PHYSICOCHEMICAL
CHARACTERIZATION
OF
TWO
NEW
NITAZOXANIDE ANALOGS WITH ANTIPARASITIC ACTIVITY.
Adriana Valladares-Méndez,1,3 Mirna García-Flores,1,3 Gabriel Navarrete-Vázquez,1 Luis M.
Orozco-Castellanos,2 Emanuel Hernandez-Nuñez,4 Julio C Rivera-Leyva.1,2 *
1
Facultad de Farmacia, Universidad Autónoma del Estado de Morelos, 62209, Cuernavaca Morelos,
México.
2
Departamento de Farmacia, Universidad de Guanajuato 36050 Guanajuato, Guanajuato, México.
3
RGV Analitica Industrial Research Laboratory 62733 Yautepec, Morelos, México.
4
Departamento de Recursos del Mar, Centro de Investigación y de Estudios Avanzados del IPN,
Unidad Mérida, 97310, Yucatán, México.
Corresponding Author:
E-mail. julio.rivera@uaem.mx
1
Facultad de Farmacia, Universidad Autónoma del Estado de Morelos, Av. Universidad 1001, col
Chamilpa, CP 62209, Cuernavaca Morelos, México.
Tel 00-52-777 39 7089, ext 7136
3
RGV Analitica Industrial Research Laboratory, Tulipanes 17, Col. Jovito Serrano, 62733 Yautepec,
Morelos, México.
Tel. 00-52-735 394 0586
1. ABSTRACT
The low oral bioavailability of drugs is a consequence of low solubility, low permeability, and low
dissolution rate in the gastrointestinal tract (GIT). For this reason, it is important to evaluate
bioavailability in the early stages of drug discovery and development. In this work, some
physicochemical properties of two new Nitazoxanide analogs (Compound 1 and Compound 2) were
evaluated. Both compounds showed limited aqueous solubility in the pH range of GIT (16 and 18
g/ml respectively) considering the physiological interval, log P values were 1.01 and 1.44, for
Compound 1 and Compound 2 respectively. These values indicate slight affinity by lipid components.
The compounds have a thiazole ring with basic character and an amide group with acidic character,
therefore they present ampholytic behavior; their pKa values were 6.029 and 9.565 for Compound 1,
and 4.945 and 8.127 for Compound 2. The apparent permeability (Papp) was evaluated using the everted
rat intestine, and the quantification was realized with a validated HPLC method with UV-Vis
absorbance. The results showed that the permeability was limited for both compounds (1.15 x 10-5 cm/s
for Compound 1 and 9.09 x 10-7 cm/s for Compound 2) compared to furosemide (1.16 x 10-5), the low
permeability standard. These new compounds were classified as low permeability and low solubility
(Class IV) according to Biopharmaceutical Classification System (BCS).
Keywords: Nitazoxanide analogs, physicochemical characterization, apparent permeability, aqueous
solubility, biopharmaceutical properties.
2. INTRODUCTION
During the early stages of drug development, it is necessary to evaluate the biological efficacy in in
vitro models, as well as their pharmacological, toxicological and pharmacokinetic properties in animal
models 1. The development of pharmaceutical products for oral administration requires a thorough
knowledge of their biopharmaceutical properties because they determine dissolution rate, intestinal
permeability, gastrointestinal transit, first pass effect, and plasma concentration of the drug2. The
evaluation of solubility is very important for the process of drug candidate molecule selection 3, since a
limited solubility could significantly decrease absorption through the intestinal barrier, the arrival at the
systemic circulation and, consequently, decreased efficacy. The evaluation of the pH-dependent
solubility profile, pKa, partition coefficient (Log P), apparent permeability, and dissolution rate of
bioactive molecules would provide information about their potential absorption 4. Nitazoxanide (NTZ,
2-acetyloxy-N-(5-nitro-2-thiazolyl) benzamide), is a prodrug derived from nitrothiazole 5, related
structurally to metronidazole (MTZ), with a broad activity spectrum of antiprotozoal, anthelmintic and
antimicrobial effect
67
; after oral administration, NTZ is metabolized in the blood by plasmatic
esterases to their primary metabolite, tizoxanide (TZ, 2-hydroxy-N-(5-nitro-2-thiazolyl) benzamide)
and a TZ glucuronide (GTZ) 8. The activity of NTZ over bacteria and anaerobic parasites is produced
by interference in energy production during the transference of electrons in the enzyme pyruvate
ferredoxin oxidoreductase (POFR)910. NTZ is practically insoluble in water (7.55 x 10-3 g/ml)11,
soluble in organic solvents as dimethyl sulfoxide (DMSO) and dimethyl formamide (DMF); NTZ and
TZ have an approximate solubility of 0.1 mg/ml in a solution 1:10 of DMSO:Phosphate Buffer pH 7.2.
