) TGI^dM) TGI^d

>100

49.59

7.83

>100

28.93

85.59

21.97

70.27

8.30

>100

36.55

>100

46.88

>100

53.55

>100

69.77

1.14

>100

69.23

>100

88.58

>100

78.60

>100

5.64

51.43

66.33

6.13

>100

32.50

>100

88.24

96.24

59.11

>100

94.80

>100

99.35

>100

61.70

>100

3.77

>100

70.18

>100

95.93

>100

97.51

>100

8.47

54.75

38.88

21.74

22.69

73.47

41.92

73.39

6.81

39.26

31.21

25.88

58.51

0.60

1549 (m, C]N). ¹H NMR (400 MHz, DMSO-d₆)

d: 0.87 (t, 3H,

(CH₂)₄eCH₃); 1.35 (m, 4H, (CH₂)₂e(CH₂)₂eCH₃); 1.69 (q, 2H,

CH₂eCH₂e(CH₂)₂eCH₃); 3.20 (t, 2H, CH₂e(CH₂)₃eCH₃); 7.41 (t, 1H,

H₆, J_6e5¼ 7.7); 7.50 (d, 1H, H₅); 7.74 (t, 1H, H_7,J_7e6¼ J_7e8¼ 7.7); 8.02

6.77

(d, 1H, H₈); 12.53 (s, 1H, OH). ¹³C NMR (100 MHz, DMSO-d₆)

d: 14.1

28.49

49.86

72.96

49.03

34.23

0.19

>100

0.49

0.71

71.48

71.30

52.76

0.69

(Se(CH₂)₄eCH₃); 22.4 (Se(CH₂)₃eCH₂eCH₃); 29.2 (SeCH₂eCH₂e

(CH₂)₂eCH₃); 29.8 (Se(CH₂)₂eCH₂eCH₂eCH₃); 31.2 (SeCH₂e

(CH₂)₃eCH₃); 120.8 (C₉); 125.8 (C₈); 126.9 (C₅); 127.4 (C₇); 135.3

(C₆); 149.3 (C₁₀); 156.9 (C₂); 162.3 (C₄). MS (m/z % abundance): 248

(M^þ, 21); 215 (20); 201 (49); 191 (33); 178 (100); 162 (33); 145 (20);

121 (44); 91 (26). Elemental Analysis for C₁₅H₁₆N₂OS Calcd/Found

(%): C: 62.90/62.58; H: 6.45/6.33; N: 11.29/11.21.

Cisplatin

>100

2.50

8.26

>100

6.63

67.50

>100

ND^f

>100

4.1.2. General procedure for compounds 2aep

To a suspension of A, 1a, 1b or 1c in POCl₃(20 mL) was added

N,N-dimethylformamide (drops). The mixture was stirred and

heated under reﬂux for 2 h and then concentrated. The solution

was then poured into ice water and the aqueous layer was extracted

with CHCl₃(3 ꢂ 20 mL). The combined organic layers were dried

over Na₂SO₄, ﬁltered, concentrated, and the chloro-derivatives

were used without puriﬁcation.

A solution of the corresponding 2-alkylthio/2-mercapto-4-

chloroquinazoline or the corresponding pyrido[2,3-d]pyrimidine

(4.0 mmol) in ethanol (25 mL) was cooled to 4 ^ꢀC and the corre-

sponding amine (4.4 mmol) was added. The mixture was stirred at

room temperature for 5e6 h, heated at 75 ^ꢀC during 15 h and was

concentrated. The residue was treated with water and the precip-

itate was collected by ﬁltration, washed with Et₂O (3 ꢂ 15 mL) and

recrystallized from the appropriate solvent.

>100

36.65

31.05

59.04

43.60

87.15

40.23

45.15

66.30

33.97

63.63

>100

86.27

9.13

12.41

>100

80.37

74.98

93.68

71.85

>100

70.14

74.86

90.94

67.32

>100

73.66

>100

0.08

5.89

23.61

52.25

39.68

16.30

4.10

11.28

7.78

3.54

37.39

0.41

8.40

>100

87.73

54.56

>100

90.81

44.89

32.75

45.39

42.34

>100

58.21

>100

36.96

>100

78.54

>100

88.21

>100

85.52

>100

73.48

69.59

79.49

75.60

>100

78.30

>100

5.20

24.40

15.34

45.53

10.32

15.45

35.66

5.84

20.81

71.25

6.84

3.70

3.72

0.57

75.59

37.12

70.90

57.36

>100

35.86

44.05

34.72

4.1.3. 4-Benzylamino-2-mercapto-quinazoline (2a)

Etoposide ND

From 4-chloro-2-mercaptoquinazoline and benzylamine. Yield:

6%; mp 208e209 ^ꢀC. IR (KBr) cm^ꢁ1: 3204 (m, NeH); 3096 (m, CeH);

Mammary gland cell culture derived from non-malignant cells.

2921 (m, CeH); 1615 (m, C]N). ¹H NMR (400 MHz, DMSO-d₆)

Bronchial epithelium cell culture derived from non-malignant cells.

Concentration that inhibits 50% of cell growth.

Concentration that inhibits 100% of cell growth.

Concentration that kills 50% of cells.

4.56 (d, 2H, CH₂eNH; J_CH2eNH¼ 5.6 Hz); 6.68 (t, 1H, NHeCH₂); 7.10

(dt, 1H, H₆, J_6e5¼ 7.9 Hz, J_6e7¼ 7.2 Hz, J_6e8¼ 1.1 Hz); 7.26 (m, 2H,

ND: No data.

H₂þ H₆); 7.36 (m, 4H, H₅þ H₃þ H₄þ H₅); 7.56 (dt, 1H, H₇,

J_7e8¼ 8.0 Hz, J_7e5¼1.6 Hz); 7.89 (dd, 1H, H₈); 10.92 (s, 1H, SH). ¹³

NMR (100 MHz, DMSO-d₆) d: 44.8 (CH₂); 118.9 (C₉); 126.8 (C₈);

127.2 (C₅); 127.9 (C₇); 128.5 (C₂þ C₆); 128.9 (C₄); 129.7 (C₃þ C₅);

0.2 mm) (MachereyeNagel GmbH & Co. KG. Postfach 101352, D-

52313 Düren, Germany) was used for Thin Layer Chromatography.

Chemicals were purchased from E. Merck (Darmstadt, Germany),

Scharlau (F.E.R.O.S.A., Barcelona, Spain), Panreac Química S.A.

(Montcada i Reixac, Barcelona, Spain), SigmaeAldrich Química, S.A.

(Alcobendas, Madrid, Spain), Acros Organics (Janssen Pharmaceu-

ticalaan 3a, 2440 Geel, Belgium) and Lancaster (Bischheime-

Strasbourg, France). The 4-hydroxy-2-methylthioquinazoline 1a

was prepared according to the literature procedure. Yield: 88%; mp

183e184 ^ꢀC. IR (KBr) cm^ꢁ1: 3163 (m, CeH); 3050e2845 (m, CeH);

134.9 (C₆); 140.1 (C₁); 151.3 (C₁₀); 155.3 (C₄); 162.8 (C₂). MS (m/z %

abundance): 267 (M^þ, 11); 251 (100); 234 (11); 178 (29); 119 (18);

106 (42); 91 (46). Elemental Analysis for C₁₅H₁₃N₃S Calcd/Found

(%): C: 67.41/67.78; H: 4.87/4.97; N: 15.73/15.60.

4.1.4. 2-Mercapto-4-(4⁰-methoxybenzyl)aminoquinazoline (2b)

From 4-chloro-2-mercaptoquinazoline and 4-methoxybenzyl-

amine. Yield: 13%; mp 222e223 ^ꢀC. IR (KBr) cm^ꢁ1: 3220 (m,

NeH); 3124 (m, CeH); 2962e2833 (m, CeH); 1604 (m, C]N). ¹H

NMR (400 MHz, DMSO-d₆)

: 3.75 (m, 4H, H₂O þ NH þ OCH₃); 4.67

1700 (m, C]O); 1618 (m, C]N). ¹H NMR (400 MHz, DMSO-d₆)

(s, 2H, CH₂eNH); 6.85 (d, 2H, H₃þ H₅, J_{3 e2}¼ J_{5 e6}¼ 7.5 Hz); 7.27

2.55 (s, 3H, SCH₃); 3.38 (br s, 1H, OH); 7.41 (dd, 1H, H₆, J_6e5¼ 8.2);

7.53 (d,1H, H₅), 7.75 (dd,1H, H₇, J_7e8¼ 8.0); 8.03 (d,1H, H₈). MS (m/z

% abundance): 192 (M^þ, 100); 119 (43); 90 (38). Elemental Analysis

for C₉H₈N₂OS Calcd/Found (%): C: 56.25/56.14; H: 4.16/4.10; N:

16.66/16.81.

(d, 2H, H₂þ H₆); 7.68 (m,1H, H₆); 7.96 (m, 2H, H₇þ H₅); 8.54 (d, 1H,

H₈, J_8e7¼ 8.4 Hz); 10.36 (s, 1H, SH). ¹³C NMR (100 MHz, DMSO-d₆)

: 44.6 (CH₂); 56.2 (OCH₃); 114.2 (C₃þ C₅); 116.9 (C₉); 125.5 (C₈);

126.4 (C₅); 126.9 (C₇); 128.9 (C₂þ C₆); 129.5 (C₆); 135.8 (C₁); 151.7

(C₁₀); 156.5 (C₄); 159.3 (C₄); 162.9 (C₂). MS (m/z % abundance): 296

290

E. Moreno et al. / European Journal of Medicinal Chemistry 47 (2012) 283e298

Fig. 3. Effects of 2o, 3a and 7h on cell death in MCF-7 cells at a concentration of 1, 5, 10, 15 and 20 mM for 24 h using the Apo-Direct kit (BD Pharmingen) based on the TUNEL

technique. The results are presented as the mean ꢃ SD of three independent experiments (duplicate wells). ¼ difference statistically very signiﬁcant with respect to the control

(p < 0.01).

(M^þ, 38); 191 (76); 175 (57); 163 (37); 136 (38); 121 (100); 91 (21);

77 (18). Elemental Analysis for C₁₆H₁₅N₃OS$0.3 HCl Calcd/Found

(%): C: 62.34/62.07; H: 4.87/4.82; N: 13.64/13.35.

91 (29); 77 (10). Elemental Analysis for C₁₆H₁₅N₃S$0.45 HCl Calcd/

Found (%): C: 64.55/64.77; H: 5.04/5.35; N: 14.12/13.91.

