Synthesis and pharmacological investigation of new N-hydroxyalkyl-2- aminophenothiazines exhibiting marked MDR inhibitory effect

Bioorganic & Medicinal Chemistry xxx (2013) xxx–xxx

Contents lists available at SciVerse ScienceDirect

Bioorganic & Medicinal Chemistry

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

Synthesis and pharmacological investigation of new N-hydroxyalkyl-

2-aminophenothiazines exhibiting marked MDR inhibitory effect

Daniella Takács ^a, Orsolya Egyed ^a, László Drahos ^a, Pál Szabó ^b, Katalin Jemnitz ^b, Mónika Szabó ^b,

Zsuzsa Veres ^b, Júlia Visy ^b, József Molnár ^c, Zsuzsanna Riedl ^a, György Hajós ^a,

⇑

^aInstitute of Organic Chemistry, Research Centre for Natural Sciences, Hungarian Academy of Sciences, Pusztaszeri út 59, H-1025 Budapest, Hungary

^bInstitute of Molecular Pharmacology, Research Centre for Natural Sciences, Hungarian Academy of Sciences, Pusztaszeri út 59, H-1025 Budapest, Hungary

^cDepartment of Medical Microbiology, University of Szeged, Dóm tér 10, H-6720 Szeged, Hungary

a r t i c l e i n f o

a b s t r a c t

Article history:

Novel N-hydroxyalkyl-2-aminophenothiazines implying a tetrazole moiety at the alkyl chain have been

synthesized by hydroboration–oxidation of dienes followed by Buchwald–Hartwig cross-coupling reac-

tion. Also, some sulfoxide and sulfone derivatives have been prepared by selective oxidations. MDR inhi-

bition studies on rat hepatocyte cell culture revealed that some derivatives exhibit marked biological

efﬁcacy exceeding that of the standard verapamil (e.g., 3h, 4h, 16). Selected derivatives were subjected

to chemical resolution to provide both enantiomers which were shown of similar activity on P-gp inter-

action measurements. The new compounds exhibited no toxicity.

Received 24 January 2013

Revised 11 April 2013

Accepted 12 April 2013

Available online xxxx

Keywords:

N-Hydroxyalkyl-2-aminophenothiazines

Sulfoxide and sulfone derivatives

MDR inhibition

Ó 2013 Published by Elsevier Ltd.

Buchwald–Hartwig cross-coupling reaction

Resolution of racemate

1. Introduction

displayed unwanted pharmacokinetic interactions. Third-genera-

tion inhibitors can speciﬁcally and potently inhibit P-gp function,

Multidrug resistance (MDR) is a major clinical obstacle in the

treatment of several cancers including hematological malignancies

and solid tumors. The ATP-binding cassette transporter P-glyco-

protein (P-gp, ABCB1), is one molecule that is involved in drug

resistance.¹Cancer cells can escape from the toxic effect of drugs

mainly due to the highly expressed membrane transporters that

pump drugs out of cells.²To overcome P-gp mediated MDR, mod-

ulators, so called P-gp inhibitors, can be used to block efﬂux pump

activity. P-gp inhibitors can be used in combination with antican-

cer drugs to enhance the effectiveness of chemotherapy against

resistant tumor cells. On the other hand, P-gp is presented in nor-

mal tissues, thus non-selective blockage of P-gp can cause unde-

sired side effects. Therefore, it is important to deliver P-gp

inhibitor only to the tumor cells (along with anticancer drug)

and limit its distribution in the body. Much effort has been devoted

to developing P-gp inhibitors to modulate MDR. However, none of

the inhibitors on the market have been successful. Three genera-

tions of inhibitors have been developed in order to reverse MDR.

First-generation inhibitors including verapamil, and cyclosporine

several of them have been tested in controlled clinical trials, but

no satisfactory results have been obtained.⁴For almost three dec-

ades, research has been conducted to overcome MDR through

pharmacological inhibition of ABC transporters with limited clini-

cal success. Therefore it is still urgent to develop some new agents

to overcome MDR.⁵

During the recent years, investigation of inhibition of multi-

drug resistance (MDR) attracted an increased interest world-wide.⁶

Among the effective compounds that proved to inhibit resistance,

some phenothiazine derivatives were found of high activity.⁷With

some of the most important such compounds involving chloro-

promazine,⁸triﬂuoperazine,⁹thioridazine,¹⁰two common struc-

tural features of these compounds should be pointed out: (i)

presence of an alkyl chain attached to the nitrogen atom of the

phenothiazine ring; (ii) presence of an amino moiety in most of

the derivatives.

Earlier, we described a novel synthetic approach to N-dienyl-

phenothiazines¹¹and, quite recently, reduction of these com-

pounds to N-butyl and N-hydroxybutyl derivatives¹²has been

reported. Even if the MDR inhibitory efﬁcacy of these new com-

pounds proved to be only moderate, further functionalization of

these derivatives seemed promising in order to discover new po-

tent MDR inhibitors among the newly synthesized phenothiazine

derivatives.

A

were limited by unacceptable toxicity.³Second-generation

agents such as dexverapamil, PSC833 had better tolerability but

⇑

Corresponding author. Tel.: +36 4381110; fax: +36 4381145.

E-mail address: hajos.gyorgy@ttk.mta.hu (G. Hajós).

0968-0896/$ - see front matter Ó 2013 Published by Elsevier Ltd.

http://dx.doi.org/10.1016/j.bmc.2013.04.034

Please cite this article in press as: Takács, D.; et al. Bioorg. Med. Chem. (2013), http://dx.doi.org/10.1016/j.bmc.2013.04.034

2

D. Takács et al. / Bioorg. Med. Chem. xxx (2013) xxx–xxx

route "A"

OCH₃

3''

NR¹R²

Cl

4''

4a''

N

2''

a

b, c

N

1''

5''

S

OH

S

10a''

0-49%

5a''

45-86%

10''

N

6''

7''

2

4

N

9a''

1

3

N

9''

1'

2'

8''

1

2

3

3'

4'

6'

5'

a) 2 equiv. HNR¹R², 5-7.5 mol% Pd-catalyst,10-15 mol% XPhos, 2 equiv. base

abs. toluene, 0.5-24 h, 80-110 °C, Ar

OCH₃

N

b) BH₃xMe₂S, abs. THF, 0°C rt

c) MeOH, 10% NaOH, 30% H₂O₂, rt, 24 h

O

S

OH

N

4f

OCH₃

Scheme 1. Synthetic route ‘A’ to N-(2-hydroxybutyl)-2-aminophenothiazines (3).

benzylation). Also, an alternative route ‘C’ was applied by reacting

5 with tert-butyldiphenylchlorosilane (^tBuPh₂SiCl) to give the

2. Results and discussion

2.1. Synthesis

In this paper we report on a well executable synthetic protocol

to N-tetrazolylbutylphenothiazines bearing various amino func-

tions in position 2 of the tricyclic ring as well as on biological eval-

uation of the new derivatives.

Table 1

Used primary/secondary amines and acid amides in the synthesis of N-(2-hydrox-

ybutyl)-2-aminophenothiazines (3) via route A (Scheme 1)

Compound 3

HNR¹R²

Yield %

Two of our recent ﬁndings provided proper starting point in or-

der to perform the above planned transformations: (a) the success-

ful reduction of phenothiazinyldienes to the related butyl

compounds by borane¹²and, (b) the convenient introduction of

amino groups into the desired position of phenothiazine by Buch-

wald–Hartwig cross-coupling reaction of the commercially avail-

able 2-chlorophenothiazine.^13a

N

a

—

NH₂

O

N

b

c

26

30

NH₂

By utilization of these structural conversions, the synthetic

strategy route ‘A’ starting from the easily accessible dienylpheno-

thiazine (1) shown in Scheme 1 seemed straightforward, that is,

Buchwald–Hartwig amination to 2 followed hydroboration–oxida-

tion to give the saturated monohydroxy product (3). As pharmaco-

logical measurements indicated that, concerning the MDR

modulatory activity, those compounds bearing a 4-methoxyphenyl

group on the N2 atom of the tetrazole ring are superior and, thus,

all further chemical transformations have been carried out with

such methoxy compounds (cf. Table 2 later in this publication).

Scheme 1 reveals these transformations proceeded in medium

to low yield only and, in some cases, decomposition was experi-

enced and no product could be isolated (Table 1). It is interesting

to note that in one case: with the diethylamino derivative 2f, the

expected product 3f was found as a minor component of the reac-

tion mixture, whereas a sulfoxide (4f) was formed in majority

(67%). A speciﬁc NMR spectral feature of this product is appearance

of two sets of signals due to the presence of two chiral centers (i.e.,

the secondary alcohol and sulfoxide moeties).

CH₃

NH₂

H

d

46

NH₂

e

f

—

NH

23

H

N

g

49

NH

O

h

i

33

49

44

—

N

O

NH

j

NH

k

l

—

The above ﬁndings prompted us to apply another synthetic

strategy and, thus, route ‘B’ was decided next (Scheme 2). This

route involved the hydroboration–oxidation of the diene (1) to

the hydroxybutyl derivative (5) as described in our recent publica-

tion,¹²followed by protection of the OH group (formation of 6 by

H

NH₂

O

m

H₃C

NH₂

Please cite this article in press as: Takács, D.; et al. Bioorg. Med. Chem. (2013), http://dx.doi.org/10.1016/j.bmc.2013.04.034

D. Takács et al. / Bioorg. Med. Chem. xxx (2013) xxx–xxx

3

Table 2

Comparison of yields obtained via routes ‘B’ and ‘C’ depicted in Scheme 2

Entry

HNR¹R²

Route ‘B’

Route ‘C’

Buchwald–Hartwig amination 7 (%)

Debenzylation 3 (%)

Buchwald–Hartwig amination 9 (%)

Desilylation 3 (%)

N

a

—

84

91

NH₂

O

N

b

c

—

93

—

84

—

NH₂

67

Quant.

CH₃

NH₂

d

64

—

H

NH₂

e

f

—

57

49

86

NH

H

N

g

—

NH

h

i

O

—

91

73

—

87

64

—

N

O

NH

99

—

33

—

j

NH

k

80

62

—

l

29

—

64

44

Quant.

96

H

NH₂

O

m

H₃C

NH₂

O-protected 2-chlorophenothiazine compound 8. Formation of the

benzyloxy and tert-butyldiphenylsilyloxy compounds (6 and 8)

was accomplished according to analogous procedures described

in the literature.^14,15

Both O-protected compounds (6 and 8) were subjected to Buch-

wald–Hartwig amination to give the amines 7 and 9, respectively.

In contrast to the poor yields experienced in route ‘A’ with the

transformation 1 ? 3 (Scheme 1), the cross-coupling amination

of 6 and 8 was found to proceed in good to excellent yields (44–

93%) in most cases: Pd₂(dba)₃or Pd(OAc)₂were used as the Pd-cat-

alysts in the presence of NaO^tBu, Cs₂CO₃or K₂CO₃as a base, the

ligand was XPhos in every case, whereas toluene or 1,4-dioxane

were used as a solvent.

In order to prepare a series of amine-containing sulfoxides (e.g.,

4f) the chloro compound 5 was ﬁrst oxidized by m-CPBA to the 2-

chlorosulfoxide 10²²(Scheme 3) and, then this compound was

subjected to Buchwald–Hartwig cross-coupling reaction with three

selected secondary amines to give 4h,i. Most interestingly, reaction

of 10 with diethylamine resulted in dehalogenation to yield the 2-

unsubstituted compound 11. The structures of 4h,i involve two

chirality centers (i.e., the secondary alcohol function and the novel

of the sulfoxide moiety) and, accordingly, in the ¹H NMR spectra

two sets of resonances corresponding to the two diastereomers ap-

peared. Attempts for further oxidation of these sulfoxides, unfortu-

nately, failed and prologued oxidation of 4h resulted in formation

of an N-oxide (12) rather than the expected sulfone.

The amino components involved primary amines (a–e), second-

ary amines (f–k), and two amides (l, m) as speciﬁed in Scheme 1.

Finally, deprotection of 7 and 9 by acidic treatment and reaction

with TBAF, respectively, by using literature procedures¹⁶yielded

the free hydroxy group containing products (3a–m). Table 2 sum-

marizes all 2-aminophenothiazines prepared by route ‘B’ or ‘C’.

Inspection of this table reveals that the best yields were obtained

with route ‘C’.

The aforementioned ﬁnding with formation of a sulfoxide (4f)

in course of the hydroboration–oxidation reaction directed our

attention to a deeper study on synthetic possibilities to such com-

pounds. Inspection of the pertinent literature revealed that rela-

tively few data on phenothiazine-sulfoxides exist. Thus, oxidation

to phenothiazinyl sulfoxides,^17–19and sulfones^20,21has been re-

ported. Also, selective procedures to sulfoxides and sulfones have

been described.²²

In order to circumvent this problem, the silylated 2-chloro

compound 8 was oxidized to a sulfoxide (13) and, then, a further

oxidation of 13 by m-CPBA under more forced conditions yielded

the O-protected 2-chlorophenothiazine sulfones (14). Subsequent

Buchwald–Hartwig amination with secondary amines to 15f,h,i

and the cleavage of protecting group provided the desired 2-

aminophenothiazinyl sulfones (16f,h,i) in high yields.

As revealed by the biological assays as discussed below, 3h

proved to be one of the most active MDR inhibitor (exceeding

the efﬁcacy of that of verapamil). As the above discussed synthesis

provided this compound as a racemic mixture, separation of the

enantiomeric pairs was decided. Among the several possible reso-

lution strategies, application of the semiequivalent method was

decided.²³To this end, 5 was ﬁrst functionalized by reaction of

the hydroxy group with maleic acid anhydride to give semiacid

(17). A solution of this compound in ethyl acetate was treated with

Please cite this article in press as: Takács, D.; et al. Bioorg. Med. Chem. (2013), http://dx.doi.org/10.1016/j.bmc.2013.04.034

4

D. Takács et al. / Bioorg. Med. Chem. xxx (2013) xxx–xxx

Cl

a half equivalent of S-(ꢀ)-phenylethylamine in order to obtain a

mixture of diastereomeric salts. The (ꢀ)-diastereomeric salt (18)

precipitated from the mixture, whereas the dissolved (ꢀ)-diaste-

reomeric salt and the (+)-monoester (19) remained in the solution.

The crystalline 18 was ﬁltered off and, after several recrystalliza-

tions, it was hydrolyzed to the (ꢀ)-monoester ((ꢀ)-17). Saponiﬁca-

tion of (ꢀ)-17 with sodium hydroxide solution under reﬂux

afforded the optically pure (ꢀ)-hydroxy compound ((ꢀ)-5) with

excellent enantiomeric purity (ee 99.9%). Similar work-up of the

solution containing mainly 19 via formation of (+)-monoester

(+)-17 resulted in isolation of the (+)-enantiomer of 5 with lower

optical purity (ee 74–98%) (Scheme 4).

hydroboration/

oxidation

S

OH

N

1

N

65%

N

5

route "B"

for derivatives

c,d,i,k,l

route "C"

OCH₃

for derivatives

a,b,e,f,h,i,l,m

^tBuPh₂SiCl, imidazole

abs. CH₂Cl₂, rt, 4 d, Ar

90%

1. NaH (60%), abs. DMF

0 °C, 1 h, Ar

2. BnBr, 0°C

75%

rt, 2.5 h

The obtained optically pure (ꢀ)-5 and (+)-5 were suitable start-

ing materials for preparation of those amines (i.e., (3f,h,i) that

seemed to be promising lead candidates on the basis of biological

evaluations (see below section). To this end, route ‘C’ was applied.

Accordingly, (ꢀ)-5 and (+)-5 were silylated to (ꢀ)-8 and (+)-8,

Buchwald–Hartwig amination yielded the O-protected amines

(ꢀ)-9f,h,i- and (+)-9f,h,i and, ﬁnally, deprotection afforded the de-

sired (ꢀ)-3f,h,i and (+)-3f,h,i (Scheme 5).

Cl

S

OBn

S

OR

N

8

6

t

R = BuPh₂Si

OCH₃

2 equiv. HNR¹R²

7.5-10 mol% Pd-catalyst

15-20 mol% XPhos

2 equiv. base

OCH₃

abs. toluene or

abs. 1,4-dioxane

1.5-43 h, 100-110°C, Ar

29-99%

2.2. P-gp interaction studies

NR¹R²

The above discussed set of N-substituted phenothiazine deriva-

tives was tested for P-gp inhibitor activity in primaty rat hepato-

cyte culture. The rate of rhodamine 123 (RH123) accumulation

was studied in the presence of these molecules and of verapamil,

a commonly used P-gp inhibitor. All compounds were studied at

NR¹R²

OR

S

OBn

S

N

10 lM concentration, and data are expressed as % of the inhibition

by verapamil.

9a,b,e,f,h,i,l,m

7c,d,i,k,l

Table 3 demonstrates the inhibitor activity of three pairs of

derivatives having hydrogen, chloro atoms, and triﬂuoromethyl

group on position 2 of the phenothiazine ring, whereas substitu-

ents on the phenyl ring attaching to N2 of the tetrazol involved

chloro, methoxy, and ethoxycarbonyl groups (1, 20a–d). These data

reveal that molecules containing 4-methoxyphenyltetrazole moi-

ety were found to be most effective, irrespective of the substituents

on position 2 of the phenothiazine ring even if the rate of inhibition

was considerably inﬂuenced by substituents on the latter position.

OCH₃

TBAF/abs. THF

0°C rt, 2-48 h

64-100%

cc. HCl/EtOH

45-70°C,4-6 d

0-100%

3a-m

Scheme 2. Synthetic route ‘B’ and ‘C’ to N-(2-hydroxybutyl)-2-aminophenothia-

zines (3).

ture of the derivatives, their inﬂuence at a lower (up to 10 mM)

concentration range was also studied. No remarkable difference

between the actions of this set of compounds was observed; the

rate of inhibition was comparable to that of verapamil. In case of

sulfoxide derivative (4f) obtained ﬁrst as an unexpected product

of hydroboration–oxidation, a slight, but signiﬁcant increase in P-

gp inhibition could be observed (Fig. 3).

This latter ﬁnding directed our attention to the study of the ef-

fect of oxidation of the sulfur atom in the phenothiazine ring as

shown in Figure 4. The number of oxygen atoms attached to the

sulfur atom had an impact on the interaction potential of the mol-

ecule. Compared to 3h, one additional oxygen atom (i.e., the sulf-

oxide 4h) slightly, but signiﬁcantly, and a second oxygen atom

(i.e., the sulfone 16h) considerably increased the accumulation of

RH123.

A

further variety of N-2-hydroxybutylphenothiazine com-

pounds, all containing a 4-methoxyphenyl group on the tetrazole

ring and differently substituted amino groups on position 2 of phe-

nothiazine is shown in Table 4. P-gp inhibitor capacity of some of

these derivatives (3f, 3h, 3m) was comparable to that of verapamil.

As revealed by the above data, the 2-butanol part of phenothi-

azine derivatives 3 was essential for a potent P-gp inhibition. This

part of the molecule contains a chirality center. As chirality of

many compounds has an impact on their protein binding, we have

investigated whether the use of stereoisomers of the most potent

derivatives alters their inhibitor property. For this purpose, prod-

ucts of resolution of two derivatives (3h,i) each isolated as pure

enantiomers ((+)-3h, (ꢀ)-3h, (+)-3i, (ꢀ)-3i) have been selected

and their activity has been compared with that of the related race-

mic form as well as with verapamil. As shown in Figure 1 there was

no signiﬁcant difference in RH123 accumulation in the presence of

either the racemic or any of the stereoisomer derivatives of these

compounds.

2.3. Cytotoxicity assays

MTT assay was used to assess cell viability, cytotoxicity. These

assays were performed with the most potent P-gp inhibitor com-

pounds (3h, 3f, 3b, 4h, 16h). The viability of the hepatocytes as

shown in Figure 5 did not change, so the observed decrease of

the P-gp activity was not due to cytotoxic effects in the presence

of any of these derivatives.

Oxidation of the sulfur atom of the phenothiazine ring further

improved the inhibitor potency of the molecules (Table 5). Two

of these derivatives (4h, 16h) proved to be signiﬁcantly better P-

gp inhibitors than verapamil.

The concentration dependence concerning P-gp interactions of

the most potent compounds was compared to that of verapamil.

No signiﬁcant difference was found between the effect of 3f, 3h

Hepatocytes were treated for 3 h with 10 lM of 3h, 3f, 3b, 4h,

16h or the vehicle (control), respectively. Data are shown as % of

and verapamil up to 50

lM (Fig. 2). In order to gain a more precise

the control.

relationship between the inhibitor capacity and molecular struc-

Please cite this article in press as: Takács, D.; et al. Bioorg. Med. Chem. (2013), http://dx.doi.org/10.1016/j.bmc.2013.04.034

D. Takács et al. / Bioorg. Med. Chem. xxx (2013) xxx–xxx

5

OCH₃

2 equiv. HNR¹R²

5-7.5 mol% Pd₂(dba)₃

10-15 mol% XPhos

NR¹R²

N

2 equiv. NaO^tBu or Cs₂CO₃

abs. toluene

Cl

N

O

N

m-CPBA

C rt, 20 min

S

OH

O

N

0 ^°

0.5-7 h, 110°C, Ar

S

OH

N

5

N

S

88%

1

2

0-78%

4h

4i

(HNR R = morpholine)

1

(HNR R²= N-methylpiperazine)

10

OCH₃

O

if HNR¹R²= HNEt₂

N

m-CPBA

N

O

N

0

°C

rt, 23 h

S

OH

4h

83%

N

12

11

OCH₃

Cl

m-CPBA

0°C rt, 6 h

m-CPBA

N

0°C

rt, 2 h

O

S

O

8

S

OR

quant.

88%

N

13

14

R = ^tBuPh₂Si

2 equiv. HNR¹R², 10 mol% Pd₂(dba)₃

20 mol% XPhos, 2 equiv. NaO^tBu,

abs. toluene, Ar, 70-90 °C, 70-90 min

52-92%

OCH₃

NR¹R²

N

NR¹R²

N

N N

1.5 equiv. TBAF

abs. THF

O

S

O

S

N

O

OH

OR

N

0 °C rt, Ar

2.5-4.5 h

78-88%

N

16f (HNR¹R²= diethylamine)

16h (HNR¹R²= morpholine)

15f (HNR¹R²= diethylamine)

15h (HNR¹R²= morpholine)

16i (HNR¹R²= N-methylpiperazine)

15i (HNR¹R²= N-methylpiperazine)

Scheme 3. Synthetic routes to preparation of sulfoxide and sulfone derivatives.

(control) for 3 h, hepatocytes were washed and incubated with

1 mg/mL MTT for 2 h. Then the cells were lysed with 1 mL of DMSO

and the absorbance was measured at 570 nm using a microplate

reader. Data are shown as % of the control.

3. Conclusion

Hydroboration–oxidation of dienylphenothiazines and Buch-

wald–Hartwig cross-coupling reaction of 2-chloro derivatives al-

lowed the synthesis a set of new N-2-hydroxybutylphenothia

zines and their sulfoxides and sulfone derivatives. The structure

of these compounds has been systematically modiﬁed at those

positions that have been shown to have an impact on the rate of

P-gp inhibitor activity of the molecule. The extent of inhibition

by the most potent derivatives slightly exceeded that of verapamil,

a well-known P-gp inhibitor. A high variety of efﬁcacy was ob-

served as a function of the quality of substituents on position 2

of the phenothiazine and the phenyltetrazole moiety. The N-2-

hydroxybutyl chain proved to be essential for the interaction with

P-gp. The sulfone derivative 16h exhibited the highest efﬁcacy.

MTT assays revealed that the new MDR inhibitory compounds

are not toxic.

4.2. Method for determination of P-gp inhibitor properties

P-gp interaction of the phenothiazine derivatives was studied in

a conventional monolayer culture of primary rat hepatocytes.

