International Journal of
Molecular Sciences
Communication
Study of Lipophilicity and ADME Properties of
1,9-Diazaphenothiazines with Anticancer Action
Beata Morak-Młodawska 1, * , Małgorzata Jeleń 1 , Emilia Martula 2 and Rafał Korlacki 3
Citation: Morak-Młodawska, B.;
Jeleń, M.; Martula, E.; Korlacki, R.
Study of Lipophilicity and ADME
Properties of
1,9-Diazaphenothiazines with
Anticancer Action. Int. J. Mol. Sci.
2023, 24, 6970. https://doi.org/
10.3390/ijms24086970
Academic Editors: Jean
Christopher Chamcheu and
Jean Fotie
Received: 26 January 2023
Revised: 3 April 2023
Accepted: 7 April 2023
Published: 9 April 2023
Copyright: © 2023 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
Int. J. Mol. Sci. 2023, 24, 6970. https://doi.org/10.3390/ijms24086970
1 Department of Organic Chemistry, Faculty of Pharmaceutical Sciences in Sosnowiec,
The Medical University of SilesiaJagiellońska 4, 41-200 Sosnowiec, Poland
2 Doctoral School, The Medical University of Silesia, 40-055 Katowice, Poland
3 Department of Electrical and Computer Engineering, University of Nebraska-Lincoln,
Lincoln, NE 68588, USA
* Correspondence: bmlodawska@sum.edu.pl
Abstract: Lipophilicity is one of the key properties of a potential drug that determines the solubil-
ity, the ability to penetrate through cell barriers, and transport to the molecular target. It affects
pharmacokinetic processes such as adsorption, distribution, metabolism, excretion (ADME). The
10-substituted 1,9-diazaphenothiazines show promising if not impressive in vitro anticancer potential,
which is associated with the activation of the mitochondrial apoptosis pathway connected with to
induction BAX, forming a channel in MOMP and releasing cytochrome c for the activation of caspases
9 and 3. In this publication, the lipophilicity of previously obtained 1,9-diazaphenothiazines was
determined theoretically using various computer programs and experimentally using reverse-phase
thin-layer chromatography (RP-TLC) and a standard curve. The study presents other physicochemi-
cal, pharmacokinetic, and toxicological properties affecting the bioavailability of the test compounds.
ADME analysis was determined in silico using the SwissADME server. Molecular targets studies
were identified in silico using the SwissTargetPrediction server. Lipinski’s rule of five, Ghose’s, and
Veber’s rules were checked for the tested compounds, confirming their bioavailability.
Keywords: lipophilicity; 1,9-diazaphenothiazines; anticancer action; ADME; Lipinski’s rule of five;
Ghose’s and Veber’s rules
1. Introduction
Lipophilicity is one of the important physicochemical descriptors and is related to
both pharmacodynamic and pharmacokinetic properties. It determines the absorption,
metabolism, distribution, excretion and toxicity of drugs (ADMET) [1–4]. It plays a sig-
nificant role in the transport of molecules across membranes. In addition, it affects their
ability to bind to plasma proteins and to bind to receptors at the target of the drug’s action.
Therefore, lipophilicity is regarded as the reference parameter for predicting the biolog-
ical activity of potential drugs. Physically, lipophilicity is described as the logarithmic
n-octanol-water partition coefficient (logP) that is characteristic of a given chemical. This
parameter has been used in studies on the quantitative relationship between the structure
and the activity (QSAR) [5–7].
According to Lipinski’s rule of five, lipophilicity is one of the most important factors
determining the bioavailability of a drug. These are criteria that a chemical must meet
in order to be considered a likely oral drug [8–10]. It has been proven that the logP > 5
lipophilicity value is associated with undesirable features of a given drug, such as tissue
accumulation, fast metabolic turnover, poor water solubility, or strong plasma protein
binding [11]. There is an exact correlation between lipophilicity and the permeability and
solubility of a bioactive compound. In 2003, a published study showed the relationship
between skin permeability and lipophilicity. It was also shown that too high values of
lipophilicity contribute to the immobilization of the compound within a given layer [12].
https://www.mdpi.com/journal/ijms
Int. J. Mol. Sci. 2023, 24, x FOR PEER REVIEW 2 of 12

Int. J. Mol. Sci. 2023, 24, 6970 2 of 11

lipophilicity contribute to the immobilization of the compound within a given layer [12].
Lipophilicity is inevitably associated with the penetration problem of blood–brain barrier
(BBB). High lipophilicity promotes the nonspecific binding of drug molecules with plasma
Lipophilicity is inevitably associated with the penetration problem of blood–brain barrier
proteins, thus contributing to the reduced penetration of these compounds through the
(BBB). High lipophilicity promotes the nonspecific binding of drug molecules with plasma
BBB. The literature data indicate that compounds with a moderate lipophilicity oscillating
proteins, thus contributing to the reduced penetration of these compounds through the
around 2 show optimal abilities to reach molecular targets [13–15].
BBB. The literature data indicate that compounds with a moderate lipophilicity oscillating
Recently, we described new 10-substituted-1,9-diazaphenothiazines possessing
around 2 show optimal abilities to reach molecular targets [13–15].
promising anticancer activity. These compounds were obtained in organic synthesis using
Recently, we described new 10-substituted-1,9-diazaphenothiazines possessing promis-
microwave radiation [16]. Such syntheses are currently highly efficient and selective
ing anticancer activity. These compounds were obtained in organic synthesis using mi-
[17,18]. These derivatives having in their structure various alkyl, alkynyl, cycloalkylami-
crowave radiation [16]. Such syntheses are currently highly efficient and selective [17,18].
noalkyl and dialkylaminoalkyl substituents were screened for their anticancer activity
These derivatives having in their structure various alkyl, alkynyl, cycloalkylaminoalkyl and
against using the glioblastoma
dialkylaminoalkyl SNB-19,
substituents were melanoma
screened C-32,
for their and breast
anticancer cancer
activity MDA-MB-231
against using the
cell lines [16,19].
glioblastoma SNB-19, melanoma C-32, and breast cancer MDA-MB-231 cell lines [16,19].
Theparent
The parentcompound
compound10H-1,9-diazaphenothiazine
10H-1,9-diazaphenothiazine 1 was
1 was veryvery active
active against
against mela-
melanoma
noma C-32 (IC50 = 3.83 µM), even more potent than cisplatin (IC50 = 13.2 µM), but inactive
C-32 (IC50 = 3.83 µM), even more potent than cisplatin (IC50 = 13.2 µM), but inactive against
against
other other
lines. lines.
The mostThe most promising
promising derivatives
derivatives in thiswere
in this group group were compounds
compounds 4 and 74with
and
7 with the propynyl and diethylaminoethyl groups
the propynyl and diethylaminoethyl groups (Figure 1). (Figure 1).

H (CH2)n NR2
N N N N N N

S S
1 7-12
R
N N N ( ) N(CH3)2
6 3 9 ( ) N N(CH3)2
2
( )3
S
N N
( ) N(C2H5)2
R 7 2 10 ( ) N
2
2 CH3 S
3 CH2CH=CH2 12 prothipendyl
H 3C N(CH3)2 ( )
4 CH2C CH 8 2
11 N
5 CH2C6H5
H3C

Figure 1.1.Structure
Figure of novel
Structure of10H-1,9-diazaphenothiazine 1 and 10-substituted
novel 10H-1,9-diazaphenothiazine 1,9-diazaphenothia-
1 and 10-substituted 1,9-
zines 2–11 and reference compound prothipendyl 12.
diazaphenothiazines 2–11 and reference compound prothipendyl 12.