4.1.7. 4-(4⁰-Methoxybenzyl)amino-2-methylthioquinazoline (2e)

From 4-chloro-2-methylthioquinazoline and 4-methoxybenzyl-

amine. Yield: 7%; mp 109e110 ^ꢀC. IR (KBr) cm^ꢁ1: 3283 (m, NeH);

4.1.5. 2-Methylthio-4-phenylquinazoline (2c)

From 4-chloro-2-methylthioquinazoline and aniline. Yield: 73%;

mp 79e80 ^ꢀC. IR (KBr) cm^ꢁ1: 3329 (m, NeH); 2931 (m, CeH); 1563,

2929 (m, CeH); 1612, (s, C]N). ¹H NMR (400 MHz, DMSO-d₆)

(s, C]N). ¹H NMR (400 MHz, DMSO-d₆)

d: 2.52 (s, 3H, SCH₃); 7.22 (t,

2.53 (s, 3H, SCH₃); 3.72 (s, 3H, OCH₃); 4.71 (d, 2H, CH₂eNH,

1H, H₄); 7.44 (t, 2H, H₃þ H₅); 7.58 (t, 1H, H₆, J_6e5¼ J_6e7¼ 8.0 Hz);

J_eCH2eNH¼ 5.8 Hz); 6.89 (d, 2H, H₃þ H₅, J_{3 e2}¼ J_{5 e6}¼ 8.6 Hz);

7.68 (d, 1H, H₅); 7.81 (d, 2H, H₂þ H₆); 7.87 (t, 1H, H_7,J_7e8¼ 8.0 Hz);

7.32 (d, 2H, H₂þ H₆); 7.43 (dd, 1H, H₆, J_6e5¼ 7.9 Hz, J_6e7¼ 7 Hz);

8.62 (d,1H, H₈); 10.51 (br s,1H, NHePh). ¹³C NMR (100 MHz, DMSO-

7.55 (d, 1H, H₅); 7.74 (dd, 1H, H_7,J_7e8¼ 8.4 Hz); 8.26 (d, 1H, H₈);

9.24, (br s, 1H, NHeCH₂). ¹³C NMR (100 MHz, DMSO-d₆)

d: 14.3

d₆)

: 14.3 (SCH₃); 113.3 (C₉þ C₂þ C₆); 118.2 (C₄); 124.2 (C₈); 126.3

(C₅); 126.8 (C₇); 129.7 (C₃þ C₅); 135.3 (C₆); 138.9 (C₁); 147.2 (C₁₀);

157.8 (C₄); 167.1 (C₂). MS (m/z % abundance): 267 (M^þ, 100); 220

(74); 129 (9); 77 (12). Elemental Analysis for C₁₅H₁₃N₃S$1.6 HCl

Calcd/Found (%): C: 54.70/54.91; H: 4.49/4.62; N: 12.91/12.63.

(SCH₃); 44.3 (CH₂); 56.5 (OCH₃); 113.3 (C₉); 113.9 (C₃þ C₅); 124.9

(C₈); 125.8 (C₅); 127.3 (C₇); 129.3 (C₂þ C₆); 132.2 (C₆); 134.6 (C₁);

148.1 (C₁₀); 159.2 (C₄þ C₄); 167.5 (C₂). MS (m/z % abundance): 311

(M^þ, 26); 191 (18); 136 (24); 121 (100); 91 (28); 77 (44). Elemental

Analysis for C₁₇H₁₇N₃OS$0.35 HCl Calcd/Found (%): C: 63.00/63.09;

H: 5.25/5.37; N: 12.97/12.88.

4.1.6. 4-Benzylamino-2-methylthioquinazoline (2d)

From 4-chloro-2-methylthioquinazoline and benzylamine.

Yield: 44%; mp 139e140 ^ꢀC. IR (KBr) cm^ꢁ1: 3210 (m, NeH); 3059

(m, CeH); 2973, (m, CeH); 1615e1567 (s, C]N). ¹H NMR (400 MHz,

4.1.8. 4-(4⁰-Methylthiobenzyl)amino-2-methylthioquinazoline (2f)

From 4-chloro-2-methylthioquinazoline and 4-methylthioben-

zylamine. Yield: 38%; mp 119e120 ^ꢀC. IR (KBr) cm^ꢁ1: 3226e2922

DMSO-d₆)

d: 2.49 (s, 3H, SCH₃); 4.79 (d, 2H, CH₂eNH,

J_eCH2eNH¼ 5.8 Hz); 7.25 (t, 1H, H₄, J_{4 e3}¼ J_{4 e5}¼ 7.2 Hz); 7.33 (t, 2H,

(m, NeH); 1625e1573 (s, C]N). ¹H NMR (400 MHz, DMSO-d₆)

H₃þ H₅, J_{3 e2}¼ J_{5 e6}¼ 7.6 Hz); 7.39 (d, 2H, H₂þ H₆); 7.46 (dd, 1H,

H₆, J_6e5¼ 8.0 Hz, J_6e7¼ 7.1 Hz); 7.57 (d, 1H, H₅); 7.76 (dd, 1H, H_7,

J_7e8¼ 8.4 Hz); 8.31 (d, 1H, H₈); 9.42, (br s 1H, NHeCH₂). ¹³C NMR

2.44 (s, 3H, PheSCH₃); 2.54 (s, 3H, SCH₃); 4.71 (d, 2H, CH₂eNH,

J_CH2eNH¼ 5.8 Hz); 7.23 (d, 2H, H₂þ H₆, J_{2 e6}¼ J_{3 e5}¼ 8.4 Hz); 7.3

(d, 2H, H₃þ H₅); 7.51 (dd, 1H, H₆, J_6e5¼ 8.2 Hz, J_6e7¼ 6.7 Hz); 7.6,

(d, 1H, H₅); 7.82 (dd, 1H, H₇, J_7e8¼ 8.2 Hz); 8.37, (d, 1H, H₈); 9.87, (br

(100 MHz, DMSO-d₆) d: 14.5 (SCH₃); 44.7 (CH₂); 117.9 (C₉); 125.7

(C₈); 126.5 (C₅); 127.4 (C₇); 128.3 (C₂þ C₆); 128.9 (C₄); 129.8

s, 1H, NHeCH₂). ¹³C NMR (100 MHz, DMSO-d₆)

d: 14.3 (heteSCH₃);

(C₃þ C₅); 134.7 (C₆); 140.5 (C₁); 151.2 (C₁₀); 155.4 (C₄); 163.1 (C₂).

15.7 (PheSCH₃); 44.7 (CH₂); 112.9 (C₉); 123.0 (C₈); 124.7 (C₅); 126.4

MS (m/z % abundance): 281 (M^þ, 100); 235 (18); 191 (38); 106 (20);

(C₇); 126.9 (C₃þ C₅); 129.1 (C₂þ C₆); 131.2 (C₆); 136.0 (C₁); 137.5

E. Moreno et al. / European Journal of Medicinal Chemistry 47 (2012) 283e298

291

Fig. 4. Compounds 2o, 3a and 7h induce cell death on MCF-7 cells. MCF-7 cells were incubated either with 10 mM of the indicated compound or vehicle (control cells) for 4, 12, 24

and 48 h. The results are presented as the mean ꢃ SD of three independent experiments (duplicate wells). ¼ difference statistically very signiﬁcant with respect to the control

(p < 0.01).

(C₄); 146.5 (C₁₀); 159.2 (C₄); 166.9 (C₂). MS (m/z % abundance): 327

(M^þ, 51); 190 (34); 175 (15); 152 (24); 137 (100); 121 (46); 103 (24);

91 (16); 77 (16). Elemental Analysis for C₁₇H₁₇N₃S₂$0.85 HCl Calcd/

Found (%): C: 56.98/56.81; H: 4.77/5.24; N: 11.73/11.71.

4.1.9. 2-Methylthio-4-(4⁰-methylselenobenzyl)aminoquinazoline

(2g)

From 4-chloro-2-methylthioquinazoline and 4-methylseleno-

benzylamine. Yield: 26%; mp 110e112 ^ꢀC. IR (KBr) cm^ꢁ1: 3204 (m,

NeH); 3096 (m, CeH); 2921 (m, CeH); 1615 (m, C]N). ¹H NMR

(400 MHz, DMSO-d₆) d: 2.31 (s, 3H, SeeCH₃); 2.46 (d, 3H, SeCH₃;

J_SCH3eCH2¼ 5.7 Hz); 4.71 (dd, 2H, CH₂ePh; J_CH2eNH¼ 12.0 Hz); 7.29

(dt, 1H, H₆); 7.40 (m, 4H, CH₂ePh); 7.53 (dd, 1H, H₅); 7.70 (dt, 1H,

H₇); 8.22 (dd, 1H, H₈); 8.94 (m, 1H, NHeCH₂). ¹³C NMR (100 MHz,

Table 3

Effects of 2o, 3a and 7h on cell cycle distribution in MCF-7 cells. Exponentially

growing cells were treated with 15 mM of the indicated compound or vehicle

DMSO-d₆) d: 7.5 (SeCH₃); 14.3 (SCH₃); 44.2 (CH₂); 113.7 (C₉); 123.8

(C₈); 125.6 (C₅); 126.9 (C₇); 129.6 (C₃þ C₅); 130.4 (C₂þ C₆); 131.0

(control) for 24 h. The changes of cell cycle phase distribution were assessed by DNA

ﬂow cytometric analysis. Results are expressed as percentages of total cell

counts. Camptothecin was used as a positive control. Results are presented as the

mean ꢃ SEM of three independent experiments (duplicate wells). ^**p < 0.01 with

respect the control.

(C₆); 135.0 (C₁); 139.2 (C₄); 150.6 (C₁₀); 159.3 (C₄); 167.7 (C₂). MS

(m/z % abundance): 375 (M^þ, 34); 281 (10); 220 (34); 200 (35); 185

(100); 176 (46); 162 (43); 146 (34); 129 (20); 121 (96); 91 (64); 77

(26). Elemental Analysis for C₁₇H₁₇N₃SSe Calcd/Found (%): C: 54.54/

55.00; H: 4.54/4.51; N: 11.23/10.91.

Compound

Control

Time SubG₁

G₀/G₁

G₂/M

4 h

5.90 ꢃ 1.36 49.38 ꢃ 4.74 19.09 ꢃ 4.54 21.60 ꢃ 5.37

2.17 ꢃ 0.24 57.34 ꢃ 1.64 17.07 ꢃ 4.00 22.11 ꢃ 3.86

2.93 ꢃ 0.54 51.83 ꢃ 5.64 12.39 ꢃ 1.54 32.37 ꢃ 7.73

12 h

24 h

4 h

12 h

24 h

3.88 ꢃ 1.75 50.42 ꢃ 3.14 18.02 ꢃ 1.88 23.88 ꢃ 4.93

2.24 ꢃ 0.19 55.42 ꢃ 2.22 19.35 ꢃ 1.22 22.99 ꢃ 3.06

2.97 ꢃ 0.27 52.91 ꢃ 7.36 11.04 ꢃ 1.32 30.20 ꢃ 2.14

Table 4

Physico-chemical and absorption properties for the most active compounds.

Compounds c Log P MW n-OHNH n-ON

Lipinski’s Absorption

4 h

12 h

24 h

3.98 ꢃ 0.51 52.09 ꢃ 4.47 23.29 ꢃ 4.33 21.21 ꢃ 5.58

2.37 ꢃ 0.53 56.21 ꢃ 1.31 18.36 ꢃ 1.39 25.63 ꢃ 1.76

3.46 ꢃ 1.80 47.77 ꢃ 3.69 15.75 ꢃ 1.29 34.67 ꢃ 5.42

donors

acceptors violations

HIA

(%)

Caco-2

(nm/sec)

3.34

0.12

6.2

328

290

396

99.12 44.04

4 h

12 h

24 h

4.42 ꢃ 0.19 46.24 ꢃ 2.72 16.66 ꢃ 1.33 28.50 ꢃ 2.78

2.13 ꢃ 0.25 55.30 ꢃ 1.93 17.01 ꢃ 1.77 25.83 ꢃ 5.28

3.28 ꢃ 0.18 46.97 ꢃ 8.28 12.20 ꢃ 0.82 34.79 ꢃ 2.14

100

46.72

98.47 56.03

c Log P, logarithm of compound partition coefﬁcient between n-octanol and water;

MW, molecular weight; n-OHNH, number of hydrogen bond donors; n-ON, number

of hydrogen bond acceptors; HIA, Human Intestinal Absorption; Caco-2, cells

derived from human colon adenocarcinoma.