Hepatocytes were prepared from male Wistar rats (200–250 g)

(Charles River, Budapest) by two-step, in situ liver collagenase per-

fusion according to the method of Seglen.²⁴Cell viability (>90%)

was determined by trypan blue exclusion. All procedures were ap-

proved by the Institutional Animal Care and Use Committee. Hepa-

tocytes were plated at a density of 1.9 ꢁ 10⁵cells/cm²in 24-well

plates precoated with rat tail collagen in Williams Medium E con-

taining 5% of fetal calf serum, 100 nM insulin, 0.1 mg/mL gentami-

cin, 30 nM Na₂SeO₃, and 0.1 lM dexamethasone. Calf serum was

4. Experimental section

4.1. Cytotoxicity assays

present for the ﬁrst 24 h then omitted. Cells were maintained at

37 °C in a humidiﬁed atmosphere of 95% air–5% CO₂. In 1 h after

plating, and every day thereafter, the medium was changed to Wil-

liams Medium E supplemented with insulin, gentamicin, dexa-

methasone, Na₂SeO₃. The RH123 accumulation assay was

performed as described previously,¹²brieﬂy, at 4 days after plating

MTT is added directly to the culture medium and is reduced by

metabolically active cells to insoluble purple formazan dye crys-

tals. The absorbance of the sample is read directly in the wells at

an optimal wavelength of 570 nm. The amount of formazan pro-

duced is in direct proportion to the viability of the cells. After treat-

cells were washed and preloaded with 5 lM RH123 in Williams’

Medium E for 30 min. Then the hepatocytes were washed three

times with an ice cold Hanks Balaced Salt Solution (HBSS) and

incubated with RH123 free Williams’ Medium E containing the

ment with the compounds indicated at 10 lM, or with the vehicle

Please cite this article in press as: Takács, D.; et al. Bioorg. Med. Chem. (2013), http://dx.doi.org/10.1016/j.bmc.2013.04.034

6

D. Takács et al. / Bioorg. Med. Chem. xxx (2013) xxx–xxx

NH₂

O

Cl

O

OH

N

O

abs. Et₃N

abs. CH₂Cl₂

0.5 equiv.

S-(-)

S

5

N

0°C

reflux

abs. EtOAc

rt, 2 h

N

9.5 h, 99.7%

N

17

OCH₃

(-)-diastereomeric salt_crystal

(-)-diastereomeric salt + (+)-monoester

+

in solution

18

19

recrystallization from EtOAc (twice)

followed by treatment with

10% aqueous HCl solution

CH₂Cl₂/MeOH

10% aqueous HCl solution

EtOAc

rt, 50 min

(twice)

rt, 4 h

99.8%

(-)-monoester (-)-17

(+)-monoester (+)-17

CH₂Cl₂

4.5 equiv. NaOH/H₂O

rt reflux, 4 h

74%

4.5 equiv. NaOH/H₂O

rt

reflux, 7.5 h

86%

Cl

OH

S

OH

S

N

(+)-hydroxy (+)-5

ee: 74-98%

OCH₃

(-)-hydroxy (-)-5

ee: 99.9+%

Scheme 4. Resolution of racemic 5 to enantiomers.

Cl

route "C"

^tBuPh₂SiCl

imidazole

Cl

S

OH

S

OR

N

abs. CH₂Cl₂

N

0°C rt, Ar

5 d, 98%

R = ^tBuPh₂Si

(-)-8 and (+)-8

OCH₃

(-)-5 and (+)-5

HNR¹R²

Entry

f

2 equiv. HNR¹R²

10 mol% Pd₂(dba)₃

20 mol% XPhos

2 equiv. NaO^tBu

abs. toluene, 110°C

16-21 h, 45-82%

NH

h

i

O

NH

N

NH

NR¹R²

1.5 equiv. TBAF

abs. THF

S

OR

N

(-)-3f,h,i and (+)-3f,h,i

N

0°C

18-22 h,

66-98%

rt, Ar

N

(-)-9f,h,i and (+)-9f,h,i

OCH₃

Scheme 5. Synthesis of (ꢀ)-3f,h,i and (+)-3f,h,i derivatives applying route ‘C’.

phenothiazine derivatives, verapamil at different concentrations,

or the vehicle as control (0.1% DMSO), respectively for 3 h. Subse-

quently, the cells were washed with ice cold HBSS and were lysed

with 0.5% of Triton X100/HBSS. The intracellular RH123 concentra-

tion was measured ﬂuorimetrically at 519/538 nm.

were run, and four wells were used for each set of conditions. Sta-

tistical analysis was performed using the Student’s t-test.

4.3. Chemistry

The results are expressed as mean SD for all experiments.

Triplicate experiments of three independent hepatocyte isolations

Melting points were determined on a Büchi apparatus and are

uncorrected. The IR spectra were recorded on a Thermo Nicolet

Please cite this article in press as: Takács, D.; et al. Bioorg. Med. Chem. (2013), http://dx.doi.org/10.1016/j.bmc.2013.04.034

D. Takács et al. / Bioorg. Med. Chem. xxx (2013) xxx–xxx

7

Table 3

Comparison of the effects of R1 and R2 of derivatives 1, 20a–e on the RH123 accumulation

R²

R¹

N

OH

N

S

N

20

R¹

R²

Compound

Inhibition of RH123 efﬂux (% of the effect of 10 lM verapamil)

H

Cl

CF₃

Cl

OCH₃

Cl

OCH₃

COOEt

20a⁷

20b⁷

20c⁷

1

<25

25<; <75

<25

25<; <75

<25

20d⁷

20e

Table 4

Comparison of inhibition of RH123 accumulation by of N-hydroxyalkyl-2-aminophe-

nothiazines 3a–m

Table 5

Comparison of inhibition of RH123 accumulation by sulfoxide and sulfone derivatives

of 2-aminopheno-thiazines 11, 10, 4f,h,i, 16f,h,i

Compound Inhibition of RH123 efﬂux (% of the effect of 10 lM verapamil)

3a

3b

3c

3d

3e

3f

3g

3h

3i

>75

>75^a

11

>75

25<; <75

ꢂ100^a

<25

10

>75

4f

4h

4i

16f

16h

16i

>75

>100^a

25<; <75

>75

ꢂ100^a

>100^a

>75

>75^a

3j

3k

3l

<25

25<; <75

>75

a

Highest activity, concentration-dependence was also studied, see Figures 2–4.

3m

ꢂ100

3f

a

200

Highest activity, concentration-dependence was also studied, see Figures 2–4.

3h

Verapamil

175

150

125

100

10

20

30

40

50

concentration (μM)

Figure 2. Comparison of RH123 accumulation in the presence of 3f, 3h, and

verapamil.

Figure 1. Comparison of P-gp interaction activity of racemic phenothiazines and

their stereoisomer counterparts.

of Organic Chemistry, Research Centre for Natural Sciences, Hun-

garian Academy of Sciences, Pusztaszeri út 59, H-1025 Budapest,

Hungary). The exact mass measurements were performed using a

Q-TOF Premier mass spectrometer (Waters Corporation, 34 Maple

St, Milford, MA, USA) in positive electrospray mode. Enantiomeric

purities of the compounds were determined by chiral HPLC meth-

od. The chiral separation was performed with a system composed

Avatar 320 FT-IR spectrometer. NMR experiments were carried out

on 300 MHz (for ¹H) and 400 MHz (for ¹H) Varian NMR SYSTEM

spectrometers by using 5 mm Z gradient auto tunable probes. Mea-

surements were performed at +25 °C in CDCl₃or DMSO-d₆. ¹H and

¹³C NMR spectra are referenced to residual solvent signals. For the

complete ¹H, ¹³C assignments standard 1D and homo and hetero-

correlation 2D measurements were performed. The elemental

analysis has been carried out with an Elementar Vario EL III appa-

ratus (at the Analytical Laboratory for Organic Chemistry, Institute

of a Jasco PU-980 pump, a Rheodyne 7125 injector (20 ll loop), a

Jasco MD 2010 Plus UV/Vis photodiode-array detector (at

260 nm) and a ChromPass chromatographic software. Successful

separation of compounds 3f, 3h, 3i, 5 (–OH derivatives) and the

protected –Cl (8) was performed on a Chiralpak AD column

Please cite this article in press as: Takács, D.; et al. Bioorg. Med. Chem. (2013), http://dx.doi.org/10.1016/j.bmc.2013.04.034

8

D. Takács et al. / Bioorg. Med. Chem. xxx (2013) xxx–xxx

3b

*

3f

100

80

60

40

20

0

3h

250

225

200

175

150

125

100

3i

4f

verapamil

3h

3f

3b

4h

16h

0.0

2.5

5.0

7.5

10.0

concentration (µM)

Figure 5. Results of the viability (MTT) assays.

Figure 3. Comparison of RH123 accumulation in the presence of 3b, 3f, 3i, 3h, 4h,

and verapamil.

2-amine (2, 4 mmol) in abs THF (40 mL) in a dried, round-bot-

tomed ﬂask equipped with a side arm and cooled to 0 °C in an ar-

gon atmosphere was added BH₃ꢁ Me₂S (1.6 mL, 16 mmol)

dropwise by injection. After addition, the resulting yellow suspen-

sion was allowed to warm up to rt and maintained at this temper-

ature for prolongued time. After the completion of the reation

(1 day, monitored by TLC), the reaction mixture was cooled to

0 °C with an ice bath, and treated with methanol (30 mL), 10%

aqueous NaOH (2 mL) and H₂O₂(30% aqueous solution, 2 mL). This

mixture was then stirred at rt for 1 day. The solution was then

poured onto ice cold water, neutralized with 10% aqueous HCl

and extracted with dichloromethane (3 ꢁ 30 mL). The combined

organic phase was dried over anhydrous Na₂SO₄, ﬁltered and con-

centrated. The residue was puriﬁed by ﬂash chromatography on

silica gel using the eluent dichloromethane then n-hexane:ethyl

acetate = 2:1. The crude product (foam) was triturated with diethyl

ether, the mixture was cooled whereupon a solid product depos-

ited. The product was collected by ﬁltration.

3h

325

300

275

250

225

200

175

150

125

100

4h

16h

*

verapamil

*

0.0

2.5

5.0

concentration (µM)

7.5

10.0

4.3.2. General procedure for preparation of 1-(2-amino-10H-

phenothiazin-10-yl)-4-[2-(4-methoxyphenyl)-2H-tetrazol-5-

yl]butan-2-ol derivatives (3a–k) as well as the related amides

(3l,m) by debenzylation of 10-{2-(benzyloxy)-4-[2-(4-

methoxyphenyl)-2H-tetrazol-5-yl]butyl}-10H-phenothiazin-2-

amine/amide derivatives (7). Route ‘B’

Figure 4. Comparison of RH123 accumulation in the presence of 3h, 4h, 16h, and

verapamil.

(250 ꢁ 4.6 mm I.D. Daicel Chemical Ind., Tokyo, Japan). The mobile

phase was 70% n-hexane, 30% 2-propanol with 0.1% diethylamine

and 98% n-hexane, 2% 2-propanol with 0.1% diethylamine for 8,

the ﬂow rate was 1 mL/min. The basic protected compounds (9f,

To the clear solution of 10-{2-(benzyloxy)-4-[2-(4-methoxy-

phenyl)-2H-tetrazol-5-yl]butyl}-10H-phenothiazin-2-amine/amide

(7, 0.08 mmol) in ethanol (1.2 mL) was added concd HCl (1.2 mL)

dropwise, very slowly and the reaction mixture was reﬂuxed for

4–6 days. The mixture was concentrated under reduced pressure.

The residue was dissolved in water (2 mL), neutralized with concd

NaHCO₃solution and extracted with dichloromethane (3 ꢁ 3 mL).

The organic phase was dried over Na₂SO₄, ﬁltered and concen-

trated. The crude product was puriﬁed by column chromatography

on silica gel using the eluent n-hexane:ethyl acetate = 2:1 then 1:1

to give the ﬁnal product as a solid.

9h, 9i) were analysed on

a

LUX-Amylose-2 column

(250 ꢁ 4.6 mm I.D. Phenomenex Inc., USA). The mobile phase was

75% n-hexane, 25% 2-propanol with 0.1% diethylamine, the ﬂow

rate was 1 mL/min. Optical rotation values were determined on

AA-10R and JASCO P-2000 polarimeters, (k = 589 nm). Chromato-

graphic separations were carried out on silica gel (60 H, Merck).

Reactions were monitored with Merck silica gel 60 F254, TLC plates

(0.25 mm thickness). BH₃ꢁ Me₂S (borane–dimethyl sulﬁde com-

plex) >90% in dimethyl sulﬁde was purchased from Sigma–Aldrich.

All the chemicals and solvents were used as supplied.

4.3.3. General procedure for preparation of 1-(2-amino-10H-

phenothiazin-10-yl)-4-[2-(4-methoxyphenyl)-2H-tetrazol-5-

yl]butan-2-ol derivatives (3a–k) as well as the related amides

(3l,m) by desilylation of 10-(2-{[tert-butyl(diphenyl)silyl]oxy}-

4-[2-(4-methoxyphenyl)-2H-tetrazol-5-yl]butyl)-10H-

4.3.1. General procedure for the synthesis of 1-(2-amino-10H-

phenothiazin-10-yl)-4-[2-(4-methoxyphenyl)-2H-tetrazol-5-

yl]butan-2-ol (3) derivatives by hydroboration–oxidation of

dienes 2. Route ‘A’

To a solution of the appropriate 10-{(1E,3Z)-4-[2-(4-methoxy-

phenyl)-2H-tetrazol-5-yl]buta-1,3-dien-1-yl}-10H-phenothiazin-

phenothiazin-2-amine/amide derivatives (9). Route ‘C’

To the solution 10-(2-{[tert-butyl(diphenyl)silyl]oxy}-4-[2-(4-

methoxyphenyl)-2H-tetrazol-5-yl]butyl)-10H-phenothiazin-2-

amine/amide (9, 0.14 mmol) in anhydrous THF (1 mL) in a dried,

Please cite this article in press as: Takács, D.; et al. Bioorg. Med. Chem. (2013), http://dx.doi.org/10.1016/j.bmc.2013.04.034

D. Takács et al. / Bioorg. Med. Chem. xxx (2013) xxx–xxx

9

round-bottomed ﬂask equipped with a side arm and cooled to 0 °C

in an argon atmosphere was added the solution of TBAF

(0.20 mmol) in abs THF (1 mL). After 48 h, the reaction mixture

was allowed to warm up to rt and the reaction the mixture was

evaporated. The residue was puriﬁed by column chromatography

on silica gel using the eluent chloroform:methanol = 50:1, then

20:1 to give the product.

3.78 (1H, dd, J = 13.0, 9.0 Hz), 3.87 (3H, s), 3.95 (1H, dd, J = 13.0,

3.2 Hz), 4.07 (1H, m), 4.15 (1H, br s), 4.29 (2H, s), 6.22–6.29 (2H,

m), 6.87–7.03 (5H, m), 7.09–7.19 (2H, m), 7.26–7.36 (5H, m),

7.96 (2H, m); d_C(75 MHz, CDCl₃) 22.1, 32.3, 48.6, 54.0, 55.9, 66.6,

102.1, 108.3, 113.7, 114.8 (2C), 116.3, 121.5 (2C), 123.1, 127.2,

127.5 (2C), 127.8, 128.1, 128.5, 128.9 (2C), 130.7, 139.4, 145.9,

146.6, 148.4 (2C), 160.6, 166.7.

4.3.4. 1-(2-{[2-(Dimethylamino)ethyl]amino}-10H-

phenothiazin-10-yl)-4-[2-(4-methoxyphenyl)-2H-tetrazol-5-

yl]butan-2-ol (3a)

4.3.7. 4-[2-(4-Methoxyphenyl)-2H-tetrazol-5-yl]-1-(2-{[(1R)-1-

phenylethyl]amino}-10H-phenothiazin-10-yl)butan-2-ol (3d)

According to route ‘A’, this compound was obtained from 10-

{(1E,3Z)-4-[2-(4-methoxyphenyl)-2H-tetrazol-5-yl]buta-1,3-dien-

According to route ‘C’, this compound was obtained from N⁰-[10-

(2-{[tert-butyl(diphenyl)silyl]oxy}-4-[2-(4-methoxyphenyl)-2H-

tetrazol-5-yl]butyl)-10H-phenothiazin-2-yl]-N,N-dimethylethane-

1,2-diamine (9a, 0.11 g) as a beige amorphous solid, 76 mg (91%);

mp 56–58 °C; (found: C, 63.01; H, 6.18; N, 18.38; C₂₈H₃₃N₇O₂S re-

quires C, 63.25; H, 6.26; N, 18.44); m/z (EI) [M+H]⁺: 532.20

(C₂₈H₃₃N₇O₂S requires 531.24); m_max(KBr)/cm^ꢀ1: 3368, 2930,

1601, 1516 and 1464; d_H(300 MHz, CDCl₃) 2.05 (1H, m), 2.21

(1H, m), 2.24 (6H, s), 2.47 (1H, m), 2.54 (2H, t, J = 7.8 Hz), 3.09–

3.22 (4H, m), 3.85 (3H, s), 3.87 (1H, m), 4.07 (1H, m), 4.18 (1H,

m), 4.25 (1H, m), 6.25–6.27 (2H, m), 6.89–7.19 (7H, m), 7.97 (2H,

m); d_C(75 MHz, CDCl₃) 21.9, 32.2, 41.1, 45.1 (2C), 53.8, 55.6,

57.8, 66.5, 101.9, 107.8, 113.0, 114.6 (2C), 116.1, 121.3 (2C),

122.8, 126.9, 127.6, 128.0, 128.2, 130.4, 145.5, 146.7, 148.6,

160.4, 166.5.

1-yl}-N-[(1R)-1-phenylethyl]-10H-phenothiazin-2-amine

(2d,

2.19 g) as beige amorphous solid, 1.04 g (46%); mixture of two dia-

stereomers; mp 63–68 °C; (found: C, 68.15; H, 6.05; N, 14.65;

C

₃₂H₃₂N₆O₂S requires C, 68.06; H, 5.71; N, 14.88); m/z (EI)

[M+H]⁺: 565.30 (C₃₂H₃₂N₆O₂S requires 564.23); m_max(KBr)/cm^ꢀ1

:

3374, 2924, 1600, 1515 and 1466; d_H(400 MHz, DMSO-d₆) 1.38

[1.37] (3H, d, J = 6.5 Hz, CH₃), 1.70 (1H, m, H3y), 2.00 [2.09] (1H,

m, H3x), 2.88 (1H, m, H4y), 3.00 (1H, m, H4x), 3.66 (1H, m, H1y),

3.80 (1H, m, H1x), 3.83 [3.90] (1H, m, H2), 3.85 (3H, s, OCH₃),

4.43 [4.46] (1H, qd, J = 6.5, 6.2 Hz, NH–CH), 4.91 [4.95] (1H, d,

J = 5.8 Hz, OH), 6.09 [6.12] (1H, dd, J = 8.6, 2.0 Hz, H3⁰⁰), 6.19

[6.21] (1H, d, J = 6.2 Hz, NH), 6.27 (1H, d, J = 2.0 Hz, H1⁰⁰), 6.71

(1H, d, J = 8.6 Hz, H4⁰⁰), 6.86 (1H, dd, J = 7.8, 7.5 Hz, H7⁰⁰), 7.00

(1H, d, J = 7.8 Hz, H9⁰⁰), 7.05 (1H, d, J = 7.8 Hz, H6⁰⁰), 7.11 (1H, dd,

J = 7.8, 7.5 Hz, H8⁰⁰), 7.15 (1H, m, H4⁰⁰⁰), 7.16 (2H, m, H3⁰+ H5⁰),

7.27 (2H, m, H3⁰⁰⁰+ H5⁰⁰⁰), 7.35 (2H, m, H2⁰⁰⁰+ H6⁰⁰⁰), 7.90 (2H, m,

H2⁰+ H6⁰); d_C(100 MHz, DMSO-d₆) 21.0 (C4), 24.5 [24.6] (CH₃),

33.0 (C3), 52.2 [52.1] (HN–CH), 52.7 (C1), 55.6 (OCH₃), 65.4

[65.3] (C2), 101.7 [101.5] (C1⁰⁰), 107.4 [107.6] (C3⁰⁰), 108.8 (C4a⁰⁰),

115.0 (C3⁰+ C5⁰), 115.9 (C9⁰⁰), 121.3 (C2⁰+ C6⁰), 122.0 (C7⁰⁰), 125.6

(C5a⁰⁰), 125.8 (C2⁰⁰⁰+ C6⁰⁰⁰), 126.4 (C4⁰⁰⁰), 126.9 (C6⁰⁰+ C8⁰⁰), 127.3

(C4⁰⁰), 128.3 (C3⁰⁰⁰+ C5⁰⁰⁰), 129.6 (C1⁰), 145.3 (C9a⁰⁰), 146.0 (C1⁰⁰⁰),

146.1 (C10a⁰⁰), 148.1 (C2⁰⁰), 160.1 (C4⁰), 166.5 (C_tetrazole).

4.3.5. 4-[2-(4-Methoxyphenyl)-2H-tetrazol-5-yl]-1-{2-[(2-

morpholin-4-ylethyl)amino]-10H-phenothiazin-10-yl}butan-2-

ol (3b)

According to route ‘A’, this compound was obtained from 10-

{(1E,3Z)-4-[2-(4-methoxyphenyl)-2H-tetrazol-5-yl]buta-1,3-dien-

1-yl}-N-(2-morpholin-4-ylethyl)-10H-phenothiazin-2-amine (2b,

2.22 g) as yellow amorphous solid, 0.6 g (26%).

According to route ‘C’, this compound was obtained from 10-(2-

{[tert-butyl(diphenyl)silyl]oxy}-4-[2-(4-methoxyphenyl)-2H-tetra-

zol-5-yl]butyl)-N-(2-morpholin-4-ylethyl)-10H-phenothiazin-2-amine

(9b, 0.12 g) as yellow amorphous solid, 85 mg (84%); mp 65–68 °C;

(found: C, 63.19; H, 6.06; N, 16.90; C₃₀H₃₅N₇O₃S requires C, 62.81;

H, 6.15; N, 17.09); m/z (EI) [M+H]⁺: 574.10 (C₃₀H₃₅N₇O₃S requires

573.25); m_max(KBr)/cm^ꢀ1: 3374, 2933, 1601, 1515 and 1464; d_H

(300 MHz, CDCl₃) 2.03 (1H, m), 2.20 (1H, m), 2.34–2.43 (4H, m),

2.58 (2H, t, J = 5.9 Hz), 2.85 (1H, br s), 3.07–3.19 (4H, m), 3.68

(4H, t, J = 4.3 Hz), 3.86 (3H, s), 3.88 (1H, m), 4.04 (1H, m), 4.20

(1H, m), 4.22 (1H, m), 6.24–6.26 (2H, m), 6.89–7.02 (5H, m), 7.13

(2H, m), 7.95 (2H, m); d_C(75 MHz, CDCl₃) 22.1, 32.5, 40.3, 53.5

(2C), 54.0, 55.9, 57.2, 66.7, 67.1 (2C), 102.1, 108.1, 113.5, 114.9

(2C), 116.4, 121.6 (2C), 123.1, 127.2, 127.9, 128.2, 128.5, 135.0,

145.7, 147.0, 148.8, 160.6, 166.7.

4.3.8. 1-[2-(Butylamino)-10H-phenothiazin-10-yl]-4-[2-(4-

methoxyphenyl)-2H-tetrazol-5-yl]butan-2-ol (3e)

According to route ‘C’, this compound was obtained from N-bu-

tyl-10-(2-{[tert-butyl(diphenyl)silyl]oxy}-4-[2-(4-methoxyphenyl)-

2H-tetrazol-5-yl]butyl)-10H-phenothiazin-2-amine (9e, 95 mg) as

amorphous solid, 32 mg (49%); mp 114–115 °C; (found: C, 65.01;

H, 6.39; N, 15.93; C₂₈H₃₂N₆O₂S requires C, 65.09; H, 6.24; N,

16.27); m/z (EI) [M+H]⁺: 517.50 (C₂₈H₃₂N₆O₂S requires 516.23);

m_max(KBr)/cm^ꢀ1: 3403, 2956, 1601, 1517 and 1466; d_H(300 MHz,

CDCl₃) 0.94 (3H, t, J = 7.2 Hz), 1.36 (2H, m), 1.56 (2H, m), 2.13

(1H, m), 2.20 (1H, m), 2.86 (1H, br s), 3.02–3.26 (5H, m), 3.81

(1H, m), 3.84 (3H, s), 4.04 (1H, dd, J = 13.2, 3.0 Hz), 4.19 (1H, m),

6.22–6.24 (2H, m), 6.89–7.19 (7H, m), 7.97 (2H, m); d_C(75 MHz,

CDCl₃) 13.9, 20.2, 21.9, 31.5, 32.2, 43.8, 53.7, 55.6, 66.4, 101.7,

107.6, 112.8, 114.6 (2C), 116.1, 121.3 (2C), 122.8, 126.9, 127.6,

128.0, 128.2, 130.4, 145.5, 146.6, 148.7, 160.4, 166.4.

