Azaphenothiazines – promising phenothiazine derivatives. An insight into nomenclature, synthesis, structure elucidation and biological properties

Accepted Manuscript Azaphenothiazines – Promising phenothiazine derivatives. An insight into nomenclature, synthesis, structure elucidation and biological properties Krystian Pluta, Małgorzata Jeleń, Beata Morak-Młodawska, Michał Zimecki, Jolanta Artym, Maja Kocięba, Ewa Zaczyńska PII:

S0223-5234(17)30531-7

DOI:

10.1016/j.ejmech.2017.07.009

Reference:

EJMECH 9572

To appear in:

European Journal of Medicinal Chemistry

Received Date: 8 May 2017 Revised Date:

5 July 2017

Accepted Date: 6 July 2017

Please cite this article as: K. Pluta, Mał. Jeleń, B. Morak-Młodawska, Michał. Zimecki, J. Artym, M. Kocięba, E. Zaczyńska, Azaphenothiazines – Promising phenothiazine derivatives. An insight into nomenclature, synthesis, structure elucidation and biological properties, European Journal of Medicinal Chemistry (2017), doi: 10.1016/j.ejmech.2017.07.009. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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ACCEPTED MANUSCRIPT Azaphenothiazines – promising phenothiazine derivatives. An insight into nomenclature, synthesis, structure elucidation and biological properties# Krystian Pluta1*, Małgorzata Jeleń1, Beata Morak-Młodawska1, Michał Zimecki2,

1 1

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Jolanta Artym2, Maja Kocięba2 and Ewa Zaczyńska2

The Medical University of Silesia, School of Pharmacy with the Division of Laboratory

Medicine, Department of Organic Chemistry, Jagiellońska 4, 41-200 Sosnowiec, Poland, 2

Institute of Immunology and Experimental Therapy, Polish Academy of Sciences,

#

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Department of Experimental Therapy, R. Weigla 12, 53-114 Wrocław, Poland. Part CLII in the series of Azinyl Sulfides.

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*Corresponding author. E-mail: [email protected] (K. Pluta).

Abstract

For the last two decades, classical phenothiazines have attracted attention of researchers, as the hitherto investigations have revealed many significant biological activities

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within this class of compounds, other than originally discovered neuroleptic ones. Important, new pharmaceutical results on phenothiazines, as 10-substituted dibenzothiazines, were recently highlighted in several reviews. Azaphenothiazines are structurally modified phenothiazines by substitution of one or both benzene rings in the phenothiazine ring system

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with the azine rings, such as: pyridine, pyridazine, pyrimidine, pyrazine, 1,2,4-triazine, quinoline, quinoxaline, benzoxazine and benzothiazine. They form over 50 different heterocyclic

azine

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systems, of tri-, tetra-, penta- and hexacyclic structures, and contain from one to even four nitrogen

atoms.

This

review

summarizes

the

methodical

knowledge

on

azaphenothiazines, referring to their nomenclature, synthesis, structure analysis and above all significant varied biological activities, examined in vitro and in vivo. It describes, in addition, current trends in the synthesis of azaphenothiazines. The influence of the azaphenothiazine ring system, the nature of the substituents, predominantly at the thiazine nitrogen atom, as well as at the azine nitrogen atom and carbon atom, on the biological activities, were also discussed. Keywords: phenothiazines, azaphenothiazines, structural analysis, structure – activity relationship, multiple biological activities. 1

ACCEPTED MANUSCRIPT 1. Introduction Phenothiazines A, linearly fused tricyclic compounds, containing the nitrogen and sulfur atoms, have been known since 19th century. The parent compound, 10H-dibenzo-1,4thiazine, was obtained the first time by Bernthsen in 1883 by thionation of diphenylamine with elemental sulfur [1]. This class of heterocyclic organic compounds became very

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important because of their interesting chemical properties and valuable biological activities. Syntheses, transformations, structures, physicochemical properties, crystallography studies and biological activity of phenothiazines were reviewed exhaustively in a monograph edited by Gupta three decades ago [2]. More recently, a retrosynthetic approach to the synthesis of

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phenothiazines was described [3].

During recent years, hundreds articles regarding these compounds were annually published

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and until now about 6000 phenothiazine derivatives have been obtained. Classical phenothiazines, substituted in position 10 with the dialkylaminoalkyl groups and additionally in position 2 with small groups, exhibit significant activities such as: neuroleptic, antiemetic, antihistaminic, antipuritic, analgesic and antihelmintic [2]. At least 100 phenothiazines were used in therapy, mainly as neuroleptics. Recent reports deal with anticancer, antiplasmid, antiviral, anti-inflammatory, and antibacterial activities, reversal of multi-drug resistance and

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potential treatment in Alzheimer’s and Creutzfeldt-Jakob diseases while they also exhibit antioxidant and antihyperlipidemic activity. Their significant activities were recently reviewed in articles and chapters of several monographs [4-15]. The phenothiazine structure can be modified to obtain new compounds by employing

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the following procedures (quite frequently used together): 1. introduction of a substituent at the thiazine nitrogen atom (position 10),

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2. introduction of a substituent at the benzene ring (positions 1-4 and 6-9), 3. oxidation of the sulfide function into sulfoxide (S-oxide) and sulfone (S-dioxide), 4. substitution of one or two benzene rings with homoaromatic and heteroaromatic rings (most often azine rings).

In the latter case, the modification with the heteroaromatic azine ring (pyridine,

pyridazine, pyrimidine, pyrazine, 1,2,4-triazine, quinoline, quinoxaline) resulted in azaphenothiazines of the azinobenzo-1,4-thiazine and diazino-1,4-thiazine structures B and C (Fig. 1). Azaphenothiazines can be tricyclic, tetracyclic, pentacyclic and hexacyclic.

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ACCEPTED MANUSCRIPT R

R

R

N

N

N

S

S

S

A

B

C

N

N

N

= azine

N

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Fig. 1

Fig. 1

In comparison with parent phenothiazines, they may contain an additional one (monoazaphenothiazines), two (diazaphenothiazines), tri (triazaphenothiazines) and four

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(tetraazaphenothiazines) nitrogen atoms. Tetracyclic and pentacyclic azaphenothiazines can form linearly and angularly fused ring system. The azaphenothiazine chemistry and medicinal

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chemistry is significant, varied, plenteous and contain syntheses, structure identifications and physicochemical and biological properties published in over 300 articles and patents. In contrast to classical phenothiazines, which contain tricyclic dibenzothiazine ring system

(sometime

modified

by

the

naphthalene

ring

to

form

benzo-

and

dibenzophenothiazines), the structures of azaphenothiazines are much more differentiated and

azine nitrogen atoms:

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consist of over 50 types, depending on a nature of the fused ring system and a location of the

a. 4 monoazaphenothiazines, b. 12 diazaphenothiazines,

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c. 6 triazaphenothiazines,

d. 10 tetraazaphenothiazines (Fig. 2) e. over 20 the benzo, dibenzo, naphtho, pyridobenzo, benzoxazino and benzothiazino

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derivatives of monoaza- and diazaphenothiazines.

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Fig. 2

The aim of this review is to clarify the nomenclature problems, describe the synthetic aspects (particularly in comparison with phenothiazines), discuss the issues of the structure

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identification, and first of all to present selected azaphenothiazines exhibiting biological activities.

2. Nomenclature

Contrary to classical phenothiazines (being dibenzothiazines), azaphenothiazines

belong to different heterocyclic systems and were designated in the chemical literature in three different ways, using more popular British and American system A (as xazaphenothiazine), less known German system B (also as x-azaphenothiazine but different atom numbering), where x is the location of the azine nitrogen atom in the fused ring system, and Chemical Abstract system C (as azino[x,y-b][1,4]benzothiazine and diazino[x,y-b;z,q-

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ACCEPTED MANUSCRIPT e][1,4]benzothiazine), where x, y, z and q are the numbers of the azine carbon atoms fused with the thiazine ring. The existence of these nomenclature systems generated misunderstandings in names and numbering of atoms in rings. Here is an example taken from the authors’ experience: dichloro compound is known in the German literature as “4,5dichlor-2,7-diazaphenothiazin” (system B) (Kop, Strell, “Über 2,7-diazaphenothiazin” [16])

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and is sometimes incorrectly considered in the English language literature as 2,7diazaphenothiazine (suggesting a classification to system A) after translation into English. In fact,

this

compound

is

1,9-dichloro-3,7-diazaphenothiazine (system

4,6-

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dichlorodipyrido[3,4-b;4’,3’-e][1,4]thiazine (system C) (Fig. 3).

A) and

Fig. 3

Looking for some azaphenothiazines in the literature, one must be aware that those systems still exist. For example oxypendyl, one of the most known 1-azaphenothiazines, is named as 10-[3-(hydroxyethyl-4-piperazinyl)propyl]-4-azaphenothiazine in The Merck Index

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[17]. Its 3-chloro analog, known as cloxypendyl, is described in the English language literature as the 2-chloro-4-azaphenothiazine type, just after its German name “2-chlor-4azaphenothiazin” [18]. In order to systematize here this differentiated and abounding subject

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regarding azaphenothiazines, as well as to compare isomeric and analogical compounds, the authors used mainly system A, and only for more complicated compounds, system C.

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3. Synthetic aspects

In comparison with the synthesis of N-substituted phenothiazine derivatives,

obtainable from commercially available 10H-phenothiazine and its analogs, the parent NHunsubstituted azaphenothiazines are not commercial products, excluding the simplest 10H-1azaphenothiazine. So, the first step in the synthesis of the azaphenothiazine derivatives is obtainment of N-unsubstituted azaphenothiazine (N = the thiazine nitrogen atom). Similarly

to

phenothiazines,

N-unsubstituted

azaphenothiazines

of

the

azinobenzothiazine structure B, were synthesized by: a. cyclization of o-aminophenyl azinyl sulfides, containing a good leaving group X and o- azidophenyl azinyl sulfides (routes Ia-b), 5

ACCEPTED MANUSCRIPT b. thionation of phenyl azinyl amines with elemental sulfur or thionyl chloride (route IIa), c. cyclization of o-mercaptophenyl azinyl amines, containing good leaving group (route IIb),

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d. reaction of o-aminobenzenethiols with o-disubstituted azines (route III) (Scheme 1).

Scheme 1

N-unsubstituted azaphenothiazines of the diazinothiazine structure C were synthesized by similar routes:

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a. cyclization of o-aminodiazinyl sulfides, containing a good leaving group X (route IV), b. thionation of diazinyl amines with elemental sulfur (route Va), c. cyclization of o-mercaptodiazinyl amines, containing a good leaving group X (route

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Vb),

d. reactions of o-aminoazinethiols with o-disubstituted azines (route VI) and osubstituted azinethiols with o-substituted azinyl amines, containing good leaving

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groups X (Scheme 2).

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Scheme 2

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Over 90% of synthesis of N-unsubstituted azaphenothiazines were obtained by the routes described above. The cyclizations of o-aminophenyl azinyl sulfides and oaminodiazinyl sulfides (routes Ia and IV) need to be worth noticing, as these reactions can proceed via the Ullmann cyclization to form azaphenothiazines B1 and C1 or can proceed via the Smiles rearrangement to o-mercaptophenyl azinyl amines and o-mercaptodiazinyl amines,

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and further to form azaphenothiazines B2 and C2 (routes IIb and Vb, Scheme 3).

Scheme 3

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The Smiles rearrangement proceeds most often in basic conditions, but is also observed in neutral or acidic conditions. Comparing to the Ullmann products B1 and C1, this rearrangement leads to azaphenothiazines B2 and C2 which possess the nitrogen atoms in other location, that guides to another class of azaphenothiazines (for example 4-aza- instead of 1-aza-, 3-aza instead of 2-aza- and 3,4-diaza- instead of 1,2-diaza- and so on). Also, the substituent Z can change the location in a similar way. It is worth noting that the reactions of disubstituted compounds (routes III, VIa and VIb) can proceed through the sulfide formation and further via the Smiles rearrangement, but the intermediates are most often not isolated. Unfortunately, not all products, obtained by application of the discussed routes, were identified with sufficient insight.

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ACCEPTED MANUSCRIPT Syntheses of azaphenothiazines, based on the discussed routes, were reviewed incompletely by Okafor in the 1970’s [19,20], partially (only monoazaphenothiazines) by Barański et al. (in Polish) in 1990 [21] and comprehensively (multiazaphenothiazines) by Pluta et al. in 2008 [22]. Contrary to a spontaneous, the Smiles rearrangement, the Ullmann cyclization needs

(Scheme 4) [23].

Scheme 4

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sometimes a copper catalyst, like in the synthesis of 4-azaphenothiazine, as recently published

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A variety of the azaphenothiazine types has driven a new approach into synthetic methods with a use of new substrates such as: azinyl bis-sulfides, disulfides, dithiins, and benzothiazines. Azinyl bis-sulfides (possessing two o-aminophenylthio groups) underwent cyclization under basic conditions to azaphenothiazines with one remaining sulfide function

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[24,25].

Scheme 5

The annulation reactions of dichlorodiazinyl sulfides represent a development of the

cyclization route of diazinyl sulfides, namely o,o’-dichloro-3,3’-diquinolinyl sulfides with ammonia, acetamide, alkyl and aryl amines leading to pentacyclic diquinothiazines (Scheme 6) [26-30].

