Download Reactions of carboxymethylalginic acid with some N

Reactions of carboxymethylalginic acid with some N-nucleophiles
© Serebrennikova+ Ekaterina Sergeevna, Moroz Marina Sergeevna, Cheremushkin Andrei
Ivanovich and Iozep* Anatoly Al’bertovich
Department of Chemical technology of drug substances and vitamins.
Saint-Petersburgs State Chemical pharmaceutical academy.
Prof. Popov str., 14. Saint-Petersburg 197376. Russia.
T. +7 (812) 234-19-38. E-mail: [email protected]
____________________________________
*Leader of the thematic; +Corresponding
Keywords: carboxymethylalginic acid, amides, hydrazides
Abstract
Reaction of H-form of carboxymethylalginic acid with aromatic amines and hydrazides of
pyridinecarboxylic acids forms corresponding N-arylamides and acylhydrazides. The influence of time,
temperature of reaction, solvent nature and structure of nucleophiles on the amount of aromatic fragments
entered into the polymer are studied.
Introduction
Carboxymethylpolysaccharides are successfully used in the synthesis of physiologically
active polymers for the acylation of amines, hydrazine and its derivatives (including biologically
active) [1, 2]. Recently, polymeric medicines based on alginic acid, which is widely used in
almost all fields of medicine and pharmacy, are of great interest. [3] However, it's low solubility
in water and aqueous solutions of acids limits the scope of it's application.
Carboxymethylderivates of alginic acid are soluble in water and could be used as acylating
agents, but there is no information about their chemical properties in the literature. Therefore, the
aim of our work was the synthesis and study of the acylating activity of carboxymethylalginic
acid in reactions with some N-nucleophiles.
Experimental part
The chemical modification of alginic acid was performed under the scheme:
ClCH2COOH, NaOH
i PrOH
[C5H5O2(OH)2(COONa)]n
I
AA (Na)
[C5H5O2(OH)2-k(OCH2COONa)k(COONa)]n
II CMAA
1 ArNH2
2 NaOH
[C5H5O2(OH)2-k(OCH2COONa)k-p (OCH2CONHAr)p (COONa)]n
IV AmCMAA
KY-2-8
[C5H5O2(OH)2-k(OCH2COOH)k(COOH)]n
III CMAA (Н)
1 ArCONHNH2
2 NaOH
[C5H5O2(OH)2-k(OCH2COONa)k-p (OCH2CONHNHCOAr)p (COONa)]n
V AcHCMAA
Carboxymethylalginic acid (CMAA - II) and it's H-form (III) was obtained as described previously
[4]. In this work we used samples of carboxymethylalginic acid with a degree of substitution from 0.60 to
1.75. The influence of carboxymethylation process on the molecular-mass distribution of alginic acid was
determined by gel chromatography.
Gel chromatography of polysaccharide samples was performed on a column K-16/40 "Reanal"
(1.6x40 cm) on Sephadex G-200 brand. The eluate was collected in portions by volume and was analyzed
quantitatively using spectrophotometry by the color reaction of sugars with phenol with presence of
concentrated sulfuric acid. The void volume was determined using blue dextran of molecular weight
1000000.
Acylation of aromatic amines and hydrazides of carboxylic acids was carried out in an organic
solvent (1-propanol, 2-butanol, isobutyl alcohol, methyl cellosolve, ethylene glycol, 1,4-dioxane)
containing 5% water. H-form of CMAA mixed with a fivefold excess of amine and heated from 50 to
100°C for 2 to 8 hours. At the end of exposure polysaccharide was precipitated with alcoholic solution of
alkali and centrifuged. To purify the product from ion-bound amine it was reprecipitated from aqueous
solutions of alkali with alcohol. The precipitate was centrifuged, washed with ethanol and dried in
vacuum at 61°C for 2 hours.
N-arylamides (IV) and acylglycosides (V) of CMAA obtained were characterized by the degree of
substitution Cs (the number of amide (hydrazide) groups, upon one monosaccharide fragment of the
polymer), and the reaction - at a degree of conversion Cc (percentage of carboxylic groups acylating ).
The amount of amide groups in the polymer was determined by UV spectroscopy, calibration curves were
built on model compounds - acetylated amines. The number of hydrazide groups in the polymer was
determined by iodometric titration results [5].