4.3.6. 1-[2-(Benzylamino)-10H-phenothiazin-10-yl]-4-[2-(4-

methoxyphenyl)-2H-tetrazol-5-yl]butan-2-ol (3c)

According to route ‘A’, this compound was obtained from N-ben-

zyl-10-{(1E,3Z)-4-[2-(4-methoxyphenyl)-2H-tetrazol-5-yl]buta-1,3-

dien-1-yl}-10H-phenothiazin-2-amine (2c, 2.10 g) as a brown oil,

0.65 g (30%).

According to route ‘B’, this compound was obtained from N-ben-

zyl-10-{2-(benzyloxy)-4-[2-(4-methoxyphenyl)-2H-tetrazol-5-yl]-

butyl}-10H-phenothiazin-2-amine (7c, 0.05 g, 0.08 mmol) as a

brown oil, 42 mg (quant.); (found: C, 67.42; H, 5.83; N, 15.06;

4.3.9. 1-[2-(Diethylamino)-10H-phenothiazin-10-yl]-4-[2-(4-

methoxyphenyl)-2H-tetrazol-5-yl]butan-2-ol (3f)

According to route ‘A’, this compound was obtained from N,N-

diethyl-10-{(1E,3Z)-4-[2-(4-methoxyphenyl)-2H-tetrazol-5-yl]bu-

ta-1,3-dien-1-yl}-10H-phenothiazin-2-amine (2f, 2.0 g) as white

amorphous solid, 0.48 g (23%).

According to route ‘C’, this compound was obtained from 10-(2-

{[tert-butyl(diphenyl)silyl]oxy}-4-[2-(4-methoxyphenyl)-2H-tetra-

zol-5-yl]butyl)-N,N-diethyl-10H-phenothiazin-2-amine (9f, 0.11 g,

0.14 mmol) as white amorphous solid, 0.14 g (86%); mp 51–

53 °C; HPLC: using the eluent n-hexane:2-propanol + 0.1% diethyl-

amine = 70:30, retention times: 9.5 min, 22.1 min; (found: C,

65.01; H, 6.23; N, 15.90; C₂₈H₃₂N₆O₂S requires C, 65.09; H, 6.24;

C₃₁H₃₀N₆O₂S requires C, 67.61; H, 5.49; N, 15.26); HRMS calcd

for C₃₁H₃₀N₆O₂S 550.2151 found: 550.2151; m/z: 534 (87%), 506

(22), 464 (6), 385 (98), 352 (26), 330 (9), 303 (100), 267 (13),

213 (28), 185 (63), 160 (15), 123 (49) and 91 (74); m_max(ﬁlm)/

cm^ꢀ1: 3381, 2928, 1600, 1515 and 1257; d_H(300 MHz, CDCl₃)

1.97 (1H, m), 2.11 (1H, m), 2.66 (1H, br s), 3.06–3.17 (2H, m),

Please cite this article in press as: Takács, D.; et al. Bioorg. Med. Chem. (2013), http://dx.doi.org/10.1016/j.bmc.2013.04.034

10

D. Takács et al. / Bioorg. Med. Chem. xxx (2013) xxx–xxx

N, 16.27); m/z (EI) [M+H]⁺: 517.50 (C₂₈H₃₂N₆O₂S requires 516.23);

m_max(KBr)/cm^ꢀ1: 2967, 2929, 1598, 1516 and 1467; d_H(400 MHz,

DMSO-d₆) 1.03 (6H, t, J = 7.1 Hz, N(CH₂)₂(CH₃)₂), 1.76 (1H, dddd,

J = 13.5, 9.3, 8.2, 5.4 Hz, H3y), 2.12 (1H, m, H3x), 2.94 (1H, ddd,

J = 15.3, 8.2, 7.4 Hz, H4y), 3.03 (1H, ddd, J = 15.3, 9.1, 5.4 Hz,

H4x), 3.28 (4H, q, J = 7.1 Hz, N(CH₂)₂(CH₃)₂), 3.79 (1H, m, H1y),

3.83 (3H, s, OCH₃), 3.96 (1H, m, H1x), 4.00 (1H, m, H2), 5.02 (1H,

d, J = 5.4 Hz, OH), 6.27 (1H, m, H3⁰⁰), 6.29 (1H, m, H1⁰⁰), 6.86 (1H,

d, J = 8.0 Hz, H4⁰⁰), 6.88 (1H, t, J = 7.5 Hz, H7⁰⁰), 7.05 (1H, d,

J = 7.5 Hz, H9⁰⁰), 7.10 (1H, d, J = 7.5 Hz, H6⁰⁰), 7.14 (1H, m, H8⁰⁰),

7.16 (2H, m, H3⁰+ H5⁰), 7.88 (2H, m, H2⁰+ H6⁰); d_C(100 MHz,

DMSO-d₆) 12.4 (N(CH₂)₂(CH₃)₂, 2C), 21.0 (C4), 33.1 (C3), 43.8

(N(CH₂)₂(CH₃)₂, 2C), 53.0 (C1), 55.6 (OCH₃), 65.6 (C2), 100.5

(C1⁰⁰), 106.5 (C3⁰⁰), 108.5 (C4a⁰⁰), 114.9 (C3⁰+ C5⁰), 116.1 (C9⁰⁰),

121.3 (C2⁰+ C6⁰), 122.1 (C7⁰⁰), 125.8 (C5a⁰⁰), 126.9 (C6⁰⁰+ C8⁰⁰),

127.6 (C4⁰⁰), 129.6 (C1⁰), 145.4 (C9a⁰⁰), 146.4 (C10a⁰⁰), 147.5 (C2⁰⁰),

160.0 (C4⁰), 166.4 (C_tetrazole).

1-yl}-2-(4-methylpiperazin-1-yl)-10H-phenothiazine (2i, 2.01 g)

as a brown oil, 1.06 g (49%).

According to route ‘B’, this compound was obtained from 10-{2-

(benzyloxy)-4-[2-(4-methoxyphenyl)-2H-tetrazol-5-yl]butyl}-2-

(4-methylpiperazin-1-yl)-10H-phenothiazine

(7i,

0.32 g,

0.50 mmol) as a brown oil, 0.09 g (33%).

According to route ‘C’, this compound was obtained from 10-(2-

{[tert-butyl(diphenyl)silyl]oxy}-4-[2-(4-methoxyphenyl)-2H-tet-

razol-5-yl]butyl)-2-(4-methylpiperazin-1-yl)-10H-phenothiazine

(9i, 51 mg) as a brown oil, 47 mg (64%); HPLC: using the eluent n-

hexane:2-propanol + 0.1% diethylamine = 70:30, retention times:

17.3, 23.8 min; (found: C, 64.06; H, 6.18; N, 17.91; C₂₉H₃₃N₇O₂S re-

quires C, 64.06; H, 6.12; N, 18.03); HRMS calcd for C₂₉H₃₃N₇O₂S

543.2416 found: 543.2434; m/z: 543 (54%), 515 (70), 464 (26),

394 (47), 310 (83), 239 (29), 207 (38), 167 (18), 108 (56) and 70

(87); m_max(ﬁlm)/cm^ꢀ1: 2966, 2843, 1594, 1514 and 1257; d_H

(300 MHz, CDCl₃) 2.06 (1H, m), 2.26 (1H, m), 2.34 (3H, s), 2.54

(4H, t, J = 5.1 Hz), 2.80 (1H, br s), 3.10–3.24 (6H, m), 3.87 (1H, m),

3.88 (3H, s), 4.07 (1H, dd, J = 13.5, 3.6 Hz), 4.20 (1H, m), 6.52–

6.56 (2H, m), 6.90–7.20 (7H, m), 7.98 (2H, m); d_C(75 MHz, CDCl₃)

22.1, 32.4, 46.3, 49.6 (2C), 54.2, 55.2 (2C), 55.9, 66.7, 105.4, 111.5,

114.9 (2C), 116.5, 116.7, 121.6 (2C), 123.2, 127.3, 127.9, 128.0,

128.2, 130.9, 145.6, 146.7, 151.8, 160.6, 166.6.

4.3.10. 1-[2-(Diphenylamino)-10H-phenothiazin-10-yl]-4-[2-(4-

methoxyphenyl)-2H-tetrazol-5-yl]butan-2-ol (3g)

According to route ‘A’, this compound was obtained from 10-

{(1E,3Z)-4-[2-(4-methoxyphenyl)-2H-tetrazol-5-yl]buta-1,3-dien-

1-yl}-N,N-diphenyl-10H-phenothiazin-2-amine (2g, 2.38 g) as

beige amorphous solid, 1.20 g (49%); mp 70–74 °C, (found: C,

70.51; H, 5.37; N, 13.55; C₃₆H₃₂N₆O₂S requires C, 70.57; H, 5.26;

4.3.13. 4-[2-(4-Methoxyphenyl)-2H-tetrazol-5-yl]-1-(2-

N, 13.72); HRMS calcd for

C

₃₆H₃₂N₆O₂S 612.2307 found:

piperidin-1-yl-10H-phenothiazin-10-yl)butan-2-ol (3j)

612.2289; m/z: 612 (23%), 584 (62), 569 (3), 488 (7), 463 (42),

436 (2), 408 (2), 379 (100), 365 (42), 347 (28), 332 (19), 304 (5),

292 (9), 269 (14), 255 (12), 239 (5), 225 (9), 210 (13), 186 (6),

167 (12), 136 (4), 122 (38), 108 (22), 91 (15), 77 (23), 63 (5) and

51 (9); m_max(KBr)/cm^ꢀ1: 2929, 1580, 1516, 1456 and 1256; d_H

(400 MHz, DMSO-d₆) 1.69 (1H, m), 2.00 (1H, m), 2.88 (1H, m),

2.98 (1H, m), 3.61 (1H, dd, J = 13.5, 6.2 Hz), 3.74 (1H, dd, J = 13.5,

5.6 Hz), 3.83 (1H, m), 3.84 (3H, s), 4.94 (1H, d, J = 5.7 Hz), 6.53–

6.56 (2H, m), 6.93–7.03 (9H, m), 7.14–7.26 (8H, m), 7.89 (2H, m);

d_C(100 MHz, DMSO-d₆) 21.0, 32.9, 52.9, 55.6, 65.3, 112.0, 115.0

(2C), 116.3, 118.0, 118.2, 121.2 (2C), 122.6 (2C), 122.9, 123.6

(4C), 124.5, 127.1, 127.4, 127.8, 129.4 (4C), 129.6, 145.0, 146.0,

147.0 (2C), 147.1, 160.1, 166.4.

According to route ‘A’, this compound was obtained from 10-

{(1E,3Z)-4-[2-(4-methoxyphenyl)-2H-tetrazol-5-yl]buta-1,3-dien-

1-yl}-2-piperidin-1-yl-10H-phenothiazin (2j, 2.0 g) as beige amor-

phous solid, 0.91 g (44%); mp 63–67 °C; (found: C, 65.81; H, 6.31;

N, 15.50; C₂₉H₃₂N₆O₂S requires C, 65.88; H, 6.10; N, 15.90); HRMS

calcd for C₂₉H₃₂N₆O₂S 528.2307 found: 528.2314; m/z: 528 (79%),

500 (50), 485 (2), 379 (22), 346 (17), 307 (4), 295 (100), 281 (95),

263 (29), 225 (11), 205 (16), 179 (5), 137 (4), 122 (30), 106 (11), 92

(4), 80 (10) and 65 (4); m_max(KBr)/cm^ꢀ1: 2931, 1595, 1516, 1464

and 1256; d_H(300 MHz, CDCl₃) 1.56 (2H, m), 1.68 (4H, m), 2.05

(1H, m), 2.24 (1H, m), 2.78 (1H, br s), 3.06–3.25 (6H, m), 3.84

(1H, m), 3.86 (3H, s), 4.04–4.21 (2H, m), 6.52–6.57 (2H, m), 6.93–

7.20 (7H, m), 7.99 (2H, m); d_C(75 MHz, CDCl₃) 21.9, 24.9, 25.8

(2C), 32.2, 51.0 (2C), 53.8, 55.6, 66.4, 105.6, 111.8, 114.6 (2C),

115.8, 116.2, 121.3 (2C), 122.9, 127.0, 127.7, 127.8, 127.9, 132.0,

145.4, 146.3, 152.5, 160.4, 166.4.

4.3.11. 4-[2-(4-Methoxyphenyl)-2H-tetrazol-5-yl]-1-(2-

morpholin-4-yl-10H-phenothiazin-10-yl)butan-2-ol (3h)

According to route ‘A’, this compound was obtained from 10-

{(1E,3Z)-4-[2-(4-methoxyphenyl)-2H-tetrazol-5-yl]buta-1,3-dien-

4.3.14. 4-[2-(4-methoxyphenyl)-2H-tetrazol-5-yl]-1-(2-

1-yl}-2-morpholin-4-yl-10H-phenothiazine

(2h,

2.05 g,

pyrrolidin-1-yl-10H-phenothiazin-10-yl)butan-2-ol (3k)

0.49 mmol) as white crystals, 0.70 g (33%).

According to route ‘C’, this compound was obtained from 10-(2-

{[tert-butyl(diphenyl)silyl]oxy}-4-[2-(4-methoxyphenyl)-2H-tet-

According to route ‘B’, this compound was obtained from 10-{2-

(benzyloxy)-4-[2-(4-methoxyphenyl)-2H-tetrazol-5-yl]butyl}-2-

pyrrolidin-1-yl-10H-phenothiazine (7k, 50 mg) as brown amor-

phous solid, 26 mg (62%); mp 154–157 °C; (found: C, 64.99; H,

6.01; N, 15.93; C₂₈H₃₀N₆O₂S requires C, 65.35; H, 5.88; N, 16.33),

m/z (EI) [M+Na]⁺: 536.30 (C₂₈H₃₀N₆O₂S requires 514.64); m_max

(KBr)/cm^ꢀ1: 3345, 2852, 1598, 1515 and 1257; d_H(300 MHz,

CDCl₃) 1.96–2.09 (5H, m), 2.21 (1H, m), 2.85 (1H, br s), 3.13–3.22

(6H, m), 3.88 (3H, s), 3.91 (1H, m), 4.09 (1H, dd, J = 13.5, 3.3 Hz),

4.23 (1H, m), 6.14–6.21 (2H, m), 6.89–7.29 (7H, m), 7.98 (2H, m);

d_C(75 MHz, CDCl₃) 22.1, 25.6 (2C), 32.4, 48.0 (2C), 54.1, 55.9,

66.7, 100.8, 107.3, 111.4, 114.8 (2C), 116.4, 121.5 (2C), 123.0,

127.1, 127.8, 128.3, 128.5, 130.7, 145.9, 146.9, 148.3, 160.6, 166.7.

razol-5-yl]butyl)-2-morpholin-4-yl-10H-phenothiazine

(9h,

0.11 g) as white crystals, 0.14 g (87%); mp 64–68 °C; HPLC: using

the eluent n-hexane:2-propanol + 0.1% diethylamine = 70:30,

retention times: 11.8 min, 17.4 min; (found: C, 63.37; H, 5.80; N,

15.48; C₂₈H₃₀N₆O₃S requires C, 63.38; H, 5.70; N, 15.84); m/z (EI)

[M+H]⁺: 531.40 (C₂₈H₃₀N₆O₃S requires 530.21); m_max(KBr)/cm^ꢀ1

:

3307, 2959, 1596, 1516 and 1466; d_H(300 MHz, CDCl₃) 2.06 (1H,

m), 2.21 (1H, m), 2.77 (1H, br s), 3.09–3.18 (6H, m), 3.83–4.19

(6H, m), 3.89 (3H, s), 3.95 (1H, br s), 6.45–6.58 (2H, m), 6.91–

7.20 (7H, m), 7.97 (2H, m); d_C(75 MHz, CDCl₃) 22.0, 32.4, 49.9

(2C), 54.2, 55.9, 66.6, 67.0 (2C), 105.1, 111.2, 114.9 (2C), 116.5,

117.2, 121.5 (2C), 123.3, 127.4, 127.7, 127.9, 128.3, 130.6, 145.6,

146.7, 151.7, 160.6, 166.6.

4.3.15. N-(10-{2-Hydroxy-4-[2-(4-methoxyphenyl)-2H-tetrazol-

5-yl]butyl}-10H-phenothiazin-2-yl)formamide (3l) (E/Z

isomers)

4.3.12. 4-[2-(4-Methoxyphenyl)-2H-tetrazol-5-yl]-1-[2-(4-

methylpiperazin-1-yl)-10H-phenothiazin-10-yl]butan-2-ol (3i)

According to route ‘A’, this compound was obtained from 10-

{(1E,3Z)-4-[2-(4-methoxyphenyl)-2H-tetrazol-5-yl]buta-1,3-dien-

According to route ‘C’, this compound was obtained from N-[10-

(2-{[tert-butyl(diphenyl)silyl]oxy}-4-[2-(4-methoxyphenyl)-2H-

tetrazol-5-yl]butyl)-10H-phenothiazin-2-yl]formamide (9l, 0.10 g,

0.14 mmol) as grey amorphous solid, 0.06 g (99%); mp 75–78 °C;

Please cite this article in press as: Takács, D.; et al. Bioorg. Med. Chem. (2013), http://dx.doi.org/10.1016/j.bmc.2013.04.034

D. Takács et al. / Bioorg. Med. Chem. xxx (2013) xxx–xxx

11

(found: C, 61.16; H, 4.61; N, 17.02; C₂₅H₂₄N₆O₃S requires C, 61.46;

H, 4.95; N, 17.20); m/z (EI) [M+H]⁺: 489.20 (C₂₅H₂₄N₆O₃S requires

488.16); m_max(KBr)/cm^ꢀ1: 3266, 1685, 1591, 1515 and 1461; d_H

(300 MHz, CDCl₃) 2.07 (1H, m), 2.21 (1H, m), 2.84 (1H, br s), 3.16

(2H, m), 3.86 (3H, s), 3.88 (1H, m), 4.07 (1H, m), 4.17 (1H, m),

6.67 [6.70] (1H, s), 6.91–7.58 (9H, m), 7.96 (2H, m), 8.33 [8.64]

(1H, d, J = 1.1, [J = 11.0] Hz); d_C(75 MHz, CDCl₃) 22.1, 32.5, 54.1,

55.9, 66.6, 108.7 [108.0], 114.6 [113.4], 114.9 (2C), 116.4, 116.5,

121.5 (2C), 123.5, 123.8, 127.7, 127.8, 128.0, 128.7, 136.9, 145.3

[145.0], 146.0 [146.9], 159.1, 162.3 [160.7], 166.6 [166.4].

methoxyphenyl)-2H-tetrazol-5-yl]butan-2-ol¹²(10), secondary

amine, base: NaO^tBu or Cs₂CO₃, and dry toluene (5 mL). The ﬂask

was ﬂushed with argon for 5 min. The resulting mixture was

heated under reﬂux with magnetic stirring for 0.5–7 h. After cool-

ing down to rt the reaction mixture was concentrated and the res-

idue was puriﬁed by ﬂash chromatography on silica gel using the

eluent dichloromethane, then n-hexane:ethyl acetate = 2:1 and

1:1 and the crude product was recrystallized from methanol to

give the ﬁnal product as yellow-white crystals.

4.3.19. 4-[2-(4-Methoxyphenyl)-2H-tetrazol-5-yl]-1-(2-

morpholin-4-yl-5-oxido-10H-phenothiazin-10-yl)butan-2-ol

(4h)

4.3.16. N-(10-{2-Hydroxy-4-[2-(4-methoxyphenyl)-2H-tetrazol-

5-yl]butyl}-10H-phenothiazin-2-yl)acetamide (3m)

According to route ‘C’, this compound was obtained from N-[10-

(2-{[tert-butyl(diphenyl)silyl]oxy}-4-[2-(4-methoxyphenyl)-2H-

tetrazol-5-yl]butyl)-10H-phenothiazin-2-yl]acetamide (9m, 0.10 g,

0.14 mmol) as grey amorphous solid, 65 mg (96%); mp 99–100 °C;

(found: C, 62.04; H, 4.99; N, 16.56; C₂₆H₂₆N₆O₃S requires C, 62.13;

H, 5.21; N, 16.72); m/z (EI) [M+H]⁺: 503.30 (C₂₆H₂₆N₆O₃S requires

This compound was obtained from the reaction of 1-(2-chloro-

5-oxido-10H-phenothiazin-10-yl)-4-[2-(4-methoxyphenyl)-2H-

tetrazol-5-yl]butan-2-ol (10, 0.30 g, 0.61 mmol), Pd₂(dba)₃(0.03 g,

0.03 mmol, 5 mol %), XPhos (0.03 g, 0.06 mmol, 10 mol %), NaO^tBu

(0.12 g, 1.21 mmol), and morpholine (0.11 mL, 1.21 mmol). The

reaction time was 0.5 h. After recrystallization, the white-yellow

crystals were collected by ﬁltration, 0.26 g (78%); mp 88–90 °C;

(found: C, 61.15; H, 5.64; N, 14.99; C₂₈H₃₀N₆O₄S requires C,

61.52; H, 5.53; N, 15.37); m/z (EI) [M+H]⁺: 547.40 (C₂₈H₃₀N₆O₄S re-

502.18);

m

_max(KBr)/cm^ꢀ1: 3306, 2934, 1666, 1515 and 1462; d_H

(400 MHz, DMSO-d₆) 1.77 (1H, dddd, J = 14.1, 9.0, 8.4, 5.4 Hz,

H3y), 2.00 (3H, s, CH₃), 2.12 (1H, m, H3x), 2.94 (1H, ddd, J = 14.8,

8.4, 7.4 Hz, H4y), 3.05 (1H, ddd, J = 14.8, 8.4, 5.4 Hz, H4x), 3.77

(1H, dd, J = 13.6, 6.5 Hz, H1y), 3.84 (3H, s, OCH₃), 3.92 (1H, dd,

J = 13.6, 5.8 Hz, H1x), 4.00 (1H, m, H2), 5.04 (1H, d, J = 5.5 Hz,

OH), 6.92 (1H, m, H7⁰⁰), 7.03 (1H, d, J = 8.0 Hz, H4⁰⁰), 7.08 (1H, d,

J = 8.1 Hz, H9⁰⁰), 7.12 (1H, dd, J = 7.8, 1.3 Hz, H6⁰⁰), 7.15–7.16 (4H,

m, H3⁰+ H5⁰+ H3⁰⁰+ H8⁰⁰), 7.40 (1H, d, J = 1.3 Hz, H1⁰⁰), 7.87 (2H,

m, H2⁰+ H6⁰), 9.90 (1H, s, NH); d_C(100 MHz, DMSO-d₆) 20.9 (C4),

23.9 (O@C–CH₃), 33.0 (C3), 52.9 (C1), 55.6 (OCH₃), 65.3 (C2),

107.1 (C1⁰⁰), 113.2 (C3⁰⁰), 115.0 (C3⁰+ C5⁰), 116.2 (C9⁰⁰), 117.6

(C2⁰⁰), 121.3 (C2⁰+ C6⁰), 122.6 (C7⁰⁰), 124.6 (C5a⁰⁰), 127.0 (C6⁰⁰),

127.1 (C4⁰⁰), 127.3 (C8⁰⁰), 129.6 (C1⁰), 139.2 (C4a⁰⁰), 144.9 (C9a⁰⁰),

145.5 (C10a⁰⁰), 160.0 (C4⁰), 166.4 (C_tetrazole), 168.3 (O@C–CH₃).

quires 546.20); m

_max(KBr)/cm^ꢀ1: 3274, 2926, 1589, 1516 and 1256;

d_H(400 MHz, DMSO-d₆) 1.86 (1H, m, H3y), 2.06 (1H, m, H3x), 2.97

(1H, m, H4y), 3.06 (1H, m, H4x), 3.29 (4H, m, N–(CH₂)₂), 3.74 (4H,

m, O–(CH₂)₂), 3.84 (3H, s, OCH₃), 4.05 (1H, m, H2), 4.34 (1H, m,

H1y), 4.48 (1H, m, H1x), 5.17 (1H, br s, OH), 6.89 (1H, m, H3⁰⁰),

6.97 [7.25] (1H, m, H1⁰⁰), 7.16 (2H, m, H3⁰+ H5⁰), 7.22 (1H, m,

H7⁰⁰), 7.58 [7.61] (1H, m, H8⁰⁰), 7.70 (1H, m, H4⁰⁰), 7.85 (1H, m,

H6⁰⁰), 7.86 [7.65] (1H, m, H9⁰⁰), 7.92 (2H, m, H2⁰+ H6⁰); d_C

(100 MHz, DMSO-d₆) 21.0 (C4), 32.0 (C3), 47.4 (N–(CH₂)₂, 2C),

54.0 [54.2] (C1), 55.6 (OCH₃), 65.9 (O–(CH₂)₂, 2C), 67.2 [67.3]

(C2), 102.0 [102.7] (C1⁰⁰), 109.1 [109.3] (C3⁰⁰), 115.0 (C3⁰+ C5⁰),

116.5 (C4a⁰⁰), 118.5 [118.0] (C9⁰⁰), 121.3 (C2⁰+ C6⁰), 121.5 (C7⁰⁰),

127.2 (C5a⁰⁰), 129.6 (C1⁰), 129.8 (C6⁰⁰), 131.1 (C4⁰⁰), 132.1 [132.3]

(C8⁰⁰), 138.8 (C9a⁰⁰), 140.4 (C10a⁰⁰), 154.2 (C2⁰⁰), 160.1 (C4⁰), 166.4

(C_tetrazole).

