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(1)OPTIMIZATION REACTION OF SOME 1, 4-DISUBSTITUTED THIOSEMICARBAZIDES WITH TUBERCULOSTATIC ACTIVITY I

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OPTIMIZATION REACTION OF SOME 1, 4-DISUBSTITUTED THIOSEMICARBAZIDES WITH TUBERCULOSTATIC ACTIVITY

I. HURJUIa,b, C. CHEPTEAc, C. F. DASCALUa, L. HURJUIb, C. PEPTUd, V. SUNELe, D.O. DOROHOIa*

a”Al. I. Cuza” University, Faculty of Physics, 11 Carol I Blv. RO-700506, Iasi, Romania

b“Gr. T. Popa” UniversityFaculty of Medicine, Biophysics and Medical Physics Dept., 16 University Street, Iasi, 700115, Romania

c”Gr. T. Popa” Medicine and Pharmacy University, Faculty of Medical Bioengineering, Biomedical Sciences Dept., 9-13 Kogalniceanu Street, RO- 700454, Iasi, Romania

d”P. Poni” Institute of Macromolecular Chemistry, 41A, Grigore Ghica Voda Alley, 700487, Iasi, Romania

e”Al. I. Cuza” University, Faculty of Chemistry, Department of Organic Chemistry, 11 Carol I Blv. RO-700506, Iasi Romania

Six new 1,4-disubstituted thiosemicarbazides were synthesized and their obtaining reactions were optimized in a 23 factorial experiment. The structural features of the analyzed compounds were established by spectral means. The tuberculostatic activity against Mycobacterium Tuberculosis has been tested for different concentrations of the studied thiosemicarbazides in DMSO + phosphate buffer in volumetric ratios 1:4. The highest tuberculostatic activity has been registered for compound IV.

(Received September 18, 2012; Accepted November 7, 2012)

Keywords: thiosemicarbazides; optimization chemical reactions; tuberculostatic activity;

1. Introduction

The 1,4-disubstituted thiosemicarbazides is a group of substances with therapeutic interest.

The tuberculostatic activity of various thiosemicarbazides [1-6] was noted by many authors, as well as stimulating action of enzymes like lipase or amylase in gastric secretions [2-4].

Other authors notify that the compounds with this structure show antiviral [2-5, 7-10], cytostatic [11,12], antibacterial [13-15], anticonvulsant [16], tuberculostatic activity [17, 18] antifungal activity [14,19, 20].

Some authors of the above mentioned works consider that the N1-N4-disubstituted thiosemicarbazides molecules consist from a toxophore group, which is an active group and a haptophore portion that allows close attachment to the cellular constituents. According to this description, the thiourea group (– NH – CS – NH –) represents toxophore group and the radicals attached to the two nitrogen atoms are haptophore portions.

The conclusions of authors cited above are the thiosemicarbazides activity depends largely on the structure haptophore portions, so the entirely molecule structure.

Based on this findings we decided to synthesize the new series of 4-substituted thiosemicarbazides based on 5-nitroindazole.

      

*Corresponding author: [email protected]

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II-VII S

II R= - C6H5; III R= - C6H4 – CH3(p); IV R= - C6H4 – OCH3(p); V R= - C6H4 – Br(p); VI R= - C6H4 – Cl(p); VII R= - C6H4 – I(p);

Scheme 1. Obtaining of 1-acyl- 4-aryl-thiosemicarbazides (II-VII)

The chemical structures of the synthesized compounds were elucidated by means of elemental and spectral analysis (FT-IR, 1H-NMR, 13C-NMR, SM).

FT-IR spectra of the studied thiosemicarbazide derivatives present an intense absorption band at 2918-3123 cm-1, specific for NH group. The absorption band from 1619-1621 cm-1 is specific for CO group, and the C=S group is revealed by an absorption band at 1137-1250 cm-1. The NO2 group is identified by υ NO2 symmetric at 1338-1343 cm-1 and by υ NO2 asymmetric at 1525-1535 cm-1. The compounds (V-VII) show FT-IR absorption bands at 625-721 cm-1. FT-IR spectra of compound IV is given in Fig.1

   

Fig. 1. The IR spectrum of 1-(5’-nitroindazol-1’-yl-acetyl)-4-(p-metoxyphenil) thiosemicarbazide IV

