Introduction

The past two decades witnessed a noticeable rise in the incidence of fungal infections with high death rates, especially among people who suffer from immunodeficiency and those who take chemotherapy [1]. Every year, more than one billion people worldwide are exposed to fungal diseases, and at least 1.6 million deaths are recorded annually, exceeding the rates of malaria [2, 3]. Over 300,000, 750,000, and 10,000 cases, respectively, of invasive aspergillosis, candidiasis, and mucormycosis have been reported in recent years [4]. Human mucormycosis is regarded as one of the most harmful invasive fungal diseases which are caused by a wide range of fungal pathogens, including Rhizopus, Mucor, and Syncephalastrum [5]. Posaconazole, amphotericin B, and isavuconazole are the common antifungal drugs for treatment of mucormycosis [6], but the increased use of antifungal medications has led to the appearance of fungal strains including L. corymbifera, C. albicans, R. arrhizus, M. circinelloids, and R. microsporus that are resistant to antifungal agents [7, 8]. Although it is scientifically proved that people with weak immunity are more likely to develop mucormycosis, incomplete diagnostic tools and treatment decisions contributed to the lack of control over its spread [9]. So, the researchers turned their compass toward the search for alternative antifungal treatments to fight drug-resistant fungus.

Mosquitoes are considered dangerous insects, as they cause human infection with many dangerous viruses that may lead to death, such as those that cause yellow fever, chikungunya, dengue fever, West Nile fever, and Japanese encephalitis [10]. Although there are many species of mosquitoes, 100 species have been identified as vectors for human and other vertebrate diseases. The most common genera are Aedes, Anopheles, and Culex, which have the ability to resist many chemical insecticides [11]. Sterilization procedures, biological control, and biopesticides are further mosquito control strategies. One of the mosquitoes that pose a threat to human health is the Anopheles mosquito (especially Anopheles stephensi), which transmits Plasmodium parasites that cause malaria from one person to another [12]. In 2013, there were an estimated 198 million malaria cases and a recorded 584,000 deaths, mostly children living in Africa. The fatality rates from malaria have, however, decreased by 47% globally and by 54% across the African continent since 2000. However, the effectiveness of chemical pesticides now in use is losing quickly due to the development of resistance in several species and strains of mosquitoes; as a result, there is a desire for more eco-friendly safe pesticides [13].

Chitosan is a natural linear poly(amino saccharide) that is characterized chemically by the presence of amino and hydroxyl groups in a  repetitive manner. These groups enable many researchers to conduct many modification reactions on the chitosan structure to produce functional chitosan derivatives [14]. These reactions combine the properties of chitosan with those of the modifying compounds. Chitosan is the deacetylation product of chitin, which is a polymer of 2- acetoamido -2-deoxy-b-D-glucopyranose monomer [15, 16]. Chitin is considered the second most abundant polymer after cellulose in terms of natural availability, as it is found in the shells of crustaceans such as shrimp, crabs, insects, and others [17]. Chitosan possesses unique properties such as desirable biodegradability, biocompatibility, non-toxicity, antimicrobial, and antioxidant properties [18, 19]. Chitin has poor solubility in organic solvents due to its rigid structure and the presence of hydrous bonds, whereas chitosan dissolves in dilute acid solutions due to the presence of amine groups and the generation of cationic polyelectrolytes [20]. Due to its cationic character [21], it was functionalized for many purposes, such as antibacterial [22, 23], antifungal [24, 25], antiviral [26, 27], and larvicidal purposes [21, 28], as well as chelating and adsorption of toxic heavy metals [18, 29] and organic pollutants [30].

Based on the above facts, the present study dealt with the development of a new modified biopolymer (coded here as PTDz-Cs) and its characterization by FT-IR, 1H/13C-NMR, elemental analysis, XRD, TGA, and XPS. The antifungal efficacy of PTDz-Cs against R. microspores, M. racemosus, L. corymbifera, and S. racemosum was determined. Also, its larvicidal efficacy against A. stephensi larvae was evaluated.

Experimental work

Chemicals and instruments

Powdered chitosan (degree of deacetylation of 70–95%) was procured from Sigma–Aldrich. Its molecular weight was about 6 × 104 determined by the viscosity method. Thiosemicarbazide (CH5N3S, white solid, M.P. 183 °C, purity 99%) and iodopropane (C3H7I, colorless liquid, purity 99%) were purchased from Sigma–Aldrich. Succinic anhydride was prepared in our lab as described previously [31]. Other chemicals and solvents like thionyl chloride (SOCl2, purity 99%), carbon disulfide (CS2, colorless liquid, purity 99.9 + %), triethylamine, dimethyl sulfoxide (DMSO), and hydrochloric acid (HCl) were obtained from Alfa Aesar and El-Nasr companies in Egypt and used as received. 5-Amino-1,3,4-Thiadiazole-2-thiol (TDz-amine) was prepared as described in [32, 33]. Elemental C-H-N-S AnalyzerVario El M (Germany) was employed to carry out the elemental analysis. Infrared spectra were measured by the Schimadzu Nicolet iS 10FT-IR Spectrometer. 1H/13C NMR spectra were done on the JNM-ECA 500 II-JEOL-JAPAN using deuterated DMSO-D6 solution. GC–MS analysis was run on Shimadzu Japan's GC–2010. X-ray diffraction (XRD) analysis was run on X′ Pert powder PAN analytical with Cu-radiation (l = 1.542 Å) at 45 K.V., 35 M.A. and scanning speed 0.02°/sec. X-ray photoelectron spectroscopy (XPS) was done on K-ALPHA (Thermo Fisher Scientific, USA) with monochromatic x-ray Al K-Alpha radiation from −10 to 1350 e.v. Spot size 400 micro m at pressure 10–9 mbar with full spectrum pass energy 200 e.v. and at narrow spectrum 50 e.v. Discovery SDT 650-Simultaneous DSC-TGA/DTA (Differential Scanning Calorimetry-Thermal Gravimetric Analysis/Differential Thermal Analysis) Instruments (USA) was employed to perform the thermal analysis (N2 & 10 °C/min heating rate).