IR spectra of the reaction products in pellets of potassium bromide were recorded on Fourier
spectrometer FSM-1201.
Results and Discussion
Studying the molecular-mass distribution of alginic acid used (Acros Organics), we have
found that after successive precipitation from aqueous solutions of alkali with hydrochloric acid
and alcohol, it consists of four major fractions of different molecular weight (elution volume 3 7, 10-14, 25-30, 36-40 ml).
After processing of the polysaccharide with alkali at room temperature for 24 hours and
then at 55°C for 1 hour a clear separation of the main fractions and the appearance of a new one
with lower molecular weight (elution volume 54-58 ml) at gel chromatogram is observed. This
suggests that alginic acid molecules of different molecular weight due to the substituents of noncarbohydrate kind, carboxyl groups or hydrogen bonds form a quite solid "associates", the
destruction of which leads to the formation of alkali polysaccharide fractions with lower
molecular weight.
Changing the properties of alginic acid with alkali cleaning is also marked at determining
the amount of carboxylic groups in the polymer. Thus, in the samples of the original and
processed with alkali alginic acid 0.75-0.78 and 0.94 moles of carboxylic groups per mole of the
polymer's monosaccharide fragment were found by conductometric titration, respectively.
Increase in the amount of carboxyl groups in the polysaccharide is a sign of increase in it's
purity.
When comparing the gel chromatograms of alginic acid and the products of its
carboxymethylation (Fig. 1) a significant change in molecular-mass distribution of
polysaccharides is not seen. Thus, as expected, a decrease in mass of low-molecular fractions
and increase in high-molecular fractions is observed.
Analysis of the samples of H-form of CMAA obtained by hydroxame method [6] has
shown that they contain up to 0.16-0.18 mol of ester groups upon monosaccharide fragment,
which are formed by intramolecular esterification when H-form of CMAA is heated during
drying.
Samples of N-arylamides CMAA synthesized are powders of white to light beige, soluble
in water and water-alkaline solutions, insoluble in alcohol. Acylhydrazides of CMAA obtained
are amorphous powders from cream to beige, slightly soluble in water, aqueous alkali solutions
and insoluble in alcohol.
In the IR spectra of the compounds obtained absorption bands, which are absent in the
spectrum of the original alginic acid, were detected at 1675-1681 and 1548-1549 cm-1. These
could be attributed to stretch vibrations of C = O («amide I») and bending vibrations NH
(«amide II») of amide group. In addition, the spectra have absorption bands at 695, 752-758,
817-831 cm-1, characteristic of the mono- and di-substituted benzene ring, respectively [7].
optical density
0,7
0,6
0,5
0,4
0,3
0,2
0,1
0
-10
1
2
0
Vсв
10
20
30
40
50
60
70
elution volume, ml
Fig.1 Molecular-mass distribution of alginic acid,
clarified by alkali (1), and carboxymethylalginic acid (2)
To determine the acylating activity of uronic acids mixture of H-form of alginic acid and
aniline was boiled for 4 hours in 1-propanol. The reaction product contained about 0.084 mole of
amide groups on the monosaccharide fragment, while the degree of substitution of the amide
obtained in the same conditions from the H-form of CMAA (Ccm = 1.75) is 0.38 mol / mol. Due
to the fact that alginic acid itself is about 5 times less in acylating activity, in some cases, the
reaction of amines with CMAA was characterized by the degree of conversion of carboxymethyl
groups.
To determine the conditions of acylation allowing to enter a required number of amide
groups into the polysaccharide, we have studied the effect of reaction time on the degree of
conversion of the carboxyl groups in the amide (Table 1).
Tab. 1. The effect of time on the results of the acylation of aniline by the H-form
of carboxymethylalginic acid (Ccm = 0.60, 1-propanol containing 5% water, 100°C)
Н-form of CMAA
Reaction Degree of substitution (Cs),
Сc of the carboxyl groups of
Degree of conversion of the
time, h
carboxymethylchitin [8]
mol/mol
carboxyl groups (Сc)*, %
2
0.22
13.8 (36.7)
15.0
3
0.29
18.1 (48.3)
21.0
4
0.31
19.4 (51.7)
23.0
6
0.30
18.8 (50.0)
23.0
8
0.32
20.0 (53.3)
23.0
*- in brackets are the values of Cc*, calculated by the number of carboxymethyl groups in
carboxymethylalginic acid
It was found that in these conditions the formation of the amide groups in CMAA is in the
first 4 hours, just as in the case carboxymethylchitin (CMC) [8]. Further, the time has no effect
on the reaction.