4.3.17. 1-[2-(Diethylamino)-5-oxido-10H-phenothiazin-10-yl]-

4-[2-(4-methoxyphenyl)-2H-tetrazol-5-yl]butan-2-ol (4f)

This compound was obtained from the hydroboration–oxida-

tion of N,N-diethyl-10-{(1E,3Z)-4-[2-(4-methoxyphenyl)-2H-tetra-

The same product was isolated from the reaction of 4-(2-(4-

methoxyphenyl)-2H-tetrazol-5-yl)-1-(2-morpholino-10H-pheno-

thiazin-10-yl)butan-2-ol (3h, 0.04 g, 0.08 mmol) and m-CPBA

(0.02 g, 0.11 mmol) in abs dichloromethane (1 mL) to give white-

yellow crystals, 0.04 g (98%).

zol-5-yl]buta-1,3-dien-1-yl}-10H-phenothiazin-2-amine^13a

(2f,

0.21 g, 0.42 mmol) as yellow amorphous solid; 0.15 g (67%); the

mixture of two diastereomers; mp 76–80 °C; (found: C, 63.01; H,

5.90; N, 15.66; C₂₈H₃₂N₆O₃S requires C, 63.14; H, 6.06; N, 15.78);

m/z (EI) [MꢀN₂]⁺: 504.00 (C₂₈H₃₂N₆O₃S requires 532.23); m_max

(KBr)/cm^ꢀ1: 3301, 2968, 1590, 1516 and 1466; d_H(400 MHz,

DMSO-d₆) 1.12 (6H, m, N(CH₂)₂(CH₃)₂), 1.84 (1H, m, H3y), 2.06

(1H, m, H3x), 2.98 (1H, m, H4y), 3.03 (1H, m, H4x), 3.43 (4H, m,

N(CH₂)₂(CH₃)₂), 3.86 (3H, s, OCH₃), 4.07 (1H, m, H2), 4.30 (1H, m,

H1y), 4.44 (1H, m, H1x), 5.00 (1H, br s, OH), 6.58 (1H, m, H3⁰⁰),

6.89 [6.56] (1H, m, H1⁰⁰), 7.16 (2H, m, H3⁰+ H5⁰), 7.19 (1H, m,

H9⁰⁰), 7.57 (1H, m, H7⁰⁰), 7.60 (1H, m, H8⁰⁰), 7.61 [7.87] (1H, m,

H4⁰⁰), 7.81 (1H, m, H6⁰⁰), 7.93 (2H, m, H2⁰+ H6⁰); d_C(100 MHz,

DMSO-d₆) 12.3 (N(CH₂)₂(CH₃)₂, 2C), 21.1 (C4), 32.3 (C3), 44.0

(N(CH₂)₂(CH₃)₂, 2C), 54.4 [54.3] (C1), 55.6 (OCH₃), 67.4 [67.2]

(C2), 98.9 [97.9] (C1⁰⁰), 106.3 [106.4] (C3⁰⁰), 113.1 [113.0] (C4a⁰⁰),

115.0 (C3⁰+ C5⁰), 117.8 [118.6] (C4⁰⁰), 121.2 (C9⁰⁰), 121.3

(C2⁰+ C6⁰), 126.6 [126.8] (C5a⁰⁰), 129.6 (C6⁰⁰), 129.7 (C1⁰), 131.7

(C7⁰⁰), 131.8 (C8⁰⁰), 140.0 [139.0] (C9a⁰⁰), 141.7 [140.7] (C10a⁰⁰),

150.7 (C2⁰⁰), 160.1 (C4⁰), 166.4 (C_tetrazole).

4.3.20. 4-[2-(4-Methoxyphenyl)-2H-tetrazol-5-yl]-1-[2-(4-

methylpiperazin-1-yl)-5-oxido-10H-phenothiazin-10-yl]butan-

2-ol (4i)

This compound was obtained from the reaction of Pd(OAc)₂

(0.01 g, 0.05 mmol, 7.5 mol %), XPhos (0.04 g, 0.09 mmol,

15 mol %), NaO^tBu (0.12 g, 1.21 mmol), 1-(2-chloro-5-oxido-10H-

phenothiazin-10-yl)-4-[2-(4-methoxyphenyl)-2H-tetrazol-5-yl]-

butan-2-ol (10, 0.30 g, 0.61 mmol) and N-methylpiperazine

(0.14 mL, 1.21 mmol). The reaction time was 0.5 h. After recrystal-

lization, the white-yellow crystals were collected by ﬁltration,

73 mg (22%); mp 211–214 °C; (found: C, 62.20; H, 5.69; N, 17.21;

C

₂₉H₃₃N₇O₃S requires C, 62.23; H, 5.94; N, 17.52); m/z (EI)

[M+H]⁺: 560.20 (C₂₉H₃₃N₇O₃S requires 559.24); m_max(KBr)/cm^ꢀ1

:

3285, 2950, 1589, 1516 and 1255; d_H(400 MHz, DMSO-d₆) 1.85

(1H, m, H3y), 2.04 (1H, m, H3x), 2.22 (3H, s, CH₃), 2.42 (4H, m,

H₃C–N–(CH₂)₂), 3.00 (1H, ddd, J = 15.4, 8.6, 7.6 Hz, H4y), 3.06

(1H, ddd, J = 15.4, 9.4. 5.4 Hz, H4x), 3.34 (4H, m, N–(CH₂)₂), 3.85

(3H, s, OCH₃), 4.03 (1H, m, H2), 4.36 (1H, dd, J = 14.4, 8.2 Hz,

H1y), 4.44 (1H, dd, J = 14.4, 3.5 Hz, H1x), 4.86 (1H, d, J = 3.5 Hz,

OH), 6.86 (1H, dd, J = 8.5, 1.3 Hz, H3⁰⁰), 6.94 (1H, d, J = 1.3 Hz,

H1⁰⁰), 7.18 (2H, m, H3⁰+ H5⁰), 7.22 (1H, dd, J = 7.9, 7.1 Hz, H7⁰⁰),

7.59 (1H, ddd, J = 8.4, 7.1, 1.4 Hz, H8⁰⁰), 7.67 (1H, d, J = 8.5 Hz,

H4⁰⁰), 7.85 (1H, dd, J = 7.9, 1.4, H6⁰⁰), 7.86 (1H, d, J = 8.4 Hz, H9⁰⁰),

4.3.18. General procedure for preparation of 1-(2-amino)-5-

oxido-10H-phenothiazin-10-yl)-4-[2-(4-methoxyphenyl)-2H-

tetrazol-5-yl]butan-2-ol derivatives (4 and 11)

A

round-bottomed ﬂask was charged with Pd-catalyst:

Pd₂(dba)₃or Pd(OAc)₂(5–7.5 mol %), ligand: XPhos (10–

15 mol %), 1-(2-chloro-5-oxido-10H-phenothiazin-10-yl)-4-[2-(4-

Please cite this article in press as: Takács, D.; et al. Bioorg. Med. Chem. (2013), http://dx.doi.org/10.1016/j.bmc.2013.04.034

12

D. Takács et al. / Bioorg. Med. Chem. xxx (2013) xxx–xxx

7.90 (2H, m, H2⁰+ H6⁰); d_C(100 MHz, DMSO-d₆) 21.0 (C4), 32.0

(C3), 45.7 (CH₃), 47.1 (N–(CH₂)₂, 2C), 54.0 (C1), 54.3 (H₃C–N–

(CH₂)₂, 2C), 55.6 (OCH₃), 67.2 (C2), 102.0 (C1⁰⁰), 109.3 (C3⁰⁰), 115.0

(C3⁰+ C5⁰), 116.0 (C4a⁰⁰), 118.6 (C9⁰⁰), 121.3 (C2⁰+ C6⁰), 121.6

(C7⁰⁰), 127.2 (C5a⁰⁰), 129.6 (C1⁰), 129.7 (C6⁰⁰), 131.1 (C4⁰⁰), 132.0

(C8⁰⁰), 138.8 (C9a⁰⁰), 141.3 (C10a⁰⁰), 154.0 (C2⁰⁰), 160.1 (C4⁰), 166.4

(C_tetrazole).

m/z: 640 (2%), 612 (14), 491 (75), 458 (2), 401 (5), 342 (2), 317 (75),

226 (25), 186 (27), 123 (27) and 91 (100); m_max(KBr)/cm^ꢀ1: 2927,

2853, 1515, 1453 and 1256; d_H(400 MHz, DMSO-d₆) 1.90 (1H, m,

H3y), 2.13 (1H, m, H3x), 2.90 (1H, m, H4y), 2.98 (1H, m, H4x),

3.85 (3H, s, OCH₃), 3.88 (1H, m, H1y), 3.90 (1H, m, H2), 4.05 (1H,

m, H1x), 4.25 (2H, m, NH–CH₂), 4.43 (1H, d, J = 12.0 Hz, O–CH1y),

4.67 (1H, d, J = 12.0 Hz, O–CH1x), 6.25–6.28 (2H, m, H3⁰⁰+ NH),

6.37 (1H, d, J = 2.1 Hz, H1⁰⁰), 6.83 (1H, d, J = 8.2 Hz, H4⁰⁰), 6.90 (1H,

dd, J = 7.8, 7.4 Hz, H7⁰⁰), 7.05 (1H, d, J = 8.3 Hz, H9⁰⁰), 7.10–7.25

4.3.21. 1-(2-Chloro-10H-phenothiazin-10-yl)-4-[2-(4-

methoxyphenyl)-2H-tetrazol-5-yl]butan-2-ol (5)

This compound was synthesized according to the literature

procedure.¹²

(10H,

m,

H3⁰+ H5⁰+ H6⁰⁰+ H8⁰⁰+ H4⁰⁰⁰+ H2⁰⁰⁰⁰+ H3⁰⁰⁰⁰+ H4⁰⁰⁰⁰

+

H5⁰⁰⁰⁰+ H6⁰⁰⁰⁰), 7.28 (2H, m, H3⁰⁰⁰+ H5⁰⁰⁰), 7.33 (2H, m, H2⁰⁰⁰+ H6⁰⁰⁰),

7.90 (2H, m, H2⁰+ H6⁰); d_C(100 MHz, DMSO-d₆) 20.7 (C4), 30.5

(C3), 46.5 (NH–CH₂), 50.7 (C1), 55.6 (OCH₃), 71.3 (O–CH₂), 73.3

(C2), 101.1 (C1⁰⁰), 107.2 (C3⁰⁰), 109.1 (C4a⁰⁰), 115.0 (C3⁰+ C5⁰),

116.2 (C9⁰⁰), 121.3 (C2⁰+ C6⁰), 122.2 (C7⁰⁰), 126.0 (C5a⁰⁰), 126.6

(C4⁰⁰⁰), 127.0 (C8⁰⁰), 127.1 (C2⁰⁰⁰+ C6⁰⁰⁰), 127.3 (C4⁰⁰), 127.6

(C6⁰⁰+ C4⁰⁰⁰⁰), 127.8 (C2⁰⁰⁰⁰+ C6⁰⁰⁰⁰), 128.0 (C3⁰⁰⁰⁰+ C5⁰⁰⁰⁰), 128.2

(C3⁰⁰⁰+ C5⁰⁰⁰), 129.6 (C1⁰), 138.5 (C1⁰⁰⁰⁰), 140.0 (C1⁰⁰⁰), 145.4 (C10a⁰⁰),

146.1 (C9a⁰⁰), 148.9 (C2⁰⁰), 160.1 (C4⁰), 166.2 (C_tetrazole).

4.3.22. 10-{2-(Benzyloxy)-4-[2-(4-methoxyphenyl)-2H-tetrazol-

5-yl]butyl}-2-chloro-10H-phenothiazine (6)

To a solution of 1-(2-chloro-10H-phenothiazin-10-yl)-4-[2-(4-

methoxyphenyl)-2H-tetrazol-5-yl]butan-2-ol (5, 0.30 g, 0.63 mmol)

in abs DMF (5 mL) in a dried, round-bottomed ﬂask equipped with

a side arm and cooled to 0 °C in an argon atmosphere was added

sodium hydride (60%, 0.30 g, 7.65 mmol). The reaction mixture be-

came a suspension. To this suspension was added benzyl bromide

(0.66 mL, 5.63 mmol) dropwise and the starting suspension turned

to a clear solution. This mixture was then stirred at rt for 2.5 h,

then the solvent was removed in vacuo. The residue was dissolved

in water (20 mL), and was neutralized by saturated NH₄Cl solution.

After extraction with dichloromethane (3 ꢁ 10 mL), the combined

organic phase was dried over Na₂SO₄, ﬁltered and evaporated.

The residue was puriﬁed by ﬂash chromatography on silica gel

using the eluent dichloromethane, then n-hexane:ethyl ace-

tate = 4:1. The crude product was recrystallized from n-hexane

and the white crystals were collected by ﬁltration, 0.27 g (75%);

mp 114–116 °C; (found: C, 65.22; H, 4.89; N, 12.11; C₃₁H₂₈ClN₅O₂S

4.3.25. 10-{2-(Benzyloxy)-4-[2-(4-methoxyphenyl)-2H-tetrazol-

5-yl]butyl}-N-[(1R)-1-phenylethyl]-10H-phenothiazin-2-amine

(7d)

This compound was obtained from the reaction of 10-{2-(ben-

zyloxy)-4-[2-(4-methoxyphenyl)-2H-tetrazol-5-yl]butyl}-2-chloro-

10H-phenothiazine (6, 0.30 g, 0.52 mmol) and (R)-phenylethyl-

amine (0.13 mL, 1.04 mmol) to give the product as a brown oil;

0.22 g (64%); a mixture of two diastereomers. The reaction time

was 1.5 h; (found: C, 71.67; H, 5.95; N, 12.53; C₃₉H₃₈N₆O₂S re-

quires C, 71.53; H, 5.85; N, 12.83); HRMS calcd for C₃₉H₃₈N₆O₂S

654.2777 found: 654.2794; m/z: 654 (79%), 626 (21), 584 (50),

505 (40), 463 (61), 433 (16), 399 (6), 379 (88), 331 (84), 281

(60), 226 (30), 167 (26), 137 (24) and 108 (100); m_max(ﬁlm)/

cm^ꢀ1: 3408, 2927, 1600, 1515 and 1454; d_H(400 MHz, DMSO-d₆)

1.38 [1.39] (3H, d, J = 7.1 Hz, CH₃), 1.90 (1H, m, H3y), 2.10 (1H,

m, H3x), 2.95 (2H, m, H4y + H4x), 3.83 (1H, m, H1y), 3.99 (1H, m,

H1x), 3.85 (3H, s, OCH₃), 3.91 (1H, m, H2), 4.41 (1H, d,

J = 11.5 Hz, O–CH1y), 4.45 (1H, m, HN–CH), 4.64 (1H, d,

J = 11.5 Hz, O–CH1x), 6.16 (1H, dd, J = 8.2, 2.2 Hz, H3⁰⁰), 6.21

[6.20] (1H, d, J = 8.5 Hz, NH), 6.29 (1H, d, J = 2.2 Hz, H1⁰⁰), 6.76

(1H, d, J = 8.2 Hz, H4⁰⁰), 6.89 (1H, ddd, J = 8.0, 7.3, 2.2 Hz, H7⁰⁰),

7.02 (1H, dd, J = 8.6, 1.4 Hz, H9⁰⁰), 7.10–7.38 (14H, m,

H3⁰+ H5⁰+ H6⁰⁰+ H8⁰⁰+ H2⁰⁰⁰+ H3⁰⁰⁰+ H4⁰⁰⁰+ H5⁰⁰⁰+ H6⁰⁰⁰+ H2⁰⁰⁰⁰+ H3-

requires C, 65.31; H, 4.95; N, 12.28); m

_max(KBr)/cm^ꢀ1: 3410, 2960,

1515, 1454 and 1252; d_H(300 MHz, CDCl₃) 2.11 (1H, m), 2.54 (1H,

m), 3.02–3.11 (2H, m), 3.85 (3H, s), 3.92 (1H, m), 4.07 (2H, m), 4.58

(1H, d, J = 11.1 Hz), 4.67 (1H, d, J = 11.1 Hz), 6.79–7.30 (14H, m),

7.90 (2H, m); d_C(75 MHz, CDCl₃) 22.4, 31.2, 51.8, 55.8, 72.9, 73.8,

114.7 (2C), 116.2, 116.3, 121.4 (2C), 122.7, 123.4, 124.5, 125.7,

127.6, 127.9 (2C), 128.3 (2C), 128.5, 129.3, 130.5, 130.8, 133.6,

138.3, 144.7, 146.8, 160.5, 166.4.

4.3.23. General procedure for preparation of 10-{2-(benzyloxy)-

4-[2-(4-methoxyphenyl)-2H-tetrazol-5-yl]butyl}-10H-

phenothiazine-2-amine/amide derivatives (7) applying

Buchwald–Hartwig cross-coupling reaction

0000

+ H4⁰⁰⁰⁰+ H5⁰⁰⁰⁰+ H6⁰⁰⁰⁰), 7.90 (2H, m, H2⁰+ H6⁰); d_C(100 MHz,

DMSO-d₆) 20.6 (C4), 24.6 (CH₃), 30.4 (C3), 50.7 (C1), 52.1 (HN–

CH), 55.6 (OCH₃), 71.3 (O–CH₂), 73.2 (C2), 101.6 (C1⁰⁰), 107.7

(C3⁰⁰), 108.9 (C4a⁰⁰), 115.0 (C3⁰+ C5⁰), 116.1 (C9⁰⁰), 121.3

(C2⁰+ C6⁰), 122.2 (C7⁰⁰), 125.7 (C5a⁰⁰), 125.8 (C2⁰⁰⁰+ C6⁰⁰⁰), 125.9

(C4⁰⁰⁰⁰), 126.4 (C4⁰⁰⁰), 127.0 (C8⁰⁰), 127.3 (C6⁰⁰), 127.4 (C4⁰⁰), 127.8

(C2⁰⁰⁰⁰+ C6⁰⁰⁰⁰), 128.0 (C3⁰⁰⁰⁰+ C5⁰⁰⁰⁰), 128.3 (C3⁰⁰⁰+ C5⁰⁰⁰), 129.6 (C1⁰),

138.5 (C1⁰⁰⁰⁰), 145.3 (C9a⁰⁰), 145.9 (C1⁰⁰⁰), 146.1 (C10a⁰⁰), 148.1

(C2⁰⁰), 160.1 (C4⁰), 166.2 (C_tetrazole).

A

round-bottomed ﬂask was charged with Pd-catalyst:

Pd₂(dba)₃(7.5 mol %), ligand: XPhos (15 mol %), 10-{2-(benzyloxy)-

4-[2-(4-methoxyphenyl)-2H-tetrazol-5-yl]butyl}-2-chloro-10H-phe-

nothiazine (6), the appropriate amine/amide, base: NaO^tBu or

K₂CO₃, and dry toluene (5 mL). The ﬂask was ﬂushed with argon

for 5 min. The resulting mixture was heated under reﬂux with

magnetic stirring for 1.5–43 h. After cooling down to rt the reaction

mixture was concentrated and the residue was puriﬁed by ﬂash

column chromatography on silica gel using the eluent dichloro-

methane, then n-hexane:ethyl acetate = 2:1 and 1:1 to give the

ﬁnal product.

4.3.26. 10-{2-(Benzyloxy)-4-[2-(4-methoxyphenyl)-2H-tetrazol-

5-yl]butyl}-2-(4-methylpiperazin-1-yl)-10H-phenothiazine (7i)

This compound was obtained from the reaction of 10-{2-(ben-

zyloxy)-4-[2-(4-methoxyphenyl)-2H-tetrazol-5-yl]butyl}-2-chloro-

10H-phenothiazine (6, 0.20 g, 0.35 mmol) and N-methylpiperazine

(0.08 mL, 0.70 mmol) to give the product as a brown oil, 0.44 g

(99%). The reaction time was 18 h. (found: C, 68.28; H, 6.53; N,

15.20; C₃₆H₃₉N₇O₂S requires C, 68.22; H, 6.20; N, 15.47); m/z (EI)

4.3.24. N-Benzyl-10-{2-(benzyloxy)-4-[2-(4-methoxyphenyl)-

2H-tetrazol-5-yl]butyl}-10H-phenothiazin-2-amine (7c)

This compound was obtained from the reaction of 10-{2-(ben-

zyloxy)-4-[2-(4-methoxyphenyl)-2H-tetrazol-5-yl]butyl}-2-chloro-

10H-phenothiazine (6, 0.30 g, 0.52 mmol) and benzylamine

(0.11 mL, 1.00 mmol) to give the product as brown amorphous so-

lid, 0.25 g (67%). The reaction time was 1.5 h; mp 40–45 °C; (found:

C, 71.28; H, 5.70; N, 13.05; C₃₈H₃₆N₆O₂S requires C, 71.22; H, 5.66;

N, 13.11); HRMS calcd for C₃₈H₃₆N₆O₂S 640.2620 found: 640.2592;

[M+H]⁺: 634.60 (C₃₆H₃₉N₇O₂S requires 633.29); m_max(ﬁlm)/cm^ꢀ1

:

3061, 2937, 2839 1583 and 1515; d_H(300 MHz, CDCl₃) 2.11 (1H,

m), 2.25 (3H, s), 2.28 (1H, m), 2.53 (4H, t, J = 5.1 Hz), 2.91–3.14

(6H, m), 3.88 (3H, s), 3.97 (1H, dd, J = 12.6, 4.8 Hz), 4.05–4.18

(2H, m), 4.60 (1H, d, J = 11.2 Hz), 4.78 (1H, d, J = 11.2 Hz), 6.48–

Please cite this article in press as: Takács, D.; et al. Bioorg. Med. Chem. (2013), http://dx.doi.org/10.1016/j.bmc.2013.04.034

D. Takács et al. / Bioorg. Med. Chem. xxx (2013) xxx–xxx

13

6.53 (2H, m), 6.80–7.26 (12H, m), 7.89–7.94 (2H, m); d_C(75 MHz,

CDCl₃) 21.5, 31.4, 43.2, 49.6 (2C), 52.1, 55.3 (2C), 55.9, 72.9, 74.2,

105.0, 111.2, 114.8 (2C), 116.1, 121.5 (2C), 122.8, 126.0, 127.1,

127.2, 127.8 (2C), 127.9, 128.0, 128.1 (2C), 128.2, 128.4, 138.6,

145.4, 146.9, 151.7, 160.5, 166.6.

the mixture was diluted with dichloromethane (175 mL) and meth-

anol (22 mL). The clear solution was washed with water (150 mL),

neutralized with saturated aqueous NaHCO₃, again with water

(150 mL) and ﬁnally with saturated aqueous NaCl solution

(150 mL). The organic phase was dried over Na₂SO₄, ﬁltered and con-

centrated. The crude product was puriﬁed by column chromatogra-

phy on silica gel using the eluent n-hexane:ethyl acetate = 6:1 and

recrystallized by n-hexane to give white crystals, 4.71 g (90%); mp

116–118 °C; HPLC: using the eluent n-hexane:2-propanol + 0.1%

diethylamine = 98:2, retention times: 12.4 min, 14.4 min; (found:

C, 66.86; H, 5.45; N, 9.67; C₄₀H₄₀ClN₅O₂SSi requires C, 66.88; H,

4.3.27. 10-{2-(Benzyloxy)-4-[2-(4-methoxyphenyl)-2H-tetrazol-

5-yl]butyl}-2-pyrrolidin-1-yl-10H-phenothiazine (7k)

This compound was obtained from the reaction of 10-{2-(ben-

zyloxy)-4-[2-(4-methoxyphenyl)-2H-tetrazol-5-yl]butyl}-2-chloro-

10H-phenothiazine (6, 0.30 g, 0.52 mmol) and pyrrolidine

(0.09 mL, 1.04 mmol) to give the product as beige amorphous solid,

0.26 g (80%). The reaction time was 1.5 h; mp 43–44 °C; (found: C,

69.86; H, 6.06; N, 13.81; C₃₅H₃₆N₆O₂S requires C, 69.51; H, 6.00;

N, 13.90): HRMS calcd for C₃₅H₃₆N₆O₂S 604.2620 found: 604.2593;

m/z: 604 (59%), 535 (40), 455 (64), 422 (13), 322 (9), 281 (100),

212 (94), 180 (29), 138 (11) and 108 (69); m_max(KBr)/cm^ꢀ1: 2838,

1599, 1515, 1459 and 1255; d_H(300 MHz, DMSO-d₆) 1.95 (4H, t,

J = 6.3 Hz), 2.13 (1H, m), 2.31 (1H, m), 3.08 (2H, m), 3.19 (4H, t,

J = 6.3 Hz), 3.88 (3H, s), 4.00 (1H, m), 4.18 (2H, m), 4.60 (1H, d,

J = 11.2 Hz), 4.80 (1H, d, J = 11.2 Hz), 6.10–6.18 (2H, m), 6.81–7.29

(12H, m), 7.91 (2H, m); d_C(75 MHz, DMSO-d₆) 21.5, 25.6 (2C),

31.4, 48.0 (2C), 52.0, 55.9, 72.9, 74.3, 100.4, 106.9, 110.6, 114.8

(2C), 115.9, 121.5 (2C), 122.6, 127.0, 127.5, 127.7, 127.8, 127.9,

128.3, 128.4 (2C), 128.5 (2C), 138.7, 145.9, 146.9, 148.2, 160.5, 166.7.