For those
For those twotwo compounds,
compounds, the the expression
expression of of H3,
H3, TP53,
TP53, CDKN1A,
CDKN1A, BCL-2,BCL-2, andand BAXBAX
genes was detected by the RT-QPCR method showing
genes was detected by the RT-QPCR method showing the induction of mitochondrial the induction of mitochondrial
apoptosis. The
apoptosis. Theresults
resultsofofgenegeneexpression
expressionanalysis
analysis indicated
indicated thatthatcompounds
compounds 4 and4 and7 se-
7
lectively reduced
selectively reducedthe theexpression
expressionofofthe theH3H3gene,
gene,and and as
as well
well the expression of the the TP53
TP53
gene, but enhanced
gene, enhancedthe theexpression
expressionofof thethe
CDKN1A
CDKN1A gene in the
gene in tested cell lines.
the tested The induc-
cell lines. The
tion was was
induction confirmed
confirmed in the BAX/BCL-2
in the BAX/BCL-2 gene
geneexpression
expressionratioratiostudies
studies of mitochondrial
of mitochondrial
apoptosis in
apoptosis in two
two cancer
cancer cell
cell lines
lines (SNB-19
(SNB-19 and and MDA-MB-231).
MDA-MB-231). In In the
the C-32
C-32 melanoma
melanoma cell cell
line, aa transcription
line, transcription gene gene activity
activity indicated
indicated aa different
different modemode of of cell
cell death.
death. The
The Annexin
Annexin V V
apoptosis
apoptosisdetection
detectionassay assayshowed
showed populations
populations corresponding
corresponding to viable, necrotic,
to viable, earlyearly
necrotic, and
late
andapoptopic
late apoptopic cells.cells.
While SNB-19
While cells cells
SNB-19 werewere
treated with with
treated compounds
compounds 4 and4 7,
andthere was
7, there
awas
slight increase
a slight in the
increase inearly and and
the early late late
cell cell
apoptosis
apoptosis populations
populations andand
a slight decrease
a slight decreasein
the viable
in the cellcell
viable population.
population. In order
In order to thoroughly
to thoroughly understand
understand thethe mechanism
mechanism of action of
of action
derivatives 4 and 7, the determination of proteins related to apoptosis
of derivatives 4 and 7, the determination of proteins related to apoptosis was performed was performed using
the Proteome
using Profiler Profiler
the Proteome Human Apoptosis Array. Twelve
Human Apoptosis Array.expressed proteins were
Twelve expressed confirmed
proteins were
in the study. We found the following proteins involved in apoptosis:
confirmed in the study. We found the following proteins involved in apoptosis: BAX, pro- BAX, pro-caspase-3,
cytochrome c, and SMAC/Diablo.
caspase-3, cytochrome Compounds Compounds
c, and SMAC/Diablo. 4 and 7 are indicated
4 and 7toare be indicated
highly likely to
to be
induce
highly BAX,likelyas tothey
induce form a channel
BAX, as theyinform
MOMP and release
a channel in MOMPcytochrome c for the
and release activationc
cytochrome
of
forcaspases 9 and 3 of
the activation thereby initiating
caspases 9 andapoptosis
3 therebyvia the intrinsic
initiating mitochondrial
apoptosis pathwaymito-
via the intrinsic [19].
These results prompted further research in the field of 1,9-diazaphenothiazines,
chondrial pathway [19]. These results prompted further research in the field of 1,9- including
pharmacokinetic analyses, in particular lipophilicity and the target analysis.
The purpose of this work is to determine the lipophilicity parameters (logPcalcd , RM0
and logPTLC ) of eleven new anticancer 10-substituted 1,9-diazaphenothiazines 1–11 by
computational programs and by the RP-TLC method; to discuss the influence of the nature
Int. J. Mol. Sci. 2023, 24, 6970 3 of 11

of the substituents; to compare the calculated data with the experimental ones; and to
analyze of the molecular descriptors and ADME properties.

2. Results
The research began with the calculation of lipophilicity parameters with the use
of eleven popular computer modules available on the VCCLAB server [20] and Swis-
sADME [21] server. These programs are based on various mathematical algorithms. The
calculated logPcalcd values for the 10-substituted 1,9-diazaphenothiazines differed depend-
ing on the substituents on the thiazine nitrogen atom and on the calculation program
(Table 1). The 10H-1,9-diazaphenothiazine 1 was characterized by the lowest lipophilicity
(logPcalcd = 1.51), which was obtained by the MlogP program. The highest lipophilicity
was obtained for the 10-benzyl-1,9-diazaphenothiazine 5 (logPcalcd = 4.75) according to the
XLOGP2 module. In contrast, derivatives with the dialkylaminoalkyl or cycloalkylamino
substituents 6–11 were characterized by lipophilicity in the range from 2.02 to 3.89.
Table 1. The calculated lipophilic parameters (logPcalcd ) for 1,9-dipyridothiazine 1–10 using the
internet data bases: VCCLAB [20] and SwissADME * [21].

No Alogps AC_Logp ALOGP MLOGP XLOGP2 XLOGP3 ILogP * XLogP * WlogP * MlogP * SILICOS-IT *
1 2.62 2.36 2.45 1.80 2.74 2.14 1.86 2.14 2.30 1.51 2.19
2 2.44 3.07 2.65 2.09 3.04 2.28 2.22 2.28 2.33 1.83 2.05
3 2.53 2.96 3.84 2.57 3.31 2.39 2.46 2.92 2.88 2.28 2.71
4 2.70 3.67 3.27 2.57 3.66 2.92 2.48 2.39 2.41 2.28 2.55
5 3.52 4.44 4.24 3.43 4.75 3.77 2.84 3.77 3.75 3.13 3.41
6 2.52 3.22 2.85 2.36 3.25 2.67 3.00 2.67 2.65 2.02 2.20
7 3.27 3.63 3.49 2.61 3.74 3.05 3.32 3.05 3.04 2.27 2.57
8 3.07 3.56 3.30 2.61 3.55 3.11 3.24 3.11 2.90 2.27 2.40
9 3.12 3.56 3.25 2.61 3.40 2.80 3.19 2.80 2.41 2.27 2.53
10 3.66 3.88 3.71 2.85 3.76 3.16 3.23 3.16 2.80 2.52 2.76
11 3.76 3.89 3.78 2.95 3.69 3.19 3.51 3.58 3.19 2.75 2.84
*-data obtained using the SwissADME server.

In the next stage, experimental measurements of the lipophilicity coefficient were

performed with the use of reversed-phase thin-layer chromatography RP-TLC. First, the
relative lipophilicity of these derivatives 1–11 was measured as expressed by the RM0
chromatographic values.
The experimental RP-TLC method provided the RM retention parameter (calculated
from RF value measurements) using the following equation:

RM = log (1/RF − 1) (1)

The RM values decreased linearly with increasing acetone concentration in the mobile
phase (r = 0.9485–0.9985). Extrapolation to zero acetone concentration provided the values
of the relative lipophilicity parameter (RM0 ), which showed the division between the
non-polar stationary phase and the polar mobile phase, according to the equation:

RM = RM0 + bC, (2)

where C is the concentration of acetone. The RM0 values of 10-substituted

1,9-diazaphenothiazines are in the range 1.1970–1.9988 (Table 2).
nt. J. Mol. Sci. 2023, 24, x FOR PEER REVIEW 4 of 12

Int. J. Mol. Sci. 2023, 24, 6970 4 of 11

Table 2. The RM0 values and b (slope) and r (correlation coefficient) of the equation RM = RM0 + bC for
compounds 1–11.
Table 2. The RM0 values and b (slope) and r (correlation coefficient) of the equation RM = RM0 + bC
Nocompounds 1–11.
for −b RM0 r
1 0.0205 1.1970 0.9916
2 No 0.0192 −b 1.3850 RM0 0.9954 r
3 1 0.0278 0.0205 1.4052 1.1970 0.9949 0.9916
4 2 0.0195 0.0192 1.4720 1.3850 0.9985 0.9954
3 0.0278 1.4052 0.9949
5 0.0205 1.7877 0.9868
4 0.0195 1.4720 0.9985
6 5 0.0170 0.0205 1.8096 1.7877 0.9973 0.9868
7 6 0.0180 0.0170 1.7934 1.8096 0.9168 0.9973
8 7 0.0228 0.0180 1.8961 1.7934 0.9319 0.9168
8 0.0228 1.8961 0.9319
9 0.0123 1.9434 0.9891
9 0.0123 1.9434 0.9891
10 10 0.0214 0.0214 1.9781 1.9781 0.9485 0.9485
11 11 0.0211 0.0211 1.9988 1.9988 0.9686 0.9686