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Scheme 6

Unparalleled, as in phenothiazines, azaphenothiazines were obtained also from benzo1,4-thiazines through a building an azine ring, such as pyridine, pyridazine, and pyrimidine

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(Scheme 7) [25,31-34].

Scheme 7

An employment of diquino-1,4-dithiins, as the thioazine substrates (Scheme 8), is

unique in the azaphenothiazine synthesis. Angularly condensed diquino-1,4-dithiin (thioquinanthrene) in the fusion reactions (without a solvent) with hydrochloride of substituted anilines, and its dihydrochloride in the reaction with aniline, led to angularly condensed tetracyclic quinobenzothiazines. A usage of dimethylthioquinanthrenium dichloride,

in

the

reaction

with

substituted

anilines

in

pyridine,

led

to

methylquinobenzothiazinium chlorides, a new unique class of azaphenothiazines of the

9

ACCEPTED MANUSCRIPT ammonium structure. As a result, methylquinobenzothiazinium chlorides were transformed into quinobenzothiazines. These syntheses proceeded through the nucleophilic opening of the

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1,4-dithiin ring with anilines and further 1,4-thiazine ring closure [28,35-41].

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Scheme 8

Next, linearly condensed diquino-1,4-dithiin of the 5,12-diaza-6,13-dithiapentacene structure in a similar fusion reaction led to linear tetracyclic quinobenzothiazines, angular pentacyclic quinonaphthothiazines and diquinothiazine [42,43]. Its isomer of the 5,7-diaza-

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6,13-dithiapentacene structure, in the reaction with acetamide and hydrochlorides of aniline

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and diaminoalkanes, gave linear pentacyclic diquinothiazines (Scheme 9) [44].

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Scheme 9

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Diazinyl disulfides are indispensable thioazine substrates in the reactions with amines. o,o’-Dinitro-4,4’-dipyridinyl disulfide in DMF, in the presence of NaOH, underwent cyclization via the Smiles rearrangement to 2,7-diazaphenothiazine [45]. In contrast, 2,2’dipyridinyl disulfides reacted with p-substituted anilines in nitromethane in the presence of

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catalyst I2/FeF3, without the Smiles rearrangement, to form 4-azaphenothiazines (Scheme 10)

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[46].

o,o’-Dichloro-3,3’-diquinolinyl

Scheme 10 disulfides

reacted

with

substituted

anilines,

naphthylamines and 6-aminoquinoline in MEGD (monomethyl ether of diethylene glycol) to form

tetracyclic

quinobenzothiazines

and

pentacyclic

diquinothiazine

and

quinonaphthothiazines (Scheme 11) [36,42,47].

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Scheme 11

A very unique route to synthesis of azaphenothiazines is a contraction of the 7-

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membered thiazepine ring in azinobenzothiazepines. Depending on the conditions, pyrimidobenzothiazepinediones led to synthesis of 10H-pyrimidobenzothiazinedione or to 4a-

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substituted pyrimidobenzothiazinediones (Scheme 12) [48].

Scheme 12

4. Structural aspects

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Not all planned syntheses led to azaphenothiazines, sometimes these reactions stopped at the stage of the rearrangement product (P1) – amine without further cyclization, due to

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strong hydrogen bonding [49] or at formation of bis-sulfides (P2), as the results of disubstitution [50,51]. As mentioned above, the thiazine ring formation can be achieved by the Ullmann cyclization or through the Smiles rearrangement, followed by cyclization to sole or both products, but sometimes the obtained azaphenothiazines were the products of other processes, such as unique double Smiles rearrangement (P3) [52-55], formation of the NazineH tautomers (P4) instead of the most common Nthiazine-H ones [56] and formation of the 4asubstituted structures (P5) [48,57-59].

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ACCEPTED MANUSCRIPT O H N

N

H2N

O Cl

N

N

N

P1

Z

S

N

=

N

H2N

N

CH3 N

N SK

Cl

S

N

N

,

NH2

P2

N N

Z = H, Cl N

N

H N

N

Z Cl

S

S

Z

N

P4

P3

Z = H, Cl, Br

S

,

N

Fig. 4

O NH

X

O P5 X = NO2, Cl, CH2OR, CH2Cl

= N

N

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N

H N

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NO2 N

Summarizing all the above considerations, the structure analysis of the resultant

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azaphenothiazines is not easy (especially discrimination of the isomeric and tautomeric structures) and needs to be performed without any doubts. The proper determination (the position of the nitrogen atoms and possible substituents) is crucial to perform correct structure – activity relationship.

The correct structures of obtained azaphenothiazines were initially determined on the

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basis of chemical ways: independent syntheses [60-62], lack of formation of the 1,2,3-triazole ring during the diazotization of the amino derivatives [63-65] and analysis of the directive influence of the functional groups in subsequent nitration [66-68]. Later, spectroscopic methods were used, first of all NMR spectra, analyzing the

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chemical shift of the H-1 proton in tricyclic azaphenothiazines [69], a broad NH proton signal due to the NH-O hydrogen bonding [51], long range coupling of the NH and H-4 protons [69] and carbon-protons (13C-H) [70]. Successively advanced NMR techniques were used in the

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structure solutions: NOE and COSY spectra in the beginning [29,40,42,71] and further twodimensional NOESY, ROESY, HSQC and HMBC spectra to assign unequivocally all proton and carbon signals [36,39,47,72-74]. Other spectroscopic methods were used on a limited scale: UV spectra to discriminate the azaphenothiazine types [75] and IR spectra to observe strong hydrogen bonding between the NH and NO2 groups [76]. With the development of X-ray analysis, the obtained azaphenothiazines were identified directly without any doubts. Only 15 types of (out of over 50) azaphenothiazines were analyzed with X-rays: 1-azaphenothiazine [77-83], 1,3-diazaphenothiazine [34,84], 1,4diazaphenothiazine [70], 2,3-diazaphenothiazine [85-87], 1,6-diazaphenothiazine [73], 1,8diazaphenothiazine [72], 2,7-diazaphenothiazine [53,88], 3,6-diazaphenothiazine [74], linear 13

ACCEPTED MANUSCRIPT quinobenzothiazine [30,89], angular quinobenzothiazine [36], angular quinobenzothiazinium salt [37], pyridoquinothiazinium salt [90], quinonaphthothiazine [47], linear diquinothiazine [30,91,92] and angular diquinothiazine [93,94]. These analyses not only confirmed the structures, assumed from other methods, but also presented the spatial arrangement of the molecules (planar or folded ring system) and the location of the substituents, mainly the N-

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substituents (equatorial or axial and the orientation towards the ring system). Having in mind that many structures of the synthesized azaphenothiazines were assigned arbitrarily, one can assume that X-ray analysis would be more relevant in the azaphenothiazine structure

5. Biological activity of azaphenothiazines

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elucidation.

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Following a search of the azaphenothiazine literature, the authors of this review estimate that at least 2000 different azaphenothiazines have been synthesized up to present, about 1,000 of these compounds were studied biologically and about 600 compounds were described, as having interesting biological activity. This review describes only compounds displaying promising biological effects, and is arranged in a way to show which of the structure elements condition various biological actions:

nature of the azaphenothiazine system (the kind of azine in the ring system, the

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a.

location of the azine nitrogen atom, the way of the ring fusion), b.

nature of the substituents at the thiazine and azine nitrogen atoms (as trivalent

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nitrogen atoms and tetravalent nitrogen atoms forming an ammonium salt), c.

nature of the substituents at the ring carbon atom, as well as the oxo group formation,

d.

nature of the thiazine ring – the sulfur atom oxidation in relation to the S-oxide and S-

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dioxide function.

The review describes also the progress in the studies on the biological activity of

azaphenothiazines related to the last sixty years. Pyridobenzothiazines 1-Azaphenothiazine Of the four isomeric pyridobenzothiazines (monoazaphenothiazines), 10-aminoalkyl derivatives of the 1-aza series have been studied most extensively for neuroleptic activity [95]. Generally, they are the most known azaphenothiazines still used in the various therapies 14

ACCEPTED MANUSCRIPT as prothipendyl 1a, isothipendyl 1b, oxypendyl 1c, cloxypendyl 1d and pipazethate 1e (Fig. 5)

Fig. 5

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[17,95,96].

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Prothipendyl, 10-(3-dimethylaminopropyl)-1-azaphenothiazine, was obtained in 1960 by Yale and Bernstein [97]. This compound is known as an antipsychotic drug under brand

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name Dominal and Tolnate [17, 96], and is still used in schizophrenia [98], treatment-resistant depression [99], acute-mania [100], unspecific sedation [101] and dementia [102]. Recently, prothipendyl was found to exhibit antiviral activity against chikungunya virus (CHIKV), a mosquito-transmitting alphavirus causing CHIK fever [103,104]. It was suspected to be administered illegally at low doses to race-horses to improve their performance [105]. Isothipendyl, 10-(2-dimethylamino-2-methylethyl)-1-azaphenothiazine, was obtained

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by Yale and Sowiński as long ago as in 1958 [106]. Due to a shorter alkyl chain between the amine and thiazine nitrogen atoms, in comparison with prothipendyl, this compound exhibits mainly antihistaminic and some anticholinergic and sedative activities. The compound has wide clinical applications for local and generalized allergic reactions, insect bites and

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radiation sickness under such brand names as: Adantol, Adanton, Selignon and Apaisyl [17, 96,107-109]. As some other drugs it binds to bovine serum albumin [107]. Similarly to

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classical phenothiazines, isothipendyl exhibited some phototoxic properties with ultraviolet A (UVA), on the other hand it reduced the erythema response to UVB radiation [108,109]. Oxypendyl,

10-[3-(hydroxyethyl-4-piperazinyl)propyl]-1-azaphenothiazine,

was

obtained by Schuler and Klebe in 1962 [110]. It exhibits antiemetic activity and is known as Pervetral [17,96].

Cloxypendyl, 3-chloro-10-[3-(hydroxyethyl-4-piperazinyl)propyl]-1-azaphenothiazine, was obtained by Gross et al. in 1968 [18]. The compound displayed potent sedative and neuroleptic properties, very good tolerance and favorable therapeutic range. Its analogs with the dimethylaminoethyl, dimethylaminopropyl, hydroxypiperazinylpropyl groups and its homopiperazine, and acetyl derivatives, as well as the dechloro derivatives, were less active.

15

ACCEPTED MANUSCRIPT Pipazethate, 2-(2-piperidinylethoxy)ethyl 1-azaphenothiazine-10-carboxylate, was obtained by Schuler, Klebe and Schlichtegroll in 1964 [111]. It is a first phenothiazine, introduced as an antitussive drug, suppressing irritative and spasmodic cough by inhibiting the excitability of the cough centre and the peripheral neural receptors in the respiratory passage, but not depressing respiration. It is known as: Lenopect, Theratuss, Selvigon Selgon and Toraxan [17,96,112]. 2

(Fig.

6)

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10-(Diisopropylaminoethylthio)carbonyl-1-azaphenothiazines

were

evaluated for a neuropharmacological activity in a mouse model. Compounds 2a-e showed significant activity with the minimum effective dose (MED50) of 0.058-1.8 mg/kg, at which reactive signs were observed in half of the mice tested. The most active derivative 2a

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exhibited also hypotensive activity in the anesthetized cat and caused severe depression in

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mice, dogs and monkeys [113].

Fig. 6

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7- and 9-halogeno derivatives (fluorine, chlorine and bromine) of 10H-3-nitro-1azaphenothiazines 3a-f (without any groups at the thiazine nitrogen atom) (Fig. 7) were screened for their antibacterial activity against E. coli, B. subtilis and S. aureus by exhibiting moderate to significant potency, as reflected by respective zones of inhibition: 7-12, 8-12 and

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8-15 mm. The most active compounds were derivatives 3c (15 mm, against S. aureus), 3b (12

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mm, against E coli) and 3a and 3e (12 mm, against B. subtilis) [114].

Fig. 7 10H-3-nitro-1-azaphenothiazines 4a-e (Fig. 8) were tested for the antimicrobial activity (against S. aureus, E. coli, A. flavus, A. niger, F. moniliformae and C. lunata). The compounds with the phenoxy (4c) and methoxy (4b and 4d) groups exhibited better activity

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ACCEPTED MANUSCRIPT than other compounds, and for selected strains, comparable to that of mycostatin, the

Fig. 8

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reference drug [115].

Substituted 10H-3-nitro-1-a zaphenothiazines and their 10-acetyl, 10-ribofuranosyl 5a-

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i and S-oxide derivatives 6a-c (Fig. 9) were tested for antimicrobial activity using a paper disc method. All compounds were more bactericidal against E. coli, S. aureus, A. niger, A. flavus,

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C. lunata and F. oxyporium than the reference drugs streptomycin and mycostatin at 100 µg/disk. The most active compounds were derivatives 6a, 6d and 6g against A. flavus (the activity ratio of 1.69, 1.80 and 1.62, in comparison with the reference drug) and 6g against A.

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niger (1.78) [116].