Camptothecin 4 h

5.39 ꢃ 0.36 47.24 ꢃ 2.81 18.36 ꢃ 0.91 23.38 ꢃ 5.00

12 h 3.06** ꢃ 0.19 59.50 ꢃ 1.14 16.24 ꢃ 2.14 20.21 ꢃ 5.19

24 h 3.05** ꢃ 0.12 58.52 ꢃ 1.15 15.27 ꢃ 2.28 20.25 ꢃ 5.45

292

E. Moreno et al. / European Journal of Medicinal Chemistry 47 (2012) 283e298

4.1.9.1. Procedure for the preparation of 4-methylselenobenzylamine

4.1.9.1.1. Preparation of 4-methylselenobenzaldehyde. A solution

of NaBH₄(22.20 mmol) in ethanol (30 mL) was added to a mixture

of dimethyl diselenide (10.60 mmol) and ethanol (30 mL) under an

N₂atmosphere and the mixture was stirred at room temperature

for 20 min. Benzaldehyde (20.14 mmol) was added and the

mixture was heated at 150 ^ꢀC during 2 h. The reaction mixture was

evaporated and extracted with dichloromethane (3 ꢂ 20 mL), the

organic phase was washed with water and dried over Na₂SO₄and

concentrated to dryness. The residue was used without puriﬁca-

4.1.11. 2-Methylthio-4-(3-phenylpropyl)aminoquinazoline (2i)

From 4-chloro-2-methylthioquinazoline and 3-phenylpropyl-

amine. Yield: 73%; mp 117e118 ^ꢀC. IR (KBr) cm^ꢁ1: 3234 (m,

NeH); 3054 (m, CeH); 2928 (m, CeH); 1568, (s, C]N). ¹H NMR

(400 MHz, DMSO-d₆) d: 1.96 (m, 2H, NHeCH₂eCH₂eCH₂ePh); 2.47

(s, 3H, SeCH₃); 2.68 (m, 2H, NHe(CH₂)₂eCH₂ePh); 3.53 (m, 2H,

NHeCH₂e(CH₂)₂ePh); 7.23 (m, 5H, Ph); 7.38 (t, 1H, H₆,

J_6e5¼ J_6e7¼ 7.7 Hz); 7.52 (d, 1H, H₅); 7.69 (t, 1H, H_7,J_7e8¼ 7.7 Hz);

8.18 (d, 1H, H₈); 8.43 (br s, 1H, NHe(CH₂)₃ePh). ¹³C NMR (100 MHz,

DMSO-d₆)

d: 14.2 (SCH₃); 30.9 (NHeCH₂eCH₂eCH₂ePh); 33.5

tion. A yellowish liquid (2.84 g, 95%) was obtained. IR (KBr) cm^ꢁ1

(NHe(CH₂)₂eCH₂ePh); 41.2 (NHeCH₂e(CH₂)₂ePh); 113.7 (C₉);

3052 (m, CeH), 1694 (s, C]O). ¹H NMR (400 MHz, DMSO-d₆)

123.8 (C₈); 124.7 (C₅); 126.9 (C₇); 128.3 (C₄); 128.7 (C₂þ C₆); 129.6

2.45 (s, 3H, SeCH₃); 7.51 (d, 2H, H₃þ H₅, J_3e2¼ J_5e6¼ 8.3 Hz); 7.76

(dd, 2H, H₂þ H₆, J_2-COH¼ J_6-COH¼ 0.4 Hz); 9.96 (s, 1H, COH).

Elemental Analysis for C₈H₈OSe Calcd/Found (%): C: 48.24/48.12;

H: 4.02/3.89.

(C₃þ C₅); 134.9 (C₆); 140.5 (C₁); 148.2 (C₁₀); 159.4 (C₄); 167.7 (C₂).

MS (m/z % abundance): 309 (M^þ, 43); 218 (20); 205 (100); 191 (21);

159 (26); 91 (25). Elemental Analysis for C₁₈H₁₉N₃S$0.1 HCl Calcd/

Found (%): C: 69.08/68.93; H: 6.12/6.48; N: 13.43/13.15.

4.1.9.1.2. Preparation of (E)-4-(methylseleno)benzaldehyde oxi-

me. To

mixture of 4-methylselenobenzaldehyde (2.84 g,

4.1.12. 2-Methylthio-4-(4-phenylbutyl)aminoquinazoline (2j)

From 4-chloro-2-methylthioquinazoline and 4-phenylbutyl-

amine. Yield: 60%; mp 80e81 ^ꢀC. IR (KBr) cm^ꢁ1: 3206 (m, NeH);

3080 (m, CeH); 2929 (m, CeH); 1569 (s, C]N). ¹H NMR (400 MHz,

14.27 mmol) and ethanol (30 mL) was added Na₂CO₃(1.74 g,

4.16 mmol) in water (10 mL). Hydroxylamine hydrochloride (1.50 g,

2.14 mmol) in water (15 mL) was added slowly to the mixture and

the solution was stirred at room temperature for 24 h. The reaction

mixture was evaporated and washed with water, ﬁltered and dried.

The residue was recrystallized from ethanol to give a white solid

(2.56 g). Yield: 83.2%; mp 185e186 ^ꢀC. IR (KBr) cm^ꢁ1: 3285 (s,

OeH); 2972e2929 (m, CeH); 1590 (s, C]N). ¹H NMR (400 MHz,

DMSO-d₆) d: 1.70 (m, 4H, NHeCH₂e(CH₂)₂eCH₂ePh); 2.49 (m, 2H,

NHe(CH₂)₃eCH₂ePh); 2.65 (s, 3H, SCH₃); 3.73 (m, 2H,

NHeCH₂e(CH₂)₃ePh); 7.21 (m, 5H, Ph); 7.60 (t, 1H, H₆,

J_6e5¼ J_6e7¼ 8.0 Hz); 7.71 (d, 1H, H₅); 7.91 (t, 1H, H_7,J_7e8¼ 8.0 Hz);

8.53 (d, 1H, H₈); 10.39 (br s, 1H, NHe(CH₂)₄ePh). ¹³C NMR

DMSO-d₆)

2.37 (s, 3H, SeCH₃); 7.41 (dd, 2H, H₃þ H₅,

(100 MHz, DMSO-d₆) d: 14.1 (SCH₃); 28.3 (NHe(CH₂)₂eCH₂

eCH₂ePh); 29.2 (NHeCH₂eCH₂e(CH₂)₂ePh); 35.6 (NHe(CH₂)₃e

CH₂ePh); 42.4 (NHeCH₂e(CH₂)₃ePh); 112.0 (C₉); 123.3 (C₈); 124.9

J_3e2¼ J_5e6¼ 6.8 Hz, J_3-SeCH3¼ J_5-SeCH3¼1.7 Hz); 7.49 (dd, 2H,

H₂þ H₆, J_2-CHN¼ J_6-CHN¼ 1.4 Hz); 8.09 (s, 1H, CH]N); 11.25 (s, 1H,

OH). Elemental Analysis for C₈H₈ONSe Calcd/Found (%): C: 44.86/

44.65; H: 4.20/4.24; N: 6.54/6.42.

(C₅); 127.2 (C₇); 127.7 (C₄); 128.6 (C₂þ C₆); 129.6 (C₃þ C₅); 135.2

(C₆); 140.1 (C₁); 148.8 (C₁₀); 158.8 (C₄); 165.8 (C₂). MS (m/z %

4.1.9.1.3. 4-Methylselenobenzylamine. To

mixture of (E)-4-

abundance): 323 (M^þ, 100); 276 (30); 218 (91); 205 (32); 191 (41);

175 (23); 145 (29); 91 (38). Elemental Analysis for C₁₉H₂₁N₃S$1.65

HCl Calcd/Found (%): C: 59.49/59.40; H: 6.91/6.30; N: 10.95/10.50.

(methylseleno)benzaldehyde oxime (0.52 g, 2.43 mmol) in ethanol

(50 mL) was added zinc (1.75 g, 27.00 mmol) in water (10 mL). 10%

Hydrochloric acid (10 mL) was added slowly with vigorous agita-

tion and the mixture was heated under reﬂux for 12 h. The mixture

was cooled to room temperature and 20% NaOH (10 mL) was added

and the product was extracted with dichloromethane (3 ꢂ 20 mL).

The organic phase was washed with water, dried over Na₂SO₄and

concentrated to dryness. The residue was used without puriﬁca-

4.1.13. 4-(5-Hydroxypentyl)amino-2-methylthioquinazoline (2k)

From 4-chloro-2-methylthioquinazoline and 5-hydroxy-1-

pentylamino. Yield: 33%; mp 140e141 ^ꢀC. IR (KBr) cm^ꢁ1: 3348 (br

s, OeH); 2979e2929 (m, CeH); 1625e1579 (s, C]N). ¹H NMR

(400 MHz, DMSO-d₆) d: 1.37e1.41 (m, 2H, NHeCH₂eCH₂eCH₂);

tion. A yellowish liquid (0.24 g, 49%) was obtained. IR (KBr) cm^ꢁ1

3272 (m, NeH); 3003 (m, CeH); 2923 (m, CeH); 1589 (s, C]N). ¹H

NMR (400 MHz, DMSO-d₆) : 2.32 (s, 3H, SeCH₃); 3.44 (br s, 2H,

H₂O þ NH₂); 3.68 (s, 2H, CH₂eNH₂); 7.25 (d, 2H, H₃þ H₅;

J_3e2¼ J_5e6¼ 8.0 Hz); 7.35 (d, 2H, H₂þ H₆); Elemental Analysis for

C₈H₁₁NSe Calcd/Found (%): C: 48.00/48.25; H: 5.50/5.34; N: 7.00/

6.79.

1.44e1.49 (m, 2H, NHeCH₂eCH₂eCH₂eCH₂); 1.61e1.66 (m, 2H,

eCH₂eCH₂eOH); 2.50 (s, 3H, SCH₃,); 3.38e3.42 (m, 2H, NHeCH₂,

J_NHeCH2¼ 5.8 Hz); 3.48e3.52 (m, 2H, CH₂OH); 4.38 (t, 1H, CH₂OH);

7.37 (t, 1H, H₆); 7.51, (d, 1H, H₅); 7.68 (t, 1H, H₇); 8.16 (d, 1H, H₈);

8.31, (t, 1H, NHeCH₂). ¹³C NMR (100 MHz, DMSO-d₆)

d: 14.2 (SCH₃);

24.0 (NHe(CH₂)₂eCH₂e(CH₂)₂eOH); 29.2 (NHeCH₂eCH₂e

(CH₂)₃eOH); 33.1 (NHe(CH₂)₃eCH₂eCH₂eOH); 41.4 (NHeCH₂e

(CH₂)₄eOH); 61.5 (NHe(CH₂)₄eCH₂eOH); 113.8 (C₉); 123.8 (C₈);

124.4 (C₅); 126.9 (C₇); 134.6 (C₆); 150.5 (C₁₀); 159.4 (C₄); 167.8 (C₂).

MS (m/z % abundance): 205 (81); 191 (100); 175 (56); 160 (64); 145

(68); 129 (37); 103 (40); 91 (18); 77 (10). Elemental Analysis for

C₁₄H₁₉N₃SO$0.1 HCl Calcd/Found (%): C: 59.86/59.78; H: 6.77/6.60;

N: 14.96/14.84.

4.1.10. 2-Methylthio-4-(2-phenylethyl)aminoquinazoline (2h)

From 4-chloro-2-methylthioquinazoline and 2-phenylethy-

lamine. Yield: 43%; mp 60e61 ^ꢀC. IR (KBr) cm^ꢁ1: 3248 (m, NeH);

3063e3024 (m, CeH); 2925 (m, CeH); 1563 (s, C]N). ¹H NMR

(400 MHz, DMSO-d₆)

d: 2.59 (s, 3H, SCH₃); 2.99 (t, 2H,

CH₂eCH₂ePh); 3.79 (m, 2H, CH₂eCH₂ePh); 7.26 (m, 5H, Ph); 7.46,

(t, 1H, H₆, J_6e5¼ J_6e7¼ 8.0 Hz); 7.57 (d, 1H, H₅); 7.77 (t, 1H, H_7,

J_7e8¼ 8.0 Hz); 8.27 (d, 1H, H₈); 9.21, (br s, 1H, NHeCH₂eCH₂ePh).