1213
.
The effect of food on drug absorption is well documented. In the case of propranolol,
propoxyphene, nitrofurantoin and phenytoin, an increment in absorption rate has been observed, and it
could explain the modification of pharmacokinetic parameters
14
. The oral administration of NTZ is
done in tablets and suspension. It has been observed that the presence of food modifies the
pharmacokinetics of TZ. A dose of 1000 mg in a tablet can increase Cmax by 29 % and the area under
the curve (AUC) by 50 % in fed versus fasting conditions. When NTZ is administered in suspension,
the presence of food can increase the AUC by 40-50 % and Cmax by 10 % 15.
Hoffman et al. showed with 1H RMN analysis that in DMSO-d6 at pH close to 6, the amine group of
NTZ predominates in the anionic form (NTZ-), while the neutral species is present in acid conditions 9.
An experimental pKa of 6.18 corresponding to the deionization of amide group was determined, and it
would explain the low solubility of NTZ in pH interval of 0 to 6, where the neutral species exist,
whereby the absorption is limited, decreasing their bioavailability significantly.
In 2009 and 2015, Chan-Bacab et al.
16
and Scior et al.
17
, designed, synthesized and evaluated the
antiparasitic activity of 2 new NTZ analogs: 4-nitro-N-(5-nitro-1,3-thiazol-2-yl)benzamide (Compound
1) and N-(5-nitrothiazol-2-yl)cinnamamide (Compound 2). Their design was based on removing the
acetoxy group and introducing an additional nitro group (NO2) to Compound 1, and a cinnamic
conjugated double bond to Compound 2. Both compounds kept the pharmacophoric group 2-amino-5nitrothiazole because it is considered that this group is responsible for antiparasitic activity 18919 (Figure
1). These compounds were tested in vitro against some parasites responsible for diarrheal diseases:
Giardia lamblia
20
, Entamoeba histolytica, the parasite that causes Chagas disease, Trypanosoma
cruzi, and the parasite responsible for skin diseases in Mexico, Leishmania mexicana 16.
Compound 1, NTZ and TZ decreased the rate of growth of T. cruzi and L. mexicana with respect to the
conventional treatment of beznidazol and pentamidine; Compound 1 was 2 fold more active than NTZ
and TZ, inhibiting the growth of T. cruzi and L. mexicana, and 6 fold more active than pentamidin
against L. mexicana (IC50 = 2.53 µM). Compound 1 has an activity similar to that of NTZ and TZ
against T. cruzi and it was 2 fold more active than beznidazole (IC50 = 15.02 µM)16.
Compound 2 showed an activity against T. vaginalis similar to NTZ (IC50=0.069 µM) that was 2 fold
higher than TZ, and 4 fold higher than metronidazole (results not reported, personal communication).
This compound presented an IC50 similar to NTZ against G. intestinalis (0.970 M and 1.21 M
respectively), and it was 5 fold more active than metronidazole (IC50 = 5.36 M). Besides, compound 2
had an amoebicidal effect, and the activity was comparable to NTZ with an IC50 = 0.45 M) 17.