Fig. 9

Similarly substituted 10H-3-nitro-1-azaphenothiazines and their 10-acetyl, 10ribofuranosyl 7 and S-oxide derivatives 8 (Fig. 10) were screened for antimicrobial activity. The ribofuranosyl derivatives had better antibacterial (against S. aureus, E. coli) and antifungal (A. flavus, A. niger and F. oxysporium) activities than their parent compounds (no data given) [117].

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Fig. 10

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10-(1-Methylpiperidinyl-3-ethyl)-1-azaphenothiazines 9 (Fig. 11) exhibited an antitubercular activity against M. tuberculosis H37Rv with MIC > 10 mg/mL in the microplate alamar blue assay. This compound showed high binding to the D2 dopamine (86%

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of ligand displacement) and 5-HT2A serotonin (85%) receptors at 10 µM [118].

Fig. 11

Ten 10-carbamoylmethyl-1-azaphenothiazines with the dialkylamino (methyl, ethyl) and heterocyclic (pyrrolyl, imidazolyl, pyrrolidinyl, 2-pyridinylamino, piperazinyl and 4methylpiperazinyl, morpholinyl and indolyl) fragments were screened for sedative and

TE D

antibacterial properties. Compounds 10a-d (Fig. 12) showed significant sedative activity, similar to diazepam in the motor coordination test, and the locomotive activity. In turn, compounds 10a-b exhibited a hypnosis activity, similar to phenobarbitone. All compounds

AC C

[119].

EP

showed mild antibacterial action against V. cholera, B. spharicus, E. faecalis and S. typhi

Fig. 12

1-Azaphenothiazines 11 and their S-oxides 12 (Fig. 13) demonstrated a mixed radical scavenging activity in both DPPH (1,1-diphenyl-2-picryl hydrazyl) and ABTS+ (2,2azinobis(3-ethylbenzothiazoline-6-sulfonic acid)) assays. Some compounds showed an increase in sulfhydryl group (GSH) assay and a significant decrease in lipid peroxidation (LPO), revealing potent antioxidant activities in Swiss albino mice (data not accessible) [120].

18

ACCEPTED MANUSCRIPT R

R N

N

N

N

Z

Z

Z = H, 8-CH3 , 8-F, 8-Br, 9-F, 8-CH3 , 9-OC2H5

S

S

R = H, COCH3,

O 11

12

Fig. 13 1-Azaphenothiazine 13 (Fig. 14), with a large multicyclic substituent (containing two

RI PT

piperidine and one pyridine rings linked by the methylene and carbonyl groups), exhibited potent affinities for the human and murine H3 receptors (Ki = 7 and 26 nM) and good AUC and exceptionally high brain/plasma ratio, which translated into a very high receptor

M AN U

promising agent for the treatment of obesity [121].

SC

occupancy in ex vivo occupancy assay in mouse. This compound can be regarded as a

TE D

Fig. 14

Twenty one 10-substituted 1-azaphenothiazines 14, containing a hexyl linker ending with acyclic and cyclic amino groups, bromine atom or nitro group and additional halogen

EP

atom in position 7 (Fig. 15), were evaluated for their activity against 6 cancer cell lines. Most of them (14a-k) exhibited strong inhibitory effect on the growth of the tested lines with IC50 <

AC C

10 µg/mL against selected lines, and were a few times (and even up to 12) more active than a standard drug actinomycin D. The most active was compound 14c (with the piperidinylhexyl group) showing IC50 = 2.27-3.8 µg/mL against lung cancer H460, malignant brain cancer T98G and thyroid cancer SNU80, respectively. Similarly active were compounds 14h (with the pyrrolidinylpiperidinylhexyl group) against H460 and SNU80 cell lines (IC50 = 2.1 and 2.3 µg/mL), 14d (thiomorpholinylhexyl group) and 14b (pyrrolidinylhexyl) against H460 line IC50 = 2.5 and 2.7 µg/mL). Derivative 14a, containing the bromohexyl group and bromine atom, was active against these three cell lines showing IC50 = 3.8-6.2 µg/mL. Compounds 14b, 14c and 14f (methylpiperazinylhexyl) were identified as the leading molecules for future studies, due to their high activity, low cytotoxicity with regard to non-malignant brain T98G

19

ACCEPTED MANUSCRIPT and lung fibroblast MRC5 cell lines, and in silico pharmacokinetic and acute toxicity studies

RI PT

[122].

Fig. 15

SC

Bis(1-azaphenothiazines) 15a-k, containing the 1,ω-alkylene and trans-1,4-but-2enylene linkers between two azaphenothiazine units and the substituents in position 7 (H, F,

M AN U

Cl, Br, Fig. 16), showed moderate to significant antimicrobial activities against selected bacterial and fungal strains. The 1,6-hexylene linker turned out to be more active than 1,4butylene. The most active compounds were derivatives 15g (-(CH2)6-, H) against S. aureus and S. epidermis, 15j (-(CH2)6-, Br) against S. epidermis and E. coli, and 15a (CH2CH=CHCH2-, H) against A. fumigatus with MIC = 6.25 µg/mL, similarly active to

TE D

ciprofloxacin and miconazole [123].

X

Z

S

N

N N

EP

N

Z

X

Z

(CH2)6

H

a

CH2CH=CHCH 2

H

g

b

CH2CH=CHCH 2

Br

h

(CH2)6

F

H

i

(CH2)6

Cl

j

(CH2)6

Br

c

X

Z

(CH2)4

d

(CH2)4

F

e

(CH2)4

Cl

f

(CH2)4

Br

S

15

AC C

Fig. 16

2-Azaphenothiazines

10-Aminoalkyl-2-azaphenothiazines (for example the most active dimethylamino-2-

methylpropyl derivative 16, Fig. 17) appeared to be generally less potent antipsychotics than their 1-aza analogs. However, their N-oxides 17 had useful CNS depressant properties [95].

20

ACCEPTED MANUSCRIPT Fig. 17 3-Azaphenothiazines 10-Dimethylaminopropyl-3-azaphenothiazine 18 had slight sedative and hypnotic

SC

Fig. 18

RI PT

properties (Fig. 18) [95].

Later, substituted 1-cyano-3-azaphenothiazin-3H(2)-ones 19, and their S-oxides 20 and S-dioxides 21 (Fig. 19) were tested for binding affinity to the benzodiazepine receptors

M AN U

and anticonvulsant activities. Most of the compounds (19a-b, 19d-f, 19h and 21b) exhibited significant binding properties with IC50 = 0.31-0.91 µM. The methylation of the thiazine nitrogen atom (19h) increased the binding. The oxidation of the sulfide function to S-oxide and S-dioxide and the introduction of the methyl group in position 4 (19g) generally diminished the affinity. The presence of the chlorine atom in position 7 or 8 did not significantly alter the binding. In the anticonvulsant assay, (the ability to inhibit

TE D

pentylenetetrazole (PTZ)-induced convulsions in mice) the most active compound was derivative 19b. Compounds 19a, 19c-d, 20 and 21a-b were less active. This time the 10-

AC C

EP

methyl derivative was inactive [31].

Fig. 19 Five 10-aminoalkyl 3-azaphenothiazines as the oxalate salts 22a-e (Fig. 20) were screened for their potential analgesic activity. These compounds exhibited good activity (5986% inhibition) in the phenylbenzoquinone writhing test in rats at a dose of 20 mg/kg, superior to that of aspirin and dextropropoxyphene at doses of 50 and 56 mg/kg. The most 21

ACCEPTED MANUSCRIPT active compound was derivative 22c with the pyrrolidinylpropyl group. The compounds with the propyl chain were more active than those with ethyl chain. The compounds with the reduced pyridine ring (with the additional 3-methyl group) were generally less active, with exception of 3-methyl-10-morpholinylpropyl-1,2,3,4-tetrahydro-3-azaphenothiazine, giving

Fig. 20

SC

RI PT

93% inhibition [124].

M AN U

3-Azaphenothiazines, as the 3-substituted ammonium salt structure, 10H-3aminoalkyl-3-azaphenothiazinium chlorides 23a-d, containing open and cyclic amine moieties (Fig. 21), were tested for a potential hypotensive effect. The most active compound was the dimethylaminopropyl derivative 23c, causing a marked fall in blood pressure of

EP

TE D

phenobarbital-anesthetized dogs [125].

Fig. 21

AC C

4-Azaphenothiazines

Various heterocycle-fused benzothiazines were screened for their potential activities as

inhibitors of allergy. 10-Benzylaminobutyl-4-azaphenothiazine S-oxide 24 (Fig. 22) with additional groups, caused 60% inhibition of the histamine release, in a rat allergy model, at oral dose of 10 mg/kg [126].

Fig. 22

22

ACCEPTED MANUSCRIPT 10-(3-Fluoro-4-methoxybenzyl)-4-azaphenothiazine 25 (Fig. 23) stronger inhibited tubulin polymerization (IC50 = 1-5 µg/mL) than the analogous benzylated phenothiazines and phenoxazines, but weaker than the reference drugs: combretastatin A-4, phenstatin and desoxypodophyllotoxin. This azaphenothiazine is considered as a novel lead compound to

RI PT

obtain new anticancer agents targeting tubulin polymerization with improved properties [127].

SC

Fig. 23

1,2-Diazaphenothiazines In

the

late

fifties

of

M AN U

Pyridazinobenzothiazines

20th

century

10-dialkylaminoalkyl-3-methoxy

1,2-

diazaphenothiazines 26 (Fig. 24) were reported to exhibit good antihistaminic activity but no

TE D

data were given [128].

Fig. 24

EP

Later 10H-4-(2-aminophenylthio)-1,2-diazaphenothiazine 27 was patented as an anti-

AC C

inflammatory agent [129].

Fig. 25

Recently, 10H-1,2-diazaphenothiazines 28a-d, possessing heterocyclic substituent such as pyrrolidinyl, piperidinyl, piperazinyl and morpholinyl in position 3 (Fig. 26), showed a lower inhibitory activity (IC50 > 287 µM) against soybean 15-lipoxygenase enzyme in comparison with pyrimidobenzothiazine [130].

23

ACCEPTED MANUSCRIPT

RI PT

Fig. 26 2,3-Diazaphenothiazines

In 1962 CIBA Company patented synthesis and properties of 10-dialkylaminoalkyl2,3-diazaphenothiazinones 29a-g (with the pyridazinone moiety in the tricyclic ring system,

SC

Fig. 27). The compounds exhibited inhibitory activity in transmission of stimuli in the central

M AN U

nervous system, as well as histaminolytic and antiparasitic effects (no data given) [131].

TE D

Fig. 27

Two groups of 10H-2,3-diazaphenothiazin-1-ones 30 and 10H-2,3-diazaphenothiazin4-ones 31, possessing mainly acyclic and cyclic dialkylaminoalkyl groups at the pyridazine

EP

nitrogen atom (Fig. 28), were described in a series of Japan patents as sedative,

AC C

antihistaminic, analgesic and anti-inflammatory agents [132-135].

Fig. 28

10-Substituted 2,3-diazaphenothiazin-1-ones were tested for their antiallergic and antiarrhythmic properties. The most active compounds were 10-substituted derivatives 32a-c (Fig. 29) exhibiting antiallergic action in mice at a dose of 25, 50 and 100 mg/kg, respectively. Derivatives 32a-b displayed antiarrhythmic actions against CHCl3-induced arrhythmia in mice at 100 and 25 mg/kg. Other compounds without the oxo group were inactive [69].

24

ACCEPTED MANUSCRIPT

Fig. 29 10H-2,3-diazaphenothiazines, possessing fused triazole moiety (linking position 1 and

RI PT

2, Fig. 30) with additional dialkylaminoalkyl and dialkylaminoalkylthio groups, were tested in biological models. The most active compounds found were derivatives 33a-d exhibiting fair analgesic activity (54-65% inhibition) in phenylquinone-induced writhing in mice at a dose of 10 mg/kg i. p. However, these compounds showed only limited anti-inflammatory activity

M AN U

SC

(25-32% inhibition) in the carrageen-induced edema test at a dose of 50 mg/kg [136].

Fig. 30

10H- and 10-substituted 2,3-diazaphenothiazin-1-one S-dioxides were tested for their

TE D

antiallergic and anti-inflammatory activities. Generally, the dialkylaminoalkyl and their Noxide derivatives were more active than the methyl compounds. In particular, derivatives 34ab and 34d (Fig. 31) exhibited analgesic activity comparable to that of phenylbutazone. Derivatives 34c-e were as strongly anti-inflammatory as phenylbutazone and more active than

EP

2-substituted isomers. The most active compounds exhibited very low ulcerogenic potential

AC C

and high LD50 values [137].

Fig. 31

Subsequently, the 4-oxo isomers 35 (Fig. 31) were evaluated for the same activities. The obtained results showed that many derivatives possessed very good analgesic activity, superior to that of phenylbutazone, and not related to nature and position of the dialkylaminoalkyl group. These compounds exhibited very low ulcerogenic potential and high LD50 values [138]. 25

ACCEPTED MANUSCRIPT 3,4-Diazaphenothiazines 10-Dialkylaminoethyl 3,4-diazaphenothiazines 36a-b (Fig. 32) showed antihistaminic

RI PT

activity approximately equal to that of antazoline [75].