4.1.14. 2-Pentylthio-4-(4⁰-methylselenobenzyl)aminoquinazoline

(2l)

From 4-chloro-2-pentylthioquinazoline and 4-methylseleno-

benzylamine. Yield: 17%; mp 179e180 ^ꢀC. IR (KBr) cm^ꢁ1: 3287 (m,

NeH); 3111 (m, CeH); 2967e2899 (m, CeH); 1595 (s, C]N). ¹H

¹³C NMR (100 MHz, DMSO-d₆)

14.9 (SCH₃); 35.0

(NHeCH₂eCH₂ePh); 43.7 (NHeCH₂eCH₂ePh); 113.1 (C₉); 124.3

(C₈); 124.9 (C₅); 126.3 (C₇); 129.0 (C₄); 129.5 (C₂þ C₆); 129.9

(C₃þ C₅); 134.9 (C₆); 140.0 (C₁); 147.7 (C₁₀); 159.2 (C₄); 167.2 (C₂).

MS (m/z % abundance): 295 (M^þ, 37); 204 (45); 191 (100); 175 (27);

160 (18); 145 (27); 129 (14); 91 (17). Elemental Analysis for

C₁₇H₁₇N₃S$0.8 HCl Calcd/Found (%): C: 62.89/62.92; H: 5.83/5.59;

N: 12.80/12.95.

NMR (400 MHz, DMSO-d₆) d: 0.82 (t, 3H, (CH₂)₄eCH₃); 1.22 (m, 4H,

(CH₂)₂e(CH₂)₂eCH₃); 1.56 (q, 2H, CH₂eCH₂e(CH₂)₂eCH₃); 2.32 (s,

3H, SeCH₃); 3.11 (t, 2H, CH₂e(CH₂)₃eCH₃); 4.82 (br s, 2H, CH₂e Ph);

7.28 (d, 2H, H₃þ H₅, J_3e2¼ J_5e6¼ 8.1 Hz); 7.37 (d, 2H, H₂þ H₆); 7.54

(t, 1H, H₆, J_6e5¼ J_6e7¼ 7.9 Hz); 7.62 (d, 1H, H₅); 7.84 (t, 1H, H₇,

E. Moreno et al. / European Journal of Medicinal Chemistry 47 (2012) 283e298

293

J_7e8¼ 7.9 Hz); 8.46 (d, 1H, H₈); 10.11 (s, 1H, NHeCH₂). ¹³C NMR

NMR

(400 MHz,

DMSO-d₆)

1.36e1.38

(m,

2H,

(100 MHz, DMSO-d₆)

: 7.3 (SeCH₃); 14.5 (Se(CH₂)₄eCH₃); 22.5

NHeCH₂eCH₂eCH₂); 1.41e1.49 (m, 2H, NHeCH₂eCH₂eCH₂eCH₂);

1.60e1.68 (m, 2H, CH₂eCH₂eOH); 2.51 (s, 3H, SCH₃); 3.38e3.42 (m,

2H, NHeCH₂); 3.49e3.56 (m, 2H, CH₂OH); 4.36 (t, 1H, CH₂eNH,

J_CH2eNH¼ 5.1 Hz); 7.39 (dd, 1H, H₆, J_6e5¼ 8.2 Hz, J_6e7¼4.4 Hz); 8.57

(t, 1H, CH₂OH, J_CH2eOH¼ 5.3 Hz); 8.61 (dd, 1H, H₅, J_5e7¼ 1.9 Hz);

(Se(CH₂)₃eCH₂eCH₃); 29.2 (SeCH₂eCH₂e(CH₂)₂eCH₃); 29.9

(Se(CH₂)₂eCH₂eCH₂eCH₃); 31.2 (SeCH₂e(CH₂)₃eCH₃); 44.1

(CH₂); 112.8 (C₉); 124.8 (C₈); 125.5 (C₅); 126.9 (C₇); 129.3 (C₃þ C₅);

130.8 (C₂þ C₆); 135.4 (C₆); 136.7 (C₁); 138.2 (C₄); 147.5 (C₁₀);

159.3 (C₄); 166.4 (C₂). MS (m/z % abundance): 430 (M^þ, 100); 384

(26); 361 (57); 200 (11); 185 (76); 170 (31); 91 (12). Elemental

Analysis for C₂₁H₂₅N₃SSe$0.3 HI Calcd/Found (%): C: 53.80/53.75;

H: 5.34/4.76; N: 8.60/8.40.

8.85 (dd, 1H, H₇). ¹³C NMR (100 MHz, DMSO-d₆)

d: 14.7 (SCH₃); 24.2

(NHe(CH₂)₂eCH₂e(CH₂)₂eOH); 29.8 (NHeCH₂eCH₂e(CH₂)₃e

OH); 32.9 (NHe(CH₂)₃eCH₂eCH₂eOH); 42.3 (NHeCH₂e(CH₂)₄e

OH); 62.3 (NHe(CH₂)₄eCH₂eOH); 102.4 (C₁₀); 121.6 (C₆); 132.9

(C₅); 156.1 (C₇); 158.8 (C₉); 159.6 (C₄); 171.2 (C₂). MS (m/z %

abundance): 281 (M^þ, 51); 191 (31); 160 (30); 103 (50); 91 (100); 77

(44). Elemental Analysis for C₁₃H₈N₄OS Calcd/Found (%): C: 56.11/

56.66; H: 6.47/6.63; N: 20.14/20.03.

4.1.15. 4-Benzylamino-2-methylthiopyrido[2,3-d]pyrimidine (2m)

From 4-chloro-2-methylthiopyrido[2,3-d]pyrimidine and ben-

zylamine. Yield: 94%; mp 194e195 ^ꢀC. IR (KBr) cm^ꢁ1: 3289 (m,

NeH); 2922 (m, CeH); 1568 (s, C]N). ¹H NMR (400 MHz, DMSO-

d₆)

: 2.46 (s, 3H, SCH₃); 4.76 (d, 2H, CH₂eNH, J_CH2eNH¼ 5.1 Hz);

4.1.19. General procedure for the preparation 3a and b

7.25e7.39 (m, 5H, Ph); 7.43 (dd, 1H, H₆, J_6e5¼ 8.2 Hz, J_6e7¼4.4 Hz);

8.66 (dd, 1H, H₅, J_5e7¼ 1.8 Hz); 8.88 (dd, 1H, H₇); 9.21, (t, 1H,

To a solution of 2,4-dichloroquinazoline or 2,4-dichloropyrido

[2,3-d]pyrimidine (3.7 mmol) in ethanol (50 mL) was added sele-

nourea (9.85 mmol). The reaction mixture was heated under reﬂux

for 5 h. The solid was ﬁltered off, washed with water (3 ꢂ15 mL)

and recrystallized from the appropriate solvent.

NHeCH₂). ¹³C NMR (100 MHz, DMSO-d₆)

d: 14.4 (SCH₃); 44.9 (CH₂);

108.5 (C₁₀); 120.5 (C₆); 128.2 (C₄); 128.6 (C₂þ C₆); 129.6

(C₃þ C₅); 133.6 (C₅); 139.6 (C₁); 156.3 (C₇); 159.1 (C₉); 160.4 (C₄);

172.0 (C₂). MS (m/z % abundance): 282 (M^þ, 39); 235 (11); 191 (40);

145 (19%); 103 (68%); 91 (100%); 77 (36%). Elemental Analysis for

C₁₅H₁₄N₄S Calcd/Found (%): C: 63.83/63.52; H: 4.96/5.15; N: 19.86/

19.68.

4.1.19.1. 2,4-Dihydroselenoquinazoline (3a). From 2,4-dichloro-

quinazoline and selenourea. Yield: 75%; mp >300 ^ꢀC. IR (KBr)

cm^ꢁ1: 3104 (m, CeH); 2941 (m, CeH); 1611 (s, C]N). ¹H NMR

(400 MHz, DMSO-d₆)

J_7e8¼ J_7e6¼ 7.9 Hz); 8.31 (d, 1H, H₈); 13.75 (s, 1H, SeH₍₄₎); 14.55 (s,

1H, SeH₍₂₎). ¹³C NMR (100 MHz, DMSO-d₆)

: 124.6 (C₉); 125.3 (C₈);

: 7.35e7.42 (m, 2H, H₅þ H₆); 7.80 (t, 1H, H₇,

4.1.16. 4-(4⁰-Methoxybenzyl)amino-2-methylthiopyrido[2,3-d]

pyrimidine (2n)

From 4-chloro-2-methylthiopyrido[2,3-d]pyrimidine and 4-

127.4 (C₅); 128.9 (C₇); 135.4 (C₆); 149.3 (C₁₀); 164.5 (C₄); 175.8 (C₂).

MS (m/z % abundance): 289 (M^þ, 48); 209 (47); 160 (52); 129 (100);

103 (38); 80 (32). Elemental Analysis for C₈H₆N₂Se₂Calcd/Found

(%): C: 42.86/43.12; H: 3.12/3.42; N: 18.75/18.94.

methoxybenzylamine. Yield: 30%; mp 169e170 ^ꢀC. IR (KBr) cm^ꢁ1

3284 (m, NeH); 2929 (CeH); 1575 (s, C]N). ¹H NMR (400 MHz,

DMSO-d₆)

CH₂eNH,

: 2.50 (s, 3H, SCH₃); 3.72 (s, 3H, OCH₃); 4.67 (d, 2H,

J_CH2eNH¼ 5.7 Hz);

6.90

(d,

2H,

H₃þ H₅,

J_{3 e2}¼ J_{5 e6}¼ 8.7 Hz); 7.31 (d, 2H, H₂þ H₆); 7.41 (dd, 1H, H₆,

4.1.19.2. 2,4-Dihydroselenopyrido[2,3-d]pyrimidine (3b). From 2,4-

dichloropyrido[2,3-d]pyrimidine and selenourea. Yield: 46%; mp

>300 ^ꢀC. IR (KBr) cm^ꢁ1: 3044 (m, CeH); 1545 (s, C]N). ¹H NMR

J_6e5¼ 8.2 Hz, J_6e7¼4.4 Hz); 8.65 (dd, 1H, H₅, J_5e7¼ 1.9 Hz); 8.85,

(dd, 1H, H₇); 9.11 (t, 1H, NHeCH₂). ¹³C NMR (100 MHz, DMSO-d₆)

14.4 (SCH₃); 44.3 (CH₂); 55.9 (OCH₃); 108.5 (C₁₀); 114.6 (C₃þ C₅);

(400 MHz, DMSO-d₆)

7.39 (ddd, 1H, H₆, J_6e5¼ 8.0 Hz,

120.9 (C₆); 129.7 (C₂þ C₆); 131.5 (C₁); 133.5 (C₅); 156.3 (C₇); 159.2

J_6e7¼4.7 Hz, J_6-SeH(4)¼ 0.5); 8.55 (ddd, 1H, H₅, J_5e7¼ 1.9 Hz, J_5-

(C₄þ C₉); 160.3 (C₄); 172.0 (C₂). MS (m/z % abundance): 312 (M ,

¼ 0.6 Hz); 8.83 (ddd,1H, H₇, J_7-SeH(4)¼ 0.6 Hz); 14.18 (s, 1H,

SeH(4)

13); 191 (21); 136 (22); 121 (100); 103 (58); 91 (31); 77 (36).

Elemental Analysis for C₁₆H₁₆N₄OS Calcd/Found (%): C: 61.54/61.31;

H: 5.13/5.43; N: 17.94/18.35.

SeH₍₄₎); 14.69 (s, 1H, SeH₍₂₎). ¹³C NMR (100 MHz, DMSO-d₆)

d: 120.7

(C₁₀); 121.8 (C₆); 131.4 (C₅); 147.6 (C₇); 155.7 (C₉); 156.3 (C₄); 163.6

(C₂). MS (m/z % abundance): 290 (M^þ, 24); 210 (26); 160 (48); 130

(52); 103 (100); 91 (33); 77 (67); 59 (37); 50 (32). Elemental

Analysis for C₇H₅N₃Se₂$1.35 HCl Calcd/Found (%): C: 24.79/24.42;

H: 1.87/1.59; N: 12.39/12.38.