The primary objective of this project was to evaluate the physicochemical properties with
biopharmaceutical relevance of 2 new NTZ analogs, Compound 1 and Compound 2 because they are
considered excellent candidates for antiparasitic drugs.
3. MATERIALS AND METHODS
DMSO, n-octanol, naproxen and furosemide (used as permeability standards) were purchased from
Sigma-Aldrich (Steinheim, Germany), methanol grade HPLC, sodium hydroxide, phenolphthalein, and
potassium biphthalate were obtained from J.T. Baker (J. T. Baker, Co. USA). Other reagents and salts
used were purchased from Fermont (Productos Químicos, Monterrey, S.A. México). Compounds 1 and
2 were synthesized in the Medicinal Chemistry Laboratory of the Pharmacy Faculty, Universidad
Autónoma del Estado de Morelos (UAEM), Mexico. Simulated Intestinal Fluid (SIF) without enzymes
was prepared as indicated in the USP 35.
3.1 Chromatographic conditions.
The two compounds identified in section 2 were analyzed using a Shimadzu chromatographic system
equipped with an auto-sampler SIL-10A, an isocratic pump model LC-10A, a UV-Vis detector model
SPD-10A and software Class VPTM 7.4 SP4. Table 1 shows the chromatographic conditions employed
for the analysis of each compound under study, including the naproxen and furosemide (high and low
permeability standards respectively)21.
3.2 Analytical Methods Validation
To validate analytical methods, specificity, linearity, repeatability, accuracy and detection and
quantification limits were evaluated following Mexican Official Standard NOM-177-SSA1-1998 and
ICH Q2 guide 22 23.
Specificity
Specificity evaluation was carried out using SIF blank solutions and SIF samples obtained from everted
rat intestine and that were injected into the HPLC to assess the extent to which endogenous constituents
might interfere at the retention time of evaluated analytes.
Linearity
To determine the linearity, standard solutions covering the analytical range in every method were
prepared. A stock solution was prepared in DMSO (1 mg/ml) of each compound and then diluted to
100 µg/ml with methanol. Thus, solutions of each compound with different concentrations were
prepared by diluting different volumes using SIF as a solvent. The curves were prepared in triplicate.
Precision and Accuracy
Assay precision of each evaluated compound was assessed by calculating the variability of control
samples with five replicates (n=5). The relative standard deviation (%RSD) was calculated with
%𝑅𝑆𝐷 =
𝑆𝐷
𝑀
𝑥 100……Equation 1
where M is the mean of the experimentally determined concentration of control samples analyzed in a
day and SD is the standard deviation of M. Accuracy was calculated by determining the relative
recovery (% RE) using the formula
𝐶
%𝑅𝐸 = 𝑇 𝑥 100 …….Equation 2
where C is the experimentally determined concentration and T is the theoretical concentration, and
absolute standard deviation (ASD) with
𝐴𝑆𝐷 = |100 − %𝑅𝐸| …..Equation 3
LOD and LOQ.
LOD and LOQ were evaluated based on the standard deviation of the response (Sb0) and the slope of
calibration curves (m). Equations 4 and 5 were used 23.
𝐿𝑂𝐷 =
𝐿𝑂𝑄 =
3.3∗𝑆𝑏°
……Equation 4
𝑚
10∗𝑆𝑏°
𝑚
……Equation 5
3.3 pKa determination by potentiometric titration
Before experimentation, the potentiometer was calibrated using standard aqueous buffers of pH 4.01,
7.0, and 10.0. Titration was carried out at 25 ± 0.5 °C using 0.01 M NaOH solutions. The Ionic strength
of solutions was maintained by using 0.02 M potassium chloride solution. Sample preparation: 0.001
equivalents of compounds 1 and 2 were dissolved individually in 100 ml of 0.02 M potassium chloride
solution. The titration was carried out in small aliquots with the help of standardized NaOH solution,
and pH-titration curves were obtained, under constant stirring of solutions. NaOH was added in small
volumes starting with 0.1 ml and up to 0.5 ml near the end point, and the pH was measured after each
addition using the Logger Pro software. Then, the pKa was calculated at pH of half neutralization
point. Each sample was titrated in triplicate, and the mean of the three readings was used in the
calculation 24.