In this case, the degree of conversion of carboxyl groups into the amide (Cc) in CMAA is
less than in CMC, but if you take into account in its calculation only carboxymethyl groups
(Cc*), it is much higher.
CMAA lower activity compared with CMC in reactions with amines could be explained by
the low reactivity of uronic acids compared with carboxymethyl groups. At the same time, we
know that the CMC reacts with nucleophiles 1.5-1.75 times worse than carboxymethyldextrane
(CMD) [2], and CMAA - about the same as the CMD [4]. Taking into account that Cc* of
CMAA in reactions with amines is 2.3 times higher than CMC, we can assume that uronic acids
catalyze the reaction and accelerate the formation of amides, or much more involved in the
reaction with aniline than in the original alginic acid.
At determining the effect of temperature on the reaction of H-form of CMAA with amines
we have found out, that the carboxyl groups of the polysaccharide acylate the amine only at
temperatures above 80°C (Table 2).
Tab. 2. Temperature effect on the results of the acylation of aniline by H-form
of carboxymethylalginic acid (Ccm = 1.75, 1-propanol containing 5% water, 4 h)
Temperature, °С
50
60
70
80
90
100
Degree of substitution (Сs), mol/mol
0.05
0.04
0.08
0.07
0.23
0.38
Degree of conversion Сc, %
1.82
1.45
2.91
2.55
8.36
13.8
Most likely, at lower temperatures ammonium salts are formed, which hinders the
acylation. At temperatures above 80°C, these salts partially or fully converted to the parent
compounds, and the more heat appears, the more carboxyl groups that can react with a
nucleophile is formed. Therefore, when the temperature rises from 80 to 100°C Cs values are
increased by more than 5-fold from 0.07 to 0.38 moles per monosaccharide fragment.
In the reaction products of H-form with aniline at temperatures below 80°C amide groups
are also detected (Cs about 0.05 moles per monosaccharide fragment), which may be due to the
presence of a small amount of polymer lactones. They do not form salts with amines and acylate
them at low temperatures.
H-forms of carboxymethylderivates of other polysaccharides (dextran (in DMSO and
dioxane) and chitin (in 1-propanol)) react similarly [2, 8].
To determine the effect of the solvent on the process of acylation of aromatic amines by the
H-form of CMAA 1-propanol, 2-butanol, isobutyl alcohol, 1,4-dioxane, ethylene glycol and
methyl cellosolve were used. They have different values of the dielectric permittivity (ε) and
containing 5% water as has been previously shown that the addition of water in case of certain
solvents increases the yield of the desired product [9].
Tab. 3. The results of acylation of aniline by H-form of CMAA in various media (Ccm = 1.75, 4 h, 100°C)
characteristics of solvents
Solvent
μ, D
ε
1,4-dioxane
0.45
2.21
2-butanol
1.65
15.8
2-methyl-1-propanol
1.63
17.7
1-propanol
1.64
20.7
methyl cellosolve
2.20
16.0
ethylene glycol
1.5÷2
34.5
μ - dipole moment; ε - dielectric permittivity
boiling point, °С
101.3
99.5
108.4
97.2
124.6
197.8
Degree of
substitution Сs,
mol/mol
0.21
0.23
0.22
0.38
0.11
0.45
Degree of
conversion Сc,
%
7.64
8.36
8.0
13.8
4.0
16.4
According to the results, there is some correlation between the results of acylation of
aniline by H-form of CMAA and dielectric permittivity of solvents. The highest degree of
conversion of the carboxyl groups in the amide (16.4%) is achieved when the reaction is
performed in ethylene glycol (ε = 34.5) and the lowest (8.36, 8.0%) - in the 2-butanol (ε = 15.8)
and isobutyl alcohol (ε = 17.7) . However, this dependence is maintained when using alcohols,
which dipole moment is about the same. When performing the reaction in esters on the contrary,
at large values of the dielectric permittivity the degree of conversion of the carboxyl groups is
minimal. For example, in methyl cellosolve (ε = 16.0) Cc = 4.0%, and in dioxane (ε = 2.21) 7.64%.