Fig. 32 Pyrimidobenzothiazines

SC

1,3-Diazaphenothiazines

In the sixties of 20th century, Knoll Company patented synthesis and properties of

M AN U

10H-1,3-diazaphenothiazines 37, substituted in the pyrimidine and benzene rings (Fig. 33). The compounds exhibited antibacterial, analgesic and anti-inflammatory activities but no data were available [139,140]. H N

Z3

N N

S Z2

Z1 = H, CH3, C6H5, (CH2)2N(C2H5)2, (CH2)3N(CH3)2, (CH2)2 N

, (CH2)2 N

O

Z2 = H, CH3 Z3 = H, Cl

TE D

37

Z1

Fig. 33

Afterwards, Wellcome Company patented synthesis and properties of over 50 10H-

EP

1,3-diazaphenothiazines 38 of the similar and new structures (Fig. 34). The compounds exhibited antimicrobial activity against S. faecalis, E. coli, S. aureus, P. vulgaris and P. aeruginosa. The compounds also showed depressant action of the tranquilizer type on mice

AC C

but no data were given [141-145]. Very recently, one of those compounds (10H-2-(pyrrolidin1-yl)-1,3-diazaphenothiazine) was reported to show moderate radical-trapping antioxidant activity [146].

Fig. 34 Next, Pfizer Company patented synthesis and properties of over 30 10H-1,3diazaphenothiazin-2,4-diones 39 and their S-oxides 40, and over 50 varied 4a-substituted 1,3-

26

ACCEPTED MANUSCRIPT diazaphenothiazin-2,4-diones 41, containing pyrimidinedione moiety in the tricyclic ring system (Fig. 35). It is worth noting that the 4a-substituted structure type is unique in the azaphenothiazine chemistry and is found only for 1,3-diazaphenothiazin-2,4-diones. Compounds 39-41 exhibited inhibitory action toward phosphodiesterase. 1,3-Dimethyl-7chloro

compounds

41,

with

the

methyl(hydroxyethyl)amino,

hydroxypiperidinyl,

Fig. 35

SC

as anti-inflammatory agents (no data were provided) [58,59].

RI PT

methylpiperidinyl, morpholinyl, methoxy and ethoxy groups for X, were particularly effective

M AN U

10-Phenylsubstituted 1,3-diazaphenothiazine S-dioxides 42a-f, possessing additional substituents in the tricyclic ring system (2,4-(NH2)2-8-Cl, Fig. 36), were tested for potential inhibition of globin proteolysis (IGP). The best results were found for compounds 42a and 42c with IGP of 83.7% and 72.6%, respectively. Compounds 42c-f were less active. The 7chloro isomers of 42a-f turned out to be inactive. All compounds exhibited a weak ability to inhibit β-hematin formation in comparison with chloroquine. Compound 42a was further

TE D

tested in infected mice with P. berghei ANKA, a chloroquine susceptible strain of murine malaria, at 20 mg/kg. This compound reduced and delayed the progress of malaria (12.7%)

AC C

EP

[34].

Fig. 36

2,4-Diazaphenothiazines

Along with 1,3-diazaphenothiazines, Knoll Company patented synthesis and properties of 2,4-diazaphenothiazines 43a-c (Fig. 37). The compounds exhibited antibacterial, analgesic and anti-inflammatory activities, but no data were provided [139,140].

27

ACCEPTED MANUSCRIPT

Fig. 37 10H-2,4-diazaphenothiazin-1,3-diones 44a-b and 4,6-dihydro-2,4-diazaphenothiazine-

RI PT

1,3-dione 45 (Fig. 38) were tested for inhibitory activity toward lipoxygenase enzyme. All compounds exhibited significant inhibitory actions in the rat peritoneal cavity resident cells assay (RPC) with IC50 values of 1.4 µM (45), 1.9 µM (44b) and 5.3 µM (44a). When tested for acute toxicity, compound 45 demonstrated high (69%) survival rate at a dose of 50 mg/kg

SC

and did not show any acute toxic symptoms at 600 mg/kg. This compound also exhibited high (69%) edema inhibitory effect in the yeast induced rat foot edema (RFE) assay at a dose of 60

M AN U

mg/kg [147].

Fig. 38

TE D

Later, 10H-2,4-diazaphenothiazin-1,3-diones 46a-b (Fig. 39) were evaluated for the antiviral activity against the MT-4 cells infected with HIV, showing IC50 values of 4.58 and

Fig. 39

AC C

EP

4.30 µM [148].

10H-1-methyl-2,4-diazaphenothiazines 47 with the heterocyclic substituents in

position 3 (pyrrolidinyl, piperidinyl, piperazinyl and morpholinyl, Fig. 40) were assessed for inhibition of soybean 15-lipoxygenase (SLO) activity. Compounds 47a-e were found the most potent with IC50 = 18-58 µM. The best activity was determined for derivative 47c with the 4methylpiperazinyl group. The exchange of the methyl into ethyl group at the piperazine nitrogen atom, decreased the activity (IC50 = 34 µM). The compounds with the hydroxypiperidinyl and phenylpiperazinyl groups appeared to be inactive. A good correlation was found between the inhibitory potency and lipophilicity of the compound [149].

28

ACCEPTED MANUSCRIPT

Fig. 40

RI PT

A few years later, 2,4-diazaphenothiazines with the same substituents Z2 (Fig. 41), were evaluated once more for SLO inhibition. The compounds without the methyl group in position 1 were weakly active or inactive. Only compound 48a, with the 4-methylpiperazinyl group, was moderately effective (IC50 = 87.7 µM). The compounds with the methyl group were more active, with best results achieved for 48b, with IC50 = 21.2 µM and the 4-

SC

ethylpiperazinyl derivative 48c (IC50 = 40.7 µM). The inhibitory activity as -logIC50 correlated

M AN U

well with the estimated inhibitory constant -logKi obtained from molecular docking [150].

Fig. 41

TE D

Similar 2,4-diazaphenothiazines heterocyclic groups in position 1 were tested for the inhibition of SLO activity. The most potent were the 4-methylpiperazinyl derivatives 49a-c (Fig. 42), among them the propyl analog 49c with IC50 = 8.9 µM. Those compounds also exhibited the best radical scavenging activity. Other compounds, containing the pyrrole,

EP

piperazine, morpholine and 4-phenylpiperazine moieties, were less active. The inhibitory activity (-logIC50) correlated well with the estimated inhibitory constant (-logKi) [151]. Very

AC C

recently, the analog of compound 49 with the pyrrolidine ring in position 3 (Z = H) was reported to be remarkably potent radical-trapping antioxidant [146].

Fig. 42 10H-2,4-diazaphenothiazin-1,3-diones (10-thiaisoalloxazine derivatives), possessing the alkoxymethyl group at the pyrimidine nitrogen atom (Fig.43), were evaluated in lymphocytes, based on the inhibitory activity against the viral-induced cytopathic activity. The most active compounds were found derivatives 50a-c, exhibiting modest inhibitory activity towards the cytopathic effect of HIV-1. Compound 50g, containing the β-ribosyl 29

ACCEPTED MANUSCRIPT substituent, was the least active. Compound 50f, in addition, was found as the least toxic among the tested derivatives. Of note, compounds 50a-c and 50f exhibited good selectivity

Fig. 43

SC

RI PT

indices (cytotoxic and inhibitory effect) [152].

M AN U

Two 10H-2,4-diazaphenothiazin-1,3-diones 51a-b, among other heterocycles, possessing the pyrimidinedione ring (Fig. 44), were investigated for their potential as trypanothione reductase inhibitors. Both compounds showed inhibition of recombinant T. cruzi TryR enzymatic activity (36% and 62%), but lesser than that of chlorpromazine. The

TE D

compounds were inactive against promastigotes of Leishmania amazonensis [153].

EP

Fig. 44

Pyrazinobenzothiazines

AC C

1,4-Diazaphenothiazines

10-Propyl-1,4-diaza-2,3-dichlorophenothiazine 52b (Fig. 45) exhibited antimicrobial

activity against S. aureus and T. mentagrophytes giving a complete control of the bacterial growth at a concentration of 500 ppm. The parent compound 52a showed anthelmintic activity, providing 90% control of the infection rate, resulting from dog hookworm larvae contained in the diet of mice, at a level of 0.06% by weight. Compound 52b showed a complete control of the aquatic pest daphnia infection spread at a dosage of 2 ppm. It also secured similar control of the two spotted spider mite invasion at a level of 500 ppm [154156].

30

ACCEPTED MANUSCRIPT

Fig. 45 10-Substituted 2,3-dichloro(or chloro-methoxy)-1,4-diazaphenothiazines 53a-j with

RI PT

various dialkylaminoalkyl groups at the thiazine nitrogen atom (Fig. 46) were tested for antimicrobial activity. The dichloro compounds 53d-f were found to be the most active giving 100% control against growth of S. aureus, C. albicans, T. mentagrophytes, A. tereus, C. pelliculos and P. pullulans at a concentration of 100 ppm. Compounds 53a-c and the chloro-

TE D

M AN U

SC

methoxy derivatives 53g-j exhibited lesser activity (at a level of 500 ppm) [157].

Fig. 46

10H-2,3-dichloro-1,4-diazaphenothiazine 5-oxide 54a and 10-benzyl-2,3-dichloro-1,4-

EP

diazaphenothiazine S-oxide 54b and S-dioxide 55 (Fig. 47) were also investigated for the antimicrobial activity. The most active derivative was found N-unsubstituted S-oxide 54a,

AC C

providing 100% growth inhibition against T. mentagrophytes, B. subtilis and A. terreus at a level of 1-10 ppm, whereas against C. albicans and S. aureus at 500 and 1000 ppm, respectively. The benzylated S-dioxide 55 demontrated similar effects at a level of 400-500 ppm. Its S-oxide analog 54b gave 95% effectiveness against downey mildew at the 450 ppm concentration [158].

Fig. 47 31

ACCEPTED MANUSCRIPT The dimethylaminopropyl derivatives of substituted 1,4-diazaphenothiazines 56a-d (Fig. 48) were studied for relative affinities for receptors relevant to neuroleptic action by measuring the displacement of radioligands from membrane binding sites in mammalian brain. The interactions with rat caudate dopamine receptors were measured using [3H]spiperone and [3H]apomorphine antagonist and agonist radioligands. The potent

RI PT

compound was proved to be the 2-chloro derivative 56a, being almost as effective (IC50 = 62.1 nM and 67.3 nM) as chlorpromazine. Other pyrazine substituted derivatives 56b-c were

M AN U

Fig. 48

SC

less potent, while the benzene substituted derivative 56d was relatively inactive [70]

Representative compounds of 10H- and 10-substituted 1,4-diazaphenothiazines and their S-oxides and S-dioxides (Fig. 49) were investigated for their biological activities in various assays. Compounds 57e and 57g-h exhibited the most potent activity in the polymorphonuclear leukocyte assay with IC50 = 0.05-1 µg/mL, while compounds 57a-b, 57f

TE D

and 57i were less active (IC50 = 1-5 µg/mL). Compounds 57a and 57f proved to be the most active in the antigen challenge inhibition with the inhibition of 85% at a dose of 10 µg/mL and 71% at 3 µg/mL, respectively. In the asthmatic rat assay, compounds 57h and 57e showed a highest potency attaining 47% inhibition at a dose of 5 mg/kg and 43% inhibition at 3

EP

mg/kg. Compound 58, an S-oxide derivative of compound 57f, showed a weaker potency (12.5%). In the platelet activating factor (PAF) induced hyperalgesia assay, compound 57a

AC C

was found to be a best inhibitor (70% at a dose of 10 mg/kg). The tested compounds were effective inhibitors of the mammalian 5-lipoxygenase enzyme system of the arachidonic acid cascade [159].

R N

N

Z S 57

N

R

Z

a

H

H

b

CH3

H

c

COCH3

H

d

CH2CO2C2H5

e

H

7-OH

f

CH3

7-OCH3

H

g

H

h

CH3

7-OH-8-Cl

i

CH3

7-OCH3-8-Cl

CH3

H3CO

N

N

S

N

O 58

7-OCH3

32

ACCEPTED MANUSCRIPT Fig. 49 A

series

of

24

10H-1,4-dibenzoazaphenothiazines

59-62,

with

different

cycloaminomethyl moieties in position 8, was evaluated for inhibition of intracellular adhesion molecule-1 (ICAM-1), which plays a pivotal role in the inflammatory and immunological responses. The cycloamine moiety was represented by the azetine, piperidine,

RI PT

morpholine, piperazine, perhydroazepine and perhydroazocine rings, possessing various additional groups (amido, acyl, carbamoyl, sulfonyl, sulfonamido and sulfonamidoalkyl (Fig. 50). Almost all compounds (except of 59e, 59f and 60a) exhibited significant inhibitory activity with IC50 = 0.27-1.4 µM.