3.4 Partition coefficient25
The octanol–water partition coefficient was determined using the traditional shake-flask technique. To
2 ml of a buffer solution (pH 7.4) containing 50 µg/ml of each compound, 2 ml of n-Octanol,
previously saturated with buffer solution, were added. The mixture was shaken for 40 min, centrifuged
at 8000 g for 10 min, and the aqueous, and organic phases were separated. Samples were analyzed by
HPLC as indicated in section 3.1. The partition coefficient (log P) was calculated according to the
equation 6:
[𝐶𝑜𝑚𝑝𝑜𝑢𝑛𝑑]𝑜𝑟𝑔
𝐿𝑜𝑔 𝑃 = 𝑙𝑜𝑔 [𝐶𝑜𝑚𝑝𝑜𝑢𝑛𝑑 ]𝑎𝑞 ……Equation 6
where [Compound] org is the drug concentration in the organic phase and [Compound] aq is the drug
concentration in the aqueous phase. Every experiment was evaluated by quintuplicate. In addition, Log
P in silico values were calculated with Molinspiration, ACD/LAB, ChemDraw Ultra and Osiris
Softwares.
3.5 Solubility profile pH-dependent
The solubility of the compounds was evaluated in the pH interval of 1 to 8 using phosphate buffer
solutions (0.01 M), 10 mg of each test compound was placed in Eppendorf microtubes with 1 ml of
each buffer solution. All tubes were maintained with constant agitation for 72 h at 37 °C, reaching
equilibrium. Filtered samples were analyzed by HPLC. Every experiment was evaluated in triplicate.
3.6 Permeability Assay
A Wistar male rat (200-250 g) was used in every evaluation. The first segment of the small intestine
was extracted (10 cm approximately) and washed with saline solution (NaCl 0.9 %) at 37 °C, the lipoid
extensions were removed and the intestine segment was invested with a handle. The intestinal sac was
tied at the top end of a cannula connected to a sample syringe, and then the lower end was tied,
introducing the prepared segment of intestine into a glass chamber containing simulated intestinal fluid
(SIF) at 37 ± 0.5°C. Oxygen was bubbled with a pump to maintain viable tissue 26.
The everted intestinal sac was filled with 1.5 ml of SIF solution; while outside the everted sac a test
compound solution (50 µg / ml) remained. The permeability standard, naproxen and furosemide (high
and low permeability respectively), were prepared in the same way. Samples of 300 µL were collected
from inside the sac with a metallic cannula at 5, 10, 15, 30, 45, 60, 90, and 120 min (Transport lumen
to serosal direction L-S), and a medium deposition was made. Similarly, an experiment with no everted
sac was used to evaluate the transport in the serosal to the luminal direction (S-L). An initial and final
sample of the exterior solution was taken. The samples were kept refrigerated until analysis by HPLC.
The permeability standards, naproxen and furosemide (high and low permeability respectively), were
prepared in the same way 27.
The apparent intestinal permeability (Papp) of test compounds was calculated according to equation 7:
𝑉
𝑃𝑎𝑝𝑝 = (𝐴∗𝐶𝑜) 𝑑𝐶/𝑑𝑡……Equation 7
where Papp= Apparent permeability (cm/s), V = Intestinal Volume (ml), A = Superficial area (cm2),
dC/dt = Concentration vs time gradient, Co = Initial concentration (µg/ml)
In all studies with animals, the international recommendations of biomedical research in animal
handling
28
and current Mexican Official Standard NOM-062-ZOO-1999 on the management of
laboratory animals were followed 29.