Further, we used 1-propanol (Tb = 97°C; η = 2.2620) as a media, because the high boiling
point and viscosity of ethylene glycol (Tb = 198°C; η = 2120) complicate the isolation and
purification of the product.
As in the reactions with other polysaccharidecarbonic acids (carboxymethyldextran
derivatives [9], chitin [8] and other polysaccharides), electron-donating substituents in the pposition of the benzene ring of amines accelerate the acylation of amines by H-form of CMAA,
and electron-accepting — slow down (Table 4). Moreover, the number of amide groups in the
samples of N-arylamides of CMAA are at linear function from σ-Hammett constants of the
substituent in the amine: y =-38.127h + 21.659, n = 5, R2 = 0.977. In this case, aniline was
excluded from the correlation, as in the reactions with it Cc of the carboxyl groups of the
polymer into amide was highest compared to other amines. This is probably due to the smaller
size of the molecule of aniline.
Tab. 4. Results of the reaction of H- form of carboxymethylalginic acid
with some aromatic amines and hydrazides
(Ccm = 1.75, 1-propanol containing 5% water, 4 h, 100°C)
N-nucleophile
4-methoxyaniline
4-ethoxyaniline
4-methylaniline
aniline
4-bromaniline
4-ethoxycarbonylaniline
isonicotinoyl hydrazide
nicotinoyl hydrazide
picolonoyl hydrazide
*- Ccm = 1.43
λmax of solutions in 0.1 n NaOH, nm
N-arylamide
N-nucleophile
(acylhydrazides) CMAA
231
245
233
244
232
241
229
238
240
247
283
265
296
303
276
286
274
300
Degree of
substitution Сs,
mol/mol
0.44*
0.46*
0.33*
0.38
0.25
0.04*
0.75
0.80
0.76
Degree of
conversion Сc,
%
18.1
18.9
13.6
13.8
9.09
1.65
27.3
29.1
27.6
Effect of the degree of carboxymethylation of alginic acid on the degree of conversion of
carboxyl groups into the amide was quite complicated. When changing the Ccm from 0 to about
0.8 percent of carboxyl groups reacted with amine increases. But then with increasing of Ccm the
degree of conversion of carboxyl groups decreases (Table 5).
Tab. 5. Effect of the degree of conversion of the carboxyl groups into the amide
on the degree of carboxymethylation of alginic acid (aniline, 1-propanol, 5% water, 4 h, 100°C)
Degree of carboxymethylation Сcm,
mol/mol
0.30
0.75
0.88
1.30
1.75
Degree of substitution Сs,
mol/mol
0.06
0.31
0.32
0.31
0.38
Degree of conversion Сc,
%
4.62
17.7
17.0
13.5
13.8
Therefore, we can assume that the appearance of the second carboxymethyl group in the
monosaccharide unit reduces their acylating activity in reactions with amines and the reactivity
of the H-form of CMAA overall. A similar dependence was found at the esterification of CMD in
autocatalysis [10].
The developed method of acylation of aromatic amines has been used to modify CMAA by
pyridinecarboxylic acid hydrazides. In this work we used the hydrazides of nicotinic, picolinic
and isonicotinic acids. As expected, hydrazides are more responsive to H-form of CMAA than
aromatic amines, the degree of conversion of the carboxyl groups of the polymer is from 27 to
29% (Table 4), due to their greater nucleophilicity.
Resume
1. H-form of carboxymethylalginic acid when heated with aromatic amines and hydrazides of
acids form substituted amides and acylhydrazides, with the degree of conversion of the
carboxyl groups of the polymer depends on the time, the reaction temperature and the kind of
the solvent and the structure of the nucleophile. Electron-donating substituents in the pposition of the benzene ring of amines accelerate the acylation and elektron-accelerating
substituents slow it down.
2. Activity of hydrazides of pyridinecarboxylic acids in reactions with H-form of
carboxymethylalginic acid is higher than aromatic amines.
References
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