SC

The effect of compounds 61 and 62 on neutrophil accumulation was next evaluated in a mouse IL-1- induced paw inflammation model at a dose of 10 mg/kg. Compounds 61b, 61d

M AN U

and 61i showed very strong inhibitory effects on neutrophil migration with the inhibition values ranging from 83.4 to 89.7%. Compounds 61c and 61k were less active with the values of 77.6 and 66.1%. Among compounds 62 derivative 62b was the most active (53.4%). Compounds 61j and 62d were found to be inactive. A selected compound 61i (with the dimethylaminosulfonamido group) also suppressed the up-regulation of other adhesion molecules, such as E-selectin with IC50 = 0.55 µM and vascular cell adhesion molecule-1

TE D

(VCAM-1) with IC50 = 0.36 µM. This compound exhibited good oral bioavailability evaluated in fed male rats. Compound 61j seems to be a promising agent in the treatment of chronic disorders, for example rheumatoid arthritis [160]. NR2

R2N

N

N

N

EP

a H

b

N

c

N

N

b S

N

3-CONH2

b NHSO2CH3

H

a 2-CONH2

N

N X

N

c CH2NHSO2CH3 d (CH2)2NHSO2CH3

S

c 4-CONH2

N

61

N

e NHSO2CF3

d

N

e

N

O

g NHSO2NH2

f

N

N CH3

i

NHSO2N(CH3)2

j

NHSO2 N

60

AC C

59

N

X

H

X

N

S

X a NHCOCH3

f NHSO2C6H5

h NHSO2NHCH3

R

H N R

N

N

N

a COCH3

k NHSO2 N

O

b SO2CH3 S 62

N

c SO2NH2 d SO2N(CH3)2

Fig. 50 Another series of 22 10H-1,4-dibenzoazaphenothiazines 63-64, substituted in position 8 with cycloamines linked by the methylene (mainly but also by the ethylene and carbonyl groups), were evaluated for ICAM-1 inhibition. Replacement of the sulfonamide and sulfonyl

33

ACCEPTED MANUSCRIPT groups in the previously prepared compounds by carboxylic group, connected with the piperidine, azabicyclooctane, azabicyclononane and oxaazabicyclononane by alkylene, Xalkylene (X = N, O, S, Fig. 51), spirocycloalkyl and aryl linkers, resulted in a number of potent adhesion molecule inhibitors. Compounds 63a-e, 63j-l, 64a-b and 64g exhibited very strong inhibitory activity with the IC50 = 0.42-1.0 µM. Compounds 63e, 63c and 63b

RI PT

piperidinealkanoic acid moiety were found to be the most active (IC50 of 0.42, 0.47 and 0.53 µM, respectively). The compounds with the azabicycloalkane moiety showed differential activity. The most active compound in this group was the azabicyclooctane derivative 64h (0.81 µM) but its isomer 64h was over 12-fold less active. The azabicyclononane derivatives

SC

64c and 64d were more strongly active (3.0 and 2.3 µM). The exchange of the methylene linker by the ethylene and carbonyl groups (analogs of compound 63d) caused a decrease in

active than compound 63e.

M AN U

the activity. Compounds 63g-i, containing heteroatom in the linker, were at least 20-fold less

Selected active compounds were evaluated for inhibition of neutrophil accumulation in the IL-1-induced paw inflammation model. The most active found were derivatives 64c (91.5% inhibition), 64b (88,8%), 63l (88.0%), 64h (83.2%), 64a (82.8%) and 64d (80.6%) at a dose of 10 mg/kg. Compounds 63a-e were less active, even at a dose of 30 mg/kg. Compound 64c was selected for further study on inhibition of other adhesion

TE D

molecules giving IC50 values of 2.5 µM (VCAM-1) and 4.5 µM. Compounds 64c and 61i were evaluated for their concentration in plasma. Compound 64c exhibited about 28-fold higher plasma concentration than compound 61i indicating that its strong inhibitory activity

EP

resulted likely from its much higher plasma concentration. Furthermore, both compounds were evaluated in the carrageenan-induced pleurisy model in the rat. Compound 64c significantly inhibited leukocyte infiltration from a dose of 1

AC C

mg/kg and was 3-fold more active than compound 61i. In a next experiment, compound 64c suppressed dose-dependently further increase in the paw swelling at doses of 2.5 to 20 mg/kg. This compound exhibited good oral bioavailability (98.5%). Due to its better pharmacokinetics in the rat, compound 64c showed significant potency in the carrageenan pleurisy model in the rat, a much higher than might be predicted from its moderate ICAM-1 inhibition compared to that of 61i [161].

34

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Fig. 51

SC

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ACCEPTED MANUSCRIPT

Tricyclic azaphenothiazines Dipyridothiazines

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1,6-Diazaphenothiazines

As early as in 1958 1,6-diazaphenothiazine 65a, possessing the dimethylaminopropyl group at the thiazine nitrogen atom, was evaluated for its pharmacological activity. This compound was more toxic and exhibited a weaker synergic action with morphine and

EP

barbiturates, as well as a weaker effect of lowering body temperature, than chlorpromazine [162]. Later, two new compounds 65b-c with the dimethylaminopropyl groups in position 10

AC C

were tested in vivo. Compound 65c evoked CNS depression characterized by hypotonia, reduced spontaneous motor activity and disorientation in rats at a dose of 300 mg/kg. Compound 65b produced salivation and a slight reduction of motor activity [52].

Fig. 52 Very recently, 10H-1,6-diazaphenothiazine and its 10-substituted derivatives with the alkyl, heteroaryl, amidoalkyl and dialkylaminoalkyl groups (Fig. 53) were tested for their

35

ACCEPTED MANUSCRIPT anticancer activity against glioblastoma SNB-19, melanoma C-32 and breast cancer MCF-7 cell lines. Most of them exhibited good anticancer activity with IC50 values lower than 10 µg/mL. The parent compound 66a (with the hydrogen atom) and compounds 66d and 66e (with the propargyl and nitropyridinyl groups), were more potent (IC50 = 4.8, 3.9 and 4.6 µg/mL, respectively) than cisplatin (7.4 µg/mL) against MCF-7 cells. Compound 66k (with

RI PT

the methylpiperazinylbutynyl group) was as active (7.5 µg/mL) as cisplatin. Against C-32 cells, compounds 66a and 66f (with the diethylaminoethyl group) were slightly more active (7.5 and 6.6 µg/mL) as cisplatin (7.8 µg/mL). Compound 66c (with the allyl group) was the most active against of SNB-19 cells with IC50 = 18.9 µg/mL. The most active compounds

SC

66a, 66e and 66f were nontoxic against normal fibroblasts HFF-1 with IC50 ≥ 50 µg/mL. The discussed 1,6-diazaphenothiazines were more active than prothipendyl (IC50 ≥ 23 µg/mL)

TE D

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[73].

Fig. 53

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1,8-Diazaphenothiazines

10H-1,8-diazaphenothiazine, and its 10-substituted derivatives with the alkyl, heteroaryl, dialkylaminoalkyl, amidoalkyl and sulfonamidoalkyl groups (Fig. 54), were

AC C

evaluated for their potential biological activities. In the proliferative response of human peripheral blood mononuclear cells (PBMC), induced by phytohemagglutinin A (PHA), compounds 67a (with the hydrogen atom) and 67e (with the dimethylaminopropyl group) showed strong activity (inhibition over 70%) at concentration of 50 g/mL. A moderate activity (about 60% inhibition) was exhibited by derivatives 67f-g and 67i-j. Compounds 67bf and 67h showed the strongest inhibition (over 85% at 5 µg/mL) of tumor necrosis factor alpha (TNF-α) production induced by lipopolysaccharide (LPS). All compounds exhibited very weak cytotoxic properties with a decrease of PBMC viability not exceeding 22%, even at 50 µg/mL. The most promising derivatives 67a, 67b (with the allyl group), 67e and 67j (with the acetamidopropyl group) were tested for anticancer activity against leukemia L-1220 and

36

ACCEPTED MANUSCRIPT colon carcinoma SW-948 cell lines. The most active compound found was 10H-derivative 67a, exhibiting comparable anticancer activity to that of cisplatin against carcinoma SW-948 at 5 µg/mL and leukemia L-1210 at 10 µg/mL. Compounds 67e and 67j showed strong

Fig. 54

SC

RI PT

activity at 10 µg/mL [72].

Transformation of the propargyl derivative 67c into the dialkylaminobutynyl

M AN U

derivatives (through the Mannich reaction) led to more potent compounds than the substrate but these activities were moderate. The most active compounds found were derivatives 67k-m with IC50 = 26.1-27.5 µg/mL, similar to that of prothipendyl. Against gliobastoma SNB-19 and ductal carcinoma T47D, compound 67m displayed antitumor activity with IC50 = 33.1

2,7-Diazaphenothiazines

TE D

and 45.8 µg/mL [163].

Compound 68a and its methyl derivative 68b significantly inhibited the proliferative response of PBMC to PHA at 10 µg/mL, whereas the parent compound inhibited also PMBC

EP

proliferation elicited by anti-CD3 antibodies. The parent compound was very strongly suppressive (72% inhibition) with regard to the secondary humoral response in vitro, already

AC C

at 1 µg/mL concentration. This compound also significantly inhibited the delayed-type hypersensitivity (DTH) response to ovalbumin in vivo in mice. The test of LPS-induced cytokine production revealed a total inhibition of the interleukin 6 (IL-6) at 100 µg/mL and moderate inhibition of the TNF-α production at 10 and 100 µg/mL. At the studied concentrations, compounds 68a-b were not toxic for mouse splenocytes [164].

37

ACCEPTED MANUSCRIPT Fig. 55 10H-2,7-diazaphenothiazine 68a and its selected 10-substituted derivatives with the methyl, aryl, aminoalkyl and amidoalkyl derivatives were also screened for their anticancer activity. After a preliminary test the most active compound 68a was tested towards about 60 cell lines including 9 types of cancer: leukemia, melanoma, non-small cell lung, colon, CNS,

RI PT

ovarian, renal, prostate and breast cancers. This compound showed significant potency against each type of cancer line. The best activity was found against non-small cell lung cancer cell lines HOP-62 and HOP-92 with IC50 values of 0.3 and 1.7 µg/mL. With relation to other kinds of cancer cell lines, compound 68a demonstrated the following IC50 values: 2.4 and 3.6

SC

µg/mL (colon 205 and HCT-116), 3.1, 3.9 and 5.4 µg/mL (renal RXF 393, 736-0 and ACHN), 4.1 µg/mL (leukemia HL-60(TB), 5.9 µg/mL (breast HS 578T), 6.5 µg/mL

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(melanoma M-14), 6.8 µg/mL (CNS SF-539 and SNB-19), 7.1 µg/mL (ovarian OVCAR-8) and 8.4 µg/mL (prostate PC-3). Against other cancer cell lines, this compound was a little less active or inactive. Other compounds were less active in the preliminary test [165]. Only methyl derivative 68b showed an antiproliferative action against renal cancer UO-31 (60% inhibition) and non-small cell lung cancer EKVX (51% inhibition) at 2.1 µg/mL and pnitrophenyl derivative 68c against renal cancer UO-31 (44% inhibition) at 3.2 µg/mL [166].

TE D

The parent compound 68a and the 2,4-dinitrophenyl and pyrrolidinylethyl derivatives 68d-e showed a significant antioxidant activity with IC50 values of 64, 107 and 125 µM, being more potent than the known antioxidant probucol (IC50 > 1 mM) in the non-enzymatic peroxidation of hepatic microsomal membrane lipids [167].

EP

Transformation of 10-propargyl derivative into the dialkylaminobutynyl derivatives (Fig. 56) led to compounds of different anticancer activity. The most active compound was

AC C

derivative 69d with the N-methylpiperazinylbutynyl substituent against carcinoma T-47D with IC50 = 9.6 µg/mL, being more potent than cisplatin and prothipendyl (46.9 and 32.3 µg/mL). This compound was also the most potent against glioblastoma SNB-19 (21.2 µg/mL). Against melanoma C-32, compounds 69a-d showed moderate activity with IC50 = 24.8-29.2 µg/mL. To elucidate its anticancer mechanism of action, the influence of compound 69d on expression of genes encoding TP53, CDKN1A, BCL-2 and BAX in the cancer cells was studied. An increase in the number of CDKN1A copies in the T-47D and SNB-19 cells suggested a possibility of involvement of the compound in the cell cycle arrest. The analysis of the gene expression ratio BCL-2/BAX in T-47D cells showed activation of mitochondrial apoptosis [163].

38

ACCEPTED MANUSCRIPT

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Fig. 56 3,6-Diazaphenothiazines

10H-3,6-diazaphenothiazine 70a and its 10-derivatives with various alkyl, heteroaryl and dialkylaminoalkyl substituents (Fig. 57) were screened for anticancer activity. The parent

SC

compound 70a exerted very strong cytotoxic action against glioblastoma SNB-19, melanoma C-32 and breast cancer MCF-7 cell lines with IC50 = 0.46 and 0.72 µg/mL. This type of 10Hdipyridothiazine appeared to be the most potent out of four diazaphenothiazines (1,6-, 1,8-

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and 2,7-) tested. A similarly strong and selective action was found for the 2-pyrimidinyl derivative 70e against breast cancer MCF-7 (IC50 = 0.73 µg/mL). Both compounds were more potent than cisplatin and prothipendyl. The dimethylaminopropyl derivative 70f showed as good activity as cisplatin (6.3 vs 7.8 µg/mL) against melanoma C-32 and moderate activity (11.3 µg/mL) against breast cancer MCF-7. Compounds 70b-d and 70g exhibited weaker

TE D

activity (28.4-32.9 µg/mL) against the selected lines. All compounds were non-toxic or almost non-toxic against normal fibroblast HFF-1cell line. The analysis of gene expressions (H3, TP53, CDKN1A, BCL-2 and BAX) confirmed the antiproliferative activity of compounds 70a and 70e, and indicated the activation of the p53 pathway in cancer cells, leading to the

EP

cell cycle arrest. The gene expression ratio BAX/BCL-2 suggested an occurrence of

AC C

mitochondrial apoptosis pathway in the MCF-7 and SNB-19 cells [74].