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signals metin g

Fig. 2. T

The 1H-NM s at 9.66-11.

group show

Fig. 3. T

The 1H-RMN s

MR spectra 02 ppm and signals at 2.3

The 13C-RMN s

spectrum of 1- thio confirm the at 7.17-8.95 30 ppm (s, 1

spectrum of 1- thio

-(5’-nitroindaz osemicarbazid

e presence o 5 ppm, respe

H) and at 3.3

-(5’-nitroinda osemicarbazid

zol-1’-yl-acety de IV

of the proto ectively arom

36 ppm (s, 2H

azol-1’-yl-acet de IV

yl)-4-(p-metox

ns bound at matic protons

H).

tyl)-4-(p-meto

xyphenil)

t nitrogen sh s. The proton

oxyphenil)

howing ns from

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Fig. 4. The mass spectrum of 1-(5’-nitroindazol-1’-yl-acetyl)-4-(p-metoxyphenil) thiosemicarbazide IV

In the 13C-NMR spectra, C=S resonates at 176.63-187.16 ppm and the signals corresponding to C=O appear at 165.58-168.89 ppm.

The mass spectrum confirms the supposed chemical structures. So, all thiosemicarbazides appear the signals of sodium adducts (M + Na), for values m/z 393, 408, 423, 472, 833, 519. In the same spectra were also identified protonated ionic species [M + H].

3. Optimization process

Considering the connection between the chemical structure and the potential biological activity of thiosemicarbazides (II-VII), we thought of notable importance to establish the optimal conditions for the additive reaction of hydrazide (I) to the six aromatic isothiocyanates.

Our preliminary researches revealed that the reaction time and temperature are factors with major influence on the reaction yield when the compounds II-VII are prepared.

In these conditions the reaction yield

was considered as being optimization indicator, while the reaction time X1t(expressed in hours) and temperature X2 T(expressed in0

C

) were considered as relevant variables.

Three values of each system variable were considered in a 32factorial experiment [25-30]

organized in order to determine the best conditions in which the studied reactions take place. In the title of the factorial experiment 3 is the number of the variation levels for each relevant variable of the system and 2 represents the number of the relevant variables taken into consideration in this study.

Let us consider the influence of the system variables and of their conjugate effects on the reaction field as being described by the polynomial (1).

The factorial programs permit to avoid a long and laborious series of experiments for determination the model coefficients

a

0,

a

i(i=1,2) and aij(i, j=1,2), usually named regression coefficients, from relation (1). The relation (1) must be applied to each studied thiosemicarbazide for the experimental data.

2 1 2 12

2 2 22

1 11 2 2 1 1

0

a x a x a x a x a x x

a

    

 (1)

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In relation (1), x1and x2 are a-dimensional variables for the reaction time (i=1) and for reaction temperature (i=2). They are defined by;

2 ,

1

 

i

X X x X

i i

i i (2)

The a-dimensional variables can be converted in real variables by using relation:

2 ,

1

X x X i

X

i i i i (3)

In relations (2) and (3), the following notations were made:

2

min

max i

i i

X

X X

 (4)

for the middle of the variable variation domain and

min

2

X

i

X

imax

X

i (5)

for the length of the variation domain.

A-dimensional variables x1and x2 are usually defined in order to facilitate simulation. For real variables of the system, X1tand X2T , which vary in the ranges:

1min 1max

1

X

,

X

X

 and

X

2

X

2min,

X

2max

, (6)

the a-dimensional variables x1and x2 have the corresponding unitary limit values.:

2 , 1

; 1

; min

min min  

 

x i

X X

x X

i

i i

i i (7)

2 , 1

; 1

; max

max max  

 

x i

X X

x X

i

i i

i i (8)

The real and the a-dimensional variables of the reactions considered here are contained in Table 1 which also contains the mass of the substances II-VII obtained in the 32factorial experiment made for each compound II-VII. The substances were arranged in Table 1 after the common reaction temperature. The a-dimensional variables are given between parentheses in Table 1.

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7 3.5 (1) 55 (1) 1.45 1.61 1.75 1.56

8 3.5 (1) 60 (0) 1.48 1.66 1.77 1.60

9 3.5 (1) 65 (1) 1.45 1.63 1.74 1.56

From the mass experimentally determined, mexp., the reaction yield can be approximated by using relation (9).