Then a calibration curve was created using the same measuring conditions. A set of
Then a calibration curve was created using the same measuring conditions. A set of
reference reference
substances I–V with I–V
substances the literature values ofvalues
with the literature logPlit. was used was
of logP in the range
used from
in the range from
lit.
1.21 to 3.54 (Table 3). The calibration curve was used to convert the values of the relative
1.21 to 3.54 (Table 3). The calibration curve was used to convert the values of the relative
lipophilicity parameter
lipophilicity RM0 of theRtested
parameter hybrids into the value of the absolute lipophilicity
M0 of the tested hybrids into the value of the absolute lipophilicity
parameterparameter
logPTLC. logP .
TLC

Table 3. RM0 and logPlit. values and b (slope) and r (correlation coefficient) of the equation RM = RM0 +
Table 3. RM0 and logPlit . values and b (slope) and r (correlation coefficient) of the equation RM = RM0
bC for standards I–V.
+ bC for standards I–V.
Parameters I II III IV V
Parameters I II III IV V
logPTLC 1.21 [22] 1.58 [22] 2.43 [23] 3.18 [22] 5.53 [22]
RM0 logPTLC1.011 1.21 [22]
1.601 1.58 [22]
2.281 2.43 [23]
2.996 3.18 [22]
3.588 5.53 [22]
RM0 1.011 1.601 2.281 2.996 3.588
−b −b 0.018 0.0180.019 0.0190.033 0.034
0.033 0.0340.044 0.044
r r 0.9964 0.9967
0.9964 0.9961
0.9967 0.9842
0.9961 0.9864
0.9842 0.9864

The equation of the standard curve with which it was possible to convert the relative
The equation of the standard curve with which it was possible to convert the relative
lipophilicity parameter RM0 into the absolute parameter logPTLC is presented in Figure 2.
lipophilicity parameter RM0 into the absolute parameter logPTLC is presented in Figure 2.

logPTLC = 0.9862RM0 + 0.2957 (r = 0.9953, s = 02124, F = 348.99, p = 0.0002)

Figure
Figure 2. The 2. Thecurve
standard standard curveprepared
equation equationwith
prepared withsubstances
standard standard substances
I–V. I–V.

The logP values for all new anticancer 10-substituted 1,9-diazphenothiazines 1–11
The logPTLC valuesTLCfor all new anticancer 10-substituted 1,9-diazphenothiazines 1–11
are collected in Table 4.
are collected in Table 4.
Table 4. The logPTLC values of investigated compounds 1–11.
Table 4. The logPTLC values of investigated compounds 1–11.
No of Investigated Compounds
No of Investigated Compounds
1 2
1 2 3 43 54 65 76 87 98 109 1110 11

logPTLC logP TLC 1.476

1.476 1.662 1.747
1.662 1.682 1.682 2.059
1.747 2.080
2.059 2.064
2.080 2.166
2.064 2.212
2.166 2.247
2.212 2.267
2.247 2.267

Simultaneously with conducting

Simultaneously the experimental
with conducting studies, thestudies,
the experimental analyzes ofanalyzes
the molecularof molecu-
descriptors, Lipinski’s, Ghose’s, and Veber’s parameters were performed using the Swis-
lar descriptors, Lipinski’s, Ghose’s, and Veber’s parameters were performed using the
sADME server (Table 5)
SwissADME [21]. (Table 5) [21].
server
ADME parameters were performed
ADME parameters using theusing
were performed PreADMET server (Table
the PreADMET 6) (Table
server [24]. 6) [24].
As shownAs inshown
Tables in
5 and 6, the
Tables compounds
5 and studied show
6, the compounds significant
studied differences
show significant in
differences in
molecularmolecular
descriptors as well asas
descriptors their
wellADME parameters.
as their All tested All
ADME parameters. derivatives, however, however,
tested derivatives,
meet the requirements of Lipinski’s five rule, and Ghose’s, and Veber’s rules [21] (Table 5).
Int. J. Mol. Sci. 2023, 24, 6970 5 of 11

Table 5. The molecular descriptor and parameters of Lipinski’s, Ghose’s, and Veber’s rules for
1,9-dipyridothiazines 1–11 and prothipendyl 12 [21].

Molecular H-Bond H-Bond Rotatable Molar P-gp Lipinski’s Ghose’s Veber’s

No TPSA
Mass (M) Acceptors Donors Bonds Refractivity Substrate Rules Rules Rules
1 201 2 1 0 58.63 63.11 + + + +
2 215 2 0 0 63.54 54.32 + + + +
3 241 2 0 2 72.68 54.32 - + + +
4 239 2 0 1 71.31 54.32 + + + +
5 291 2 0 2 88.02 54.32 + + + +
6 286 3 0 4 85.66 57.56 + + + +
7 300 3 0 5 90.47 57.56 + + + +
8 300 3 0 4 90.47 57.56 - + + +
9 298 3 0 3 92.27 57.56 + + + +
10 312 3 0 3 97.07 57.07 + + + +
11 326 3 0 3 101.88 57.56 + + + +

Table 6. The ADME activities predicted for 1,9-dipyridothiazines 1–11 and prothipendyl 12 [24].

No 1 2 3 4 5 6 7 8 9 10 11 12
BBB 1.02 2.36 3.06 3.36 4.45 2.40 3.17 2.97 2.91 1.26 0.85 2.40
Caco-2 25.41 27.81 25.07 24.30 25.39 22.55 22.61 22.81 24.14 24.00 23.92 22.55
HIA 95.81 98.05 97.67 97.71 97.37 97.78 97.66 97.66 97.66 97.57 97.49 97.78
MDCK 59.28 52.80 26.47 9.12 3.73 37.63 80.31 26.77 60.18 52.03 9.17 37.63
PPB 99.77 93.89 88.37 88.12 96.53 68.57 78.56 74.82 74.86 82.47 80.83 68.57
SP −3.36 −3.24 −3.14 −2.92 −2.94 −3.50 −3.32 −3.36 −3.76 −3.62 −3.58 −3.50

The values of the relative lipophilicity parameter RM0 of the tested molecules 1–11
were correlated with the ADME parameters determined in silico. The results are presented
in Table 7.

Table 7. The correlation of the RM0 values with the predicted ADME parameters.

No of ADME
Equation r
Compound Activities
BBB = 14.86 RM0 3 + 54.304 RM0 2 − 57.146 RM0 +
1–11 BBB 0.8616
17.105
Caco-2 = 37.616 RM0 3 − 178.2 RM0 2 + 273.43
1–11 Caco-2 0.7191
RM0 − 110.91
1–11 HIA HIA = 7.29 RM0 2 + 24.557 RM0 + 77.334 0.7962
MDCK = 529.07 RM0 3 + 2648.5 RM0 2 + 93449
1–11 MDCK 0.6205
RM0 – 35,341
1–11 PPB PPB = 30.856 RM0 2 − 125.38 RM0 + 205.96 0.7335
1–11 SP SP = −3.0033 RM0 2 + 9.2228 RM0 − 10.128 0.8115

An analysis of the relationship between the lipophilicity and the polarity of the studied
molecules 1–11 was also performed using the BOILED-Egg method of estimating the
penetration through the brain or intestines, as an accurate predictive model [21,25]. This
analysis is presented in Figure 3. The tested compounds are within the range of good
permeability through the blood–brain barrier (BBB) (yellow area) and the proper binding
to the human blood albumin (HIA) (white area). Additionally, compounds that can become
substrates for p-glycoprotein (blue points) and two derivatives 3, 8 that show a negative
result (red points) have been indicated in the Figure 3.
Int. J. Mol. Sci. 2023, 24, x FOR PEER REVIEW 6 of 12

Int. J. Mol. Sci. 2023, 24, 6970 substrates for p-glycoprotein (blue points) and two derivatives 3, 8 that show a negative
6 of 11
result (red points) have been indicated in the Figure 3.

Figure 3.
Figure Graph of
3. Graph of the
the dependence
dependence of
of lipophilicity
lipophilicity on
on the
the polarity
polarity of
of the
the studied
studied molecules
molecules 1–11,
1–11,
determined by the BOILED-Egg method [21].
determined by the BOILED-Egg method [21].