4. RESULTS AND DISCUSSION
4.1 Analytical Methods Validation
Specificity
In Figure 2, we may see the typical chromatograms of analyzed compounds where the corresponding
signals do not show interference with another signal. Blank runs of SIF solutions and SIF samples
obtained from the everted sac were completely free of interferences. The retention time for Compound
1 and Compound 2 was 4.66 min and 5.2 min, respectively.
Linearity
Table 2 summarizes the quantification intervals of analytical methods for evaluating new compounds,
as well as the equation of the straight line of calibration curves.
It is possible to observe that in each quantification method, the regression coefficient is higher than
0.99 (r >0.99), which indicates that the methods fulfill the requirements of linearity.
Repeatability and Accuracy
Table 3 presents the repeatability and accuracy evaluations of quantification methods for compounds
under study, and the variability was below 6.16 %. The results allowed us to conclude that the methods
are repeatable. Likewise, each method showed a relative recovery higher than 91.4%, and an absolute
standard deviation of 8.57 %.
Limit of Detection (LOD) and Limit of Quantification (LOQ)
In Table 3, we may appreciate the LOD and LOQ in the concentrations interval used in each analytical
method under analysis conditions established. Each LOQ of each quantification curve is close to their
lowest concentration, and they showed good accuracy and precision. LOD in all methods varies
between 0.06 to 0.75 g/ml.
4.2 Chemical Properties
4.2.1 pKa Determination
In Table 4 we show the pKa values determined for evaluated compounds. Both new compounds
contain a thiazol group in their structure, which has basic behavior and whose pKa value is 6.029 for
Compound 1 and 4.954 for Compound 2, values are very similar to 2-aminothiazole (5.28)
30
.
Previously it has been reported that alkylic substituents increase the value of pKa. Compounds 1 and 2
present a nitro group in 5 position, and it could be affecting the protonation grade in the thiazole ring 30.
In the same way, both compounds have an amide group on position 2, whose proton has acidic
character. The corresponding values of ionization of this functional group are 9.565 for Compound 1
and 8.127 for Compound 2.
A second nitro group in Compound 1 could produce an inductive effect that increases the basic
character of thiazolic ring and also decreases the acid character of the amide group, and how it is
observed in Compound 2. The dissociation equilibrium and speciation diagrams corresponding to the
compounds evaluated are presented in Figure 3.
Both compounds behave as ampholytes, and their neutral species are present in the pH interval 5.0 to
8.0 (physiological interval), where both compounds have the highest probability to be absorbed in the
small intestine. The neutral form for Compound 1 predominates in pH ranging 5 to 7, so its absorption
is favored in the final segment of the duodenum and ileum. To Compound 2, their neutral form
predominates on the pH interval of 4-6, indicating that the absorption would be favored in the duodenal
segment.
4.2.2 Partition Coefficient (Log P)
Table 4 shows the theoretical and experimental values of Log P for the compounds studied. All
calculated values of Compound 1 are in the range 1.5 to 2.0, with an experimental value of 1.0. The
values corresponding to Compound 2 range from 0.78 to 2.8, with an experimental value of 1.44. The
estimated values indicate that Compound 2 has a higher lipophilic character than Compound 1,
although both compounds have a high affinity for lipophilic environments. This difference could be
related directly to the presence of an additional double bond and the absence of the nitro group in the
substituent of Compound 2, making it less polar than Compound 1. Both compounds meet the Lipinski
rules, and thus it could predict a high degree of absorption 4, although the solubility could be a limiting
parameter.
4.2.3 Solubility
Figure 4 shows the pH-dependent aqueous solubility profiles of new NTZ analogs.
It can be seen that in the range pH 0-4, Compound 1 shows the highest value of solubility (14 µg/ml
approximately, Fig. 7a), whereas in pH 5 to 9 it is less soluble (5 g/ml).
Compound 2 shows the highest solubility in pH interval from 1.5 to 2.5 (17.60 µg/ml), while the pH
ranging from 4 to 6.8 (duodenal pH), a decreased solubility is observed (2.0 µg/ml). The highest
solubility of both compounds is in the pH interval of 1 to 4. However, in the highest pH values,
solubility decreases significantly.