5.61; N, 9.75); m

_max(KBr)/cm^ꢀ1: 3070, 2931, 1516, 1456 and 1255;

d_H(300 MHz, CDCl₃) 1.09 (9H, s), 2.13 (1H, m), 2.25 (1H, m), 3.05

(2H, t, J = 7.8 Hz), 3.80–3.82 (2H, m), 3.86 (3H, s), 4.31 (1H, m),

6.14 (1H, m), 6.59 (1H, m), 6.80–6.83 (3H, m), 6.90–7.00 (4H, m),

7.32–7.45 (6H, m), 7.71–7.77 (4H, m), 7.87 (2H, m); d_C(75 MHz,

CDCl₃) 19.6, 20.7, 27.3 (3C), 32.7, 52.0, 55.9, 68.8, 114.7 (2C),

116.1, 116.3, 121.4 (2C), 122.8, 123.2, 124.5, 125.7, 127.5, 127.8,

128.0 (2C), 128.1 (2C), 128.3, 130.1, 130.2, 130.8, 133.4 (2C), 134.0,

136.1 (2C), 136.2 (2C), 144.3, 147.2, 160.4, 166.7.

4.3.30. General procedure for the preparation of the 10-(2-

{[tert-butyl(diphenyl)silyl]oxy}-4-[2-(4-methoxyphenyl)-2H-

tetrazol-5-yl]butyl)-10H-phenothiazin-2-amine/amide

derivatives (9) applying Buchwald–Hartwig amination

A

round-bottomed ﬂask was charged with Pd-catalyst:

4.3.28. N-(10-{2-(Benzyloxy)-4-[2-(4-methoxyphenyl)-2H-

tetrazol-5-yl]butyl}-10H-phenothiazin-2-yl)formamide (7l) (E/Z

isomers)

This compound was obtained from the reaction of 10-{2-(ben-

zyloxy)-4-[2-(4-methoxyphenyl)-2H-tetrazol-5-yl]butyl}-2-

chloro-10H-phenothiazine (6, 0.30 g, 0.52 mmol) and formamide

(0.04 mL, 1.04 mmol) to give the product as beige amorphous solid,

87 mg (29%); the mixture of amide rotamers. The reaction time

was 43 h; mp 63–67 °C; (found: C, 66.34; H, 5.49; N, 14.12;

Pd₂(dba)₃or Pd(OAc)₂(10 mol %), ligand: XPhos (20 mol %), 10-(2-

{[tert-butyl(diphenyl)silyl]oxy}-4-[2-(4-methoxyphenyl)-2H-tetra-

zol-5-yl]butyl)-2-chloro-10H-phenothiazine

(8),

appropriate

amine/amide, base: NaO^tBu or K₂CO₃), and dry toluene or 1,4-diox-

ane (5 mL). The ﬂask was ﬂushed with argon for 5 min. The result-

ing mixture was heated at reﬂux with magnetic stirring for 4–31 h.

After cooling down to rt the reaction mixture was concentrated

and the residue was puriﬁed by ﬂash column chromatography on

silica gel using the eluent dichloromethane, then n-hexane:ethyl

acetate = 2:1 and 1:1 to give the product.

C

₃₂H₃₀N₆O₃S requires C, 66.42; H, 5.23; N, 14.52); m/z (EI)

[M+H]⁺: 579.30 (C₃₂H₃₀N₆O₃S requires 578.21); m_max(KBr)/cm^ꢀ1

:

2930, 2855, 1694, 1591 and 1516; d_H(400 MHz, DMSO-d₆) 1.97

(1H, m, H3y), 2.18 (1H, m, H3x), 3.00 (2H, m, H4y + H4x), 3.86

(3H, s, OCH₃), 3.98 (1H, m, H2), 3.99 (1H, m, H1y), 4.16 (1H, m,

H1x), 4.52 [4.50] (1H, m, O–CHy), 4.73 [4.70] (1H, m, O–CHx),

6.95 [6.93] (1H, m, H7⁰⁰), 7.08–7.30 (8H, m, H4⁰⁰+ H6⁰⁰+ H8⁰⁰+

H2⁰⁰⁰+ H3⁰⁰⁰+ H4⁰⁰⁰+ H5⁰⁰⁰+ H6⁰⁰⁰), 7.12 (1H, m, H9⁰⁰), 7.15 (1H, m,

H3⁰⁰), 7.16 (2H, m, H3⁰+ H5⁰), 7.51 [7.00] (1H, s, H1⁰⁰), 7.86 (2H,

m, H2⁰+ H6⁰), 8.2 (8.8) (1H, d, J = 1.1, [J = 11.0] Hz, O@C–H), 10.2

(10.1) (1H, d, J = 1.1, [J = 11.0] Hz, NH); d_C(100 MHz, DMSO-d₆)

20.7 (C4), 30.5 (C3), 50.8 (C1), 55.5 (OCH₃), 71.3 (O–CH₂), 73.4

(C2), 107.4 [105.8] (C1⁰⁰), 111.8 (C4a⁰⁰), 113.5 (C3⁰⁰), 115.0

(C3⁰+ C5⁰), 116.4 (C9⁰⁰), 119.4 (C4⁰⁰), 121.2 (C2⁰+ C6⁰), 122.8

[119.4] (C7⁰⁰), 127.3 (C8⁰⁰+ C6⁰⁰), 127.4 (C5a⁰⁰), 127.5 (C4⁰⁰⁰), 127.7

(C2⁰⁰⁰+ C6⁰⁰⁰), 128.0 (C3⁰⁰⁰+ C5⁰⁰⁰), 129.6 (C1⁰), 138.4 [138.1] (C1⁰⁰⁰),

144.8 [144.7] (C9a⁰⁰), 145.4 [145.2] (C10a⁰⁰), 146.3 (C2⁰⁰), 160.0

(C4⁰), 162.6 (C=O), 166.2 (C_tetrazole).

4.3.31. N⁰-[10-(2-{[tert-Butyl(diphenyl)silyl]oxy}-4-[2-(4-

methoxyphenyl)-2H-tetrazol-5-yl]butyl)-10H-phenothiazin-2-

yl]-N,N-dimethylethane-1,2-diamine (9a)

This compound was obtained from the reaction of 10-(2-{[tert-

butyl(diphenyl)silyl]oxy}-4-[2-(4-methoxyphenyl)-2H-tetrazol-5-

yl]butyl)-2-chloro-10H-phenothiazine (8, 0.20 g, 0.28 mmol) and

N,N-dimethylethane-1,2-diamine (0.06 mL, 0.56 mmol) to give

the product as brown amorphous solid, 0.18 g (84%). The reaction

time was 4 h; mp 56–58 °C; (found: C, 68.43; H, 6.38; N, 12.53;

C₄₄H₅₁N₇O₂SSi requires C, 68.63; H, 6.68; N, 12.73); m/z (EI)

[M+H]⁺: 770.30 (C₄₄H₅₁N₇O₂SSi requires 769.36); m_max(KBr)/

cm^ꢀ1: 3382, 2932, 1601, 1516 and 1459; d_H(300 MHz, CDCl₃)

1.08 (9H, s), 2.14 (1H, m), 2.22 (1H, m), 2.23 (6H, s), 2.52 (2H, t,

J = 5.8 Hz), 2.98–3.07 (4H, m), 3.81–3.84 (2H, m), 3.86 (3H, s),

4.10 (1H, m), 4.36 (1H, m), 5.86 (1H, d, J = 1.8 Hz), 6.10–6.17 (2H,

m), 6.75–6.84 (3H, m), 6.97–7.03 (3H, m), 7.35–7.47 (6H, m),

7.72–7.80 (4H, m), 7.90 (2H, m); d_C(75 MHz, CDCl₃) 19.6, 20.7,

27.3 (3C), 32.6, 41.4, 45.4 (2C), 51.8, 55.8, 58.2, 69.0, 101.7,

107.2, 112.5, 114.7 (2C), 115.7, 121.5 (2C), 122.4, 126.9, 127.3,

127.5, 127.9 (2C), 128.0 (2C), 128.3, 130.0, 130.1, 130.8, 133.6,

134.5, 136.2 (2C), 136.3 (2C), 145.1, 147.2, 148.8, 160.4, 166.9.

4.3.29. 10-(2-{[tert-Butyl(diphenyl)silyl]oxy}-4-[2-(4-

methoxyphenyl)-2H-tetrazol-5-yl]butyl)-2-chloro-10H-

phenothiazine (8)

A

solution of 1-(2-chloro-10H-phenothiazin-10-yl)-4-[2-(4-

methoxyphenyl)-2H-tetrazol-5-yl]butan-2-ol (5, 3.50 g,

7.29 mmol) in anhydrous dichloromethane (175 mL) in a dried,

round-bottomed ﬂask equipped with a side arm and cooled to 0 °C

in an argon atmosphere was treated with imidazole (1.49 g,

21.88 mmol) in small portions. tert-Butyldiphenylchlorosilane

(7.30 mL, 28.13 mmol) was added dropwise to the mixture, which

became a suspension. The resulting reaction was allowed to warm

up to rt and stirred for 4 days. After completion of the reaction,

4.3.32. 10-(2-{[tert-Butyl(diphenyl)silyl]oxy}-4-[2-(4-

methoxyphenyl)-2H-tetrazol-5-yl]butyl)-N-(2-morpholin-4-

ylethyl)-10H-phenothiazin-2-amine (9b)

This compound was obtained from the reaction of 10-(2-{[tert-

butyl(diphenyl)silyl]oxy}-4-[2-(4-methoxyphenyl)-2H-tetrazol-5-

yl]butyl)-2-chloro-10H-phenothiazine (8, 0.10 g, 0.14 mmol) and

2-morpholinoethanamine (0.04 mL, 0.27 mmol) to give the product

Please cite this article in press as: Takács, D.; et al. Bioorg. Med. Chem. (2013), http://dx.doi.org/10.1016/j.bmc.2013.04.034

14

D. Takács et al. / Bioorg. Med. Chem. xxx (2013) xxx–xxx

as white amorphous solid, 0.17 g (78%). The reaction time was 31 h;

mp 64–67 °C; (found: C, 67.90; H, 6.98; N, 11.97; C₄₆H₅₃N₇O₃SSi re-

quires C, 68.03; H, 6.58; N, 12.07); m/z (EI) [M+H]⁺: 812.60

(C₄₆H₅₃N₇O₃SSi requires 811.37); m_max(KBr)/cm^ꢀ1: 2957, 2855,

1601, 1516 and 1460; d_H(300 MHz, CDCl₃) 1.08 (9H, s), 2.15 (1H,

m), 2.25 (1H, m), 2.48 (4H, t, J = 3.9 Hz), 2.60 (2H, t, J = 5.4 Hz),

2.95–3.15 (4H, m), 3.74 (4H, t, J = 3.9 Hz), 3.82 (2H, m), 3.85 (3H,

s), 4.10 (1H, br s), 4.36 (1H, m), 5.87 (1H, d, J = 1.8 Hz), 6.10–6.17

(2H, m), 6.76–6.85 (3H, m), 7.00–7.03 (3H, m), 7.33–7.47 (6H, m),

7.73–7.88 (6H, m); d_C(75 MHz, CDCl₃) 19.6, 20.7, 27.3 (3C), 32.6,

40.2, 51.8, 53.6 (2C), 55.9, 57.4, 67.2 (2C), 69.0, 101.6, 107.3,

112.8, 114.7 (2C), 115.7, 121.4 (2C), 122.5, 126.9, 127.2, 127.5,

127.9 (2C), 128.0 (2C), 128.3, 130.0, 130.1, 130.8, 133.5, 134.6,

136.2 (2C), 136.3 (2C), 145.1, 147.3, 148.5, 160.4, 166.9.

yl]butyl)-2-chloro-10H-phenothiazine (8, 0.60 g, 0.83 mmol) and

morpholine (0.15 mL, 1.67 mmol) to give the product as white

amorphous solid, 0.59 g (91%). The reaction time was 18 h; mp

63–66 °C; HPLC: using the eluent n-hexane:2-propanol + 0.1%

diethylamine = 75:25, retention times: 18.6 min, 19.5 min;

(found: C, 68.98; H, 6.52; N, 10.53; C₄₄H₄₈N₆O₃SSi requires C,

68.72; H, 6.29; N, 10.93); m/z (EI) [M+H]⁺: 769.60 (C₄₄H₄₈N₆O₃S-

Si requires 768.33); m

_max(KBr)/cm^ꢀ1: 2958, 2855, 1595, 1516 and

1461; d_H(300 MHz, CDCl₃+ DMSO-d₆) 1.06 (9H, s), 2.08 (1H, m),

2.22 (1H, m), 2.92–3.03 (6H, m), 3.77 (4H, t, J = 4.8 Hz), 3.85 (2H,

m), 3.87 (3H, s), 4.31 (1H, m), 6.09 (1H, m), 6.24 (1H, d,

J = 2.1 Hz), 6.44 (1H, dd, J = 8.4, 2.1 Hz), 6.76–6.79 (2H, m), 6.89

(1H, d, J = 8.4 Hz), 6.96–7.05 (3H, m), 7.30–7.46 (6H, m), 7.69–

7.77 (4H, m), 7.83 (2H, m); d_C(75 MHz, CDCl₃+ DMSO-d₆) 12.8,

24.3, 32.0 (3C), 37.4, 54.3 (2C), 56.8, 60.7, 71.6 (2C), 73.6,

103.1, 109.4, 113.7 (2C), 114.6, 114.8, 120.3 (2C), 121.6, 125.4,

126.1, 126.5, 126.8 (2C), 126.9 (2C), 128.9, 129.1, 129.4, 132.0,

133.2, 134.9 (2C), 135.0 (2C), 143.6, 145.8, 150.5 (2C), 159.4,

165.5.

4.3.33. N-Butyl-10-(2-{[tert-butyl(diphenyl)silyl]oxy}-4-[2-(4-

methoxyphenyl)-2H-tetrazol-5-yl]butyl)-10H-phenothiazin-2-

amine (9e)

This compound was obtained from the reaction of 10-(2-{[tert-

butyl(diphenyl)silyl]oxy}-4-[2-(4-methoxyphenyl)-2H-tetrazol-5-

yl]butyl)-2-chloro-10H-phenothiazine (8, 0.20 g, 0.28 mmol) and

n-butylamine (0.06 mL, 0.56 mmol) to give the product as beige

amorphous solid, 0.12 g (57%). The reaction time was 21 h; mp

46–50 °C; (found: C, 70.38; H, 6.79; N, 10.83; C₄₄H₅₀N₆O₂SSi requires

C, 69.99; H, 6.67; N, 11.13); m/z (EI) [M+H]⁺: 755.40 (C₄₄H₅₀N₆O₂SSi

requires 754.35); m_max(KBr)/cm^ꢀ1: 3408, 2957, 1601, 1516 and

1460; d_H(300 MHz, CDCl₃) 0.97 (3H, t, J = 7.2 Hz), 1.08 (9H, s), 1.40

(2H, m), 1.55 (2H, m), 2.13 (1H, m), 2.23 (1H, m), 2.95–3.06 (4H,

m), 3.26 (1H, br s), 3.83 (2H, m), 3,85 (3H, s), 4.35 (1H, m), 5.74

(1H, s), 6.09–6.18 (2H, m), 6.74–6.84 (3H, m), 7.00–7.03 (3H, m),

7.32–7.48 (6H, m), 7.73–7.89 (6H, m); d_C(75 MHz, CDCl₃) 13.9,

19.4, 20.3, 20.5, 27.0 (3C), 31.6, 32.3, 43.7, 51.6, 55.6, 68.6, 101.4,

106.5, 112.0, 114.4 (2C), 115.4, 121.2 (2C), 122.2, 126.7, 127.0,

127.3, 127.6 (2C), 127.8 (2C), 128.0, 129.7, 129.8, 130.5, 133.2,

134.3, 136.0 (2C), 136.1 (2C), 145.0, 146.7, 148.3, 160.1, 166.6.

4.3.36. 10-(2-{[tert-Butyl(diphenyl)silyl]oxy}-4-[2-(4-

methoxyphenyl)-2H-tetrazol-5-yl]butyl)-2-(4-methylpiperazin-

1-yl)-10H-phenothiazine (9i)

This compound was obtained from the reaction of 10-(2-{[tert-

butyl(diphenyl)silyl]oxy}-4-[2-(4-methoxyphenyl)-2H-tetrazol-5-

yl]butyl)-2-chloro-10H-phenothiazine (8, 0.60 g, 0.83 mmol) and

N-methylpiperazine (0.19 mL, 1.67 mmol) to give the product as

beige amorphous solid, 0.48 g (73%). The reaction time was 18 h;

mp 58–60 °C; HPLC: using the eluent n-hexane:2-propanol + 0.1%

diethylamine = 75:25, retention times: 20.5 min, 23.5 min; (found:

C, 68.79; H, 6.63; N, 12.24; C₄₅H₅₁N₇O₂SSi requires C, 69.11; H,

6.57; N, 12.54); m/z (EI) [M+H]⁺: 782.30 (C₄₅H₅₁N₇O₂SSi requires

781.36); m_max(KBr)/cm^ꢀ1: 2932, 2855, 1596, 1516 and 1461; d_H

(300 MHz, CDCl₃) 1.07 (9H, s), 2.11 (1H, m), 2.28 (1H, m), 2.36

(3H, s), 2.53 (4H, t, J = 4.8 Hz), 3.02–3.07 (6H, m), 3.83 (2H, m),

3.85 (3H, s), 4.34 (1H, m), 5.99 (1H, m), 6.27 (1H, d, J = 2.1 Hz),

6.44 (1H, dd, J = 8.4, 2.1 Hz), 6.73–6.77 (2H, m), 6.90 (1H, d,

J = 8.4 Hz), 6.96–7.01 (3H, m), 7.31–7.45 (6H, m), 7.73–7.79 (4H,

m), 7.86 (2H, m); d_C(75 MHz, CDCl₃) 19.6, 20.8, 27.2 (3C), 32.7,

46.4, 49.5 (2C), 52.1, 55.3 (2C), 55.9, 69.1, 104.8, 110.8, 114.7

(2C), 115.9, 116.0, 121.5 (2C), 122.6, 126.9, 127.0, 127.6, 127.9

(2C), 128.1 (2C), 129.9, 130.2, 130.8, 133.3, 134.6, 136.1 (2C),

136.3 (2C), 144.7, 147.3, 151.6 (2C), 160.4, 166.9.

4.3.34. 10-(2-{[tert-Butyl(diphenyl)silyl]oxy}-4-[2-(4-

methoxyphenyl)-2H-tetrazol-5-yl]butyl)-N,N-diethyl-10H-

phenothiazin-2-amine (9f)

This compound was obtained from the reaction of 10-(2-{[tert-

butyl(diphenyl)silyl]oxy}-4-[2-(4-methoxyphenyl)-2H-tetrazol-5-

yl]butyl)-2-chloro-10H-phenothiazine (8, 1.00 g, 1.39 mmol) and

diethylamine (0.17 mL, 2.79 mmol) to give the product as white

amorphous solid, 0.92 g (87%). The reaction time was 4 h; mp 56–

57 °C; HPLC: using the eluent n-hexane:2-propanol + 0.1% diethyl-

amine = 75:25, retention times: 9.6 min, 10.2 min; (found: C,

69.68; H, 6.68; N, 10.75; C₄₄H₅₀N₆O₂SSi requires C, 69.99; H, 6.67;

N, 11.13); m/z (EI) [M+H]⁺: 755.50 (C₄₄H₅₀N₆O₂SSi requires

754.35); m_max(KBr)/cm^ꢀ1: 2964, 2856, 1598, 1516 and 1466; d_H

(300 MHz, CDCl₃) 1.07 (9H, s), 1.09 (6H, t, J = 6.9 Hz), 2.13 (1H,

m), 2.27 (1H, m), 3.07 (2H, t, J = 7.2 Hz), 3.23 (4H, q, J = 6.9 Hz),

3.86 (2H, m), 3.88 (3H, s), 4.36 (1H, m), 5.91 (1H, d, J = 8.4 Hz),

6.06 (1H, d, J = 2.4 Hz), 6.23 (1H, dd, J = 8.4, 2.7 Hz), 6.67–6.75

(2H, m), 6.85 (1H, d, J = 8.7 Hz), 6.98–7.00 (3H, m), 7.32–7.45 (6H,

m), 7.73–7.79 (4H, m), 7.88 (2H, m); d_C(75 MHz, CDCl₃) 12.8 (2C),

19.6, 20.9, 27.2 (3C), 32.7, 44.8 (2C), 52.2, 55.9, 69.2, 100.8, 107.4,

111.0, 114.7 (2C), 115.8, 121.4 (2C), 122.3, 126.8, 127.3, 127.5,

127.9 (2C), 128.0 (2C), 128.2, 129.9, 130.1, 130.8, 133.4, 134.6,

136.1 (2C), 136.3 (2C), 144.8, 147.8, 148.2, 160.4, 166.9.

4.3.37. N-[10-(2-{[tert-Butyl(diphenyl)silyl]oxy}-4-[2-(4-

methoxyphenyl)-2H-tetrazol-5-yl]butyl)-10H-phenothiazin-2-

yl]formamide (9l) (E/Z isomers)

This compound was obtained from the reaction of 10-(2-{[tert-

butyl(diphenyl)silyl]oxy}-4-[2-(4-methoxyphenyl)-2H-tetrazol-5-

yl]butyl)-2-chloro-10H-phenothiazine (8, 0.20 g, 0.28 mmol) and

formamide (0.02 mL, 0.56 mmol) to give the product as beige

amorphous solid, 0.14 g (67%). The reaction time was 22 h; mp

68–71 °C; (found: C, 67.36; H, 5.85; N, 11.37; C₄₁H₄₂N₆O₃SSi re-

quires C, 67.74; H, 5.82; N, 11.56); m/z (EI) [M+H]⁺: 727.50

(C₄₁H₄₂N₆O₃SSi requires 726.28); m_max(KBr)/cm^ꢀ1: 3309, 2930,

2856, 1697 and 1561; d_H(300 MHz, DMSO-d₆) 0.95 (9H, s), 2.05

(2H, m), 2.96 (2H, m), 3.84 (3H, s), 3.94 (2H, m), 4.19 (1H, m),

6.35 [6.40] (1H, d, J = 8.1 Hz), 6.84–6.87 (2H, m), 7.03 (2H, m),

7.10–7.15 (3H, m), 7.29–7.40 (7H, m), 7.57–7.59 (4H, m), 7.78

(2H, m), 8.24 [8.72] (1H, d, J = 1.1, [J = 11.0] Hz, O@C–H), 10.1

[10.0] (1H, d, J = 1.1, [J = 11.0] Hz, NH); d_C(75 MHz, DMSO-d₆)

18.8, 19.9, 26.8 (3C), 32.2, 51.5, 55.6, 68.6, 107.1, 113.5, 114.9

(2C), 115.7, 118.7, 121.1 (2C), 122.7, 124.8, 127.2 (2C), 127.3,

127.6 (2C), 127.7 (2C), 129.6, 129.7, 129.9, 132.8 (2C), 135.3 (2C),

135.4 (2C), 138.1, 143.9, 145.9, 159.6, 160.0, 165.9.