Using SwissTargetPrediction
Using SwissTargetPrediction server
server [26],
[26], the
the molecular
molecular targets
targets that
that are
are likely
likely to
to be
be
achieved by reached tested 1,9-diazphenothiazines were also predicted (Table 8).
achieved by reached tested 1,9-diazphenothiazines were also predicted (Table 8). These These
include the
include the family
family CG
CG and
and AG
AG protein,
protein, kinase,
kinase, protease,
protease, and
and cytochrome
cytochrome 450.
450.

Table 8.
Table Probable target
8. Probable target classes
classes of
of 1,9-dipyridothiazines 1–11 [26].
1,9-dipyridothiazines 1–11 [26].

No No ProbableProbable
TargetsTargets
1 1 Family
Family C G C Gunclasificated
protein, protein, unclasificated protein,
protein, enzyme enzyme
2 2 Proteaze,CFamily
Proteaze, Family C G protein,
G protein, cytochrome
cytochrome 450 450
3 Cytochrome 450, kinase, electrochemical transporter
3 4
Cytochrome 450,Unclasificated
kinase, electrochemical transporter
protein, enzyme, kinase
4 5 Unclasificated protein, enzyme, kinase
Phosphodiesterase, enzyme, kinase
5 6 Phosphodiesterase, enzyme,
Kinase, protease, kinase
family AG protein
6 7 Enzyme, protease, family
Kinase, protease, family AG protein AG protein
8 Family AG protein, kinase, ligand-gate ion channel
7 Enzyme, protease, family AG protein
9 Family AG protein, ligand-gate ion channel, kinase
8 10 Family AGFamily
protein,
AGkinase,
protein,ligand-gate ionchannel,
ligand-gate ion channelkinase
9 11 Family AGFamily
protein,
AGligand-gate ion channel,
protein, ligand-gate kinasekinase
ion channel,
10 Family AG protein, ligand-gate ion channel, kinase
11
3. Discussion Family AG protein, ligand-gate ion channel, kinase
The attention of this work was focused on the evaluation of lipophilicity, physico-
3. Discussion
chemical properties, and molecular targets of new anticancer-active 10-substituted 1,9-
The attention of 1–11,
diazaphenothiazines this work
whichwas have focused
differentonsubstituents
the evaluation of thiazine
at the lipophilicity, physico-
nitrogen atom:
chemical
the alkyl, properties, and molecular
alkenyl, alkynyl, targets of and
dialkylaminoalkyl, new cycloalkylaminoalkyl.
anticancer-active 10-substituted 1,9-
The synthesis,
diazaphenothiazines
the structure, and high1–11, whichpotential
biological have different
of thesesubstituents at the
derivatives have thiazine
been nitrogen
previously doc-
atom:
umented the [19].
alkyl, These
alkenyl, alkynyl, dialkylaminoalkyl,
compounds showed significant and cycloalkylaminoalkyl.
and The syn-
highly promising anticancer
activitythe
thesis, as determined
structure, and in vitro
high against
biologicalthepotential
glioblastoma SNB-19,
of these the melanoma
derivatives C-32,
have been and
previ-
the breast
ously cancer MDA-MB-231
documented cell lines. The
[19]. These compounds mostsignificant
showed active, 1,9-diazaphenothiazines
and highly promising4anti- and
7, wereactivity
cancer analyzedas in terms of the
determined expression
in vitro againstofthe
genes affecting the
glioblastoma neoplastic
SNB-19, process (H3,
the melanoma C-
TP53,
32, andCDKN1A, BCL-2,
the breast cancer BAX). These studies
andMDA-MB-231 showed
cell lines. The the activation
most of the mitochondrial
active, 1,9-diazaphenothia-
apoptotic
zines 4 and pathway
7, were and the destruction
analyzed in terms of ofthe
normal histoneofformation.
expression genes affecting the neoplastic
Lipophilic studies were started with
process (H3, TP53, CDKN1A, BCL-2, and BAX). These analyzes in studies
silico using available
showed VCCLAB
the activation ofand
the
SwissADME web
mitochondrial servers.pathway
apoptotic These studies
and theuse various mathematical
destruction modules
of normal histone described on
formation.
the websites of the above servers. The obtained results of the calculated lipophilicity fall into
a wide range of values. This is most likely the result of the use of different computational
models. The most lipophilic compound was derivative 5 (logPcalcd = 4.75) with a benzyl
substituent. The least lipophilic compounds were native 10H-1,9-diazaphenothiazine 1
 Int. J. Mol. Sci. 2023, 24, x FOR PEER REVIEW 7 of 12

Lipophilic studies were started with analyzes in silico using available VCCLAB and
SwissADME web servers. These studies use various mathematical modules described on
Int. J. Mol. Sci. 2023, 24, 6970 7 of 11
the websites of the above servers. The obtained results of the calculated lipophilicity fall
into a wide range of values. This is most likely the result of the use of different computa-
tional models. The most lipophilic compound was derivative 5 (logPcalcd = 4.75) with a ben-
zyl
(logP substituent. The least lipophilic compounds were native 10H-1,9-diazaphenothiazine
calcd = 1.51) with a small hydrogen atom at position 10. The results of these studies are
1 (logP
included calcd = 1.51)1,
in Table with
anda the
small hydrogenvisualization
graphical atom at position 10. The
of the results of
calculated these
logP studies
values of each
are included in Table 1, and the graphical visualization of the calculated
compound is shown in Figures 3 and 4. Such large differences in the obtained values logP values of of
the each compound is shown in Figures 3 and 4. Such large differences in the obtained values
lipophilicity parameter were reported before [27–29] and, as in these previous studies,
of the lipophilicity parameter were reported before [27–29] and, as in these previous stud-
accurate experimental measurments are needed in order to narrow them down.
ies, accurate experimental measurments are needed in order to narrow them down.