Overall, the studies of the characterization of physical and chemical parameters suggest that both
compounds are very slightly dissolved in aqueous media. The lower solubility was found for pH values
higher than 5. For their Log P values, it is observed that the compounds have the capacity to cross the
cellular membrane in a very limited way, which may be affecting the plasmatic concentrations after
oral administration 4.
4.2.4 Apparent Permeability (Papp)
Figure 5 shows the comparative transport of the compounds tested and the permeability standards
(furosemide and naproxen respectively) across the everted rat intestine in both the luminal-serosal (LS) and serosal-luminal (S-L) directions. The apparent permeability and the comparative ratio, with
respect to furosemide of all evaluated compounds, can be found in Table 5. In L-S transport, naproxen
has a permeation rate higher than furosemide (1.33 fold). Compound 1 shows a similar permeation to
furosemide (0.99) while Compound 2 shows a 10-fold lower permeability.
These results show that Compounds 1 and 2 have a low permeability, because their respective value is
similar to furosemide, low permeability standard (Class IV), according to the Biopharmaceutical
Classification System.
The in situ rat intestinal perfusion model is a commonly used technique for evaluating the permeability
of drugs and new chemical entities
31
. Human perfusion studies have shown an excellent correlation
with a rat in situ intestinal permeability for a variety of compounds
32
. Amidon et al.
33
also reported
that the permeability results of nine drugs used in rat in situ model were well correlated with humans
with regards to whether or not drugs were subject to active uptake or absorbed by simple passive
diffusion
34
. The S-L transport can be appreciated in Figure 8b. The permeability of each evaluated
compound is lesser than L-S transport, and this determination indicates that the absorption is favored in
L-S direction. The results observed in our experiments show that new NTZ analogs have a low
permeability in the rat everted intestinal sac model, despite having a log P greater than 1. We can
conclude that the Compounds 1 and 2 will have absorption problems if they are administered orally.
The observed low permeability values may be caused by low solubility and interactions between
transport systems in L-S, and could explain the difference between L-S and S-L transports.
5. CONCLUSIONS
The analytical methods for Compounds 1 and 2 were adequate because they fulfilled selectivity,
linearity, repeatability, and accuracy criteria, and they allowed us to quantify the molecules under study
in Log P, solubility, and apparent permeability assays.
Compounds 1 and 2 showed low solubility in the physiological pH interval (pH 4-8), and they could
have disposition problems for absorption during GIT transit. Compound 1 will be in the neutral form in
pH interval highest to 5, favoring the absorption process in ileum; however it would have a low
solubility at the same pH interval, diminishing their availability for absorption. Compound 2 presents
the same behavior as Compound 1, and it shows similar problems for absorption process in the
duodenal tract. Data of log P and Papp in the rat everted intestinal sac indicate that these compounds
should be absorbed in vivo although some active transport mechanism might be involved. In addition,
it would be of value to determine their pharmacokinetic behavior in other species to establish its
therapeutic potential.
6. ACKNOWLEDGEMENTS
Part of this work was carried out at the premises of the Universidad of Guanajuato and RGV Analítica
Industrial Research Laboratory. The authors acknowledge the support received by both institutions,
whether as economic support PROMEP 2011 Program as well as an internal grant from the Faculty of
Pharmacy of Universidad Autonoma del Estado de Morelos, Universidad de Guanajuato and RGV
Analitica Industrial Research Laboratory. Likewise, the support of the Direccion General de Apoyo a la
Investigacion y al Posgrado (DAIP) of Universidad de Guanajuato for the revision of the manuscript in
English is appreciated.
7. CONFLICTS OF INTEREST
The authors declare that no conflict of interest exists.
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Table 1. Chromatographic conditions for the analysis of Nitazoxanide analogs (Compounds 1 and 2).
Compound
Column
Mobile Phase
 (nm)
Naproxen
µ Bondapak TM C18.