4.3.35. 10-(2-{[tert-Butyl(diphenyl)silyl]oxy}-4-[2-(4-

methoxyphenyl)-2H-tetrazol-5-yl]butyl)-2-morpholin-4-yl-

10H-phenothiazine (9h)

This compound was obtained from the reaction of 10-(2-{[tert-

butyl(diphenyl)silyl]oxy}-4-[2-(4-methoxyphenyl)-2H-tetrazol-5-

Please cite this article in press as: Takács, D.; et al. Bioorg. Med. Chem. (2013), http://dx.doi.org/10.1016/j.bmc.2013.04.034

D. Takács et al. / Bioorg. Med. Chem. xxx (2013) xxx–xxx

15

4.3.38. N-[10-(2-{[tert-Butyl(diphenyl)silyl]oxy}-4-[2-(4-

methoxyphenyl)-2H-tetrazol-5-yl]butyl)-10H-phenothiazin-2-

yl]acetamide (9m)

was made alkaline with 10% aqueous Na₂CO₃and extracted with

dichloromethane (3 ꢁ 0.5 mL). The organic phase was dried over

Na₂SO₄, ﬁltered and concentrated. The crude product was puriﬁed

by column chromatography on silica gel using the eluent chloro-

form:methanol = 15:1, then 10:1 to give white-grey crystals,

34 mg (83%); mp 152–154 °C; (found: C, 59.80; H, 5.30; N, 14.84;

This compound was obtained from the reaction of 10-(2-{[tert-

butyl(diphenyl)silyl]oxy}-4-[2-(4-methoxyphenyl)-2H-tetrazol-5-

yl]butyl)-2-chloro-10H-phenothiazine (8, 0.20 g, 0.28 mmol) and

acetamide (0.03 g, 0.56 mmol) to give the product as beige amor-

phous solid, 0.09 g (44%). The reaction time was 22 h; mp 73–

76 °C; (found: C, 68.04; H, 5.69; N, 11.16; C₄₂H₄₄N₆O₃SSi requires

C, 68.08; H, 5.99; N, 11.34); m/z (EI) [M+H]⁺: 741.70 (C₄₂H₄₄N₆O₃SSi

requires 740.30); m_max(KBr)/cm^ꢀ1: 2958, 2930, 2856, 1516 and

C

₂₈H₃₀N₆O₅S requires C, 59.77; H, 5.37; N, 14.94); m/z (EI)

[M+H]⁺: 563.50 (C₂₈H₃₀N₆O₅S requires 562.20); m_max(KBr)/cm^ꢀ1

:

2963, 2927, 1588, 1515 and 1260; d_H(400 MHz, DMSO-d₆) 1.86

(1H, m, H3y), 2.08 (1H, m, H3x), 2.87 (2H, m, H3y⁰⁰⁰+ H5y⁰⁰⁰), 2.96

(1H, m, H4y), 3.07 (1H, m, H4x), 3.79 (2H, m, H2y⁰⁰⁰+ H6y⁰⁰⁰), 3.85

(3H, s, OCH₃), 4.08 (1H, m, H2), 4.14 (2H, m, H3x⁰⁰⁰+ H5x⁰⁰⁰), 4.44

(2H, m, H2x⁰⁰⁰+ H6x⁰⁰⁰), 4.47 (1H, m, H1y), 4.55 (1H, m, H1x), 5.17

(1H, d, J = 6.2 Hz, OH), 7.16 (2H, m, H3⁰+ H5⁰), 7.32 [7.31] (1H, m,

H7⁰⁰), 7.68 [7.71] (1H, m, H8⁰⁰), 7.91 (2H, m, H2⁰+ H6⁰), 7.95 [7.78]

(1H, m, H9⁰⁰), 7.97 [8.06] (1H, m, H6⁰⁰), 8.02 [8.07] (1H, m, H3⁰⁰),

8.07 [7.96] (1H, m, H4⁰⁰), 8.72 [8.77] (1H, m, H1⁰⁰); d_C(100 MHz,

DMSO-d₆) 21.0 (C4), 32.2 (C3), 53.3 (C1), 55.6 (OCH₃), 61.7

(C2⁰⁰⁰+ C6⁰⁰⁰), 66.7 (C3⁰⁰⁰+ C5⁰⁰⁰), 66.8 (C2), 110.9 [111.2] (C1⁰⁰),

114.7 [114.3] (C3⁰⁰), 115.0 (C3⁰+ C5⁰), 118.3 [117.7] (C9⁰⁰), 121.3

(C2⁰+ C6⁰), 122.3 [122.4] (C7⁰⁰), 125.9 [125.3] (C2⁰⁰), 129.6 (C1⁰),

129.6 [130.2] (C6⁰⁰), 130.7 [129.6] (C4⁰⁰), 130.8 [130.3] (C5a⁰⁰),

132.7 [132.9] (C8⁰⁰), 138.8 [138.4] (C9a⁰⁰), 139.4 [138.9] (C10a⁰⁰),

159.2 [159.1] (C4a⁰⁰), 160.1 (C4⁰), 166.3 (C_tetrazole).

t

1463; d_H(400 MHz, DMSO-d₆) 0.97 (9H, s, Bu), 1.90 (1H, m, H3y),

2.00 (3H, s, O@C–CH₃), 2.10 (1H, m, H3x), 2.97 (2H, m,

H4y + H4x), 3.84 (3H, s, OCH₃), 3.93 (2H, m, H1y + H1x), 4.20 (1H,

m, H2), 6.34 (1H, d, J = 6.8 Hz, H9⁰⁰), 6.86–6.87 (2H, m, H7⁰⁰+ H8⁰⁰),

7.01 (1H, d, J = 8.1 Hz, H4⁰⁰), 7.06 (1H, dd, J = 6.8, 2.0 Hz, H6⁰⁰), 7.13

(1H, dd, J = 8.1, 2.0 Hz, H3⁰⁰), 7.16 (2H, m, H3⁰+ H5⁰), 7.31 (4H, m,

H3⁰⁰⁰+ H5⁰⁰⁰+ H3⁰⁰⁰⁰+ H5⁰⁰⁰⁰), 7.37 (1H, s, H1⁰⁰), 7.40 (2H, m,

H4⁰⁰⁰+ H4⁰⁰⁰⁰), 7.60 (4H, m, H2⁰⁰⁰+ H6⁰⁰⁰+ H2⁰⁰⁰⁰+ H6⁰⁰⁰⁰), 7.81 (2H, m,

H2⁰+ H6⁰), 9.90 (1H, s, NH); d_C(100 MHz, DMSO-d₆) 18.8 (C-^tBu),

19.8 (C4), 23.9 (O@C–CH₃), 26.8 (^tBu, 3C), 32.1 (C3), 51.4 (C1), 55.6

(OCH₃), 68.3 (C2), 106.8 (C1⁰⁰), 113.3 (C3⁰⁰), 114.9 (C3⁰+ C5⁰), 115.7

(C9⁰⁰), 117.8 (C2⁰⁰), 121.2 (C2⁰+ C6⁰), 122.7 (C7⁰⁰), 124.9 (C5a⁰⁰),

127.2 (C4⁰⁰+ C6⁰⁰+ C8⁰⁰), 127.6 (C3⁰⁰⁰+ C5⁰⁰⁰), 127.7 (C3⁰⁰⁰⁰+ C5⁰⁰⁰⁰),

129.6 (C1⁰), 129.7 (C4⁰⁰⁰), 129.8 (C4⁰⁰⁰⁰), 132.8 (C1⁰⁰⁰+ C1⁰⁰⁰⁰), 135.4

(C2⁰⁰⁰+ C6⁰⁰⁰+ C2⁰⁰⁰⁰+ C6⁰⁰⁰⁰), 139.2 (C4a⁰⁰), 143.9 (C9a⁰⁰), 145.8 (C10a⁰⁰),

160.0 (C4⁰), 165.9 (C_tetrazole), 168.2 (O@C–CH₃).

4.3.42. General procedure for the preparation of O-tert-

butyldiphenylsilyl protected sulfoxide and sulfone derivatives

(13, 14)

4.3.39. 1-(2-Chloro-5-oxido-10H-phenothiazin-10-yl)-4-[2-(4-

methoxyphenyl)-2H-tetrazol-5-yl]butan-2-ol (10)

To the solution of the appropriate protected hydroxy compound

(8 or 13, 0.56 mmol) in abs dichloromethane, m-CPBA (0.12 g,

0.72 mmol)/abs dichloromethane (6 mL) was added dropwise at

0 °C. The clear purple reaction mixture became a white suspension.

After completion of the reaction (2–6 h), the mixture was cooled to

0 °C, water was added and stirred for 45 min. It was made alkaline

with 10% aqueous Na₂CO₃and extracted with dichloromethane

(three times). The organic phase was dried over Na₂SO₄, ﬁltered

and concentrated. The crude product was puriﬁed by column chro-

matography on silica gel using the eluent n-hexane:ethyl ace-

tate = 2:1 ill. 1:1 to give white crystals.

This compound was prepared as described in the literature.¹²

4.3.40. 4-[2-(4-Methoxyphenyl)-2H-tetrazol-5-yl]-1-(5-oxido-

10H-phenothiazin-10-yl)butan-2-ol (11)

This compound was obtained from reaction of 1-(2-chloro-5-

oxido-10H-phenothiazin-10-yl)-4-[2-(4-methoxyphenyl)-2H-tet-

razol-5-yl]butan-2-ol (10, 0.30 g, 0.61 mmol) and diethylamine

(0.12 mL, 1.21 mmol) by using the reaction conditions with prepa-

ration of derivatives 4h,i (see above). The reaction time was 7 h.

After column chromatography the crude product was recrystal-

lized from ethyl acetate and white crystals were collected by ﬁltra-

tion, 0.11 g (39%); mp 167–169 °C; (found: C, 62.25; H, 4.70; N,

14.89; C₂₄H₂₃N₅O₃S requires C, 62.46; H, 5.02; N, 15.17); m/z (EI)

4.3.43. 10-(2-{[tert-Butyl(diphenyl)silyl]oxy}-4-[2-(4-

methoxyphenyl)-2H-tetrazol-5-yl]butyl)-2-chloro-10H-

phenothiazine 5-oxide (13)

[M+H]⁺: 462.20 (C₂₄H₂₃N₅O₃S requires 461.15); m_max(KBr)/cm^ꢀ1

:

This compound was obtained from 10-(2-{[tert-butyl(diphe-

nyl)silyl]oxy}-4-[2-(4-methoxyphenyl)-2H-tetrazol-5-yl]butyl)-2-

chloro-10H-phenothiazine (8, 0.40 g) to give the product as white

crystals, 0.36 g (88%); mp 64–68 °C; (found: C, 65.28; H, 5.60; N,

9.17; C₄₀H₄₀ClN₅O₃SSi requires C, 65.42; H, 5.49; N, 9.54); m/z

(EI) [M+H]⁺: 734.30 (C₄₀H₄₀ClN₅O₃SSi requires 733.23); m_max

(KBr)/cm^ꢀ1: 2931, 1581, 1516, 1456 and 1256; d_H(300 MHz,

CDCl₃) 0.93 (9H, s), 2.04 (1H, m), 2.17 (1H, m), 2.99 (2H, m), 3.88

(3H, s), 4.10–4.52 (3H, m), 6.62 (1H, m), 7.00 (2H, m), 7.07–7.45

(10H, m), 7.54–7.80 (6H, m), 7.89 (2H, m); d_C(75 MHz, CDCl₃)

19.4, 20.8, 27.0 (3C), 32.5, 51.6, 55.9, 69.2, 114.7 (2C), 116.3,

116.5, 117.5, 121.5 (2C), 122.6, 122.7, 123.0, 124.5, 126.2, 128.0

(2C), 128.1 (2C), 129.5, 130.3, 130.6, 131.4, 131.9, 132.7, 133.2,

136.0 (4C), 138.8, 141.0, 160.5, 166.2.

3374, 2926, 1585, 1515 and 1462; d_H(400 MHz, DMSO-d₆) 1.84

(1H, m, H3y), 2.06 (1H, m, H3x), 2.96 (1H, m, H4y), 3.07 (1H, m,

H4x), 3.86 (3H, s, OCH₃), 4.06 (1H, m, H2), 4.42 (1H, m, H1y),

4.51 (1H, m, H1x), 5.16 (1H, br s, OH), 7.18 (2H, m, H3⁰+ H5⁰),

7.28 [7.29] (2H, m, H3⁰⁰+ H7⁰⁰), 7.67 [7.65] (2H, m, H2⁰⁰+ H8⁰⁰),

7.92 (2H, m, H2⁰+ H6⁰), 7.93 [7.76] (4H, m, H1⁰⁰+ H4⁰⁰+ H6⁰⁰+ H9⁰⁰);

d_C(100 MHz, DMSO-d₆) 21.0 (C4), 32.1 (C3), 53.3 (C1), 55.6 (OCH₃),

67.2 (C2), 115.0 (C3⁰+ C5⁰), 118.3 [117.7] (C1⁰⁰+ C9⁰⁰), 121.3

(C2⁰+ C6⁰), 122.0 [121.8] (C3⁰⁰+ C7⁰⁰), 125.3 [125.9] (C4a⁰⁰+ C5a⁰⁰),

129.6 (C1⁰), 130.1 [130.0] (C4⁰⁰+ C6⁰⁰), 132.5 [132.7] (C2⁰⁰+ C8⁰⁰),

139.2 [138.5] (C9a⁰⁰+ C10a⁰⁰), 160.1 (C4⁰), 166.4 (C_tetrazole).

4.3.41. 4-[2-(4-Methoxyphenyl)-2H-tetrazol-5-yl]-1-[5-oxido-2-

(4-oxidomorpholin-4-yl)-10H-phenothiazin-10-yl]butan-2-ol

(12)

To a solution of 4-[2-(4-methoxyphenyl)-2H-tetrazol-5-yl]-1-

(2-morpholin-4-yl-5-oxido-10H-phenothiazin-10-yl)butan-2-ol

(4h, 0.04 g, 0.07 mmol) in abs dichloromethane (1 mL) m-CPBA

(0.02 g, 0.11 mmol)/abs dichloromethane (0.5 mL) was added

dropwise at 0 °C. The clear purple reaction mixture became a white

suspension. After completion of the reaction (23 h), the mixture

was cooled to 0 °C, water was added and stirred for 45 min. It

4.3.44. 10-(2-{[tert-Butyl(diphenyl)silyl]oxy}-4-[2-(4-

methoxyphenyl)-2H-tetrazol-5-yl]butyl)-2-chloro-10H-

phenothiazine 5,5-dioxide (14)

This compound was obtained from 10-(2-{[tert-butyl(diphe-

nyl)silyl]oxy}-4-[2-(4-methoxyphenyl)-2H-tetrazol-5-yl]butyl)-2-

chloro-10H-phenothiazine 5-oxide (13, 0.42 g, 0.35 mmol) to give

the product as white crystals, 0.43 g (quant.); mp 62–67 °C;

Please cite this article in press as: Takács, D.; et al. Bioorg. Med. Chem. (2013), http://dx.doi.org/10.1016/j.bmc.2013.04.034

16

D. Takács et al. / Bioorg. Med. Chem. xxx (2013) xxx–xxx

(found: C, 64.40; H, 5.47; N, 9.27; C₄₀H₄₀ClN₅O₄SSi requires C,

64.02; H, 5.37; N, 9.33); m/z (EI) [M+H]⁺: 750.30 (C₄₀H₄₀ClN₅O₄SSi

requires 749.23); m_max(KBr)/cm^ꢀ1: 2930, 1585, 1516, 1461 and

1256; d_H(300 MHz, CDCl₃) 1.00 (9H, s), 1.96 (1H, m), 2.14 (1H,

m), 2.98 (2H, t, J = 7.5 Hz), 3.88 (3H, s), 4.10–4.43 (3H, m), 6.66

(1H, d, J = 8.1 Hz), 7.00 (2H, m), 7.13–7.28 (4H, m), 7.37–7.45

(6H, m), 7.68–7.71 (4H, m), 7.85–7.97 (4H, m); d_C(75 MHz, CDCl₃)

19.5, 20.7, 27.1 (3C), 32.0, 51.7, 55.9, 69.1, 114.7 (2C), 117.1, 117.2,

121.5 (2C), 122.8 (2C), 123.6, 124.1, 125.2, 125.9, 128.1 (2C), 128.2

(2C), 130.3, 130.4, 130.7, 133.0, 133.2, 133.5, 136.1 (4C), 139.4,

141.0, 143.1, 160.5, 166.2.

116.9, 121.5 (2C), 122.0, 123.1, 125.1, 126.7, 128.0 (2C), 128.2

(2C), 130.2, 130.4, 130.5, 132.4, 133.0, 134.0, 136.0 (2C), 136.2

(2C), 141.4, 144.0, 154.7, 160.5, 166.4.

4.3.48. 10-(2-{[tert-Butyl(diphenyl)silyl]oxy}-4-[2-(4-

methoxyphenyl)-2H-tetrazol-5-yl]butyl)-2-(4-methylpiperazin-

1-yl)-10H-phenothiazine 5,5-dioxide (15i)

This compound was obtained from the reaction of 10-(2-{[tert-

butyl(diphenyl)silyl]oxy}-4-[2-(4-methoxyphenyl)-2H-tetrazol-5-

yl]butyl)-2-chloro-10H-phenothiazine 5,5-dioxide (14, 0.20 g,

0.27 mmol) and N-methylpiperazine (0.06 mL, 0.53 mmol). The

reaction time was 70 min. After ﬂash chromatography the product

was isolated as beige amorphous solid, 0.20 g (92%); mp 81–86 °C;

(found: C, 66.31; H, 6.29; N, 11.94; C₄₅H₅₁N₇O₄SSi requires C,

66.39; H, 6.31; N, 12.04); m/z (EI) [M+H]⁺: 814.30 (C₄₅H₅₁N₇O₄SSi

requires 813.35); m_max(KBr)/cm^ꢀ1: 3418, 2932, 1593, 1516 and

1456; d_H(300 MHz, CDCl₃) 1.00 (9H, s), 1.98 (1H, m), 2.14 (1H,

m), 2.38 (3H, s), 2.56 (4H, t, J = 4.8 Hz), 2.98 (2H, t, J = 7.2 Hz),

3.28 (4H, t, J = 4.8 Hz), 3.88 (3H, s), 4.10 (1H, m), 4.21 (1H, m),

4.41 (1H, m), 6.39 (1H, d, J = 6.9 Hz), 6.52 (1H, s), 6.66 (1H, d,

J = 9.0 Hz), 6.97–7.13 (4H, m), 7.36–7.47 (6H, m), 7.71–7.79 (5H,

m), 7.85–7.92 (3H, m); d_C(75 MHz, CDCl₃) 19.5, 20.7, 27.1 (3C),

31.9, 46.2, 47.9 (2C), 51.4, 54.9 (2C), 55.9, 69.2, 101.4, 109.8,

114.7 (2C), 116.3, 116.9, 121.5 (2C), 122.0, 123.1, 125.0, 126.9,

128.0 (2C), 128.2 (2C), 130.2, 130.4, 130.7, 132.4, 133.0, 134.1,

136.1 (2C), 136.2 (2C), 141.3, 144.2, 154.5, 160.4, 166.4.

4.3.45. General procedure for preparation of 10-(2-{[tert-butyl

(diphenyl)silyl]oxy}-4-[2-(4-methoxyphenyl)-2H-tetrazol-5-

yl]butyl)-2-amino-10H-phenothiazine 5,5-dioxide derivatives (15)

A

round-bottomed ﬂask was charged with Pd-catalyst:

Pd₂(dba)₃(10 mol %), ligand: XPhos (20 mol %), 10-(2-{[tert-butyl

(diphenyl)silyl]oxy}-4-[2-(4-methoxyphenyl)-2H-tetrazol-5-yl]bu-

tyl)-2-chloro-10H-phenothiazine 5,5-dioxide (14), secondary

amine, base: NaO^tBu, and dry toluene (5 mL). The ﬂask was ﬂushed

with argon for 5 min. The resulting mixture was heated under re-

ﬂux with magnetic stirring for 70–90 min. After cooling down to

rt the reaction mixture was concentrated and the residue was puri-

ﬁed by ﬂash chromatography on silica gel using the eluent dichlo-

romethane, then n-hexane:ethyl acetate = 2:1 and 1:1 to give the

ﬁnal product as beige amorphous solid.

4.3.46. 10-(2-{[tert-Butyl(diphenyl)silyl]oxy}-4-[2-(4-

methoxyphenyl)-2H-tetrazol-5-yl]butyl)-N,N-diethyl-10H-

phenothiazin-2-amine 5,5-dioxide (15f)

4.3.49. General procedure for the preparation of 1-(2-amino-

5,5-dioxido-10H-phenothiazin-10-yl)-4-[2-(4-methoxyphenyl)-

2H-tetrazol-5-yl]butan-2-ol derivatives (16)

This compound was obtained from the reaction of 10-(2-{[tert-

butyl(diphenyl)silyl]oxy}-4-[2-(4-methoxyphenyl)-2H-tetrazol-5-

yl]butyl)-2-chloro-10H-phenothiazine 5,5-dioxide (14, 0.35 g,

0.47 mmol) and diethylamine (0.10 mL, 0.93 mmol). The reaction

time was 70 min. After ﬂash chromatography the product was iso-

lated as beige amorphous solid, 0.11 g (58%); mp 71–75 °C; (found:

C, 66.87; H, 6.42; N, 9.89; C₄₄H₅₀N₆O₄SSi requires C, 67.15; H, 6.40;

N, 10.68); m/z (EI) [M+H]⁺: 787.70 (C₄₄H₅₀N₆O₄SSi requires

786.34); m_max(KBr)/cm^ꢀ1: 3430, 2931, 1594, 1516 and 1471; d_H

(300 MHz, CDCl₃) 1.00 (9H, s), 1.18 (6H, t, J = 6.9 Hz), 2.04 (1H,

m), 2.16 (1H, m), 3.02 (2H, t, J = 7.5 Hz), 3.38 (4H, q, J = 6.9 Hz),

3.88 (3H, s), 4.06–4.41 (3H, m), 6.21–6.25 (2H, m), 6.45 (1H, d,

J = 9.0 Hz), 6.97–7.04 (4H, m), 7.36–7.50 (6H, m), 7.72–7.74 (5H,

m), 7.86–7.89 (3H, m); d_C(75 MHz, CDCl₃) 12.5 (2C), 19.3, 20.6,

26.8 (3C), 31.6, 44.6 (2C), 51.2, 55.6, 69.2, 97.0, 106.4, 113.1,

114.4 (2C), 116.5, 121.4 (2C), 122.6, 123.9, 125.0, 127.0, 127.8

(2C), 128.0 (2C), 130.0, 130.1, 130.5, 131.8, 132.8, 133.9, 135.8

(2C), 136.0 (2C), 141.0, 144.5, 151.1, 160.1, 166.3.

To the solution of 10-(2-{[tert-butyl(diphenyl)silyl]oxy}-4-[2-(4-

methoxyphenyl)-2H-tetrazol-5-yl]butyl)-2-amino-10H-phenothia-

zine 5,5-dioxide derivative (15, 0.22 mmol) in anhydrous THF

(15 mL) in a dried, round-bottomed ﬂask equipped with a side

arm and cooled to 0 °C in an argon atmosphere was added the solu-

tion of TBAF (0.33 mmol)/abs THF (1 mL). The reaction mixture was

allowed to warm up to rt. After the completion of the reaction as

speciﬁed (2.5–4.5 h) the reaction mixture was evaporated. The res-

idue was puriﬁed by column chromatography on silica gel using the

eluent n-hexane:ethyl acetate = 2:1 then 1:1 to give the product.