. . exp

m

calc

m

(9)

In (9)

m

calc.is the computed mass of the corresponding compound for the same quantity of the initial precursor as in the experiment.

Table 1 continued Experimental quantities obtained in reactions for determining the reaction yields of compounds IV and VII.

Nr. X1

 

h;

 

x1

 

0

 

2 2

C

;

x

X

m

 

g ;(IV) 00 .

.2

m

calc

 

g;(VII) m

48 .

.2

m

calc 1 1.5 h (x11) 500

C

(x11) 1.51 1.91 2 1.5 h (x1 1) 550

C

(x10) 1.54 1.96 3 1.5 h (x11) 600

C

(x11) 1.51 1.92 4 2.0 h (x1 0) 500

C

(x11) 1.53 1.94 5 2.0 h (x1 0) 550

C

(x10) 1.59 1.99 6 2.0 h (x1 0) 600

C

(x11) 1.52 1.94 7 2.5 h (x11) 500

C

(x11) 1.51 1.91 8 2.5 h (x1 1) 550

C

(x10) 1.55 1.96 9 2.5 h (x11) 600

C

(x1 1) 1.50 1.92

The regression coefficients

a

0,

a

i( i=1,2) and aij(i, j=1,2) can be evaluated by the least (smallest) square method [24,25,28] using the data from Tables 1. They are given in Table 2. The values of the regression coefficients can indicate the degree of influence of the corresponding parameter and, their sign indicates an increase (+) or a decrease (-) in the reaction yield determined by the corresponding parameter (or by the conjugate influence of two parameters).

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Table 2 Regression coefficients from (1) with the data of Table 1.

Compound a0 a1 a2 a12 a11 a22

II 81.44 0.562 0.232 -0.273 -2.318 -1.188

III 87.07 0.33 0.453 0.24 -0.96 -2.11

IV 78.96 0.067 -0.132 -0.11 -1.297 -2.312

V 80.55 0.285 -0.187 -0.175 -1.765 -1.3

VI 79.81 0.017 -0.075 0.087 -0.723 -1.698

VII 80.08 0.007 0.11 0 -0.887 -1.867

Let us make t-student test in order to establish the relevance of the regression coefficients from Table 2. In this purpose, three supplemental measurements were made in the center of the variation domain of the relevant variables. The average value of the reaction yield in the center of the variation domain was computed with formula (10) and it is given in Table 3.

Table 3 Reaction yield in the center of the variation domain, average yield; square average deviation and precision.

Compound 12

3

S

P

II 81.16 81.75 81.50 81.47 0.0026 0.017

III 86.90 87.05 87.20 87.05 0.0225 0.050

IV 79.01 79.50 79.20 79.24 0.0025 0.017

V 80.50 80.69 80.92 80.70 0.0623 0.083

VI 79.56 79.80 79.98 79.78 0.0444 0.070

VII 80.10 80.15 80.80 80.35 0.1325 0.121

3

3 2

1  

  (10)

The square average deviation for the supplemental experiments was estimated by using equation (11).

 

2

3 1

  2

i i S

 (11)

The precision of measurements can be estimated by the formula (12).

N

P

S

(12)

The total number of the experimental data in a 32factorial experiment is N 9.

If one supposes that all regression coefficients from relation (1) were determined with the same precision,

P

, (given by relation (12), the t-student test for each regression coefficient can be calculated with relation (13)

p P j

a

tjj ; 1,2,.., (13)

The values of the t-student coefficients are given in Table 4. Let us suppose that the maximal value of the t-student test indicating the dependence of the reaction yield on the corresponding parameter is equal to unity.

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Fig. 5. Three dimensional representation of the compound IV reaction yield vs. the a-dimensional

time and temperature of the chemical reaction

Fig. 6. Three dimensional representation of the compound VII reaction yield vs. the a-dimensional

time and temperature of the chemical reaction

Table 4 tij-student test for regression coefficients from relation (1) with the data of Table 1.