Log P
5

4.5

3.5

2.5

1.5

1
1 2 3 4 5 6 7 8 9 10 11

LogPTLC Alogps AC_Logp ALOGP

MLOGP XLOGP2 XLOGP3 ILogP*
XLogP* WlogP* MlogP* SILICOS-IT*

Figure 4. Graphical visualization of calculated logP values (using SwissADME models) of the tested
Figure 4. Graphical visualization of calculated logP values (using SwissADME models) of the
compounds with comparison of logPTLC (plotted in red). *-data obtained using the SwissADME
tested compounds with comparison of logPTLC (plotted in red). *-data obtained using the
server.
SwissADME server.
Experimental studies were performed to determine the relative lipophilicity param-
Experimental
eter RM0 according studies were performed
to the methodology to determine
described in sectionthe relative
2 and 4. Thelipophilicity parameter
tested derivatives
RM0areaccording to thebymethodology
characterized described
rather lower (below 2) RM0in parameters,
Sections 2 and which 4. The
are intested derivatives
the range from are
1.197 to 1.9781.
characterized The drug
by rather lowerprothipendyl
(below 2) R 12,
M0which is a representative
parameters, which are of in 1-azaphenothia-
the range from 1.197
zines, was used as a reference substance in the tests. It can be seen that
to 1.9781. The drug prothipendyl 12, which is a representative of 1-azaphenothiazines, the presence of an was
usedadditional secondsubstance
as a reference nitrogen atom in the
in the phenothiazine
tests. It can be seen structure
that thesubstantially
presence reduces the
of an additional
lipophilicity of these derivatives.
second nitrogen atom in the phenothiazine structure substantially reduces the lipophilicity
Subsequently, a calibration curve was made to determine the absolute lipophilicity
of these derivatives.
parameter logP. In this process, the following reference substances with a known lipo-
Subsequently, a calibration curve was made to determine the absolute lipophilicity
philicity parameter logP were used: acetanilide I, acetophenone II, 4-bromoacetophenone
parameter logP. In this
III, benzophenone IV,process, the following
anthracene V, for whichreference substances
the literature logPlit with
valuesa known lipophilic-
ranged from
ity 1.21
parameter logP were
to 5.53 (Table used: acetanilide I, acetophenone II, 4-bromoacetophenone III,
3) [22,23].
benzophenone IV, anthracene V, for which the literature logPlit values ranged from 1.21 to
5.53 (Table 3) [22,23].
Using the calibration curve equation (Figure 2), the relative lipophilicity parameter
RM0 was converted to the absolute values logP, which are summarized in Table 4. They
are in the range from 1.476 to 2.247 (Table 4). The lowest parameter is characterized by
10H-1,9-diazaphenothiazine 1, and the highest parameter has the derivative 11 containing
a cycloaminoalkyl substituent. However, these values differ from the computer-calculated
parameters, which is also shown in Figure 4. The results obtained depend on the type of
substituent on the thiazine nitrogen atom. When analyzing the lipophilicity in terms of
anticancer activity, it should be noted that the most anticancer-active compounds 4 and 7
have a similar lipophilicity value with the other derivatives, which is in the range from 1.4
 in
inin the
inthethe
the range
range
range
range from
from
from
from 1.476
1.476
1.476
1.476 to
to
to 2.247
to2.247
2.247
2.247 (Table
(Table
(Table
(Table 4).
4). The
4).
4). TheThe
The lowest
lowest
lowest
lowest parameter
parameter
parameter
parameter isisischaracterized
ischaracterized
characterized
characterized by
bybyby10H-
10H-
10H-
10H-
1,9-diazaphenothiazine
1,9-diazaphenothiazine
1,9-diazaphenothiazine
1,9-diazaphenothiazine 1,1,1,1,
and
andand
and thethe the
the highest
highest
highest
highest parameter
parameter
parameter
parameter has
hashas
has the
thethe
the derivative
derivative
derivative
derivative 11
1111
11 containing
containing
containing
containing aaaa
cycloaminoalkyl
cycloaminoalkyl
cycloaminoalkyl
cycloaminoalkyl substituent.
substituent.
substituent.
substituent. However,
However,
However,
However, these
these
these
these values
values
values
values differ
differ
differ
differ from
fromfrom
from the
the the
the computer-calculated
computer-calculated
computer-calculated
computer-calculated
parameters,
parameters,
parameters,
parameters, which
which
which
which isisisalso
isalso
also
also shown
shown
shown
shown inin
inFigure
inFigure
Figure
Figure 4.4.4.4.
The
TheThe
The results
results
results
results obtained
obtained
obtained
obtained depend
depend
depend
depend onon the
on
on the type
the
the typetype
type of
of
ofof
substituent
substituent
substituent
substituent on
on
onon the
thethe
the thiazine
thiazine
thiazine
thiazine nitrogen
nitrogen
nitrogen
nitrogen atom.
atom.
atom.
atom. When
When
When
When analyzing
analyzing
analyzing
analyzing the
the lipophilicity
the
the lipophilicity
lipophilicity
lipophilicity in
in terms
in
in terms
terms
terms of
of
ofof
Int. J. Mol. Sci. 2023, 24, 6970 8 of 11
anticancer
anticancer
anticancer
anticancer activity,
activity,
activity,
activity, itititshould
itshould
should
should be
bebebenoted
noted
noted
noted that
that
that
that the
thethe
the most
most
most
most anticancer-active
anticancer-active
anticancer-active
anticancer-active compounds
compounds
compounds
compounds 444and
4and
and
and 7777
have
havehave
have aaasimilar
asimilar
similar
similar lipophilicity
lipophilicity
lipophilicity
lipophilicity value
value
value
value with
with
with
with the
the other
the
the other
other
other derivatives,
derivatives,
derivatives,
derivatives, which
which
which
which isisisin
isin
in the
in the range
the
the range
range
range from
fromfrom
from 1.4
1.4
1.4
1.4
to
toto 2.3.
to2.3.
2.3.
2.3. This
This is,
This
This is, therefore,
is,
is, therefore,
therefore,
therefore, an
anananindication
indication
indication
indication that
that
that
that the
thethe
the lipophilicity
lipophilicity
lipophilicity
lipophilicity parameter
parameter
parameter
parameter isisisonly
isonly
only
only one
one
one
one of
ofof many
ofmany
many
many
to
factors
factors
factors2.3.affecting
factors This
affecting is, therefore,
affecting
affecting the
the
the
the biological
biological an indication
biological
biological potential.
potential.
potential.
potential. that the lipophilicity parameter is only one of many
factors
ItItItcan
Itcanaffecting
can
can be
be
beconcluded
be the
concluded
concluded
concluded biological
that
thatthat
that the
the
thethepotential.
group
group
group
group of
ofof10-substituted
of10-substituted
10-substituted
10-substituted 1,9-diazaphenothiazines
1,9-diazaphenothiazines
1,9-diazaphenothiazines
1,9-diazaphenothiazines isisismod-
ismod-
mod-
mod-
erately
erately
erately
erately It can be concluded
lipophilic
lipophilic
lipophilic
lipophilic compared
compared
compared
compared that
toto
to the
to
the
the the
the group
isomeric
isomeric
isomeric
isomeric of 1,6-,
10-substituted
1,6-,
1,6-,
1,6-, 1,8-,
1,8-,
1,8-,2,7
1,8-,2,72,7
2,7and
and1,9-diazaphenothiazines
and
and 3,6-diazaphenothiazines
3,6-diazaphenothiazines
3,6-diazaphenothiazines
3,6-diazaphenothiazines is moder-[29–
[29–
[29–
[29–
33].
33].ately
33].
33]. AAAA lipophilic
comparative
comparative
comparative
comparative compared summary
summary
summary
summary to ofthe
ofofof isomeric
selected
selected
selected
selected 1,6-,
derivatives
derivatives
derivatives1,8-, 2,7
derivatives with andthe
with
with
with 3,6-diazaphenothiazines
thethe
the following
following
following
following substituents,
substituents,
substituents,
substituents, [29–33].
hy-
hy-hy-
hy-
A
drogen
drogencomparative
drogen
drogen atomatom
atom
atom (A(A
(A(A summary
derivatives),
derivatives),
derivatives),
derivatives), ofallyl
selected
allyl
allyl
allyl (B(B(B derivatives
derivatives),
(B derivatives),
derivatives),
derivatives), with
propargylthe following
propargyl
propargyl
propargyl (C(C
(C derivatives)
(C substituents,
derivatives)
derivatives)
derivatives) and and
and
and hydrogen
dimethyl-
dimethyl-
dimethyl-
dimethyl-
atom
aminopropyl
aminopropyl
aminopropyl
aminopropyl (A derivatives),
(D(D
(D(D allyl (B
derivatives),
derivatives),
derivatives),
derivatives), isisisderivatives),
ispresented
presented
presented
presented in
ininpropargyl
in
TableTable
Table
Table 9and
999and (Cvisualized
and
and derivatives)
visualized
visualized
visualized and dimethylamino-
graphically
graphically
graphically
graphically in
in
inin
FigureFigure
Figure
Figure
propyl
5.5.5.5. (D derivatives), is presented in Table 9 and visualized graphically in Figure 5.

Table
Table
Table
Table
Table 9.
9.9.9.
9. Comparison
Comparison
Comparisonofof
Comparison
Comparison of
the
of
of the
the
the
the lipophilicity
lipophilicity
lipophilicity
lipophilicity
lipophilicity parameter
parameter
parameter
parameter
parameter logP
logP
logP
logP
logP
TLCTLC
TLCofof
TLC
TLC
of
of
of selected
selected
selected
selected isomeric
isomeric
selectedisomeric diazaphenothiazines.
diazaphenothiazines.
isomericdiazaphenothiazines.
isomeric diazaphenothiazines.
diazaphenothiazines.