Fig. 57

3,7-Diazaphenothiazines 10H-3,7-diazaphenothiazine 71a and its diethylaminoethyl and dimethylaminopropyl derivatives 71b-c (Fig. 58) exhibited antihistaminic activity. Derivative 71c was found the most potent, the parent compound 71a was a little less potent and derivative 71b was several times less active [168]. 39

ACCEPTED MANUSCRIPT

Fig. 58

1,3,6-Triazaphenothiazines and 1,3,9-triazaphenothiazines

RI PT

Pyridopyrimidothiazines

Substituted 10H-1,3,6-triazaphenothiazines 72a-f and 10H-1,3,9-triazaphenothiazines 73a-c, mostly with the amino group or chlorine atom (Fig. 59), were screened for their CNS

SC

depressant activity in mice and rats. All the compounds decreased the motor activity and rate of respiration within 30 min. The application of compound 73a (with the amino group) led to

M AN U

a significant decrease in the spontaneous motor activity. Compounds 72b (with two chlorine atoms) and 73a were the only compounds which exerted any significant effect on barbital sodium-induced sleeping time. Except of compounds 72f and 73b, all compounds increased hexobarbital sodium-induced sleeping time. Compounds 72a-e and 73a are suggested to inhibit liver microsomal activity. None of the compounds protected mice against tonic convulsion induced by strychnine, pentylenetetrazole and maximal electroshock seizures

TE D

(MES). However, compounds 72f and 73b prolonged the onset to strychnine and MES, respectively. The results indicated that only compounds 72b and 73a had a direct effect on the

AC C

EP

central nervous system, but in higher doses in comparison with chlorpromazine [64].

Fig. 59

Tetracyclic azaphenothiazines Quinobenzothiazines

A. Linearly fused Quino[3,2-b]benzothiazines (benzo[b]-1-azaphenothiazines)

40

ACCEPTED MANUSCRIPT About 80 linearly fused quno[3,2-b]benzothiazines with the hydrogen atom and alkyl, aminoalkyl, amidoalkyl, sulfonamidoalkyl and chloroethylureidoethyl groups at the thiazine nitrogen atom (in position 6), and additional substituents (CH3, F, Cl, Br, CF3, SCH3) in the benzene ring in positions 8-10, were screened for their action on PHA-induced proliferative response of PBMC, LPS-induced TNF-α production in whole blood cells and viability of

M AN U

SC

RI PT

PBMC in culture [169,170].

Fig. 60

Some compounds (Fig. 60) exhibited very strong antiproliferative activity, lack or low toxicity and inhibitory action on TNF-α production. The most active and low toxic

TE D

compounds were tested for anticancer activity against epidermal A-431, lymphoma L1210 and colon SW-948 tumor cell lines. Compounds 74a-c (with the acetylaminobutyl and chloroethylureidoalkyl groups) were as active as cisplatin attaining IC50 = 1.2 µg/mL for 74a against SW-948 tumor cell line. Those compounds were supposed to act on DNA via

EP

intercalation (the X-ray analysis for 74a unexpectedly revealed linearly fused tetracyclic ring system to be planar [90]) or alkylurea alkylation of proteins (74b-c) [169]. very

strongly

antiproliferative

N-unsubstituted

compound

was

6H-9-

AC C

A

fluoroquinobenzothiazine which completely blocked response of PBMC (100% inhibition at 10 µg/mL) and exhibited a moderate cytotoxicity [167]. Interestingly, the introduction of a substituent in position 6 decreased the compound toxicity. Further experiments (proliferation of PBMC, TNF-α production) enabled to select two compounds (74d-e) for anticancer activity against tumor lines (A-431, L1210, SW-948 and CX-1). The compounds were similarly active as cisplatin with the following IC50 values: 74d, 2.28, 2.84 and 10.83 µg/mL against L1210, SW-948 and CX-1, respectively, and 74e, 9.65 and 10.67 µg/mL against A431 and CX-1 [170].

41

ACCEPTED MANUSCRIPT Further experiments were carried out to study potential therapeutic utility in prevention of alogeneic graft rejection. The results of two-way mixed lymphocyte reaction of PBMC showed that compound 74d inhibited the proliferative response of lymphocytes to a similar degree as cyclosporine A at 10 µg/mL. The same compound moderately inhibited interleukin 2 (IL-2)-induced growth of CTLL-2 cell line. The inhibition of prostaglandin (PG)

of compound 74d on PHA-induced PBMC proliferation [170].

RI PT

synthesis by indomethacin or block of PG receptors did not interfere with the inhibitory effect

6-Substituted quinobenzothiazines were screened in silico and in vivo for their acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) inhibitory activity. Based on

SC

the docking procedure, nine most promising compounds, exhibiting the best fit for the screening complexes, were further studied. Three compounds displayed good BuChE and moderate AChE inhibitory activity. Two of them (74f-g), with the 1-methyl-2-

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piperazinylethyl group at the thiazine nitrogen atom, showed almost complete (99.9%) BuChE inhibition at 10 µM and IC50 values of 122.2 and 24.4 nM, respectively. Compound 74b, with the chloroethylureidopropyl group, was less active giving 80.0% BuChE inhibition [171].

Compounds 74a and 74d were evaluated for their potential immunosuppressive effect

TE D

in the model of delayed type hypersensitivity (DTH) to ovalbumin and in foot pad inflammation, induced in mice. Compound 74a significantly suppressed both the eliciting phase and the effectual DTH foot pad reaction [172]. Selected quinobenzothiazines exhibited modest antimicrobial activity (against S.

EP

aureus and E. coli), weaker than the angularly fused analogs [173]. Selected 6H-quinobenzothiazines 74h-j showed strong antioxidant activity in nonenzymatic lipid peroxidation of rat hepatic microsomal membrane lipids with IC50 = 3 and 2

AC C

µg/mL, respectively, for these two compounds [43].

Quino[6,7-b]benzothiazines (pyrido[2,3-b]phenothiazines) 1,4-Dihydro-11-methyl-3-carboxy-4-oxoquino[6,7-b]benzothiazine

(pyrido[2,3-

b]phenothiazine) 75 (Fig. 61), containing the 4-quinolone-3-carboxylic acid fragment, exhibited antibacterial activity against Gram-positive and Gram-negative bacterial strains but no data were disclosed in the patent [174].

42

ACCEPTED MANUSCRIPT

Fig. 61

Quino[3,4-b]benzothiazines (benzo[a]-1-azaphenothiazines)

RI PT

B. Angularly fused

12H- and 12-substituted quino[3,4-b]benzothiazines 76a-i with additional substituents (CH3, F, Cl, Br) in position 9 (Fig. 62) exhibited significant anticancer activity against amelanotic melanoma C-32 with IC50 = 5.6-9.4 µg/mL. The most active were derivatives 76f

SC

and 76i with a potency similar to that of cisplatin. The glioblastoma SNB-19 was less affected by the compounds. Compounds 76a and 76f-i showed IC50 values in the range of 6.7-12.4

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µg/mL. Compounds 76b-e were unexpectedly inactive. The compounds with the methyl group in position 11 appeared to be inactive against both lines, probably due to a steric hindrance. It seems that the dialkylaminoalkyl substituents at the thiazine nitrogen atom play

Fig. 62

AC C

EP

TE D

a crucial role in the anticancer activity [40].

Another group of 12H and 12-substituted quino[3,4-b]benzothiazines 77a-h, with the

additional substituents (F, CF3 and SCH3, Fig. 63) in positions 8-10, were tested for their anticancer activity against breast cancer MCF-7 and MDA-MB-23, and glioblastoma SNB-19 cell lines. All the discussed compounds were as active as cisplatin. The most effective was derivative 77g, possessing the methylpiperidinylethyl group at the thiazine nitrogen atom and the fluorine atom in position 9, with IC50 values in the range of 5.92-6.96 µg/mL against all cell lines. A little less active were derivatives 77b, 77f and 77h, bearing the propargyl, dimethylaminopropyl and benzyl group at the thiazine nitrogen atom, with IC50 values: 77b, 7.71 µg/mL (MDA), 77f, 7.43 and 7.91 µg/mL, 77h, 7.59 and 8.71 µg/mL (MDA, MCF-7,

43

ACCEPTED MANUSCRIPT respectively). It is worth noting that derivative 77a, which possessed only hydrogen atom at the thiazine nitrogen atom (and 8-CF3), showed a significant activity with IC50 values of 9.76

SC

Fig. 63

RI PT

and 10.0 µg/mL against MCF-7 and SNB-19 cell lines [36].

Comparing trifluoromethylquinobenzothiazines 77a-c with their 10-isomers, the first

linearly

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compounds mentioned were twice more active than their isomers. In contrast to very active 6H-9-fluoroquino[3,2-b]benzothiazine

[170],

its

angularly

fused

12H-9-

fluoroquino[3,4-b]benzothiazine (R = H, Z = 9-F) was less active. The activity seems to depend on a nature of the substituent and a location in the fused ring system [36]. 12H-quino[3,4-b]benzothiazin-6-ones, containing the 2-quinolone moiety incorporated

TE D

in to the tetracyclic ring system and additional substituents (Cl, Fig. 64), were evaluated for their antioxidant, antibacterial and antimycobacterial activities. Compounds 78a-b showed protective potentialities at concentration of 1 µM against tert-butyl hydroperoxide-induced oxidative damage, measured as the human liver cell death rate (hepatoma HepG2 cell line)

EP

[175].

Compounds 78d-e were potent against multidrug-resistant M. tuberculosis H37Rv

AC C

with MIC > 6.25 µg/mL. A more active compound 78e (GI = 67% at 6.25 µg/mL) was more potent than coumarinic phenothiazine analogs [176]. Compounds 78a-c exhibited a moderate activity against S. aureus (MIC ≥ 200 µg/disc) but lacked activity against E. coli [177].

Fig. 64

44

ACCEPTED MANUSCRIPT Disubstituted in both benzene rings quino[3,4-b]benzothiazines 79a-f (with CH3, CF3, OCH3, Br, Fig. 65) showed interesting antioxidant activities as measured by levels of reduced glutathione (GSH: 4.50-5.01 nM/mg tissue) and lipid peroxidation (LPO: 6.42-6.81 nM/mg tissue) in livers of Swiss albino mice. Compounds 79e-f exhibited significant antioxidant activity measured in DPPH• and ABTS• + assays. The antioxidant activities were attributed to

Fig. 65

SC

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the tetracyclic ring system containing the N-H bond [178].

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Quino[7,8-b]benzothiazines (pyrido[2,3-a]phenothiazines)

Another type of fused compounds, 12H-quino[7,8-b]benzothiazin-4-ones 80a-d, containing the 1-cyclopropyl-6-fluoro-4-quinolone-3-carboxylic acid moiety (Fig. 66), were screened for antibacterial activity against Gram-positive and Gram-negative bacterial strains. Their activity was higher than that of tetracycline. The most active was the parent compound

TE D

80a with MIC = 0.75 µg/mL against E. coli, and 3 and 6 µg/mL against B. cereus and S. typhimurine. The methyl derivative 80b was strongly active against B. cereus and S. typhimurine (MIC = 1.5 µg/mL), and against methicillin-resistant S. aureus (MIC = 1.5 µg/mL). The methoxy derivative 80c was less active, exhibiting good effects against S.

EP

typhimurine and MRSA (MIC = 6 and 25 µg/mL). Only the chloro derivative 80d was moderately active (MIC ≥ 100 µg/mL) against all strains. The virtual screening, using ligand-

AC C

protein docking modeling, predicted the compounds to be potential inhibitors of the topoisomerase IV enzyme [179].

Fig. 66 5-Alkylquino[3,4-b]benzothiazinium salts

45

ACCEPTED MANUSCRIPT 5-Alkylquino[3,4-b]benzothiazinium salts are unique in the azaphenothiazine chemistry, due to an original synthesis and the ammonium fragment incorporated in the tetracyclic ring system [37]. The demethylation led to the mentioned above 12Hquino[3,4b]benzothiazines 76 [40]. 5-Methylquino[3,4-b]benzothiazinium chlorides 81a-j, with additional substituents in positions 9-11 (CH3, F, Cl, Br, NH2 and OH, Fig. 67), showed

RI PT

antiproliferative activities against colon ACT116 and Lewis lung carcinoma LLC cell lines. The most active were the parent compound 81a and derivative 81i (with the amino group) with IC50 values of 2.3 and 2.2 µg/mL against LLC and ACT116, respectively. Other compounds were a little less active than doxorubicin, with IC50 = 4.4-8.8 µg/mL. An

SC

increased length of the alkyl chain at the quinoline nitrogen atom, from the methyl to butyl and decyl and the change of the chloride anion into bromide, decreased the activity (IC50 = 14.0-19.6 µg/mL). The introduction of the substituent (CH3, NH2 and OH) in position 11

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caused an inactivity of the compounds, probably due to a steric hindrance [39].