(3.9 x 300 mm)
Acetic acid 1%:methanol (45:55 v/v)
272
Furosemide
Symmetry C18.
(4.6 x 250 mm)
Acetic acid 1%:methanol
(55:45 v/v)
278
Compound 1
Zorbax CN (4.6 mm x 250
mm, Agilent, Inc.)
Acetate buffer pH 3.5/methanol
(40:60 v/v)
255
Compound 2
Zorbax CN (4.6 mm x 250
mm, Agilent Inc.)
Acetate buffer pH 3.5/methanol
(40:60 v/v)
255
Table 2. Parameters of the quantification curves of the Nitazoxanide analogs studied.
Compound
Quantification
interval
(µg/ml)
1-50
Slope
Intercept
Regression
Coefficient
92645.45
26171.80
0.99992
Furosemide
1-50
93149.467
16049.451
0.99977
Compound 1
3-50
56752.56
-13053.63
0.99887
Compound 2
3-50
13568.861
-14838.44
0.99969
Naproxen
Table 3. Repeatability, accuracy and limits of detection (LOD) and quantification (LOQ) of each
analytic method evaluated of analytical methods for evaluated compounds (n=5).
Nominal
%RSD
Concentration
(µg/ml)
2
1.99
Naproxen
10
2.46
20
1.42
2
6.16
Furosemide
10
1.10
20
2.16
6
0.64
Compound 1
13
0.61
30
0.60
6
3.63
Compound 2
13
2.37
30
2.52
Mean experimental
Concentration
(µg/ml)
1.83
10.21
20.33
1.71
10.04
21.27
5.79
12.46
29.48
5.45
12.26
30.61
% Recovery ASD (%)
91.43
102.13
101.67
95.42
100.40
106.33
96.45
95.88
98.26
97.29
95.78
104.46
8.57
2.69
1.68
4.58
1.03
6.33
3.55
4.12
1.74
3.12
4.22
4.46
LOD
LOQ
(g/ml) (g/ml)
0.06
0.18
0.12
0.38
0.75
1.69
0.48
1.45
Table 4. Determined pKa (mean ± SD, n=3) and theoretical (in silico) and experimental Log P values
for Compounds 1 and 2
pKa
Log P
Compound
pKa1
pKa2
1
6.029 ± 0.106
9.565 ± 0.062
2
4.945 ± 0.162
8.127 ± 0.206
1
Molinpiration
MiLog P
1.92
ACD/Labs
Log P
1.63  0.86
ChemDraw
Ultra
1.57
Osiris
cLogP
2.03
Experimental
Log P
1.01
2
2.60
2.65  0.86
2.81
0.78
1.44
Table 5. Permeability study using the everted intestinal sac technique
Compound
Naproxen
Everted
intestine
(L-S)
1.54 x 10 -5
Non-Everted
intestine
(S-L)
5.17 x 10 -6
Furosemide
1.16 x 10 -5
7.99 x 10 -6
Compound 1
1.15 x 10 -5
2.07 x 10 -6
Compound 2
9.09 x 10 -7
UQ
*Permeability units: cm/s, UQ: Unquantifiable
Figure 1. Chemical structure of NTZ (FW: 307.28), Compound 1 (FW: 294.24) and Compound 2 (FW:
275.83).
Figure 2. Typical chromatograms of Compound 1 and 2. a) SIF solution blank, Compound 1 and
Compound 2 in SIF solution; b) SIF sample blank; Compound 1 and Compound 2 samples.
Figure 3. Speciation diagram and dissociation equilibrium of Compound 1; (a) and Compound 2 (b)
Figure 4. pH-dependent solubility profile of new NTZ analogs. (a) Compound 1; (b) Compound 2 (n =
3).
Figure 5. Comparative studies of apparent permeability of naproxen, furosemide, Compounds 1 and 2.
(a) Serosal-Luminal transport (S-L); (b) Luminal-Serosal transport (L-S) (Mean ±SD; n = 3).