1,9-Diaza
1,9-Diaza
1,9-Diaza
1,9-Diaza
1,9-Diaza 1,6-Diaza
1,6-Diaza
1,6-Diaza[30]
1,6-Diaza
1,6-Diaza [30]
[30]
[30]
[30] 1,8-Diaza
1,8-Diaza
1,8-Diaza
1,8-Diaza
1,8-Diaza [31][31] 2,7-Diaza
[31]
[31]
[31] 2,7-Diaza [32,33]
2,7-Diaza
2,7-Diaza
2,7-Diaza [32,33] 3,6-Diaza
[32,33]
[32,33]
[32,33] 3,6-Diaza [30]
3,6-Diaza
3,6-Diaza
3,6-Diaza [30]
[30]
[30]
[30]
RRRR RRRR RRRR RRRR RRRR
Substituent
Substituent
Substituent
Substituent
Substituent NNNN NNNN NNNN NNNN NNNN NNNN NNNN NNNN NNNN
RRRR NNNN NNNN
NNNN NNNN
SSSS NNNN SSSS SSSS SSSS NNNN SSSS
A.
A.
A. HH
A. HH 1.476
1.476
1.476
1.476 2.316
2.316
2.316
2.316 1.759
1.759
1.759
1.759 1.540
1.540
1.540
1.540 0.952
0.952
0.952
0.952
A. H 1.476 2.316 1.759 1.540 0.952
B.B.CH
B.
B. CH
B. CH2CH=CH
CH
CH22CH=CH
2CH=CH
CH=CH
2 CH=CH2
2 22 2 1.682
1.682
1.682
1.682
1.682 3.008
3.008
3.008
3.008
3.008 2.313
2.313
2.313
2.313
2.313 1.940
1.940
1.940
1.940
1.940 1.329
1.329
1.329
1.329
1.329
Int. J. C.
C.C.
Mol.CH
CH
C.
C. CH
CHCH
Sci. 2CCH
2CCH
22CCH
CCH
22023, CCH 1.747
24, x FOR PEER REVIEW1.747
1.747
1.747
1.747 1.574
1.574
1.574
1.574
1.574 2.186
2.186
2.186
2.186
2.186 1.654
1.654
1.654
1.654
1.654 1.342
1.342
1.3429
1.342
1.342 of 12
D.
D. D.
CH
D. CH CH
CH
2 CH
2CH CH
CH CH
D. CH2CH2CH2N(CH3)2
2 2 2 CH
2
2 CH
CH 2 N(CH
2 2
2 N(CH
N(CH
N(CH 3 )
3 )
3
2 )
2
3 )
2 2 2.080
2.080
2.080
2.080
2.080 1.156
1.156
1.156
1.156
1.156 1.832
1.832
1.832
1.832
1.832 1.940
1.940
1.940
1.940
1.940 1.404
1.404
1.404
1.404
1.404

Among
Among
Among
Among the
the 10H-dipyridothiazines
the
the 10H-dipyridothiazines
10H-dipyridothiazines
10H-dipyridothiazines (derivatives
(derivatives
(derivatives
(derivatives A)
A) shown
A)
A) shown
shown
shown asasthe
the
as
as dark
the
the dark
dark
dark blue
blue
blue
blue curve
curve
curve
curve (Fig-
(Fig-
(Fig-
(Fig-
ureComparison
ure ure
ure 5),
5),
5),
5), of lipophilicity values
10H-3,6-diazaphenothiazine
10H-3,6-diazaphenothiazine
10H-3,6-diazaphenothiazine
10H-3,6-diazaphenothiazine isisisthe
istheofleast
the
the selected
least least
least lipophilic
lipophilicisomeric
lipophilic
lipophilic and
and
and
and 10H-1,6-diazaphenothiazine
10H-1,6-diazaphenothiazine
10H-1,6-diazaphenothiazine
10H-1,6-diazaphenothiazine
isisisthe
isthe most
the
the most
most
most lipophilic.
lipophilic.
lipophilic.
lipophilic. AAAsimilar
Asimilar
similar
similar
dipyridothiazines relationship
relationship
relationship
relationship exists
exists
exists
exists for
for derivatives
for
for derivatives
derivatives
derivatives with
with an
with
with an allyl
an
an allyl substituent
allyl
allyl substituent
substituent
substituent
(B)
(B) (B)
(B) (red
(red curve).
(red
(red curve).
curve).
curve). The
The
The
The least
least
least
least lipophilic
lipophilic
lipophilic
lipophilic dipyridothiazine
dipyridothiazine
dipyridothiazine
dipyridothiazine with
with
with
with aaapropargyl
apropargyl
propargyl
propargyl substituent
substituent
substituent
substituent isisisis
3,6-
3,6-
3,6-
3,6-
3.5
diazaphenothiazine
diazaphenothiazine
diazaphenothiazine
diazaphenothiazine and
andand
and the
thethe
the most
most
most
most lipophilic
lipophilic
lipophilic
lipophilic isisisis
1,8-diazaphenothiazine,
1,8-diazaphenothiazine,
1,8-diazaphenothiazine,
1,8-diazaphenothiazine, as
as shown
as
as shown
shown
shown by
bybyby the
the
the
the
3
green
green
green
green curve.
curve.
curve.
curve. Among
Among
Among
Among the
the derivatives
the
the derivatives
derivatives
derivatives with
withwith
with aaadimethylaminopropyl
adimethylaminopropyl
dimethylaminopropyl
dimethylaminopropyl substituent
substituent
substituent
substituent (derivatives
(derivatives
(derivatives
(derivatives
D),
D), D),
D), the
the lowest
the
the lowest
lowest
lowest lipophilicity
lipophilicity
lipophilicity
lipophilicity isisischaracterized
ischaracterized
characterized
characterized bybyby
by 1,6-diazaphenothiazine
1,6-diazaphenothiazine
1,6-diazaphenothiazine
1,6-diazaphenothiazine and
andand
and the
the highest
the
the highest
highest
highest by
byby
by
2.5 1,9-diazaphenothiazine
1,9-diazaphenothiazine
1,9-diazaphenothiazine
1,9-diazaphenothiazine (yellow
(yellow
(yellow
(yellow curve).
curve).
curve).
curve). ItItItis
Itisisalso
isalso
also
also aaaproof
aproof
proof
proof that
that lipophilicity
that
that lipophilicity
lipophilicity
lipophilicity depends
depends
depends
depends on
onon
on both
both
both
both
the
the the
the diazaphenothiazine
diazaphenothiazine
diazaphenothiazine
diazaphenothiazine system
system
system
system and
and
and
and the
the the
the type
typetype
type of
of
of substituent
ofsubstituent
substituent
substituent atat the
atatthe thiazine
the
the thiazine
thiazine
thiazine nitrogen
nitrogen
nitrogen
nitrogen atom.
atom.
atom.
atom.
2

1.5

0.5

0
1 2 3 4 5

Deriv.A Deriv. B Deriv. C Deriv. D

Figure 5.5. Graphical visualization of

Graphical visualization of logP
logPTLC values of the selected derivatives (A–D) of 1,9- 1,6-, 1,8-
Figure TLC values of the selected derivatives (A–D) of 1,9- 1,6-, 1,8-,
, 2,7- and 3,6-diazaphenothiazines [30–33].
2,7- and 3,6-diazaphenothiazines [30–33].

The molecular
Among descriptors of all (derivatives
the 10H-dipyridothiazines 1,9-diazaphenothiazines
A) shown as the were
darkthoroughly analyzed
blue curve (Figure 5),
10H-3,6-diazaphenothiazine is the least lipophilic and 10H-1,6-diazaphenothiazine Veber
against the requirements of the five rules of Lipinski, and the rules of Ghose and is the
(Tablelipophilic.
most 5). All tested
A derivatives meet the exists
similar relationship requirements of the Lipinski
for derivatives with anrule of substituent
allyl five, and of
the (red
(B) Ghose and Veber
curve). rules.
The least These results
lipophilic indicate thatwith
dipyridothiazine the tested 1,9-diazaphenothiazine
a propargyl substituent is 3,6-
derivatives can become
diazaphenothiazine anddrugs and lipophilic
the most specificallyisorally active drugs.
1,8-diazaphenothiazine, as shown by the
greenADME
curve. analyzes
Among thewere performed
derivatives for athe
with tested compounds using
dimethylaminopropyl PreADMET
substituent server
(derivatives
[24]. The results obtained in comparison to the reference compound, prothipendyl
D), the lowest lipophilicity is characterized by 1,6-diazaphenothiazine and the highest 12, turn
by
out to be quite interesting (Table 6). The tested compounds have the blood–brain
1,9-diazaphenothiazine (yellow curve). It is also a proof that lipophilicity depends on both barrier
penetration
the index BBB insystem
diazaphenothiazine the range
andfrom 0.85of
the type tosubstituent
4.55, for most of them
at the significantly
thiazine differ-
nitrogen atom.
ent from that of the descriptors
The molecular reference compound 12 (2.40). The differences
of all 1,9-diazaphenothiazines wereinthoroughly
blood–brain barrier
analyzed
penetration
against appear to depend
the requirements of theonfive
the rules
structure of the tested
of Lipinski, and derivative.
the rules ofCaco-2
Ghosecellandperme-
Veber
ability was different among the tested compounds. All tested compounds exhibited a high
HIA index, which was in the range from 95 to 98. The permeability of MDCK cells was
variable and ranged from 3.73 to 80.31. These values differ significantly in the studied
group of compounds and depend on the type of substituent in the 1,9-diazaphenothiazine
system. Similar differences were observed for the PPB parameter. All test compounds
Int. J. Mol. Sci. 2023, 24, 6970 9 of 11