TE D

Fig. 67

5-Methylquino[3,4-b]benzothiazinium chlorides 81i-j, possessing the amino and piperidinyl groups in position 9 (Fig. 68) were further tested against C-32 and SNB-19 cell lines. Whereas derivative 81i showed significant antiproliferative effect at 10 µg/mL against

EP

both cell lines, much more active 81j exhibited similar effect at 0.1 µg/mL. The latter compound exhibited a cytotoxic effect at 1 µg/mL against both lines. The antiproliferative

AC C

activity was confirmed in the analysis of a gene encoding the histone H3. Both compounds influenced cell cycle regulatory genes (TP53 and CDKN1A) expression and protein products of the genes involved in mitochondrial apoptosis pathway (BAX and BCL-2 expression). The more active compound 81j caused a significant change in total oxidative status (TOS) and promoted the oxidative activity by increasing malonodialdehyde (MDA) concentration in both cell lines. Compound 81i, in turn, affected only TOS in C-32 and MDA in SNB-19 cell line. Both compounds also increased the superoxide dismutase (SOD) activity [180]. Compounds 81a-d and 81f were also screened for their antibacterial activity. The highest activity was found for 81d, 81a and 81f (MIC = 6, 7 and 14 µg/mL, respectively) against E. coli, for 81d, 81a, 81e and 81b (MIC = 4, 6, 9 and 11 µg/mL) against E. faecalis.

46

ACCEPTED MANUSCRIPT Compound 81e was active (MIC = 26 µg/mL) against S. aureus. The change of the methyl group in butyl and decyl, and the chloride anion into bromide, decreased the antibacterial action [181]. Another series of 9-11-substituted 5-methyl-12H-quinobenzothiazinium chlorides with the alkoxy, amino and aminoalkoxy groups (Fig. 68) were tested for their anticancer activity

RI PT

against C-32, SNB-19 and MDA-MB-231 cell lines, demonstrating IC50 values in the range of 0.5-7.5, 0.7-11.5 and 0.5-12 µg/mL, respectively. The most active compounds were those with the amino (82c-d) and aminoalkoxy (82g-j) groups (IC50 ≤ 3.0 µg/mL), being significantly more active than cisplatin (up to 20-fold). The active compounds were examined

SC

toward transcriptional activity of genes encoding (H3, p53, BCL-2 and BAX) suggesting a commitment of the exposed cells to stepped-up regulatory processes, and other processes beside apoptosis in the anticancer activity. Compounds 82d and 82h bound to DNA in

TE D

M AN U

amounts enabling DNA intercalation with ethidium bromide [182].

Fig. 68

Other tetracyclic azaphenothiazines

EP

Pyridonaphthothiazines (benzo[j]-1-azaphenothiazines)

AC C

Two 6-substituted pyridonaphthothiazin-5-ones 83a-b, with the chlorine and pbromophenyl groups (Fig. 69), were tested for antioxidant activity. They showed comparable activity to ascorbic acid, the reference compound, in the hydrogen peroxide scavenging test (inhibition up to 99.99%). In an in vivo experiment involving albino rats, compound 83a exhibited higher inhibition (77.9%) of hydrogen peroxide by catalase than ascorbic acid (71.9%), showing potential abilities to promote catalase activities in the organism. Both azaphenothiazines were more potent (3.8 and 4.0 µM) than ascorbic acid (4.2 µM) in the lipid peroxidation test, measured by determination of malondialdehyde (MDA) level. The results suggested that these compounds can prevent or minimize lipid peroxidation and cell damage by free radicals. The compounds were also found to be slightly hepatotoxic to the animals [183]. 47

ACCEPTED MANUSCRIPT

Fig. 69 Benzo[b]-1,4-diazaphenothiazines

RI PT

Various 11- and 12-substituted benzo-1,4-thiazines 84 and 85, with the dialkylaminoalkyl groups (Fig. 70), were biologically screened. The compounds were claimed to have valuable antiallergic, serotonin-antagonistic, anticonvulsive, adrenolytic and sedative

R

R N

S

N

Z

R = (CH2)2N(CH3)2, (CH2)3N(CH3)2

N

N

S

N

85

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N

Z

Z

(CH2)3 N

Z

84

SC

activities (no data available) [56].

, (CH2)3 N

, (CH2)3 N

O

Z = H, Cl, Br

Fig. 70

Pyridoquinothiazines Benzo[a]-3,6-diazaphenothiazines and

12-substituted

benzo-3,6-diazaphenothiazines

86a-c

(with

the

TE D

12H-

dimethylaminopropyl and piperidinylethyl groups at the thiazine nitrogen atom, Fig. 71) exhibited growth inhibitory activity against cancer cell lines SNB-19 and C-32 with IC50

AC C

EP

values of 7.3-10.2 and 6.5-8.7 µg/mL [40].

Fig. 71

Benzo[a]-3,6-diazaphenothiazinium salts 5-Methyl-12H-benzo-3,6-diazaphenothiazinium

chloride

87

showed

mild

antiproliferative activity against cancer cell lines HCT116 and LLC with IC50 values of 17.2 and 13.2 µg/mL [39]. This compound exhibited also antibacterial activity against E. coli, E. faecalis and S. aureus strains with MIC values of 25, 31 and 52 µg/mL, respectively, but a weaker one than the most active 5-methyl-12H-benzo-3-azaphenothiazinium chlorides [181].

48

ACCEPTED MANUSCRIPT

Fig. 72

Pyridonaphthothiazines (benzo[j]-4-azaphenothiazines)

RI PT

Pentacyclic azaphenothiazines

Furanonaphthopyridothiazine 88, containing the rifampicin moiety (Fig. 73), was tested as an inhibitor of M. tuberculosis growth. This compound exhibited excellent

SC

antibacterial activity with MIC value of 0.005 µg/mL, being more potent than rifampicin

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[184].

Fig. 73

azaphenothiazines

TE D

Quinonaphthothiazines dibenzo[b,h]1-azaphenothiazines and dibenzo[b,j]1-

Isomeric, angularly condensed quinonaphthothiazines 89 and 90 (Fig. 74) were tested for their anticancer activity against glioblastoma SNB-19, melanoma C-32 and breast cancer

EP

T47D cell lines. The anticancer activity was dependent on a nature of the substituents and the ring fusion between the thiazine and the naphthalene rings. 7-Substituted compounds 89 were

AC C

more active than14-substituted isomers 90. The most active compound was the diethylaminoethyl derivative 89e, reaching IC50 values of 0.98, 0.80 and 5.9 µg/mL against respective lines. Compounds 89a (against SNB-19) and 89d (against SNB-19 and C-32) were similarly potent as chlorpromazine and cisplatin. Compounds 89d and 90e were more active than cisplatin against T47D cells [47].

49

ACCEPTED MANUSCRIPT Fig. 74 The parent compounds (89a and 90a) showed strong antioxidant action in the non-enzymatic lipid peroxidation of rat hepatic microsomal membrane lipid with IC50 values of 6 and 2 µg/mL, respectively, being more potent than trolox and probucol [43].

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Diquinothiazines A. Linearly fused

Diquino[3,2-b;2’,3’-e]thiazines (dibenzo[a,i]-1,9-azaphenothiazines)

SC

Four 6-substituted diquinothiazines 91a-d, possessing the diethylaminoethyl (91a), dimethylaminopropyl (91b), chloroethylureidoethyl (91c) and p-toluenesulfonylaminoethyl (91d, Fig. 75) were tested for the effects on the proliferative response of PBMC to PHA.

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Compounds 91a-d were distinctly inhibitory at concentration as low as 10 µg/mL, and compound 91b exhibited strong activity even at 1 µg/mL. This compound inhibited the proliferative response of PBMC, stimulated with anti-CD3 antibodies, at 10 µg/mL, similarly

EP

TE D

as chlorpromazine [164].

Fig. 75

6-Substituted diquinothiazines, possessing the alkyl, aryl, aminoalkyl and their acyl

AC C

and sulfonyl derivatives, were screened for their anticancer activity. After a preliminary test the most active compounds 91a-d were further tested on 55-60 cell lines, including nine types of cancer: leukemia, melanoma, non-small cell, colon, CNS, ovarian, renal, prostate and breast cancer [165].

The compounds exhibited very potent and selective anticancer activities, with the strongest actions corresponding to IC50 values of 1.8-0.087 µM, the TGI (total growth inhibition) values of 1.2-3.2 µM and LC50 values of 1.1-5.2 µM for selected cancer cell line. Each of those lines were affected strongly by at least one compound. The most active compound was the half-mustard derivative 91c. It exhibited the strongest effect on the melanoma cell line SK-MEL-5 showing IC50 = 87 nM (corresponding to 40 ng/mL). 50

ACCEPTED MANUSCRIPT Somewhat lower actions (IC50 = 0.25-1.0 µM) were observed for selected cancer lines: leukemia (CCRF-CEM and MOLT-4), colon (HCT-116), CNS (SNB-75 and SF-295), prostate (PC-3), non-small cell lung (NCI-H460 and HOP-92), ovary (IGROV1, OVCAR-4 and OVCAR-5) and breast (MDA-MB-460). The dialkylaminoalkyl derivatives 91a and 91b were very active (IC50 ≤ 1.0 µM)

RI PT

against cancer cell lines: ovary (IC50 = 0.19 and 0.29 µM, respectively), non-small cell lung (HOP-92), breast (MCF-7) and leukemia (91b, RPMT-8226 and MOLT-4). The ptoluenesulfonamidoethyl derivative 91d was less active, providing best results against renal cancer cell line 736-0 (IC50 = 0.48 µM), CNS and breast cancer cell lines SNB-75 and HS

SC

578T (IC50 = 1.7 µM) [165].

For many other cancer cell lines, these compounds were less active, weakly active or just inactive. Other substituted 6-diquinothiazines displayed quite good anticancer activity in

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a preliminary test. Butyl derivative 91e showed strong inhibition (93, 86 and 78%) against breast cancer MCF-7 and T-47D and renal UO-31 at 10 µM. Against the latter cancer line, other derivatives 91f-i (with the benzyl, p-chlorophenyl, p-nitrophenyl and 2-pyridinyl substituents) demonstrated inhibitory activity in the range of 57-73%. Derivatives 91g and 91i exhibited also anticancer activity (63 and 61% growth inhibition) against the MCF-7 and

TE D

melanoma SK-MEL-2 lines, respectively [166].

To test the compound toxicity for normal cells, the effects of these compounds on the viability of mouse splenocytes were determined together with chlorpromazine and cyclosporine A, as the reference drugs. Compounds 91b and, unexpectedly, chlorpromazine

EP

were toxic, compounds 91a and cyclosporine A less toxic, whereas compounds 91c-d were minimally toxic [165].

AC C

6-Substituted diquinothiazines were screened in silico and in vivo for their acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) inhibitory activities. Based on the docking procedure, six promising compounds, exhibiting a best fit fo the screening complexes were further studied. Two compounds displayed good BuChE and moderate AChE inhibitory activity. Compound 91j, with the 1-methyl-2-piperazinylethyl group at the thiazine nitrogen atom, showed almost complete (99.8%) BuChE inhibition at 10 µM. Its inhibitory potency (IC50 = 11.8 nM) was comparable to that of reference tacrine (IC50 = 5.0 nM). Less active compound was 91k with the chloroethylureidopropyl group (83.2%, 2.9 µM) [171]. A. Angularly fused Diquino[3,2-b;6’,5’-e][1,4]thiazine (benzo[b]pyrido[3,2-h]-1-azaphenothiazine)

51

ACCEPTED MANUSCRIPT 7H-Diquinothiazine 92 (Fig. 76) showed significant antioxidant activity with the IC50 = 16 µM, in non-enzymatic lipid peroxidation of rat hepatic microsomal membrane lipid,

Fig. 76

RI PT

stronger than trolox and probucol [43].

Diquino[3,4-b;4’,3’-e][1,4]thiazine (dibenzo[a,j]-3,7-diazaphenothiazine)

SC

14H-Diquinothiazine 93 (Fig. 77), unlike isomeric, linearly fused 6H-diquinothiazine 91 (R = H), exhibited very strong antioxidant activity with IC50 = 1 µM, stronger than trolox

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and probucol. The introduction of the alkyl (methyl) and aryl (p-fluorophenyl) group at the thiazine nitrogen atom strongly decreased the activity (IC50 > 500 µM) [43].

TE D

Fig. 77

Pyrazolonaphthyridobenzothiazine

Linearly condensed 11H-pyrazolonaphthyridobenzothiazine 94 (Fig. 78), possessing

EP

the amino, phenyl and p-methoxyphenyl groups, among other pyrazolonaphthyridines, exhibited moderate anticancer activity against the hepatocellular carcinoma HePG2 cell line (IC50 = 19.1 µM) in comparison with doxorubicin (IC50 = 5.0 µM). The compound showed a

AC C

weaker activity (IC50 = 34.5-36.5) against other cancer cell lines (MCF-7, HCT-116 and PC3). The molecular docking technique was used to predict the DNA-binding affinity revealing good binding modes (∆G = -30.3 kcal/mol) [185].