4.1.17. 4-(4⁰-Methylthiobenzyl)amino-2-methylthiopyrido[2,3-d]

pyrimidine (2o)

From 4-chloro-2-methylthiopyrido[2,3-d]pyrimidine and 4-

methylthiobenzylamine. Yield: 4%; mp 157e158 ^ꢀC. IR (KBr)

cm^ꢁ1: 3229 (m, NeH); 2921 (m, CeH); 1579 (s, C]N). ¹H NMR

4.1.20. General procedure for the preparation 3cef

a solution of the corresponding 2,4-dihydroselenoqui-

(400 MHz, DMSO-d₆)

: 2.44 (s, 3H, PheSCH₃); 2.48 (s, 3H,

nazoline or 2,4-dihydroselenopyrido[2,3-d]pyrimidine (2.8 mmol)

in 0.4 N NaOH (40 mL) was added the corresponding alkyl iodide

(6.11 mmol) and the mixture was heated under reﬂux for 24 h. The

reaction mixture was cooled and acetic acid was added to give pH

5e6. The precipitate was ﬁltered off and recrystallized.

heteSCH₃); 4.71 (d, 2H, CH₂eNH, J_CH2eNH¼ 5.4 Hz); 7.18 (d, 2H,

H₃þ H₅, J_{3 e2}¼ J_{5 e6}¼ 8.6 Hz); 7.33 (d, 2H, H₂þ H₆); 7.46 (dd,1H,

H₆, J_6e5¼ 4.4 Hz, J_6e7¼ 8.3 Hz); 8.68 (dd, 1H, H_5,J_5e7¼ 1.8 Hz); 8.78

(dd, 1H, H₇); 9.25 (t, 1H, NHeCH₂). ¹³C NMR (100 MHz, DMSO-d₆)

14.9 (heteSCH₃); 16.2 (PheSCH₃); 44.6 (CH₂); 103.4 (C₁₀); 121.9

(C₆); 126.3 (C₃þ C₅); 127.5 (C₂þ C₆); 131.1 (C₁); 133.4 (C₅); 138.6

4.1.20.1. 2,4-Dimethylselenoquinazoline (3c). From 2,4-dihydrosele-

noquinazoline and methyl iodide. Yield: 61%; mp 61e63 ^ꢀC. IR (KBr)

cm^ꢁ1: 3160 (m, CeH); 2982e2858 (m, CeH); 1608 (s, C]N). ¹H

(C₄); 156.6 (C₇); 158.8 (C₉); 159.5 (C₄); 171.8 (C₂). MS (m/z %

abundance): 326 (M^þ, 39); 282 (26); 191 (49); 137 (64); 121 (89);

103 (100); 91 (86); 77 (54). Elemental Analysis for C₁₆H₁₆N₄S₂$0.45

HCl Calcd/Found (%): C: 55.64/55.45; H: 4.64/4.99; N: 16.26/16.50.

NMR (400 MHz, DMSO-d₆) d: 2.53 (s, 3H, SeCH₃₍₄₎); 2.56 (s, 3H,

SeCH₃₍₂₎); 7.61 (ddd, 1H, H₇, J_7e8¼ 8.2 Hz, J_7e6¼ 7.0 Hz,

J_7e5¼1.2 Hz); 7.77 (dd, 1H, H₈, J_8e6¼ 1.2 Hz); 7.93 (ddd, 1H, H₆,

4.1.18. 4-(5-Hydroxypentyl)amino-2-methylthiopyrido[2,3-d]

pyrimidine (2p)

From 4-chloro-2-methylthiopyrido[2,3-d]pyrimidine and 5-

hydroxy-1-pentylamine. Yield: 14%; mp 135e136 ^ꢀC. IR (KBr)

cm^ꢁ1: 3302 (m, NeH); 2933 (m, CeH); 1600e1577 (s, C]N). ¹H

J_6e5¼ 7.9 Hz); 7.95 (dd,1H, H₅). ¹³C NMR (100 MHz, DMSO-d₆)

d: 6.7

(SeCH₃₍₄₎); 7.6 (SeCH₃₍₂₎); 124.2 (C₉); 126.1 (C₈); 127.9 (C₅); 129.4

(C₇); 135.7 (C₆); 148.7 (C₁₀); 164.6 (C₄); 170.8 (C₂). MS (m/z %

abundance): 318 (M^þ, 100); 303 (32); 238 (36); 223 (79); 208 (44);

143 (67); 129 (58); 121 (21); 103 (47); 95 (36); 77 (20); 51 (8).

294

E. Moreno et al. / European Journal of Medicinal Chemistry 47 (2012) 283e298

Elemental Analysis for C₁₀H₁₀N₂Se₂Calcd/Found (%): C: 37.97/

37.86; H: 3.16/3.03; N: 8.86/8.72.

(100 MHz, DMSO-d₆) d: 116.6 (C₉); 123.8 (C₈); 126.7 (C₅); 128.9 (C₇);

133.8 (C₆); 149.1 (C₁₀); 161.8 (C₄); 173.1 (C₂). MS (m/z % abundance):

225 (M^þ, 15); 191 (24); 137 (79); 121 (83); 103 (34); 91 (100); 77

(53); 69 (58); 57 (84). Elemental Analysis for C₈H₇N₃Se Calcd/Found

(%): C: 42.86/43.12; H: 3.12/3.42; N: 18.75/18.94.

4.1.20.2. 2,4-Dipentylselenoquinazoline (3d). From 2,4-dihydros-

elenoquinazoline and pentyl iodide. Yield: 45%; oil. IR (KBr)

cm^ꢁ1: 3156 (m, CeH); 2987e2862 (m, CeH); 1638 (s, C]N). ¹H

NMR (400 MHz, DMSO-d₆)

: 0.88 (t, 6H, (CH₂)₄eCH_3(2,4)); 1.38 (m,

4.1.21.2. 4-Amino-2-hydroselenopyrido[2,3-d]pyrimidine (4b). From

4-amino-2-chloropyrido[2,3-d]pyrimidine and selenourea. Yield:

68%; mp >300 ^ꢀC. IR (KBr) cm^ꢁ1: 3337e3278 (m, NeH); 3152 (m,

8H, (CH₂)₂e(CH₂)₂eCH_3(2,4)); 1.79 (q, 4H, CH₂eCH₂e(CH₂)₂e

CH_3(2,4)); 3.24 (t, 2H, CH₂e(CH₂)₃eCH_3(2,4)); 3.34 (t, 2H,

CH₂e(CH₂)₃eCH_3(2,4)); 7.60 (ddd, 1H, H₆, J_6e5¼ 8.1 Hz, J_6e7¼ 7.0 Hz,

J_6e8¼ 1.2 Hz); 7.74 (dd, 1H, H₅, J_5e7¼ 1.2 Hz); 7.89e7.93, (m, 2H,

CeH); 1647 (s, C]N). ¹H NMR (400 MHz, DMSO-d₆)

d: 7.40 (m, 1H,

H₆); 8.51 (d, 1H, H₅); 8.73 (m, 3H, H₇þ NH₂); 13.17 (s, 1H, SeH). ¹³

H₇þ H₈). ¹³C NMR (100 MHz, DMSO-d₆)

d: 14.1 (See(CH₂)₄e

NMR (100 MHz, DMSO-d₆) d: 106.4 (C₁₀); 121.1 (C₆); 134.8 (C₅);

CH_3(2,4)); 22.5 (See(CH₂)₃eCH₂eCH_3(2,4)); 26.6 (SeeCH₂eCH₂e

(CH₂)₂eCH_3(2,4)); 30.2 (SeeCH₂e(CH₂)₃eCH_3(2,4)); 32.3 (See

(CH₂)₂eCH₂eCH₂eCH_3(2,4)); 124.3 (C₉); 126.1 (C₈); 127.8 (C₅); 129.7

(C₇); 135.7 (C₆); 149.0 (C₁₀); 164.6 (C₄); 171.8 (C₂). MS (m/z %

abundance): 429 (M^þ, 16); 360 (26); 290 (33); 209 (61); 145 (25);

129 (100); 103 (36); 69 (39); 55 (28). Elemental Analysis for

C₁₈H₂₆N₂Se₂Calcd/Found (%): C: 50.47/50.42; H: 6.07/6.28; N: 6.54/

6.19.

153.2 (C₇); 156.2 (C₉); 159.0 (C₄); 181.7 (C₂). MS (m/z % abundance):

235 (M^þ, 14); 191 (27); 137 (88); 121 (77); 103 (38); 91 (100); 77

(54); 65 (43); 55 (59). Elemental Analysis for C₇H₆N₄Se$0.25 HCl

Calcd/Found (%): C: 35.84/35.80; H: 2.56/2.97; N: 23.90/24.15.

4.1.22. General procedure for compounds 4cef

To a solution of the corresponding 4a or 4b (4 mmol) in ethanol

(25 mL) was added the corresponding alkyl iodide (6 mmol) and

the mixture was heated under reﬂux for 1.5 h. The solvent was

removed in vacuo. The residue was treated with water (30 mL) and

extracted with dichloromethane (3 ꢂ 25 mL). The combined

organic layers were dried over Na₂SO₄, ﬁltered and concentrated.

The corresponding solids were puriﬁed in the appropriate solvent.

4.1.20.3. 2,4-Dimethylselenopyrido[2,3-d]pyrimidine (3e). From 2,4-

dihydroselenopyrido[2,3-d]pyrimidine and methyl iodide. Yield:

23%; mp 298e299 ^ꢀC. IR (KBr) cm^ꢁ1: 1592 (s, C]N). ¹H NMR

(400 MHz, DMSO-d₆)

7.61 (dd, 1H, H₆, J_6e5¼ 8.2 Hz, J_6e7¼4.4 Hz); 8.44 (dd, 1H, H₅); 9.11

(dd, 1H, H₇). ¹³C NMR (100 MHz, DMSO-d₆)

: 9.1 (SeCH₃₍₄₎); 9.6

d: 2.55 (s, 3H, SeCH₃₍₄₎); 2.59 (s, 3H, SCH₃₍₂₎);

4.1.22.1. 4-Amino-2-methylselenoquinazoline (4c). From 4a and

(SeCH₃₍₂₎); 121.1 (C₁₀); 121.9 (C₆); 131.0 (C₅); 148.5 (C₇); 155.0 (C₉);

156.4 (C₄); 157.8 (C₂). MS (m/z % abundance): 324 (M^þ, 16); 254

(50); 179 (30); 156 (32); 140 (100); 127 (51); 116 (75); 76 (44); 50

(23). Elemental Analysis for C₉H₉N₃Se₂$0.05 HI Calcd/Found (%): C:

33.39/33.08; H: 2.80/2.74; N: 12.99/13.05.

methyl iodide. Yield: 4%; mp 213e214 ^ꢀC. IR (KBr) cm^ꢁ1

3386e3306 (m, NeH); 3125 (m, CeH); 2927 (m, CeH); 1646 (s, C]

N). ¹H NMR (400 MHz, DMSO-d₆)

: 2.40 (s, 3H, SeCH₃); 7.40 (ddd,

1H, H₆, J_6e5¼ 8.2 Hz, J_6e7¼ 7.0 Hz, J_6e8¼ 1.3 Hz); 7.52 (dd, 1H, H₅,

J_5e7¼ 1.2 Hz); 7.71 (ddd, 1H, H₇, J_7e8¼ 8.2 Hz); 7.89 (s, 2H, NH₂);

8.14 (dd,1H, H₈). ¹³C NMR (100 MHz, DMSO-d₆)

d: 8.1 (SeCH₃); 116.3

4.1.20.4. 2,4-Dipentylselenopyrido[2,3-d]pyrimidine (3f). From 2,4-

dihydroselenopyrido[2,3-d]pyrimidine and pentyl iodide. Yield:

19%; oil. IR (KBr) cm^ꢁ1: 1594 (m, C]N). ¹H NMR (400 MHz, DMSO-

(C₉); 122.3 (C₈); 126.4 (C₅); 128.5 (C₇); 134.1 (C₆); 149.6 (C₁₀); 161.3

(C₄); 163.1 (C₂). MS (m/z % abundance): 239 (M^þ, 64); 159 (100);

145 (64); 129 (30); 117 (36); 91 (31). Elemental Analysis for

C₉H₉N₃Se Calcd/Found (%): C: 45.38/45.60; H: 3.78/3.78; N: 17.65/

17.39.

d₆)

0.86 (t, 6H, (CH₂)₄eCH_3(2,4)); 1.35 (m, 8H,

(CH₂)₂e(CH₂)₂eCH_3(2,4)); 1.70 (q, 4H, CH₂eCH₂e(CH₂)₂eCH_3(2,4));

3.19 (t, 2H, CH₂e(CH₂)₃eCH₃₍₄₎); 3.38 (t, 2H, CH₂e(CH₂)₃eCH₃₍₂₎);

6.57 (dd, 1H, H₆, J_6e5¼ 8.2 Hz, J_6e7¼4.2 Hz); 7.76 (dd, 1H, H₅,

4.1.22.2. 4-Amino-2-pentylselenoquinazoline (4d). From 4a and

pentyl iodide. Yield: 4%; mp 92e94 ^ꢀC. IR (KBr) cm^ꢁ1: 3312 (m,

NeH); 3135 (m, CeH); 2949e2862 (m, CeH); 1646 (f, C]N). ¹H

J_5e7¼ 1.7 Hz); 8.40 (dd, 1H, H₇). ¹³C NMR (100 MHz, DMSO-d₆)

14.2 (See(CH₂)₄eCH_3(2,4)); 22.5 (See(CH₂)₃eCH₂eCH_3(2,4)); 27.0

(SeeCH₂eCH₂e(CH₂)₂eCH_3(2,4)); 30.3 (SeeCH₂e(CH₂)₃eCH_3(2,4));

32.1 (See(CH₂)₂eCH₂eCH₂eCH_3(2,4)); 121.4 (C₁₀); 122.3 (C₆); 131.4

(C₅); 148.4 (C₇); 155.4 (C₉); 156.9 (C₄); 160.2 (C₂). MS (m/z %

abundance): 430 (M^þ, 34); 287 (100); 179 (45); 156 (32); 103 (23);

77 (31). Elemental Analysis for C₁₇H₂₅N₃Se₂Calcd/Found (%): C:

47.55/47.62; H: 5.83/5.62; N: 9.79/9.85.

NMR (400 MHz, DMSO-d₆) d: 0.88 (t, 3H, (CH₂)₄eCH₃); 1.36 (m, 4H,

(CH₂)₂e(CH₂)₂eCH₃); 1.76 (q, 2H, CH₂eCH₂e(CH₂)₂eCH₃); 3.13 (t,

2H, CH₂e(CH₂)₃eCH₃); 7.38 (ddd, 1H, H₆, J_6e5¼ 8.2 Hz,

J_6e7¼ 7.0 Hz, J_6e8¼ 0.8 Hz); 7.49 (dd, 1H, H₅, J_5e7¼ 1.0 Hz); 7.70,

(ddd, 1H, H₇, J_7e8¼ 8.0 Hz); 7.83 (br s, 2H, NH₂); 8.12 (dd, 1H, H₈).

¹³C NMR (100 MHz, DMSO-d₆)

d: 14.5 (See(CH₂)₄eCH₃); 21.7

(See(CH₂)₃eCH₂eCH₃); 26.6 (SeeCH₂eCH₂e(CH₂)₂eCH₃); 30.4

(SeeCH₂e(CH₂)₃eCH₃); 32.5 (See(CH₂)₂eCH₂eCH₂eCH₃); 113.7

(C₉); 124.7 (C₈); 127.5 (C₅); 129.8 (C₇); 133.9 (C₆); 150.9 (C₁₀); 161.5

(C₄); 165.3 (C₂). MS (m/z % abundance): 295 (M^þ, 27); 252 (24); 239

(44); 225 (90); 214 (49); 159 (58); 145 (100); 129 (28); 121 (81); 103

(24); 91 (37). Elemental Analysis for C₁₃H₁₇N₃Se Calcd/Found (%): C:

53.07/53.50; H: 5.78/5.71; N: 14.28/13.88.

4.1.21. General procedure for compounds 4a and b

A suspension of 2,4-dichloroquinazoline or 2,4-dichloropyrido

[2,3-d]pyrimidine (5 mmol) in 25% ammonia (30 mL) was heated

under reﬂux for 1.5 h. The resulting precipitate was ﬁltered off and

washed with water (4 ꢂ15 mL). The corresponding intermediate 4-

amino-2-chloroquinazoline or pyrido[2,3-d]pyrimidine was used

without puriﬁcation and was treated with selenourea in ethanol

(20 mL) in a stoichiometric ratio of 1:1.2, respectively. The mixture

was heated during 4 h and then cooled. The resulting precipitate

was ﬁltered off and recrystallized.

4.1.22.3. 4-Amino-2-methylselenopyrido[2,3-d]pyrimidine

(4e). From 4b and methyl iodide. Yield: 47%; mp 279e280 ^ꢀC. IR

(KBr) cm^ꢁ1: 3347e3304 (m, NeH); 3120 (m, CeH); 2928 (m, CeH);

1647 (s, C]N). ¹H NMR (400 MHz, DMSO-d₆)

d: 2.49 (s, 3H, SeCH₃);

4.1.21.1. 4-Amino-2-hydroselenoquinazoline (4a). From 4-amino-2-

chloroquinazoline and selenourea. Yield: 16%; mp 280e282 ^ꢀC. IR

(KBr) cm^ꢁ1: 3345 (m, NeH); 3141 (m, CeH); 1669 (s, C]N). ¹H NMR

7.58 (dd, 1H, H₆, J_6e5¼ 8.0 Hz, J_6e7¼4.5 Hz); 8.71 (dd, 1H, H₅,

J_5e7¼ 1.5 Hz); 8.90 (br s, 2H, NH₂); 8.92 (dd, 1H, H₇). ¹³C NMR

(100 MHz, DMSO-d₆) d: 6.7 (SeCH₃); 108.4 (C₁₀); 121.0 (C₆); 134.3

(400 MHz, DMSO-d₆)

: 7.69 (ddd, 1H, H₆); 8.00 (m, 2H, H₅þ H₆);

(C₅); 156.7 (C₇); 159.3 (C₉); 161.4 (C₄); 169.9 (C₂). MS (m/z %

8.44 (d, 1H, H₈); 9.48 (s, 1H, NH₂); 9.28 (s, 1H, NH₂). ¹³C NMR

abundance): 253 (M^þ, 6); 240 (45); 191 (24); 160 (100); 145 (42);

E. Moreno et al. / European Journal of Medicinal Chemistry 47 (2012) 283e298

295

137 (29); 121 (49); 103 (60); 91 (64); 76 (53); 64 (29); 55 (42).

Elemental Analysis for C₈H₈N₄Se$0.1 HI Calcd/Found (%): C: 38.14/

38.33; H: 3.18/3.27; N: 22.25/22.40.

42.00 mmol) in toluene at 25% (15 mL) was added dropwise over

a period of 1 h. After the addition was complete, the mixture was

heated under reﬂux and worked up as described above for 6a. The

crude residue was puriﬁed by silica gel column chromatography

(EtOAc/hexane 5:95) to give 0.85 g (15%) of 6b as an oil. IR (KBr)

cm^ꢁ1: 2927 (m, CeH); 2139 (s, eN]C]Se); ¹H NMR (400 MHz,

4.1.22.4. 4-Amino-2-pentylselenopyrido[2,3-d]pyrimidine (4f). From

4b and pentyl iodide. Yield: 73%; mp 183e184 ^ꢀC. IR (KBr) cm^ꢁ1

2946e2917 (m, CeH); 1610 (s, C]N). ¹H NMR (400 MHz, DMSO-d₆)

: 0.87 (t, 3H, (CH₂)₄eCH₃); 1.35 (m, 4H, (CH₂)₂e(CH₂)₂eCH₃); 1.77

DMSO-d₆) d: 3.73 (s, 3H, OCH₃); 3.88 (s, 2H, CH₂ePh); 6.88 (d, 2H,

H₃þ H₅, J_3e2¼ J_5e6¼ 8.7 Hz); 7.17 (d, 2H, H₂þ H₆); Elemental

Analysis for C₉H₉NOSe Calcd/Found (%): C: 47.79/47.51; H: 3.98/

4.22; N: 6.19/6.09.

(q, 2H, CH₂eCH₂e(CH₂)₂eCH₃); 3.16 (t, 2H, CH₂e(CH₂)₃eCH₃); 7.75

(dd, 1H, H₆, J_6e5¼ 8.0 Hz, J_6e7¼4.2 Hz); 8.23 (br s, 2H, NH₂); 8.60

(dd, 1H, H₅, J_5e7¼ 1.2 Hz); 8.87 (dd, 1H, H₇). ¹³C NMR (100 MHz,

DMSO-d₆)

: 14.7 (See(CH₂)₄eCH₃); 22.6 (See(CH₂)₃eCH₂eCH₃);

4.1.27. Preparation of 4-methylthiobenzylisoselenocyanate 6c

27.2 (SeeCH₂eCH₂e(CH₂)₂eCH₃); 30.4 (SeeCH₂e(CH₂)₃eCH₃);

32.0 (See(CH₂)₂eCH₂eCH₂eCH₃); 108.3 (C₁₀); 121.2 (C₆); 135.0

(C₅); 156.0 (C₇); 159.3 (C₉); 162.4 (C₄); 170.9 (C₂). MS (m/z %

abundance): 296 (M^þ, 26); 226 (100); 215 (48); 146 (49); 121 (79);

103 (47). Elemental Analysis for C₁₂H₁₆N₄Se$0.25 HI Calcd/Found

(%): C: 44.04/44.39; H: 4.89/5.09; N: 17.12/16.85.

To a mixture of 5c (4.25 g, 23.50 mmol) and triethylamine

(13.10 mL, 94.00 mmol) in dry toluene (50 mL) was added selenium

powder (2.78 g, 35.00 mmol). A solution of phosgene (3.70 mL,

35.00 mmol) in toluene at 25% (15 mL) was added dropwise over

a period of 1 h. After the addition was complete, the mixture was

heated under reﬂux and worked up as described above for 6a. The

crude residue was puriﬁed by silica gel column chromatography

(EtOAc/hexane 5:95) to give 0.85 g (15%) of 6c as an oil. IR (KBr)

cm^ꢁ1: 2927 (m, CeH); 2139 (s, N]C]Se); ¹H NMR (400 MHz,

4.1.23. Preparation of 4-methoxybenzylformamide 5b

a stirred solution of 4-methoxybenzylamine (3.93 mL,

30 mmol) was added ethyl formate (2.45 mL, 24.2 mmol). The

reaction mixture was stirred and heated at 150 ^ꢀC for 12 h. The

reaction mixture was cooled to room temperature and the

precipitate was collected by ﬁltration, dried and washed with ethyl

ether to give 5b as a white solid (4.4 g). Yield: 88%; mp 79e80 ^ꢀC; IR

(KBr) cm^ꢁ1: 3286 (s, NeH); 3012 (m, CeH); 2943e2834 (m, CeH);

DMSO-d₆) d: 3.73 (s, 3H, OCH₃); 3.88 (s, 2H, CH₂ePh); 6.88 (d, 2H,

H₃þ H₅, J_3e2¼ J_5e6¼ 8.7 Hz); 7.17 (d, 2H, H₂þ H₆); Elemental

Analysis for C₉H₉NOSe Calcd/Found (%): C: 47.79/47.51; H: 3.98/

4.22; N: 6.19/6.09.