(Table 5). All tested derivatives meet the requirements of the Lipinski rule of five, and of
the Ghose and Veber rules. These results indicate that the tested 1,9-diazaphenothiazine
derivatives can become drugs and specifically orally active drugs.
ADME analyzes were performed for the tested compounds using PreADMET server [24].
The results obtained in comparison to the reference compound, prothipendyl 12, turn
out to be quite interesting (Table 6). The tested compounds have the blood–brain barrier
penetration index BBB in the range from 0.85 to 4.55, for most of them significantly different
from that of the reference compound 12 (2.40). The differences in blood–brain barrier pene-
tration appear to depend on the structure of the tested derivative. Caco-2 cell permeability
was different among the tested compounds. All tested compounds exhibited a high HIA
index, which was in the range from 95 to 98. The permeability of MDCK cells was variable
and ranged from 3.73 to 80.31. These values differ significantly in the studied group of
compounds and depend on the type of substituent in the 1,9-diazaphenothiazine system.
Similar differences were observed for the PPB parameter. All test compounds have a very
similar and substantially low SP parameter compared to the reference compound.
The ADME parameters (BBB, Caco-2, HIA, MDCK, PPB, SP) determined in silico
were correlated with the relative lipophilicity parameter RM0 (Table 8). Correlations were
obtained as polynomial equations of the second and third degree with the r values in the
range from 0.6205 to 0.8616. The obtained results are an indication that lipophilicity is one
of many factors that can directly affect the biological activity. The obtained correlations also
show that lipophilicity is dependent on the conformation of molecules, hydrogen bonds,
ionic interactions as well as interactions related to van der Waals forces.
Using the SwissADME server [21], we performed an analysis of the relationship
between the lipophilicity of the tested 1,9-diazaphenothiazines 1–11 and their polarity.
The BOILED-Egg method was used, which estimates permeation through the brain and
intestines. This analysis is shown in Figure 3. All tested compounds fall within the range of
good permeability across the blood–brain barrier (BBB) as indicated by the yellow region
and normal binding to human blood albumin (HIA) by the white region. In addition,
derivatives 1, 2, 4–7, 9–11 that can become p-glycoprotein substrates (blue points) and two
derivatives 3, 8 that give a negative result (red points) are indicated.
At the latest stage, the determination of molecular targets was performed using the
SwissTargetPrediction server (Table 8) in order to confirm their anticancer potential. The
group of 10-substituted 1,9-diazaphenothiazines may affect the activity of the family CG
and AG protein, kinase, protease, and cytochrome 450. The obtained results are promising
and are expected to inspire further research at the in vitro level.

4. Materials and Methods

4.1. Reagents
10-Substituted 1,9-diazaphenothiazines 1–11 were obtained in the reactions described re-
cently [16]. The reference compound prothipendyl (chemical name: 10-dimethylaminopropyl-1-
azaphenothiazine) 12 (AWD Pharma Germany) was used.
The following reagents were used in the experimental studies to prepare the mobile
phase: acetone, (POCh, Gliwice, Poland), TRIS (tris (hydroxymethyl) aminomethane,
Fluka). In order to prepare the calibration curve, five chemical compounds with the
described lipophilicity parameter (logPlit .) were used: acetanilide (POCh, Gliwice, Poland),
acetophenone (POCh, Gliwice, Poland), 4-bromoacetophenone (Fluka, Buchs, Switzerland),
benzophenone (Fluka, Buchs, Switzerland), antracene (POCh, Gliwice, Poland).

4.2. Chromatographic Procedure

The RP-TLC method was used to determine the experimental lipophilicity according
to the literature [30–33]. Modified silica gel RP 18F254S (Merck) was used as the stationary
phase and a mixture of tris(hydroxymethyl)aminomethane (TRIS) (0.2 M, buffer pH = 7.4)
with acetone as the mobile phase with the range of concentrations from 40 to 70% (v/v)
in 5% increments. The compounds 1–12 and the standards I-V were dissolved in ethanol
Int. J. Mol. Sci. 2023, 24, 6970 10 of 11

(2.0 mg/mL) and 2 µL of each solution was spotted. Spots were observed under UV light
at λ = 254 nm. Each measurement was performed in triplicate and then the RF values
were calculated.

4.3. Theoretical Lipophilicity and ADMET Parameters, Target Prediction

The calculated lipophilicity was determined using various internet servers: VC-
CLAB [20] and SwissADME [21] including: Alogps, AC_Logp, ALOGP, MLOGP, XLOGP2,
XLOGP3, ILopP, XlogP, WlogP, MlogP, SILICOS-IT. The molecular descriptor and ADME
parameters were calculated using SwissADME and PreADME server [21,24]. Target predic-
tion was determined by the SwissTargetPrediction server [26].

5. Conclusions
The presented research results show the lipophilicity of the group of 10-substituted
1,9-diazaphenothiazines showing high anticancer potential, associated with the disruption
of the function of the p53 protein and the proper functioning of histones, which was
documented in previous studies. Lipophilicity as a parameter determining the reaching
of the molecules to the biological target was determined theoretically by computational
methods, and experimentally using reverse-phase thin-layer chromatography (RP-TLC).
The compounds tested were moderately lipophilic compared to the previously reported
1,6-, 1,8-, 2,7-, and 3,6-diazaphenothiazine derivatives with analogous substituents. In addi-
tion, ADME parameters that are indisputably related to lipophilicity have been determined.
The new derivatives followed the rules of Lipinski, Ghose, and Veber, indicating that they
may become oral drugs in the future. Further studies of this group of compounds in order
to fully determine the pharmacological potential of these derivatives have been planned.

Author Contributions: B.M.-M. developed the concept of the work, carried out the synthetic work,
interpreted the results, wrote, and edited original draft. M.J., E.M. contributed to the synthesis and
purification of selected compounds, formal analysis, and edited the manuscript. R.K. contributed
to formal analysis and edited the manuscript. All authors have read and agreed to the published
version of the manuscript.
Funding: This research was funded by The Medical University of Silesia in Katowice, grant PCN-1-
041/K/2/F and PCN-1-043/K/2/F.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Not applicable.
Conflicts of Interest: The authors declare no conflict of interest.
Sample Availability: Samples of the compounds 1–12 are available from the authors.