4.3.50. 1-[2-(Diethylamino)-5,5-dioxido-10H-phenothiazin-10-

yl]-4-[2-(4-methoxyphenyl)-2H-tetrazol-5-yl]butan-2-ol (16f)

This compound was obtained from the reaction of 10-(2-{[tert-

butyl(diphenyl)silyl]oxy}-4-[2-(4-methoxyphenyl)-2H-tetrazol-5-

yl]butyl)-N,N-diethyl-10H-phenothiazin-2-amine 5,5-dioxide (15f,

0.25 g, 0.32 mmol) and TBAF (0.12 g, 0.48 mmol)/abs THF (1 mL).

The reaction time was 2.5 h. After column chromatography yellow

amorphous solid were isolated, 0.14 g (78%); mp 49–52 °C; (found:

C, 61.13; H, 5.48; N, 14.98; C₂₈H₃₂N₆O₄S requires C, 61.30; H, 5.88;

N, 15.32); m/z (EI) [M+H]⁺: 549.30 (C₂₈H₃₂N₆O₄S requires 548.22); m_max

(KBr)/cm^ꢀ1: 2968, 2928, 1593, 1516 and 1470; d_H(400 MHz, DMSO-

d₆) 1.12 (6H, t, J = 6.9 Hz, N(CH₂)₂(CH₃)₂), 1.84 (1H, dddd, J = 14.2,

9.4, 9.0, 5.0 Hz, H3y), 2.07 (1H, m, H3x), 2.99 (1H, ddd, J = 15.2, 9.0,

7.1 Hz, H4y), 3.08 (1H, ddd, J = 15.2, 9.4, 5.2 Hz, H4x), 3.42 (4H, m,

N(CH₂)₂(CH₃)₂), 3.85 (3H, s, OCH₃), 4.15 (1H, m, H2), 4.24 (1H, dd,

J = 15.0, 4.8 Hz, H1y), 4.32 (1H, dd, J = 15.0, 7.3 Hz, H1x), 5.22 (1H, d,

J = 5.3 Hz, OH), 6.63 (1H, m, H3⁰⁰), 6.64 (1H, s, H1⁰⁰), 7.16 (2H, m,

H3⁰+ H5⁰), 7.25 (1H, dd, J = 7.9, 7.3 Hz, H7⁰⁰), 7.62 (1H, m, H8⁰⁰), 7.65

(1H, m, H4⁰⁰), 7.69 (1H, d, J = 8.6 Hz, H9⁰⁰), 7.87 (1H, dd, J = 7.9,

1.4 Hz, H6⁰⁰), 7.90 (2H, m, H2⁰+ H6⁰); d_C(100 MHz, DMSO-d₆) 12.2

(N(CH₂)₂(CH₃)₂, 2C), 21.0 (C4), 32.6 (C3), 44.0 (N(CH₂)₂(CH₃)₂, 2C),

53.5 (C1), 55.7 (OCH₃), 66.8 (C2), 97.5 (C1⁰⁰), 106.3 (C3⁰⁰), 111.2

(C4a⁰⁰), 115.0 (C2⁰+ C6⁰), 117.8 (C9⁰⁰), 121.3 (C2⁰+ C6⁰+ C7⁰⁰), 121.8

(C6⁰⁰), 124.0 (C4⁰⁰), 125.3 (C5a⁰⁰), 129.6 (C1⁰), 132.5 (C8⁰⁰), 141.4 (C9a⁰⁰),

143.0 (C10a⁰⁰), 150.8 (C2⁰⁰), 160.1 (C4⁰), 166.4 (C_tetrazole).

4.3.47. 10-(2-{[tert-Butyl(diphenyl)silyl]oxy}-4-[2-(4-

methoxyphenyl)-2H-tetrazol-5-yl]butyl)-2-morpholin-4-yl-

10H-phenothiazine 5,5-dioxide (15h)

This compound was obtained from the reaction of 10-(2-{[tert-

butyl(diphenyl)silyl]oxy}-4-[2-(4-methoxyphenyl)-2H-tetrazol-5-

yl]butyl)-2-chloro-10H-phenothiazine 5,5-dioxide (14, 0.20 g,

0.27 mmol) and morpholine (0.05 mL, 0.53 mmol). The reaction

time was 90 min. After ﬂash chromatography the product was iso-

lated as beige amorphous solid, 0.20 g (92%); mp 56–60 °C; (found:

C, 65.85; H, 6.09; N, 10.30; C₄₄H₄₈N₆O₅SSi requires C, 65.97; H,

6.04; N, 10.49); m/z (EI) [M+H]⁺: 801.50 (C₄₄H₄₈N₆O₅SSi requires

800.32); m_max(KBr)/cm^ꢀ1: 2930, 1694, 1516, 1464 and 1256; d_H

(300 MHz, CDCl₃) 0.98 (9H, s), 2.04 (1H, m), 2.13 (1H, m), 2.97

(2H, t, J = 7.5 Hz), 3.10–3.18 (4H, m), 3.83 (4H, m), 3.87 (3H, s),

4.09–4.43 (3H, m), 6.47–6.67 (3H, m), 6.98–7.45 (11H, m), 7.70–

7.93 (7H, m); d_C(75 MHz, CDCl₃) 19.5, 20.6, 27.0 (3C), 32.0, 48.2

(2C), 51.5, 55.9, 66.8 (2C), 69.2, 101.3, 109.5, 114.7 (2C), 116.8,

Please cite this article in press as: Takács, D.; et al. Bioorg. Med. Chem. (2013), http://dx.doi.org/10.1016/j.bmc.2013.04.034

D. Takács et al. / Bioorg. Med. Chem. xxx (2013) xxx–xxx

17

4.3.51. 4-[2-(4-Methoxyphenyl)-2H-tetrazol-5-yl]-1-(2-morpholin-

4-yl-5,5-dioxido-10H-phenothiazin-10-yl)butan-2-ol (16h)

The crude product (foam) was triturated with n-hexane, the mix-

ture was cooled whereupon crystals deposited. The product was

ﬁltered off to yield yellow crystals, 4.98 g (quant.); mp 56–60 °C;

(found: C, 57.81; H, 3.96; N, 11.99; C₂₈H₂₄ClN₅O₅S requires C,

58.18; H, 4.18; N, 12.12); m/z (EI) [M+H]⁺: 578.30 (C₂₈H₂₄ClN₅O₅S

requires 577.12); m_max(KBr)/cm^ꢀ1: 2925, 2361, 1516, 1458 and

1256; d_H(300 MHz, CDCl₃) 2.29 (1H, m, H3y), 2.39 (1H, m, H3x),

3.07 (2H, t, J = 7.8 Hz, H4y + H4x), 3.88 (3H, s, OCH₃), 4.08 (1H,

dd, J = 13.8, 6.6 Hz, H1y), 4.17 (1H, dd, J = 13.8, 6.3 Hz, H1x), 5.56

(1H, m, H2), 6.21 (1H, d, J = 12.9 Hz, CH@CH–COOH), 6.40 (1H, d,

J = 12.9 Hz, CH@CH–COOH), 6.90–7.20 (10H, m, H3⁰+ H5⁰+ H1⁰⁰+

H3⁰⁰+ H4⁰⁰+ H6⁰⁰+ H7⁰⁰+ H8⁰⁰+ H9⁰⁰+ COOH), 7.93 (2H, m, H2⁰+

H6⁰); d_C(75 MHz, CDCl₃) 21.4 (C4), 29.9 (C3), 49.8 (C1), 55.9

(OCH₃), 72.6 (C2), 114.9 (C3⁰+ C5⁰), 116.4 (CH), 116.6 (CH), 121.5

(C2⁰+ C6⁰), 123.4 (CH), 124.0 (CH), 125.3 (C), 126.5 (C), 127.9

(CH), 128.2 (CH), 128.6 (CH), 128.7 (CH), 130.4 (C), 133.7 (C),

136.1 (CH), 144.3 (C), 146.5 (C), 160.8 (C4⁰), 164.7 (COO), 165.6

(C_tetrazole), 167.0 (COOH).

This compound was obtained from the reaction of 10-(2-{[tert-

butyl(diphenyl)silyl]oxy}-4-[2-(4-methoxyphenyl)-2H-tetrazol-5-

yl]butyl)-2-morpholin-4-yl-10H-phenothiazine 5,5-dioxide (15h,

0.18 g, 0.22 mmol) and TBAF (0.09 g, 0.33 mmol)/abs THF (1 mL).

The reaction time was 4.5 h. After column chromatography yellow

amorphous solid were isolated, 0.10 g (82%); mp 71–76 °C. The air

sensitive product was stored in an argon atmosphere; (found: C,

59.41; H, 5.34; N, 14.93; C₂₈H₃₀N₆O₅S requires C, 59.77; H, 5.37;

N, 14.94); m/z (EI) [M+H]⁺: 563.30 (C₂₈H₃₀N₆O₅S requires 562.20);

m_max(KBr)/cm^ꢀ1: 2924, 2853, 1593, 1516 and 1468; d_H(400 MHz,

DMSO-d₆) 1.81 (1H, m, H3y), 2.04 (1H, m, H3x), 2.97 (1H, m,

H4y), 3.06 (1H, m, H4x), 3.30 (4H, m, N–(CH₂)₂), 3.74 (4H, m, O–

(CH₂)₂), 3.84 (3H, s, OCH₃), 4.10 (1H, m, H2), 4.31 (1H, m, H1y),

4.36 (1H, m, H1x), 5.18 (1H, d, J = 5.3 Hz, OH), 6.92 (1H, m, H3⁰⁰),

7.01 (1H, s, H1⁰⁰), 7.16 (2H, m, H3⁰+ H5⁰), 7.28 (1H, m, H7⁰⁰), 7.65

(1H, m, H8⁰⁰), 7.71 (1H, m, H9⁰⁰), 7.72 (1H, m, H4⁰⁰), 7.89 (1H, m,

H6⁰⁰), 7.90 (2H, m, H2⁰+ H6⁰); d_C(100 MHz, DMSO-d₆) 20.9 (C4),

32.4 (C3), 47.3 (N–(CH₂)₂, 2C), 53.1 (C1), 55.6 (OCH₃), 65.8 (O–

(CH₂)₂, 2C), 66.6 (C2), 101.4 (C1⁰⁰), 109.1 (C3⁰⁰), 114.5 (C4a⁰⁰), 115.0

(C2⁰+ C6⁰), 117.9 (C9⁰⁰), 121.3 (C2⁰+ C6⁰+ C6⁰⁰), 121.6 (C7⁰⁰), 123.6

(C5a⁰⁰), 125.0 (C4⁰⁰), 129.6 (C1⁰), 132.7 (C8⁰⁰), 141.4 (C9a⁰⁰), 142.8

(C10a⁰⁰), 154.2 (C2⁰⁰), 160.1 (C4⁰), 166.3 (C_tetrazole).

4.3.54. Resolution of racemic (2Z)-4-{1-[(2-chloro-10H-

phenothiazin-10-yl)methyl]-3-[2-(4-methoxyphenyl)-2H-

tetrazol-5-yl]propoxy}-4-oxobut-2-enoic acid (17) with S-(ꢀ)-

phenylethylamine^23d

4.3.54.1. Diastereomeric salt_crystal(18).

Racemic 4-((1-(2-

chloro-10H-phenothiazin-10-yl)-4-(2-(4-methoxyphenyl)-2H-tet-

razol-5-yl)butan-2-yl)oxy)-4-oxobut-2-enoic acid (17, 5.72 g,

9.90 mmol) was dissolved in ethyl acetate (60 mL), and S-(ꢀ)-

phenylethylamine (0.64 mL, 4.95 mmol) was added to the solution.

The reaction mixture was stirred at rt for 2 h. Precipitation of the

diastereomeric salt_crystal(18) commenced in a few minutes. The

precipitated diastereomer salt was collected by ﬁltration and

washed with ethyl acetate (3 ꢁ 10 mL). The white crystals were

recrystallized from hot ethyl acetate and cooled slowly (recrystal-

lization from ethyl acetate was repeated once again) to give 4-((1-

(2-chloro-10H-phenothiazin-10-yl)-4-(2-(4-methoxyphenyl)-2H-

tetrazol-5-yl)butan-2-yl)oxy)-4-oxobut-2-enoic acid (S)-phenyl-

ethylamine salt 18 as white crystals, 2.49 g (75%); mp 147–

4.3.52. 4-[2-(4-Methoxyphenyl)-2H-tetrazol-5-yl]-1-[2-(4-

methylpiperazin-1-yl)-5,5-dioxido-10H-phenothiazin-10-

yl]butan-2-ol (16i)

This compound was obtained from the reaction of 10-(2-((tert-

butyldiphenylsilyl)oxy)-4-(2-(4-methoxyphenyl)-2H-tetrazol-5-yl)

butyl)-2-(4-methylpiperazin-1-yl)-10H-phenothiazine-5,5-dioxide

(15i, 0.20 g, 0.25 mmol) and tetrabutylammonium ﬂuoride (0.10 g,

0.37 mmol)/abs THF (1 mL). The reaction time was 4.5 h. The crude

product was puriﬁed by column chromatography on silica gel

using the eluent chloroform:methanol = 15:1 then 10:1. Yellow

amorphous solid were isolated, 0.13 g (88%); mp 74–80 °C. The

air sensitive product was stored in an argon atmosphere; (found:

C, 60.41; H, 5.70; N, 16.75; C₂₉H₃₃N₇O₄S requires C, 60.50; H,

5.78; N, 17.03); m/z (EI) [M+H]⁺: 576.20 (C₂₉H₃₃N₇O₄S requires

575.23); m_max(KBr)/cm^ꢀ1: 2937, 2842, 1593, 1516 and 1455; d_H

(400 MHz, DMSO-d₆) 1.83 (1H, m, H3y), 2.05 (1H, m, H3x), 2.21

(3H, s, H₃C–N–(CH₂)₂), 2.42 (4H, m, H₃C–N–(CH₂)₂), 2.97 (1H, m,

H4y), 3.07 (1H, m, H4x), 3.33 (4H, m, H₃C–N–(CH₂)₂), 3.84 (3H, s,

OCH₃), 4.10 (1H, m, H2), 4.30 (2H, m, H1y + H1x), 5.18 (1H, d,

J = 5.3 Hz, OH), 6.92 (1H, m, H3⁰⁰), 6.98 (1H, s, H1⁰⁰), 7.16 (2H, m,

H3⁰+ H5⁰), 7.28 (1H, m, H7⁰⁰), 7.64 (1H, m, H8⁰⁰), 7.69 (1H, m,

H4⁰⁰), 7.71 (1H, m, H9⁰⁰), 7.88 (1H, m, H6⁰⁰), 7.90 (2H, m,

H2⁰+ H6⁰); d_C(100 MHz, DMSO-d₆) 20.9 (C4), 32.4 (C3), 45.6 (N–

CH₃), 47.0 (N–(CH₂)₂, 2C), 53.1 (C1), 54.3 (H₃C–N–(CH₂)₂, 2C),

55.7 (OCH₃), 66.6 (C2), 101,4 (C1⁰⁰), 109.3 (C3⁰⁰), 114.0 (C4a⁰⁰),

115.0 (C2⁰+ C6⁰), 117.9 (C9⁰⁰), 121.3 (C2⁰+ C6⁰), 121.5 (C7⁰⁰), 121.9

(C6⁰⁰), 123.6 (C4⁰⁰), 125.1 (C5a⁰⁰), 129.6 (C1⁰), 132.7 (C8⁰⁰), 141.4

(C9a⁰⁰), 142.8 (C10a⁰⁰), 154.1 (C2⁰⁰), 160.1 (C4⁰), 166.3 (C_tetrazole).

150 °C; ½a ²_D⁵

ꢀ 4:90 = (c 0.368, methanol); (found: C, 61.51; H,

ꢃ

5.03; N, 11.67; C₃₆H₃₅ClN₆O₅S requires C, 61.84; H, 5.05; N,

12.02); m_max(KBr)/cm^ꢀ1: 3421, 2934, 2540, 1515 and 1460.

4.3.55. (ꢀ)-4-{1-[(2-Chloro-10H-phenothiazin-10-yl)methyl]-3-

[2-(4-methoxyphenyl)-2H-tetrazol-5-yl]propoxy}-4-oxobut-2-

enoic acid ((ꢀ)-17)

The recrystallized diastereomeric salt (18, 2.36 g, 3.37 mmol)

was dissolved in a mixture of methanol (52 mL) and dichlorometh-

ane (15 mL) and stirred at rt. To the reaction mixture 10% aqueous

HCl solution (14.13 mL) was added. After 4 h, the phases were sep-

arated and the organic phase was extracted with 10% aqueous HCl

solution (2 ꢁ 5 mL), dried over Na₂SO₄, ﬁltered and concentrated.

The crude product was triturated with n-hexane and yellow crys-

tals were collected by ﬁltration, 1.94 g (99.8%); ½a _D²⁵

ꢀ 11:00 (c

ꢃ

0.310, chloroform); (found: C, 57.81; H, 3.96; N, 11.99;

C

₂₈H₂₄ClN₅O₅S requires C, 58.18; H, 4.18; N, 12.12); m_max(KBr)/

4.3.53. (2Z)-4-{1-[(2-Chloro-10H-phenothiazin-10-yl)methyl]-3-

[2-(4-methoxyphenyl)-2H-tetrazol-5-yl]propoxy}-4-oxobut-2-

enoic acid (17)

cm^ꢀ1: 2929, 1516, 1458, 1256 and 1170.

4.3.56. (ꢀ)-1-(2-Chloro-10H-phenothiazin-10-yl)-4-[2-(4-

methoxyphenyl)-2H-tetrazol-5-yl]butan-2-ol ((ꢀ)-5)

To a solution of racemic 1-(2-chloro-10H-phenothiazin-10-yl)-

4-[2-(4-methoxyphenyl)-2H-tetrazol-5-yl]butan-2-ol (5, 4.15 g,

8.64 mmol) in abs dichloromethane (30 mL), abs triethylamine

(2.59 mL, 18.70 mmol) was added and the mixture was stirred at

rt. After 30 min, maleic anhydride (1.59 g, 16.17 mmol) was added

to the mixture and it was heated up and reﬂuxed for 9.5 h. The

mixture was then cooled to rt, the pH was adjusted to acidic with

10% aqueous HCl solution. After separation, the organic phase was

washed with water, dried over Na₂SO₄, ﬁltered and concentrated.

(ꢀ)-4-{1-[(2-Chloro-10H-phenothiazin-10-yl)methyl]-3-[2-(4-

methoxyphenyl)-2H-tetrazol-5-yl]propoxy}-4-oxobut-2-enoic

acid ((ꢀ)-17, 1.75 g, 3.02 mmol) was dissolved in dichloromethane

(37 mL) at rt and the solution of NaOH (0.54 g, 13.6 mmol)/H₂O

(2 mL) was added to the mixture then reﬂuxed for 7.5 h. After cool-

ing, water (30 mL) was added to the mixture, it was acidiﬁed with

10% aqueous HCl solution and extracted with dichloromethane

(3 ꢁ 20 mL). The organic phase was dried over Na₂SO₄, ﬁltered

Please cite this article in press as: Takács, D.; et al. Bioorg. Med. Chem. (2013), http://dx.doi.org/10.1016/j.bmc.2013.04.034

18

D. Takács et al. / Bioorg. Med. Chem. xxx (2013) xxx–xxx

and concentrated. The crude product was puriﬁed by ﬂash chroma-

tography on silica gel using the eluent n-hexane:ethyl acetate = 2:1

then 1:1. The product (foam) was triturated with diethyl ether and

the mixture was cooled whereupon crystals deposited. The prod-

uct, white crystals were collected by ﬁltration, 1.25 g (86%); mp

4.3.60. (ꢀ)-1-[2-(Diethylamino)-10H-phenothiazin-10-yl]-4-[2-

(4-methoxyphenyl)-2H-tetrazol-5-yl]butan-2-ol ((ꢀ)-3f)

This compound was obtained from (ꢀ)-10-(2-{[tert-butyl(di-

phenyl)silyl]oxy}-4-[2-(4-methoxyphenyl)-2H-tetrazol-5-yl]bu-

tyl)-N,N-diethyl-10H-phenothiazin-2-amine

((ꢀ)-9f,

0.20 g,

90–92 °C; ½a ²_D⁵

ꢃ

ꢀ 21:30 (c 0.282, chloroform); ee: 99.9+%; HPLC:

0.26 mmol). The crude product was puriﬁed by ﬂash chromatogra-

phy on silica gel using the eluent dichloromethane, then n-hex-

ane:ethyl acetate = 2:1 to give white amorphous solid, 0.09 g

using

the

eluent n-hexane:2-propanol + 0.1% diethyl-

amine = 70:30, retention time: 15.2 min; (found: C, 59.72; H,

4.70; N, 14.30; C₂₄H₂₂ClN₅O₂S requires C, 60.06; H, 4.62; N,

14.59); m/z (EI) [M+H]⁺: 480.20 (C₂₄H₂₂ClN₅O₂S requires 479.12);

m_max(KBr)/cm^ꢀ1: 3422, 2929, 1515, 1461 and 1259.

(66%); ee: 99.6%; ½a _D²⁵

ꢀ 9:98 (c 0.259, chloroform); HPLC: using

ꢃ

the eluent n-hexane:2-propanol + 0.1% diethylamine = 70:30,

retention time: 22.1 min.

4.3.57. (+)-4-{1-[(2-Chloro-10H-phenothiazin-10-yl)methyl]-3-

[2-(4-methoxyphenyl)-2H-tetrazol-5-yl]propoxy}-4-oxobut-2-

enoic acid ((+)-17)

4.3.61. (ꢀ)-4-[2-(4-Methoxyphenyl)-2H-tetrazol-5-yl]-1-(2-

morpholin-4-yl-10H-phenothiazin-10-yl)butan-2-ol ((ꢀ)-3h)

This compound was obtained from (S)-(ꢀ)-10-(2-{[tert-butyl(-

diphenyl)silyl]oxy}-4-[2-(4-methoxyphenyl)-2H-tetrazol-5-yl]bu-

The mother liquor obtained after separation of crystalline 18

was evaporated to give a solid (19, 3.44 g) containing a mixture of

the non-reacted part of 17, that is, (+)-17 and, additionally, traces

of 18. This mixture was dissolved in ethyl acetate (20 mL), stirred

at rt and, then, 10% aqueous HCl solution (1.35 mL) was added (this

amount of HCl was calculated as an excess for the assumed amount

of 18 being present in the mixture 19). After 50 min, the phases

were separated and the organic phase was washed with 10% aque-

ous HCl solution (2 ꢁ 0.5 mL), dried over Na₂SO₄, ﬁltered and con-

centrated. The crude product (foam) was triturated with n-

hexane, yellow crystals deposited and were collected by ﬁltration.

As the mass spectrum still indicated the presence of the reagent

S-(ꢀ)-phenylethylamine, the hydrolysis was repeated again. Yield:

3.07 g (107%, calculated for the expected amount of (+)-monoester:

tyl)-2-morpholin-4-yl-10H-phenothiazine

((ꢀ)-9h,

0.31 g,

0.40 mmol). The crude product was puriﬁed by ﬂash chromatogra-

phy on silica gel using the eluent n-hexane:ethyl acetate = 4:1 to

give beige amorphous solid, 0.21 g (98%); ee: 99.9+%;

½

a ²_D⁵

ꢃ

ꢀ 15:24 (c 0.289, chloroform). HPLC: using the eluent n-hex-

ane:2-propanol + 0.1% diethylamine = 70:30, retention time:

17.4 min.

4.3.62. (ꢀ)-4-[2-(4-Methoxyphenyl)-2H-tetrazol-5-yl]-1-[2-(4-

methylpiperazin-1-yl)-10H-phenothiazin-10-yl]butan-2-ol

((ꢀ)-3i)

This compound was obtained from (ꢀ)-10-(2-{[tert-butyl(di-

phenyl)silyl]oxy}-4-[2-(4-methoxyphenyl)-2H-tetrazol-5-yl]bu-

(+)-17); for the crude product: ½a _D²⁵

þ 14:00 (c 0.470, methanol);

ꢃ

tyl)-2-(4-methylpiperazin-1-yl)-10H-phenothiazine

((ꢀ)-9i,

m

_max(KBr)/cm^ꢀ1: 2931, 1516, 1458, 1256 and 1170.