4.1.28. General procedure for compounds 7aed

1645 (s, C]O). ¹H NMR (400 MHz, DMSO-d₆)

d: 3.73 (s, 3H, OCH₃);

The corresponding isoselenocyanate (2.40 mmol) was added

over 30 min to a stirred solution of 2-aminobenzonitrile or 2-

amino-3-cyanopyridine (2.40 mmol) in dry pyridine (30 mL).

After the addition was complete, the solution was stirred for 24 h at

100 oC. The solution was ﬁltered in order to remove the selenium

powder and then poured onto crushed ice and extracted with

dichloromethane (4 ꢂ15 mL). The combined organic layers were

dried over Na₂SO₄, ﬁltered and concentrated. The corresponding

solids were puriﬁed from the appropriate solvent.

4.22 (d, 2H, CH₂eNH); 6.89 (d, 2H, H₃þ H₅, J_3e2¼ J_5e6¼ 8.5 Hz);

7.19 (d, 2H, H₂þ H₆); 8.1 (s, 1H, HeC]O); 8.43 (s, 1H, CH₂eNH);

Elemental Analysis for C₉H₁₁NO₂Calcd/Found (%): C: 65.45/65.07;

H: 6.66/6.37; N: 8.48/8.29.

4.1.24. Preparation of 4-methylthiobenzylformamide 5c

a stirred solution of 4-methylthiobenzylamine (4.23 g,

27.60 mmol) was added dropwise ethyl formate (2.15 mL,

29.00 mmol). The reaction mixture was stirred at 150 oC for 12 h.

The reaction mixture was cooled to room temperature and the

precipitate was collected by ﬁltration, dried and washed with ethyl

ether to give 5c as a white solid (4.4 g). Yield: 85%; mp 87e89 ^ꢀC; IR

(KBr) cm^ꢁ1: 3282 (s, NeH); 3068 (m, CeH); 2918e2884 (m, CeH);

4.1.28.1. 4-Benzylamino-2-hydroselenoquinazoline (7a). From 2-

aminobenzonitrile and 6a. Yield: 31%; mp 193e194 ^ꢀC. IR (KBr)

cm^ꢁ1: 3259 (m, NeH); 3172e3120 (m, CeH); 2934 (m, CeH); 1615 (s,

C]N).¹H NMR (400 MHz, DMSO-d₆)

d: 5.98 (br s, 2H, CH₂eNH); 7.21

1652 (s, C]O). ¹H NMR (400 MHz, DMSO-d₆)

d: 2.45 (s, 3H, SCH₃);

(t, 1H, H₆); 7.29 (m, 5H, Ph); 7.40 (d, 1H, H₅); 7.60 (t, 1H, H₇); 8.09 (d,

4.25 (d, 2H, CH₂eNH); 7.25 (m, 4H, H₂þ H₃þ H₅þ H₆); 8.12 (s, 1H,

HeC]O); 8.49 (s, 1H, CH₂eNH); Elemental Analysis for C₉H₁₁NOS

Calcd/Found (%): C: 59.7/59.83; H: 6.08/5.81; N: 7.73/7.60.

1H, H₈,); 9.62 (s, 1H, NHeCH₂); 12.68 (br s, 1H, SeH). ¹³C NMR

(100 MHz, DMSO-d₆) d: 53.9 (CH₂); 116.1 (C₉); 124.8 (C₈); 125.9 (C₅);

126.4 (C₇); 127.7 (C₄); 128.1 (C₂þ C₆); 129.3 (C₃þ C₅); 134.2 (C₆);

137.6 (C₁); 149.7 (C₁₀); 155.4 (C₄); 175.2 (C₂). MS (m/z % abundance):

4.1.25. Preparation of benzylisoselenocyanate 6a

315 (M^þ, 13); 235 (30); 145 (25); 129 (34); 117 (23); 103 (36);

91 (100); 77 (29); 65 (34); 51 (24). Elemental Analysis for

C₁₅H₁₃N₃Se Calcd/Found (%): C: 57.32/56.88; H: 4.14/4.15; N: 13.37/

12.96.

To a cold mixture of 5a (1.35 g, 10.0 mmol) and triethylamine

(5.56 mL, 40.00 mmol) in dry toluene (50 mL) was added selenium

powder (1.18 g, 15.00 mmol). A solution of phosgene (1.57 mL,

15.00 mmol) in toluene at 25% (15 mL) was added dropwise over

a period of 1 h. After the addition was complete, the mixture was

heated under reﬂux for 24 h. The mixture was cooled and ﬁltered,

and the solvent was evaporated to yield the crude product, which

was puriﬁed by silica gel column chromatography (hexane 100%) to

afford 0.23 g (12%) of 6a as a viscous oil. IR (KBr) cm^ꢁ1: 2142 (s, N]

4.1.28.2. 2-Hydroseleno-4-(4⁰-methoxybenzyl)aminoquinazoline

(7b). From 2-aminobenzonitrile and 6b. Yield: 8%; mp 197e198 ^ꢀC.

IR (KBr) cm^ꢁ1: 3263 (m, NeH); 3172e3128 (m, CeH); 2924 (m,

CeH); 1605 (s, C]N). ¹H NMR (400 MHz, DMSO-d₆)

d: 3.70 (s, 3H,

OCH₃); 5.93 (br s, 2H, CH₂eNH); 6.84 (d, 2H, H₃þ H_{5 ,}

C]Se). ¹H NMR (400 MHz, DMSO-d₆)

d: 5.08 (s, 2H, CH₂ePh); 7.34

J_{3 e2}¼ J_{5 e6}¼ 8.4 Hz); 7.29 (t, 2H, H₆, J_6e5¼ J_6e7¼ 8.1 Hz); 7.33 (d,

(m, 3H, H₃þ H₄þ H₅); 7.43 (d, 2H, H₂þ H₆, J_2e3¼ J_6e5¼ 6.8 Hz);

Elemental Analysis for C₈H₇NSe Calcd/Found (%): C: 48.98/49.33; H:

3.57/3.96; N: 7.14/7.00.

2H, H₂þ H₆); 7.38 (d, 1H, H₅); 7.58 (t, 1H, H₇, J_7e8¼ 8.0 Hz); 8.08 (d,

1H, H₈); 9.63 (s, 1H, NHeCH₂); 12.66, (s, 1H, SeH). ¹³C NMR

(100 MHz, DMSO-d₆)

: 53.1 (CH₂); 55.6 (OCH₃); 114.1 (C₃þ C₅);

116.2 (C₉); 123.5 (C₈); 124.9 (C₅); 125.8 (C₇); 129.7 (C₂þ C₆); 134.5

4.1.26. Preparation of 4-methoxybenzylisoselenocyanate 6b

(C₆); 136.1 (C₁); 149.2 (C₁₀); 153.4 (C₄); 159.3 (C₄); 175.2 (C₂). MS

(m/z % abundance): 345 (M^þ, 7); 264 (14); 121 (100); 91 (26); 77

(24). Elemental Analysis for C₁₆H₁₅N₃OSe Calcd/Found (%): C: 55.81/

55.67; H: 4.36/4.36; N: 12.21/12.32.

To a mixture of 5b (4.40 g, 26.00 mmol) and triethylamine

(14.84 mL, 104.00 mmol) in dry toluene (50 mL) was added sele-

nium powder (3.16 g, 40.00 mmol). A solution of phosgene (4.2 mL,

296

E. Moreno et al. / European Journal of Medicinal Chemistry 47 (2012) 283e298

4.1.28.3. 2-Hydroseleno-4-(4⁰-methylthiobenzyl)aminoquinazoline

(7c). From 2-aminobenzonitrile and 6c. Yield: 63%; mp

170e172 ^ꢀC. IR (KBr) cm^ꢁ1: 3250 (m, NeH); 3016 (CeH); 2963 (m,

4.1.29.3. 4-(4⁰-Methylthiobenzyl)amino-2-methylselenoquinazoline

(7g). From 7c and methyl iodide. Yield: 50%; mp 160e161 ^ꢀC. IR

(KBr) cm^ꢁ1: 3245 (m, NeH); 3045 (CeH); 2960 (m, CeH); 1634 (s,

CeH); 1617 (s, C]N). ¹H NMR (400 MHz, DMSO-d₆)

d: 2.48 (s, 3H,

C]N). ¹H NMR (400 MHz, DMSO-d₆)

d: 2.45 (s, 3H, SeCH₃); 2.52 (s,

SCH₃); 5.90 (br s, 2H, CH₂eNH); 7.19 (d, 2H, H₂þ H₆); 7.3 (m, 3H,

3H, SCH₃); 5.46 (br s, 2H, CH₂eNH); 7.24 (s, 4H,

H₆þ H₃þ H₅); 7.4 (d, 1H, H₅, J_5e6¼ 8.1 Hz); 7.6 (t, 1H, H₇,

H₂þ H₃þ H₅þ H₆); 7.6 (t, 1H, H₆, J_6e5¼ J_6e7¼ 7.8 Hz); 7.62 (d, 1H,

J_7e6¼ J_7e8¼ 7.8 Hz); 8.14 (d, 1H, H₈); 9.63 (s, 1H, NHeCH₂); 12.65,

H₅); 7.86 (t, 1H, H₇, J_7e8¼ 8 Hz); 8.37 (d, 1H, H₈); 9.69 (s, 1H,

(s, 1H, SeH). ¹³C NMR (100 MHz, DMSO-d₆)

: 15.8 (SCH₃); 53.2

eNHeCH₂). ¹³C NMR (100 MHz, DMSO-d₆)

d: 11.5 (SeCH₃); 15.7

(CH₂); 116.1 (C₉); 126.2 (C₈); 126.9 (C₅); 127.3 (C₇); 128.3 (C₃þ C₅);

(SCH₃); 53.4 (CH₂); 116.5 (C₉); 126.3 (C₈); 126.9 (C₅); 127.4 (C₇);

129.5 (C₂þ C₆); 134.4 (C₆); 135.6 (C₁); 136.3 (C₄); 149.3 (C₁₀);

154.9 (C₄); 175.3 (C₂). MS (m/z % abundance): 360 (M^þ, 4); 281 (59);

235 (17); 191 (39); 175 (21); 161 (24); 137 (47); 106 (48); 121 (100);

91 (100); 77 (38). Elemental Analysis for C₁₆H₁₅N₃SSe Calcd/Found

(%): C: 42.10/42.20; H: 3.29/3.56; N: 9.21/9.20.

127.9 (C₃þ C₅); 129.2 (C₂þ C₆); 133.6 (C₆); 134.7 (C₁); 137.1 (C₄);

151.2 (C₁₀); 155.8 (C₄); 161.0 (C₂). MS (m/z % abundance): 375 (M^þ,

5); 281 (13); 159 (18); 137 (100); 121 (46); 91 (39); 77 (23).

Elemental Analysis for C₁₇H₁₇N₃SSe∙0.8 HI Calcd/Found (%): C:

42.84/42.62; H: 3.68/4.03; N: 8.82/9.19.