Fig. 78

52

ACCEPTED MANUSCRIPT The pyridothiazino and pyrimidothiazino derivatives of pyrimidobenzothiazines Pyridothiazinobenzopyrimidothiazine and pyrimidothiazinobenzopyrimidothiazine The

antimicrobial

property

of

linearly

condensed

pyrimidobenzothiazines

(1,3-

diazaphenothiazines) 95 and 96 with the pyridothiazine and pyrimidothiazine rings (Fig. 79) were evaluated. Compound 96 was more potent than compound 95 and exhibited significant

RI PT

activities against E. coli (MIC = 0.1689 mg/mL) and A. niger (0.1380 mg/mL) similarly to

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Fig. 79

SC

those of ciprofloxacin (0.1677 mg/mL) and ketoconazole (0.1356 mg/mL), respectively [186].

Hexacyclic azaphenothiazines 5,6-Ethylenediquinothiazinium chloride

Hexacyclic 5,6-ethylenediquinothiazinium chloride 97 (Fig. 80) is an exceptional

TE D

diarenothiazinium salt of the ammonium structure, as the ethylene group links both thiazine and azine nitrogen atoms [29]. This compound showed strong antiproliferative activity in the proliferative response of PBMC to PHA at 10 and 1 µg/mL [162]. This compound was tested using about 60 cell lines including 9 types of cancer: leukemia, melanoma, non-small cell,

EP

colon, CNS, ovarian, renal, prostate and breast cancer and showed significant potency against each type of the cancer line. The best antiproliferative IC50 values were found against the following cancer cell lines: colon COLO 205 (1.3 µg/mL), melanoma MALME-3M and SK-

AC C

MEL-5 (1.3 and 1.5 µg/mL), breast MCF-7 (3.2 µg/mL) leukemia SR (4.9 µg/mL), renal RXF 393 (5.0 µg/mL), non-small cell lung NCI-H460 and EKVX (7.4 and 7.6 µg/mL), ovarian OVCAR-3 (7.8 µg/mL), CNS SF-268 (8.5 µg/mL) and prostate PC-3 (10.0 µg/mL) [165].

Fig. 80 Quinazolinoquinobenzothiazines

53

ACCEPTED MANUSCRIPT Substituted 17H-quinazolinoquinobenzothiazin-6-ones (possessing a fused ring system, containing the quinoline and quinazolinone moieties, Fig. 81) were tested for antioxidant activity. Compounds 98a-c were found to be the most active in the DPPH (45.647.3% inhibition) and ABTS+· (nearly full scavenging) test. They show significant antioxidant activity measured by determining GSH and LPO (6.8 and 4.5-4.6 nM/mg tissue, respectively)

RI PT

in the livers of Swiss albino mice. The activity was attributed to the presence of the

The

benzoxazino,

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Fig. 81 pyrimidoxazino

benzophenothiazines,

and

pyridonaphthothiazines

(benzoxazinonaphthobenzothiazines,

SC

hexacyclic ring system with the N-H bond and the methyl and methoxy groups [187].

benzoxazinonaphthopyrimidothiazines,

benzothiazino and

derivatives

of

pyrimidonaphthothiazines

benzoxazinonaphthopyridothiazines,

pyrimidoxazinonaphthobenzothiazines

and

pyrimidoxazinonaphthopyridothiazines)

TE D

Angularly condensed azaphenothiazines 99-103, containing azine rings, such as: pyridine, pyrimidine, oxazine and additional thiazine (Fig. 82), were screened for antimicrobial activity against various Gram-positive and Gram-negative bacteria and fungi.

EP

All compounds demonstrated bactericidal action against at least few strains. Their activity depended on type of the ring system as well as on type and location of the substituents. Compound 99a was the most active against B. subtilis with MIC = 0.0457 mg/mL. Against B.

AC C

cereus, compound 102a showed the highest activity (MIC = 0.0661 mg/mL) followed by compounds 103 (0.0771 mg/mL), 101a (0.0871 mg/mL) and 100a (0.0874 mg/mL). Compounds 100a, 101a, 102a and 103 (MIC = 0.0871-0.0971 mg/mL) were more active than ciprofloxacin (0.1677 mg/mL) against E. coli. Compound 102a was more active against A. niger fungus (MIC = 0.0794 mg/mL) than ketoconazole (0.1356 mg/mL). Compound 99a was the most active against C. albicans fungus (MIC = 0.1000 mg/mL) [188].

54

Fig. 82 The

benzothiazino,

pyridothiazino

pyridonaphthothiazines

and and

pyrimidothiazino

derivatives

of

pyrimidonaphthothiazines

benzothiazinonaphthopyrimidothiazines,

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(benzothiazinonaphthopyridothiazines,

SC

RI PT

ACCEPTED MANUSCRIPT

pyridothiazinonaphthopyridothiazines and pyrimidothiazinonaphthopyrimidothiazines) Their thiazine analogs 104-107 (Scheme 83), possessing the thiazine ring instead of oxazine as in 99-103, were tested for antimicrobial actions against the same bacteria and fungi. All compounds showed significant antibacterial and antifungal activities. Compound

TE D

107 was found to be more active against Gram-positive bacteria (B. subtilis, B. cereus and S. aureus) and fungi (C. albicans and A. niger) than ciprofloxacin and ketoconazole. Compound 104 showed the same activity against B. subtilis as ciprofloxacin. Compound 107 exhibited the highest MIC values against S. aureus (0.0398 mg/mL), B. cereus (0.0505 mg/mL) and B.

EP

subtilis (0.0633 mg/mL). This compound showed higher antifungal activity against A. niger (0.0603 mg/mL) than ketoconazole (0.1356 mg/mL). Compound 105 was more active (0.1445

AC C

mg/mL) than ciprofloxacin (0.1677 mg/mL) against E. coli and nearly as active (0.0724 mg/mL) as ketonazole against C. albicans (0.0622 mg/mL). Compound 106 showed high activity against S. aureus (0.0794 mg/mL) and A. niger (0.0912 mg/mL) [188,189].

Fig. 83

55

ACCEPTED MANUSCRIPT Summary Azaphenothiazines are structurally modified phenothiazines, formed by substitution of one or both benzene rings with the azine rings, such as: pyridine, pyridazine, pyrimidine, pyrazine, 1,2,4-triazine, quinoline, quinoxaline, benzooxazine and benzothiazine. They constitute over 50 different heterocyclic systems bearing tricyclic, tetracyclic, pentacyclic and

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hexacyclic structures. When the azine nitrogen atoms are concerned, they may form monoaza-, diaza-, triaza and tetraazaphenothiazines. Due to the structure diversity, azaphenothiazines required new methods of synthesis, different from those of the classical phenothiazines. It seems that the synthesis problems are the reasons that not all possible azaphenothiazine

SC

systems have been identified. As the synthesis of azaphenothiazines can proceed via the Ullmann cyclization or the Smiles rearrangement, not all resultant products were

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unequivocally identified. In the recent years, this problem was solved by the 2D NMR spectra and X-ray analyses. In contrast to classical phenothiazines, where only one nomenclature system is used (system A), and the derivatives are simply named as x-phenothiazines, three nomenclature

systems

in

structurally

misunderstandings.

different

azaphenothiazines

lead

to

some

Azaphenothiazines possess the substituents R most often at the thiazine nitrogen atom

TE D

but also at the azine nitrogen atoms, when the azine ring contains the oxo group (one or two as in cases of the pyridine, pyridazine, pyrimidine and quinoline rings). In the case of lack of the oxo group, the molecules form sometimes azaphenothiazinium salts. Azaphenothiazines possess also the substituents Z at the benzene carbon atom. In a few cases the substituent can

EP

be formed in position 4a (the common carbon atom for the thiazine and benzene rings). Those three structure types do not have equivalents in classical phenothiazines.

AC C

Not all synthesized azaphenothiazines have been explored in the biological models. The azaphenothiazine ring systems appeared to be valuable pharmacophoric scaffolds, as many of NH-azaphenothiazines (having only hydrogen atom at the thiazine nitrogen atom) exhibited significant biological activities. In other cases, the nature of the substituent R at the thiazine nitrogen atom (most often) and the substituent Z in the benzene or azine rings, conditioned the biological activity. The oxidation of the thiazine sulfur atom to S-oxides and S-dioxides exceptionally enhances the biological action. The most biologically effective substituents are as follows: aminoalkyl, cycloaminoalkyl, amino, cycloamino, acyl, carbamoylalkyl, amidoalkyl, sulfonamidoalkyl and alkyl group as methyl, allyl, propargyl and benzyl

56

ACCEPTED MANUSCRIPT Selected 1-azaphenothiazines, such as prothipendyl, isothipendyl, oxypendyl and pipazethate derivatives, have been still used as antipsychotic, antihistaminic, antiemetic and antitussive drugs. Other azaphenothiazines in in vitro and in vivo experimental models exhibited a wide range of promising biological actions, such as anticancer (against various cancer cells), anti-inflammatory, antibacterial, antimycobacterial, antifungal, antiviral (against

RI PT

HIV-1 and CHIKV), analgesic, 15-lipoxygenase inhibitory, trypanothione reductase inhibitory, antiasthmatic, cardiovascular, antiarrhythmic, hypotensive, antiswelling, antiobesity, anthelmintic, antipsychotic and antihistaminic. In those studies many of azaphenothiazines were found to be more active than the reference drugs.

SC

One has to realize that not all possible tricyclic types of azaphenothiazines (not to mention tetra-, penta- and hexacyclic) have been identified, not all known azaphenothiazines have been biologically investigated, and a considerable part of known azaphenothiazines have

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only hydrogen atom at the thiazine nitrogen atom. Having these limitations in mind, we are of opinion that this class of heterocyclic compounds is worthy further exploration with an ultimate goal of finding new lead compounds, which might be beneficial in therapy. References

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EP

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AC C

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ACCEPTED MANUSCRIPT [8] J.J. Aaron, M.D. Gaye Seye, S. Trajkovska, N. Motohashi, Bioactive Phenothiazines and Benzo[a]phenothiazines: spectroscopic studies and biological and biomedical properties and applications, Top Heterocycl. Chem., Springer-Verlag, Berlin, 16 (2009) 153-231. [9] G. Sudeshna, K. Parimal, Muliple non-psychiatric effect of phenothiazines: A review, Eur. J. Pharmacol. 648 (2010) 6-14.

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SC

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AC C

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RI PT

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SC

Farmaco 27 (1972) 643-655.

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TE D

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EP

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AC C

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identification,

X-ray

study

and

anticancer

activities

quinobenzothiazines, J. Mol. Struct. 1122 (2016) 62-71.

RI PT

[36] K. Pluta, M. Szmielew, M. Suwińska, M. Latocha, Synthesis, spectroscopic structure of

new

angularly

fused

[37] A. Zięba, A. Maślankiewicz, K. Suwińska, Azinyl Sulfides, Part LXIII. 1-Alkyl-4-

SC

(arylamino)quinolinium-3-thiolates and 7-Alkyl-12H-quino[3,4-b]-1,4-benzothiazinium salts, Eur. J. Org. Chem. 2000, 2947-2953.

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transformation into new diazaphenothiazine derivatives, Heterocycles 68 (2006) 459-503. [39] A. Zięba, A. Sochanik, A. Szurko, M. Rams, A. Mrozek, P. Cmoch, Synthesis and in vitro antiproliferative activity of 5-alkyl-12(H)-quino[3,4-b] [1,4]benzothiazinium salts, Eur. J. Med. Chem. 45 (2010) 4733-4739.

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4163.

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novel 12(H)-quino[3,4-b][1,4]benzothiazine derivatives, Med. Chem. Res. 22 (2013) 4158-

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activity

of

novel

phenyl

ring-substituted

5-alkyl-12(H)quino[3,4-

EP

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AC C

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ACCEPTED MANUSCRIPT [47] M. Jeleń, K. Pluta, K. Suwinska, B. Morak-Młodawska, M. Latocha, A. Shkurenko, Quinonaphthothiazines, syntheses, structures and anticancer activities, J. Mol. Struct. 1099 (2015) 10-15. [48]

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Hiramitsu,

Y.

Maki,

S.

Senda,

Pyrimido[1,4]benzothiazines

and

pyrimido[1,5]benzothiazepines. Part 3. Novel ring-contraction and ring-opening of

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pyrimido[1,5]benzothiazepines, J. Chem. Soc. Perkin I (1978) 717-721. [49] Y. Maki, Sulfur-containing pyridine derivatives. LIII. Smiles rearrangement in pyridine derivatives and synthesis of azaphenothiazine derivatives, Yakugaku Zasshi 77 (1957) 485490.

SC

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ACCEPTED MANUSCRIPT Highlights The phenothiazine system was modified with the azine rings to form azaphenothiazines. Azaphenothiazines contain of tri-, tetra-, penta- and hexacyclic ring systems. At least 600 azaphenothiazines showed therapeutically promising, biological activities. The most active azaphenothiazines exhibited stronger actions than the reference drugs.

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This review describes properties of azaphenothiazines based on 170 references.