0.39 g, 0.49 mmol). The crude product was puriﬁed by ﬂash chro-

matography on silica gel using the eluent chloroform:metha-

nol = 30:1, 15:1 then 10:1 to give a brown oil, 0.25 g (95%); ee:

4.3.58. (+)-1-(2-Chloro-10H-phenothiazin-10-yl)-4-[2-(4-

methoxyphenyl)-2H-tetrazol-5-yl]butan-2-ol ((+)-5)

99.8%; ½a ²_D⁵

ꢀ 4:42 (c 0.328, chloroform); HPLC: using the eluent

ꢃ

n-hexane:2-propanol + 0.1% diethylamine = 70:30, retention time:

The (+)-monoester ((+)-17, 2.84 g, 4.90 mmol) was dissolved in

abs dichloromethane (59 mL) at rt and the solution of NaOH

(0.88 g, 22.09 mmol) in water (2.6 mL) was added to the reaction

mixture. The mixture was stirred and heated up to reﬂux. After

completion (4 h), the mixture was allowed to cool down to rt,

water (40 mL) was added to the solution, acidiﬁed with 10% aque-

ous HCl solution and extracted with dichloromethane (3 ꢁ 15 mL).

The combined organic phase was dried over Na₂SO₄, ﬁltered and

concentrated. The residue was puriﬁed by ﬂash chromatography

on silica gel using the eluent n-hexane:ethyl acetate = 2:1 then

1:1. The crude product (foam) was triturated with diethyl ether,

the mixture was cooled, and the white crystals were collected by

ﬁltration (twice), total yield: 1.85 g (74%) (m₁= 1.57 g, ee:

23.8 min.

4.3.63. (+)-1-[2-(Diethylamino)-10H-phenothiazin-10-yl]-4-[2-

(4-methoxyphenyl)-2H-tetrazol-5-yl]butan-2-ol ((+)-3f)

This compound was obtained from (+)-10-(2-{[tert-butyl(diphe-

nyl)silyl]oxy}-4-[2-(4-methoxyphenyl)-2H-tetrazol-5-yl]butyl)-

N,N-diethyl-10H-phenothiazin-2-amine

((+)-9f,

0.29 g,

0.39 mmol). The crude product was puriﬁed by column chroma-

tography on silica gel using the eluent chloroform to give a white

amorphous solid, 0.18 g (88%); ee: 74%; ½a _D²⁵

þ 9:32 (c 0.352, chlo-

ꢃ

roform); HPLC: using the eluent n-hexane:2-propanol + 0.1% dieth-

ylamine = 70:30, retention time: 9.5 min.

89% + m₂= 0.28 g, ee: 94%); mp 90–92 °C; ½a _D²⁵

þ 21:20 (c 0.358,

ꢃ

chloroform (m₂)). HPLC: using the eluent n-hexane:2-propa-

nol + 0.1% diethylamine = 70:30, retention time: 10.1 min; (found:

C, 59.97; H, 4.68; N, 14.45; C₂₄H₂₂ClN₅O₂S requires C, 60.06; H,

4.62; N, 14.59); m/z (EI) [M+H]⁺: 480.20 (C₂₄H₂₂ClN₅O₂S requires

479.12); m_max(KBr)/cm^ꢀ1: 3422, 2929, 1515, 1461 and 1259.

4.3.64. (+)-4-[2-(4-Methoxyphenyl)-2H-tetrazol-5-yl]-1-(2-

morpholin-4-yl-10H-phenothiazin-10-yl)butan-2-ol ((+)-3h)

This compound was obtained from (+)-10-(2-{[tert-butyl(diphe-

nyl)silyl]oxy}-4-[2-(4-methoxyphenyl)-2H-tetrazol-5-yl]butyl)-2-

morpholin-4-yl-10H-phenothiazine ((+)-9h, 0.28 g, 0.36 mmol).

The crude product was puriﬁed by ﬂash chromatography on silica

gel using the eluent chloroform, then chloroform:methanol = 50:1

4.3.59. General procedure for the preparation of (ꢀ)- and (+) 1-

(2-amino-10H-phenothiazin-10-yl)-4-[2-(4-methoxyphenyl)-

2H-tetrazol-5-yl]butan-2-ol derivatives ((ꢀ)-3 and (+)-3) by

desilylation of 10-(2-{[tert-butyl(diphenyl)silyl]oxy}-4-[2-(4-

methoxyphenyl)-2H-tetrazol-5-yl]butyl)-10H-phenothiazin-2-

amine derivatives ((ꢀ)-9 and (+)-9)

to give white amorphous solid, 0.16 g (84%); ee: 73.4%; ½a _D²⁵

þ 9:92

ꢃ

(c 0.277, chloroform); HPLC: using the eluent n-hexane:2-propa-

nol + 0.1% diethylamine = 70:30, retention time: 11.8 min.

4.3.65. (+)-4-[2-(4-Methoxyphenyl)-2H-tetrazol-5-yl]-1-[2-(4-

methylpiperazin-1-yl)-10H-phenothiazin-10-yl]butan-2-ol ((+)-

3i)

This compound was obtained from (+)-10-(2-{[tert-butyl(diphe-

nyl)silyl]oxy}-4-[2-(4-methoxyphenyl)-2H-tetrazol-5-yl]butyl)-2-

The procedure followed the aforementioned preparation of

racemic 1-(2-amino-10H-phenothiazin-10-yl)-4-[2-(4-methoxy-

phenyl)-2H-tetrazol-5-yl]butan-2-ol derivatives (3) by desilylation

of 10-(2-{[tert-butyl(diphenyl)silyl]oxy}-4-[2-(4-methoxyphenyl)-

2H-tetrazol-5-yl]butyl)-10H-phenothiazin-2-amine derivatives (9).

(4-methylpiperazin-1-yl)-10H-phenothiazine

((+)-9i,

0.39 g,

Please cite this article in press as: Takács, D.; et al. Bioorg. Med. Chem. (2013), http://dx.doi.org/10.1016/j.bmc.2013.04.034

D. Takács et al. / Bioorg. Med. Chem. xxx (2013) xxx–xxx

19

0.49 mmol). The crude product was puriﬁed by column chromatog-

raphy on silica gel using the eluent chloroform:methanol = 30:1,

15:1 then 10:1 to give a brown oil, 0.22 g (83%), ee: 72%;

0.55 mmol) and morpholine (0.10 mL, 1.11 mmol). The crude prod-

uct was puriﬁed by column chromatography on silica gel using the

eluent n-hexane:ethyl acetate = 4:1 to give beige amorphous solid,

½

a ²_D⁵

ꢃ

þ 6:21 (c 0.346, chloroform); HPLC: using the eluent n-hex-

0.35 g (82%); mp 72–80 °C; ½a _D²⁵

þ 11:13 (c 0.289, chloroform);

ꢃ

ane:2-propanol + 0.1% diethylamine = 70:30, retention time:

HPLC: using the eluent n-hexane:2-propanol + 0.1% diethyl-

amine = 75:25, retention time: 18.6 min; m_max(KBr)/cm^ꢀ1: 3429,

2957, 2855, 1595 and 1516.

17.3 min.

4.3.66. General procedure for the preparation of (ꢀ)- and (+)-10-

(2-{[tert-butyl(diphenyl)silyl]oxy}-4-[2-(4-methoxyphenyl)-2H-

tetrazol-5-yl]butyl)-2-chloro-10H-phenothiazine ((ꢀ)-8) and

(+)-8)

The procedure followed the aforementioned preparation of

racemic 10-(2-{[tert-butyl(diphenyl)silyl]oxy}-4-[2-(4-methoxy-

phenyl)-2H-tetrazol-5-yl]butyl)-2-chloro-10H-phenothiazine (8).

4.3.72. (ꢀ)-10-(2-{[tert-Butyl(diphenyl)silyl]oxy}-4-[2-(4-

methoxyphenyl)-2H-tetrazol-5-yl]butyl)-2-(4-methylpiperazin-

1-yl)-10H-phenothiazine ((ꢀ)-9i)

This compound was obtained from the reaction of (ꢀ)-10-(2-

{[tert-butyl(diphenyl)silyl]oxy}-4-[2-(4-methoxyphenyl)-2H-tet-

razol-5-yl]butyl)-2-chloro-10H-phenothiazine

((ꢀ)-8,

0.40 g,

0.56 mmol) and N-methyl-piperazine (0.12 mL, 1.11 mmol). The

crude product was puriﬁed by column chromatography on silica

gel using the eluent chloroform:methanol = 20:1, 15:1 then 10:1

to give beige amorphous solid, 0.31 g (71%); mp 66–74 °C;

4.3.67. (ꢀ)-10-(2-{[tert-Butyl(diphenyl)silyl]oxy}-4-[2-(4-

methoxyphenyl)-2H-tetrazol-5-yl]butyl)-2-chloro-10H-

phenothiazine ((ꢀ)-8)

½

a ²_D⁵

ꢃ

þ 11:50 (c 0.264, chloroform). HPLC: using the eluent n-hex-

This compound was obtained from (ꢀ)-1-(2-chloro-10H-pheno-

thiazin-10-yl)-4-[2-(4-methoxyphenyl)-2H-tetrazol-5-yl]butan-2-

ol ((ꢀ)-5, 1.60 g, 3.33 mmol) as white crystals, 2.35 g (98%); ee:

ane:2-propanol + 0.1% diethylamine = 75:25, retention time:

20.5 min; m_max(KBr)/cm^ꢀ1: 2932, 2855, 1584, 1516 and 1461.

99.9+%; ½a ²_D⁵

þ 31:20 (c 0.353, chloroform); HPLC: using the eluent

ꢃ

n-hexane:2-propanol + 0.1% diethylamine = 98:2; retention time:

14.5 min; m_max(KBr)/cm^ꢀ1: 3071, 2932, 1516, 1456 and 1255.

4.3.73. (+)-10-(2-{[tert-Butyl(diphenyl)silyl]oxy}-4-[2-(4-

methoxyphenyl)-2H-tetrazol-5-yl]butyl)-N,N-diethyl-10H-

phenothiazin-2-amine ((+)-9f)

4.3.68. (+)-10-(2-{[tert-Butyl(diphenyl)silyl]oxy}-4-[2-(4-

methoxyphenyl)-2H-tetrazol-5-yl]butyl)-2-chloro-10H-

phenothiazine ((+)-8)

This compound was obtained from the reaction of (+)-10-(2-

{[tert-butyl(diphenyl)silyl]oxy}-4-[2-(4-methoxyphenyl)-2H-tet-

razol-5-yl]butyl)-2-chloro-10H-phenothiazine

((+)-8,

0.50 g,

This compound was obtained from (+)-1-(2-chloro-10H-pheno-

thiazin-10-yl)-4-[2-(4-methoxyphenyl)-2H-tetrazol-5-yl]butan-2-

ol ((+)-5, 1.60 g, 3.33 mmol) as white crystals, 2.35 g (98%); ee:

0.70 mmol) and diethylamine (0.15 mL, 1.40 mmol). The crude

product was puriﬁed by column chromatography on silica gel

using the eluent n-hexane:ethyl acetate = 2:1 to give beige amor-

74%; ½a ²_D⁵

ꢀ 22:80 (c 0.440, chloroform); HPLC: using the eluent

ꢃ

phous solid, 0.34 g (65%); mp 65–70 °C; ee: 78%; ½a _D²⁵

ꢀ 6:42 (c

ꢃ

n-hexane:2-propanol + 0.1% diethylamine = 98:2, retention time:

12.4 min; m_max(KBr)/cm^ꢀ1: 3070, 2958, 1516, 1456 and 1255.

0.265, chloroform); HPLC: using the eluent n-hexane:2-propa-

nol + 0.1% diethylamine = 75:25, retention time: 10.2 min; m_max

(KBr)/cm^ꢀ1: 2964, 2856, 1598, 1516 and 1466.

4.3.69. General procedure for the preparation of 10-(2-{[tert-

butyl(diphenyl)silyl]oxy}-4-[2-(4-methoxyphenyl)-2H-tetrazol-

5-yl]butyl)-10H-phenothiazin-2-amine derivatives ((ꢀ)-9 and

(+)-9)

The procedure followed the aforementioned preparation of

racemic 10-(2-{[tert-butyl(diphenyl)silyl]oxy}-4-[2-(4-methoxy-

phenyl)-2H-tetrazol-5-yl]butyl)-10H-phenothiazin-2-amine (9).

4.3.74. (+)-10-(2-{[tert-Butyl(diphenyl)silyl]oxy}-4-[2-(4-

methoxyphenyl)-2H-tetrazol-5-yl]butyl)-2-morpholin-4-yl-

10H-phenothiazine ((+)-9h)

This compound was obtained from the reaction of (+)-10-(2-

{[tert-butyl(diphenyl)silyl]oxy}-4-[2-(4-methoxyphenyl)-2H-tet-

razol-5-yl]butyl)-2-chloro-10H-phenothiazine

((+)-8,

0.40 g,

0.56 mmol) and morpholine (0.10 mL, 1.11 mmol). The crude prod-

uct was puriﬁed by ﬂash chromatography on silica gel using the

eluent n-hexane:ethyl acetate = 4:1 to give beige amorphous solid,

4.3.70. (ꢀ)-10-(2-{[tert-Butyl(diphenyl)silyl]oxy}-4-[2-(4-

methoxyphenyl)-2H-tetrazol-5-yl]butyl)-N,N-diethyl-10H-

phenothiazin-2-amine ((ꢀ)-9f)

0.34 g (79%); mp 78–80 °C; ½a _D²⁵

ꢀ 11:57 (c 0.271, chloroform);

ꢃ

This compound was obtained from the reaction of (ꢀ)-10-(2-

{[tert-butyl(diphenyl)silyl]oxy}-4-[2-(4-methoxyphenyl)-2H-tet-

HPLC: using the eluent n-hexane:2-propanol + 0.1% diethyl-

amine = 75:25, retention time: 19.5 min; m_max(KBr)/cm^ꢀ1: 2958,

2856, 1596, 1516 and 1462.

razol-5-yl]butyl)-2-chloro-10H-phenothiazine

((ꢀ)-8,

0.50 g,

0.67 mmol) and diethylamine (0.15 mL, 1.40 mmol). The crude

product was puriﬁed by column chromatography on silica gel

using the eluent n-hexane:ethyl acetate = 2:1 to give brown amor-

4.3.75. (+)-10-(2-{[tert-Butyl(diphenyl)silyl]oxy}-4-[2-(4-

methoxyphenyl)-2H-tetrazol-5-yl]butyl)-2-(4-methylpiperazin-

1-yl)-10H-phenothiazine ((+)-9i)

This compound was obtained from the reaction of (+)-10-(2-

{[tert-butyl(diphenyl)silyl]oxy}-4-[2-(4-methoxyphenyl)-2H-tet-

phous solid, 0.24 g (45%); ee: 99.9+%; mp 64–70 °C; ½a _D²⁵

þ 7:39 (c

ꢃ

0.299, chloroform); HPLC: using the eluent n-hexane:2-propa-

nol + 0.1% diethylamine = 75:25, retention time: 9.6 min; m_max

(KBr)/cm^ꢀ1: 2964, 2930, 1598, 1516 and 1466.

razol-5-yl]butyl)-2-chloro-10H-phenothiazine

((+)-8,

0.40 g,

0.56 mmol) and N-methylpiperazine (0.12 mL, 1.11 mmol). The

crude product was puriﬁed by column chromatography on silica

gel using the eluent chloroform:methanol = 20:1, 15:1 then 10:1

to give beige amorphous solid, 0.33 g (75%); mp 65–72 °C;

4.3.71. (ꢀ)-10-(2-{[tert-Butyl(diphenyl)silyl]oxy}-4-[2-(4-

methoxyphenyl)-2H-tetrazol-5-yl]butyl)-2-morpholin-4-yl-

10H-phenothiazine ((ꢀ)-9h)

This compound was obtained from the reaction of (ꢀ)-10-(2-

½

a ²_D⁵

ꢃ

ꢀ 7:75 (c 0.299, chloroform); HPLC: using the eluent n-hex-

{[tert-butyl(diphenyl)silyl]oxy}-4-[2-(4-methoxyphenyl)-2H-tet-

ane:2-propanol + 0.1% diethylamine = 75:25, retention time:

razol-5-yl]butyl)-2-chloro-10H-phenothiazine

((ꢀ)-8,

0.40 g,

23.5 min; m_max(KBr)/cm^ꢀ1: 2932, 2855, 1584, 1516 and 1461.

Please cite this article in press as: Takács, D.; et al. Bioorg. Med. Chem. (2013), http://dx.doi.org/10.1016/j.bmc.2013.04.034

20

D. Takács et al. / Bioorg. Med. Chem. xxx (2013) xxx–xxx

4.3.76. Ethyl 4-(5-{3-hydroxy-4-[2-(triﬂuoromethyl)-10H-

phenothiazin-10-yl]butyl}-2H-tetrazol-2-yl)benzoate (20e)

To a solution of ethyl 4-(5-{(1Z,3E)-4-[2-(triﬂuoromethyl)-10H-

phenothiazin-10-yl]buta-1,3-dien-1-yl}-2H-tetrazol-2-yl)benzo-

ate¹¹(0.56 g, 1 mmol) in abs THF (10 mL) in a dried, round-bot-

tomed ﬂask equipped with a side arm and cooled to 0 °C in an

argon atmosphere was added BH₃ꢁ Me₂S (0.4 mL, 4 mmol) drop-

wise by injection. After addition, the resulting yellow suspension

was allowed to warm up to rt and maintained at this temperature

for prolongued time. After the completion of the reation (1 day,

monitored by TLC), the reaction mixture was evaporated, then

methanol (15 mL) was given to the residue, and stirred for 3 weeks.

The reaction mixture was contentrated in vacuo and puriﬁed by

ﬂash chromatography on silica gel using the eluent dichlorometh-

ane, then n-hexane:ethyl acetate = 2:1. The crude product (foam)

was triturated with diethyl ether and few drops of n-hexane, the

mixture was cooled whereupon crystals deposited. The product

was collected by ﬁltration as white crystals, 0.11 g (19%); mp

107–108 °C; (found: C, 58.50; H, 4.30; N, 12.84; C₂₇H₂₄F₃N₅O₃S re-

quires: C, 58.37; H, 4.35; N, 12.61); m/z (EI) [M+H]⁺: 556.30

(C₂₇H₂₄F₃N₅O₃S requires 555.16); m_max(KBr)/cm^ꢀ1: 3394, 1720,

1425, 1272 and 1125; d_H(300 MHz, CDCl₃) 1.44 (3H, t,

J = 7.2 Hz), 2.08 (1H, m), 2.25 (1H, m), 2.63 (1H, br s), 3.17–3.25

(2H, m), 3.90–4.18 (3H, m), 4.43 (2H, q, J = 7.2 Hz), 6.94–7.28

(7H, m), 8.14–8.24 (4H, m); d_C(75 MHz, CDCl₃) 14.3, 21.8, 28.0,

32.1, 53.9, 61.5, 66.3, 112.6, 116.4, 119.4 (2C), 119.8, 123.8,

125.6, 127.8, 127.9, 128.0, 130.0, 130.4, 131.0 (2C), 131.3; 139.5,

144.2, 145.9, 165.4, 166.9.

References and notes

1. Leslie, E. M.; Deeley, R. G.; Cole, S. P. C. Toxicol. Appl. Pharmacol. 2005 , 204 , 216.

2. Xu, Y.; Zhi, F.; Xu, G.; Tang, X.; Lu, S.; Wu, J.; Hu, Y. Biosci. Rep. 2012 , 32 , 559.

3. Ferry, D. R.; Traunecker, H.; Kerr, D. J. Eur. J. Cancer 1996 , 32A , 1070.

4. Thomas, H.; Coley, H. M. Cancer Control 2003 , 10 , 159.

5. Tiwari, A. K.; Sodani, K.; Dai, C. L.; Ashby, C. R., Jr.; Chen, Z. S. Curr. Pharm.

Biotechnol. 2011 , 12 , 570.

6. (a) Gatti, L.; Cossa, G.; Beretta, G. L.; Zaffaroni, N.; Perego, P. Curr. Med. Chem.

2011 , 18 , 4237; (b) Shukla, S.; Ohnuma, S.; Ambudkar, S. V. Curr. Drug Targets

2011 , 12 , 621.

7. Pluta, K.; Morak-Mlodawska, B.; Jelen, M. Eur. J. Med. Chem. 2011 , 46 , 3179.

8. Charpentier, P. U.S. Patent 2,645,640, 1953; Chem. Abstr. 1955, 49, 16278.

9. Craig, P.; Nodiff, E.; Lafferty, E. J.; Ullyot, G. J. Org. Chem. 1957 , 22 , 709.

10. Bourquin, J.-P.; Schwarb, G.; Gamboni, G.; Fischer, R.; Ruesch, L.; Guldimann, S.;

Theus, V.; Schenker, E. Helv. Chim. Acta 1958 , 41 , 1072.

11. Nagy, I.; Riedl, Z.; Hajós, G.; Messmer, A.; Gyémánt, N.; Molnár, J. ARKIVOC

2004 , 7 , 177.

12. Takács, D.; Nagy, I.; Bombicz, P.; Egyed, O.; Jemnitz, K.; Riedl, Z.; Molnár, J.;

Amaral, L.; Hajós, G. Bioorg. Med. Chem. 2012 , 20 , 4258.

13. (a) Takács, D.; Egyed, O.; Drahos, L.; Riedl, Z.; Hajós, G. Tetrahedron Lett. 2012 ,

53 , 5585; (b) Guram, A. S.; Rennels, R. A.; Buchwald, S. D. L. Angew. Chem., Int.

Ed. 1995 , 34 , 1348.

14. Agnelli, F.; Sucheck, S. J.; Marby, K. A.; Rabuka, D.; Yao, S.-L.; Sears, P. S.; Liang,

F.-S.; Wong, C.-H. Angew. Chem., Int. Ed. 2004 , 43 , 1562.

15. Garbaccio, R. M.; Stachel, S. J.; Baeschlin, D. K.; Danishefsky, S. J. J. Am. Chem.

Soc. 2001 , 123 , 10903.

16. (a) Hu, Q.-Y.; Rege, P. D.; Corey, E. J. J. Am. Chem. Soc. 2004 , 126 , 5984; (b)

Petersen, R. V.; Gisvold, O. J. Am. Pharm. Assoc. 1956 , 45 , 572.

17. Gritsenko, A. S.; Zhuraviev, S. V.; Panova, E. D. Khim. Geterotsikl. Soedin. 1967 , 3 ,

668.

18. Gilman, H.; Ranck, R. O. J. Org. Chem. 1958 , 23 , 1903.

19. Gilman, H.; Nelson, D. J. Am. Chem. Soc. 1953 , 75 , 5422.

20. McKillop, A.; Kemp, D. Tetrahedron 1989 , 45 , 3299.

21. Babudri, F.; Di Nunno, L.; Florio, S. Tetrahedron Lett. 1983 , 24 , 3883.

22. Tosa, M.; Paizs, C.; Majdik, C.; Poppe, L.; Kolonits, P.; Silberg, I. A.; Novák, L.;

Irimie, F.-D. Heterocycl. Commun. 2001 , 7 , 277.

23. (a) Pope, W. J.; Peachy, S. J. J. Chem. Soc. 1899 , 75 , 1066; (b) Whitney, T. A. J. Org.

Chem. 1980 , 45 , 4214; (c) Mofaddel, N.; Bouaziz, R. Bull. Soc. Chim. Fr. 1991 , 128 ,

773; (d) Kiss, V.; Egri, G.; Bálint, J.; Ling, I.; Barkóczi, J.; Fogassy, E. Tetrahedron:

Asymmetry 2006 , 17 , 2220.

Acknowledgments

Financial support from the Hungarian Scientiﬁc Research Fund

OTKA-NK77784 and Szeged Foundation for Cancer Research is

gratefully acknowledged. The results have been achieved in the

frame of the COST action BM0701 ‘ATENS’.

24. Seglen, P. O. Methods Cell Biol. 1976 , 13 , 29.

Please cite this article in press as: Takács, D.; et al. Bioorg. Med. Chem. (2013), http://dx.doi.org/10.1016/j.bmc.2013.04.034

Article Doi

Synthesis and pharmacological investigation of new N-hydroxyalkyl-2- aminophenothiazines exhibiting marked MDR inhibitory effect

DOI: 10.1016/j.bmc.2013.04.034

Source and publish data:

Authors:

Article abstract of DOI:10.1016/j.bmc.2013.04.034

Full text of DOI:10.1016/j.bmc.2013.04.034

Products guided by the article

R&D Labs maybe for 1442371-68-7

Relevant to this article

Hot Product