4.1.28.4. 4-Benzylamino-2-hydroselenopyrido[2,3-d]pyrimidine

(7d). From 2-amino-3-cyanopyridine and 6a. Yield: 7%; mp

225e227 ^ꢀC. IR (KBr) cm^ꢁ1: 3339 (m, NeH); 3080e3012 (m, CeH);

4.1.29.4. 4-Benzylamino-2-pentylselenoquinazoline (7h). From 7a

and pentyl iodide. Yield: 64%; mp 177e179 ^ꢀC. IR (KBr) cm^ꢁ1

3334e3306 (m, NeH); 3131 (m, CeH); 2938 (m, CeH); 1645 (s, C]

2940 (m, CeH); 1637 (s, C]N). ¹H NMR (400 MHz, DMSO-d₆)

N). ¹H NMR (400 MHz, DMSO-d₆)

d: 0.86 (t, 3H, (CH₂)₄eCH₃); 1.33

6.00 (br s, 2H, CH₂eNH); 7.20e7.38 (m, 6H, H₆þ Ph); 8.53 (d, 1H,

H₅); 8.58 (d, 1H, H₇); 9.90 (s, 1H, NHeCH₂); 13.10 (s, 1H, SeH). ¹³

NMR (100 MHz, DMSO-d₆) : 53.3 (CH₂); 108.9 (C₁₀); 121.9 (C₆);

(m, 4H, (CH₂)₂e(CH₂)₂eCH₃); 1.75 (q, 2H, CH₂eCH₂e(CH₂)₂eCH₃);

3.32 (t, 2H, CH₂e(CH₂)₃eCH₃); 5.71 (s, 2H, CH₂ePh); 7.35 (m, 5H,

CH₂ePh); 7.77 (m, 2H, H₆þ H₅); 8.06 (t, 1H, H₇); 8.53 (d, 1H, H₈);

126.6 (C₄); 127.1 (C₂þ C₆); 128.6 (C₃þ C₅); 131.0 (C₁); 134.6 (C₅);

153.7 (C₇); 156.8 (C₉); 157.3 (C₄); 175.0 (C₂). MS (m/z % abundance):

327 (M^þ, 9); 236 (27); 137 (23); 103 (31); 91 (100); 77 (30).

Elemental Analysis for C₁₄H₁₂N₄Se Calcd/Found (%): C: 53.23/52.91;

H: 3.81/4.02; N: 17.71/17.54.

10.12 (br s, 1H, NHeCH₂). ¹³C NMR (100 MHz, DMSO-d₆)

d: 14.9

(See(CH₂)₄eCH₃); 22.4 (See(CH₂)₃eCH₂eCH₃); 29.4 (SeeCH₂e

CH₂e(CH₂)₂eCH₃); 31.3 (SeeCH₂e(CH₂)₃eCH₃); 32.2 (See(CH₂)₂e

CH₂eCH₂eCH₃); 53.2 (CH₂); 114.0 (C₉); 126.3 (C₈); 126.9 (C₅); 127.5

(C₇); 128.3 (C₄); 128.7 (C₂þ C₆); 129.1 (C₃þ C₅); 135.1 (C₆); 138.0

(C₁); 146.2 (C₁₀); 155.0 (C₄); 158.2 (C₂). MS (m/z % abundance): 385

4.1.29. General procedure for compounds 7eei

(M^þ,10); 314 (100); 234 (64); 207 (55); 145 (10); 129 (24); 106 (22);

91 (68). Elemental Analysis for C₂₀H₂₃N₃Se$1 HI Calcd/Found (%): C:

46.87/47.25; H: 4.49/4.49; N: 8.20/8.63.

The appropriate alkyl iodide was added during 25 min to

a solution of the corresponding 4-benzylamino-2-hydroseleno-

quinazoline derivative 7aec (1.27 mmol) in ethanol (30 mL) and

DMF (4 mL). The mixture was heated at 70 ^ꢀC during 1.5 h. The

solvent was removed in vacuo. The resulting residue was washed

with water (3 ꢂ 25 mL) and recrystallized from the appropriate

solvent.

4.1.29.5. 4-(4⁰-Methylthiobenzyl)amino-2-pentylselenoquinazoline

(7i). From 7c and pentyl iodide. Yield: 37%; mp 169e170 ^ꢀC. IR

(KBr) cm^ꢁ1: 3321 (m, NeH); 3131 (m, CeH); 2958e2892 (m, CeH);

1589 (f, C]N). ¹H NMR (400 MHz, DMSO-d₆)

d: 0.86 (t, 3H,

(CH₂)₄eCH₃); 1.34 (m, 4H, (CH₂)₂e(CH₂)₂eCH₃); 1.78 (q, 2H,

CH₂eCH₂e(CH₂)₂eCH₃); 2.50 (s, 3H, SCH₃); 3.31 (t, 2H,

CH₂e(CH₂)₃eCH₃); 5.53 (s, 2H, CH₂ePh); 7.25 (m, 4H, Ph); 7.76 (m,

2H, H₆þ H₅); 8.07 (t, 1H, H₇); 8.53 (d, 1H, H₈); 10.32 (s, 1H,

4.1.29.1. 4-Benzylamino-2-methylselenoquinazoline (7e). From 7a

and methyl iodide. Yield: 31%; mp 195e196 ^ꢀC. IR (KBr) cm^ꢁ1: 3309

(m, NeH); 3186e3038 (m, CeH); 1651 (m, C]N). ¹H NMR

(400 MHz, DMSO-d₆)

: 2.57 (s, 3H, SeCH₃); 5.58 (br s, 2H,

NHeCH₂). ¹³C NMR (100 MHz, DMSO-d₆)

d: 14.6 (See(CH₂)₄eCH₃);

CH₂eNH); 7.35 (m, 5H, Ph); 7.74 (t, 1H, H₇); 7.79 (d, 1H, H₈); 8.06, (t,

15.5 (SCH₃); 22.4 (See(CH₂)₃eCH₂eCH₃); 29.2 (SeeCH₂eCH₂e

(CH₂)₂eCH₃); 31.3 (SeeCH₂e(CH₂)₃eCH₃); 31.8 (See(CH₂)₂eCH₂e

CH₂eCH₃); 53.1 (CH₂); 113.8 (C₉); 126.2 (C₈); 126.6 (C₅); 127.3 (C₇);

1H, H₆); 8.53 (d, 1H, H₅); 10.06 (s, 1H, NHeCH₂). ¹³C NMR (100 MHz,

DMSO-d₆) d: 11.3 (SeCH₃); 53.5 (CH₂); 113.8 (C₉); 125.8 (C₈); 126.4

(C₅); 127.1 (C₇); 127.9 (C₄); 128.4 (C₂þ C₆); 128.9 (C₃þ C₅); 134.8

127.9 (C₃þ C₅); 128.7 (C₂þ C₆); 134.8 (C₆); 136.1 (C₁); 139.0 (C₄);

146.2 (C₁₀); 155.0 (C₄); 157.9 (C₂). MS (m/z % abundance): 431 (M^þ,

9); 360 (43); 280 (7); 253 (28); 233 (9); 137 (100); 121 (11); 91 (9).

Elemental Analysis for C₂₁H₂₅N₃SSe$0.9 HI Calcd/Found (%): C:

46.22/45.91; H: 4.58/4.60; N: 7.70/7.74.

(C₆); 138.7 (C₁); 147.2 (C₁₀); 155.6 (C₄); 157.8 (C₂). MS (m/z %

abundance): 327 (M^þ, 25); 314 (23); 234 (27); 207 (21); 191 (20);

137 (53); 121 (24); 103 (44); 91 (100); 77 (37). Elemental Analysis

for C₁₆H₁₅N₃Se∙HI Calcd/Found (%): C: 42.10/42.20; H: 3.29/3.56; N:

9.21/9.20.

4.2. Cytotoxic and antiproliferative activities

4.1.29.2. 4-(4⁰-Methoxybenzyl)amino-2-methylselenoquinazoline

(7f). From 7b and methyl iodide. Yield: 31%; mp 190e191 ^ꢀC. IR

(KBr) cm^ꢁ1: 3329 (m, NeH); 3175e3058 (m, CeH); 1658 (m, C]N).

The cytotoxic effect of each substance was tested at ﬁve different

concentrations between 0.01 and 100 mM. Each substance was

¹H NMR (400 MHz, DMSO-d₆)

: 2.58 (s, 3H, SeCH₃); 3.72 (s, 3H,

initially dissolved in DMSO at a concentration of 0.1 M and serial

dilutions were prepared using culture medium. The plates with

cells from the different lines, to which medium containing the

substance under test was added, were incubated for 72 h at 37 oC in

a humidiﬁed atmosphere containing 5% CO₂. Human tumor cell

lines were provided by the European Collection of Cell Cultures

(ECACC) or the American Type Culture Collection (ATCC). Four cell

lines were used: one human lymphocytic leukemia (CCRF-CEM)

and three human solid tumors, one colon carcinoma (HT-29), one

lung carcinoma (HTB-54) and one breast adenocarcinoma (MCF-7).

CCRF-CEM, HT-29 and HTB-54 cells were grown in RPMI 1640

OCH₃); 5.94 (br s, 2H, CH₂eNH); 6.80 (d, 2H, H₃þ H₅,

J_{3 e2}¼ J_{5 e6}¼ 8.4 Hz); 7.23 (t, 1H, H₇, J_7e8¼ J_7e6¼ 8.0 Hz); 7.31 (d,

2H, H₂þ H₆); 7.42 (d,1H, H₈); 7.61 (t,1H, H₆, J_6e5¼ 8.0 Hz); 8.18, (d,

1H, H₅); 9.68 (s, 1H, NHeCH₂). ¹³C NMR (100 MHz, DMSO-d₆)

: 11.1

(SeeCH₃); 52.8 (CH₂); 55.8 (OCH₃); 114.2 (C₃þ C₅); 116.2 (C₉);

124.9 (C₈); 125.8 (C₅); 126.4 (C₇); 129.4 (C₂þ C₆); 134.7 (C₆); 135.7

(C₁); 149.9 (C₁₀); 152.9 (C₄); 158.8 (C₄); 161.2 (C₂). MS (m/z %

abundance): 359 (M^þ, 45); 191 (20); 91 (100); 77 (47). Elemental

Analysis for C₁₇H₁₇N₃OSe Calcd/Found (%): C: 56.98/56.82; H: 4.75/

4.87; N: 11.73/11.52.

E. Moreno et al. / European Journal of Medicinal Chemistry 47 (2012) 283e298

297

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medium (Invitrogen) supplemented with 10% fetal calf serum,

2 mM -glutamine, 100 units/mL penicillin, 100 g/mL strepto-

mycin and 10 mM HEPES buffer (pH ¼ 7.4). MCF-7 cells were grown

in EMEM medium (Clonetics) supplemented with 10% fetal calf

serum, 2 mM L-glutamine, 100 units/mL penicillin and 100 mg/mL

streptomycin. 184B5 cells were grown in Hams F-12/DMEM (50:50)

supplemented as described by Li et al. [61]. BEAS-2B were grown in

25 mL fetal bovine serum (FBS), 5 mL insulinetransferrinesodium

selenite (ITS), 1 mL hydrocortisone, 10 mL sodium pyruvate, 5 mL

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glutamine, 5 mL penicillin/gentamicin, 10

mL epidermal growth

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determined by the MTT method. Results are expressed as GI₅₀, the

concentration that reduces by 50% the growth of treated cells with

respect to untreated controls, TGI, the concentration that

completely inhibits cell growth, and LC₅₀, the concentration that

kills 50% of the cells. Data were obtained from at least 3 indepen-

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4.3. Evaluation of cell cycle progression and cell death

For breast adenocarcinoma MCF-7 cells, the cell death status

and cell cycle analysis of the cells were determined using the Apo-

Direct kit (BD Pharmingen), based on the TUNEL technique, under

the conditions described by the manufacturer. Brieﬂy, for ﬁxation

step cells were suspended in 1% paraformaldehyde in PBS (pH ¼ 7.4)

at a concentration of 1 ꢂ10⁶cells/mL, incubated on ice for 1 h,

collected by centrifugation, washed, adjusted to 1 ꢂ10⁶cells/mL in

70% ice-cold ethanol and incubated at ꢁ20 ^ꢀC for 30 min. After

ﬁxation, cells were recovered by centrifugation, washed, resus-

pended in FITC dUTP-DNA labeling solution and incubated for 1 h at

37 ^ꢀC. Cells were then rinsed, resuspended in PI/RNase staining

buffer, incubated in the dark for 30 min at RT and analyzed using

a Coulter Epics XL ﬂow cytometer.

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Acknowledgments

The authors wish to express their gratitude to the Ministerio de

Educación y Ciencia, Spain (SAF 2009-07744) and to the CAN

Foundation for ﬁnancial support for the project. We also thank the

Department of Education of the Navarra Government for the award

of a grant (E.M.).

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