References
1. Constantinescu, T.; Lungu, C.N.; Lung, I. Lipophilicity as a Central Component of Drug-Like Properties of Chalchones and
Flavonoid Derivatives. Molecules 2019, 24, 1505. [CrossRef] [PubMed]
2. Giaginis, C.; Tsopelas, F.; Tsantili-Kakoulidou, A. The Impact of Lipophilicity in Drug Discovery: Rapid Measurements by Means
of Reversed-Phase HPLC. In Rational Drug Design. Methods in Molecular Biology; Mavromoustakos, T., Kellici, T.F., Eds.; Springer:
New York, NY, USA, 2018; Volume 1824, pp. 217–228.
3. Kamel, M.S.; Belal, A.; Aboelez, M.O.; Shokr, E.K.; Abdel-Ghany, H.; Mansour, H.S.; Shawky, M.A.; Abd El Aleem Ali Ali
El-Remaily, M. Microwave-Assisted Synthesis, Biological Activ-ity Evaluation, Molecular Docking, and ADMET Studies of Some
Novel Pyrrolo [2,3-b] Pyrrole Derivatives. Molecules 2022, 27, 2061. [CrossRef]
4. Erckes, V.; Steuer, C. A story of peptides, lipophilicity and chromatography—Back and forth in time. RSC Med. Chem. 2022, 22,
676–687. [CrossRef] [PubMed]
5. Ginex, T.; Vazquez, J.; Gilbert, E.; Herrero, E.; Luque, F.J. Lipophilicity in drug design: An overview of lipophilicity descriptors in
3D-QSAR studies. Future Med. Chem. 2019, 11, 1177–1193. [CrossRef]
6. Kempińska, D.; Chmiel, T.; Kot-Wasik, A.; Mróz, A.; Mazerska, Z.; Namieśnik, J. State of the art and prospects of methods for
determination of lipophilicity of chemical compounds. Trends Anal. Chem. 2019, 113, 54–73. [CrossRef]
Int. J. Mol. Sci. 2023, 24, 6970 11 of 11

7. Dołowy, M.; Jampilek, J.; Bober-Majnusz, K. A comparative study of the lipophilicity of metformin and phenformin. Molecules
2021, 26, 6613. [CrossRef] [PubMed]
8. Manto Chagasa, C.; Mossa, S.; Alisaraie, L. Drug metabolites and their effects on the development of adverse reactions: Revisiting
Lipinski’s Rule of Five. Int. J. Pharm. 2018, 549, 133–149. [CrossRef]
9. Lobo, S. Is there enough focus on lipophilicity in drug discovery? Expert Opin. Drug Discov. 2020, 15, 261–263. [CrossRef]
10. Piir, G.; Kahn, I.; García-Sosa, A.T.; Sild, S.; Ahte, P.; Maran, U. Best Practices for QSAR Model Reporting: Physical and Chemical
Properties, Ecotoxicity, Environmental Fate, Human Health, and Toxicokinetics Endpoints. Environ. Health Perspect. 2018, 126, 126001.
[CrossRef]
11. Tsaioun, K. Evidence-based absorption, distribution, metabolism, excretion (ADME) and its interplay with alternative toxicity
methods. ALTEX 2016, 33, 343–358. [CrossRef]
12. Halder, A.K.; Moura, A.S.; Cordeiro, M.N.D.S. QSAR modelling: A therapeutic patent review 2010-present. Expert Opin. Ther. Pat.
2018, 28, 467–476. [CrossRef] [PubMed]
13. Alqahtani, M.S.; Kazi, M.; Alsenaidy, M.A.; Ahmad, M.A. Advances in Oral Drug Delivery. Front. Pharm. 2021, 19, 618411.
[CrossRef]
14. Kathuria, H.; Handral, H.K.; Cha, S.; Nguyen, D.T.P.; Cai, J.; Cao, T.; Wu, C.; Kang, F. Enhancement of Skin Delivery of Drugs
Using Proposome Depends on Drug Lipophilicity. Pharmaceutics 2021, 13, 1457. [CrossRef] [PubMed]
15. Han, S.; Mei, L.; Quach, T.; Porter, C.; Trevaskis, N. Lipophilic Conjugates of Drugs: A Tool to Improve Drug Pharmacokinetic
and Therapeutic Profiles. Pharm. Res. 2021, 38, 1497–1518. [CrossRef]
16. Morak-Młodawska, B.; Pluta, K.; Latocha, M.; Jeleń, M.; Kuśmierz, D.; Suwińska, K.; Shkurenko, A.; Czuba, Z.; Jurzak, M.
10H-1,9-diazaphenothiazine and its 10-derivatives: Synthesis, characterisation and biological evaluation as potential anticancer
agents. J. Enzyme Inhib. Med. Chem. 2019, 34, 1298–1306. [CrossRef]
17. Ahmed, S.A.; Kamel, M.S.; Aboelez, M.O.; Ma, X.; Al-Karmalawy, A.A.; Mousa, S.A.S.; Shokr, E.K.; Abdel-Ghany, H.; Belal, A.;
El Hamd, M.A.; et al. Thieno[2,3-b]thiophene Derivatives as Potential EGFRWT and EGFRT790M Inhibitors with Antioxidant
Activities: Microwave-Assisted Synthesis and Quantitative In Vitro and In Silico Studies. ACS Omega 2022, 7, 45535–45544.
[CrossRef]
18. Elkanzi, N.A.A.; Kadry, A.M.; Ryad, R.M.; Bakr, R.B.; Abd El Aleem Ali Ali El-Remaily, M.; Ali, M.A. Efficient and Recoverable
Bio-Organic Catalyst Cysteine for Synthesis, Docking Study, and Antifungal Activity of New Bio-Active 3,4-Dihydropyrimidin-
2(1H)-ones/thiones Under Microwave Irradiation. ACS Omega 2022, 26, 22839–22849. [CrossRef] [PubMed]
19. Morak-Młodawska, B.; Pluta, K.; Jeleń, M. Phenothiazines Modified with the Pyridine Ring as Promising Anticancer Agents. Life
2021, 11, 206. [CrossRef]
20. Available online: http://www.vcclab.org (accessed on 20 November 2022).
21. Available online: http://swissadme.ch (accessed on 11 November 2022).
22. Bodor, N.; Gabanyi, Z.; Wong, C.K. A new method for the estimation of partition coefficient. J. Am. Chem. Soc. 1989, 111,
3783–3786. [CrossRef]
23. Mannhold, R.; Cruciani, G.; Dross, K.; Rekker, R. Multivariate analysis of experimental and computational descriptors of
molecular lipophilicity. J. Comput. Mol. Des. 1998, 12, 573–581. [CrossRef]
24. Available online: http://preadmet.bmdrc.org (accessed on 22 September 2022).
25. Daina, A.; Zoete, V. A BOILED-Egg To Predict Gastrointestinal Absorption and Brain Penetration of Small Molecules. ChemMed-
Chem 2016, 6, 1117–1121. [CrossRef] [PubMed]
26. Available online: http://www.swisstargetprediction.ch/ (accessed on 14 November 2022).
27. Morak-Młodawska, B.; Jeleń, M. Lipophilicity and Pharmacokinetic Properties of New Anticancer Dipyridothiazine with
1,2,3-Triazole Substituents. Molecules 2022, 27, 1253. [CrossRef] [PubMed]
28. Morak-Młodawska, B.; Pluta, K.; Jeleń, M. Evaluation of the Lipophilicity of New Anticancer 1,2,3-Triazole-Dipyridothiazine
Hybrids Using RP TLC and Different Computational Methods. Processes 2020, 8, 858. [CrossRef]
29. Kadela-Tomanek, M.; Jastrz˛ebska, M.; Chrobak, E.; B˛ebenek, E. Lipophilicity and ADMET Analysis of Quinoline-1,4-
quinoneHybrids. Pharmaceutics 2023, 15, 34. [CrossRef]
30. Morak-Młodawska, B.; Pluta, K.; Jeleń, M. Lipophilicity of New Anticancer 1,6- and 3,6-diazaphenothiazines by of Use RP TLC
and Different Computational Methods. J. Chrom. Sci. 2018, 56, 376–381. [CrossRef]
31. Morak-Młodawska, B.; Pluta, K.; Jeleń, M. Estimation of the Lipophilicity of New Anticancer and Immunosuppressive 1,8-
Diazaphenothiazine Derivatives. J. Chrom. Sci. 2015, 53, 462–466. [CrossRef]
32. Morak-Młodawska, B.; Pluta, K.; Latocha, M.; Jeleń, M.; Kuśmierz, D. Synthesis and anticancer and lipophilic properties of 10-
dialkylaminobutynyl derivatives of 1,8- and 2,7-diazaphenothiazines. J. Enzym. Inhib. Med. Chem. 2016, 31, 1132–1138. [CrossRef]
33. Morak, B.; Nowak, M.; Pluta, K. Determination of the Lipophilicity Parameters RM0 and Log P of New Azaphenothiazines by
Reversed-Phase Thin-Layer Chromatography. J. Liq. Chromatogr. Relat. Technol. 2007, 30, 1845–1854. [CrossRef]

Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual
author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to
people or property resulting from any ideas, methods, instructions or products referred to in the content.