Compound a0 a1 a2 a12 a11 a22

II 4790.6 33.1 13.7 16.10 136.4 69.88

III 1741.4 6.6 9.1 4.8 19.2 42.2

IV 4644.7 3.94 7.76 6.47 76.29 136

V 970 3.43 2.25 2.10 21.26 15.66

VI 1140.1 0.24 1.07 1.24 10.34 24.25

VII 661.8 0.057 0.11 0.91 7.33 15.43

If one supposes that the highest value of tijcoefficients for the significant parameters is the unity, the final formulae obtained in optimization of the synthesis reactions for the studied compounds II- VII, after applying t-student test are written below:

2 2

II

81.44 0.562x

1

0.232x

2

0.273x x

1 2

2.318 x

1

1.188 x

2

      (14)

2 2

III

87.07 0.33x

1

0.453x

2

0.240x x

1 2

0.960 x

1

2.11x

2

      (15)

2 2

IV

78.96 0.067 x

1

0.130 x

2

0.110 x x

1 2

1.297 x

1

2.318 x

2

      (16)

2 2

V

80.55 0.285x

1

0.187 x

2

0.175x x

1 2

1.765x

1

1.300

2

      (17)

2 2

VI

79.81 0.750x

2

0.087 x x

1 2

0.723x

1

1.698 x

2

     (18)

2 2

VII

80.08 0.887 x

1

1.867 x

2

   (19)

The maximal yield values of the studied reactions are given in Table 5.

Table 5 Extreme values resulting from optimization.

Compound x1extr x2extr ηextr

II 0.116 0.084 81.482

III 0.186 0.117 87.127

IV 0.027 -0.029 78.962

V 0.084 -0.077 80.569

VI 0.010 -0.021 79.810

VII 0.003 0.029 80.081

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The highest value for the reaction yield was obtained for compound IV, the most active from the tuberculostatic action point of view.

4. Tuberculostatic activity

We assessed the tuberculostatic activity in order to evaluate the relationship between the chemical structure and the pharmacodynamic activity.

The compounds II - VII were tested against Mycobacterium tuberculosis, and their activity compared with that of the isonicotinic acid hydrazide (INH), showing that they do have

tuberculostatic activity.

The serial dilution technique was applied using the Youmans medium in bovine serum.

H37Rv microbial strain hominic variety was used, as inoculums in a 10-2 mg/5ml concentrated culture medium [31].

The compounds were tested in different concentrations of 5, 10, 20, 30 and 40 μg /ml.

Due to solubility difficulties of these compounds in saline, we had to dissolve them in nonpolar organic solvents (DMSO or DMF) prior to testing. We used 100 μg substance in 1 ml solvent made of DMSO and phosphate buffer, in a ratio of 1:4 (V/V).

The readings were done at 6 and 15 days after inoculations.

We found that the most active thiosemicarbazide is compound IV : 1-(5'-nitroindazole-1' - yl-acetyl)-4-(p-methoxyphenyl)-thiosemicarbazide and this shows a minimum inhibitory concentration of 10 μg/ml similar to that of the reference tuberculostatic. The next most active is compound III: 1-(5' - nitroindazole - 1' -yl-acetyl) - 4 - (p-tolyl)-thiosemicarbazide (CMI = 25 μg/ml).

Table 6. Tuberculostatic activity of compounds II - V against Mycobacterium tuberculosis, in vitro, compared to that of the isonicotinic acid hydrazide (INH).

Compou nd

Thiosemicarbazides concentration in culture medium (μg/mL) CMI

(μg/m L) 5 10 20 30 40 6day

s 15da

ys 6day s 15da

ys 6day s 15da

ys 6day s 15da

ys 6day s 15da

ys

II ++ ++ ++ ++ + + + - - - 35

III ++ +++ +++ +++ ++ ++ ++ ++ - - 45

IV ++ ++ + + - - - 25

V ++ ++ - - - 10

INH - - - 10 - No growth; + reduced growth; ++ moderate growth; +++ intense growth.

Analysis of the structure and the in vitro antituberculostatic activity of the investigated compounds shows:

- substitution of nitrogen bound in position 4 in the phenyl ring of the thiosemicarbazides leads to changes in the antibacterial activity. Substitution with methoxy group in the para position of the benzene ring of the compound IV leads to an increase in activity, while the presence of a methyl group in the same position makes compound III less active.

- none of the compounds have any tuberculostatic activity below 10 μg/ml.

- presence of bromine, like in compound II of the thiosemicarbazides, leads to a decrease in the antibacterial activity.

5. Conclusions

By using a 32factorial experiment the trials for obtaining the most efficient reaction conditions for obtaining the compounds II-VII studied in this paper were minimized. The highest

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