Synthesis of 5-(propylthio)-1,3,4-thiadiazol-2-amine (PTDz-amine)

TDz-amine (0.1 mol, 13.3 g) was suspended in absolute ethanol (20 mL) in an Erlenmeyer conical flask. Potassium hydroxide (0.1 mol) was added portion by portion and stirred for half an hour at 0 °C, followed by the addition of propyl iodide (0.1 mol) [34], and stirred for an additional 8 h at 23–25 °C. The solid product was filtered, washed with water, and recrystallized by a mixture of ethanol and water to give yellowish-white crystals of PTDz-amine.

Synthesis of 1,3,4-thiadiazol carboxylic acid derivative (PTDz-acid)

PTDz-amine (0.01 mol) was dissolved in dry benzene (15 mL); then, the solution of succinic anhydride (0.01 mol) in dry benzene (15 mL) was added dropwise to the amine solution. The solution was continuously and vigorously stirred for 3 h at 23–25 °C, after which the precipitate was separated via suction filtration. Air drying, washing by ethanol, and recrystallization from an ethanol/benzene mixture were done sequentially on the product. The physical and analytical data of the PTDz-Acid product are offered in Table 1.

Table 1 Physical and analytical data for PTDz-Acid
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Synthesis of chitosan derivative (PTDz-Cs)

PTDz-Acid (0.01 mol) was dissolved in anhydrous dichloromethane (DCM, 100 mL) in an ice bath, and then, the thionyl chloride (SOCl2, 0.013 mol) was added dropwise. The mixture was stirred under reflux for 2 h at 70–80 °C. DCM was then evaporated under reduced pressure. The natural polysaccharide (chitosan, 0.01 mol, dispersed in 25 mL anhydrous DCM) was added to the crude acid chloride in 25 mL anhydrous DCM in the presence of triethyl amine (TEA, 0.03 mol) as an HCl scavenger. The reaction mixture was stirred overnight at 70 °C. Furthermore, the mixture is cooled and then filtered, and the precipitate is taken up and washed three times with DCM to give the new functionalized chitosan derivative, as shown in Scheme 1.

Scheme 1
scheme 1

Synthesis diagram of major reactions to get new functionalized chitosan derivative (PTDz-Cs)

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Antifungal activity

The antifungal activity of PTDz-Cs and Cs only was evaluated against R. microspores (accession no. MG518370.1), M. racemosus (accession no. MG547571.1), L. corymbifera (accession no. MK300698.1), and S. racemosum (accession no. MK621186.1) which were isolated in our previous studies [35,36,37,38]. The agar well diffusion was executed in compliance with the Clinical Laboratory Standard Institute (document M51-A2) [26]. The fungal suspension was prepared in sterilized phosphate buffer solution (PBS) pH 7.0, and after being counted in a cell counter chamber, the inoculum was adjusted to 107 spores/mL. On agar MEA plates, one milliliter of the fungal suspension (107 spores/mL) was streaked evenly. Wells (8 mm) were cut using a sterile cork borer; then, 100 µl of PTDz-CS, Cs, and amphotericin B (AMB) as standard antifungal was transferred to each well individually and kept at 4 ℃ for 2 h, followed by incubation at 30 ℃ for 3 days. Then, the inhibition zones were observed and determined. In order to evaluate the minimum inhibitory concentration (MIC) of PTDz-CS and Cs, the broth microdilution method was used in accordance with the CLSI M27-A3 document with modifications proposed by Rojas et al. [39].

Toxicity against mosquitoes malaria vectors

Mosquito rearing

Eggs of A. stephensi were produced by the Medical Entomology Institute (MEI) field station in Dokki (Giza, Egypt). The eggs were placed in plastic containers (18 × 13 × 4 cm3) including 500 mL of deionized water, and then kept in laboratory conditions [R.H. 75–85%, Temp. 27 ± 2 °C, 14:10 (L:D) photoperiod], waiting for the larvae to hatch [40, 41]. Larvae stage feeding each day was performed by a mixture of crushed dog biscuits (Pedigree, USA) and hydrolyzed yeast (Sigma-Aldrich, Germany) in a 3:1 ratio (w:w), and water was renewed every 2 days. Breeding was monitored daily inside containers closed with a piece of muslin cloth to prevent the arrival of foreign mosquitoes. Dead individuals are removed from containers, and pupae are collected daily. Pupae are transferred to glass beakers containing 500 mL of water. Glass beakers containing mosquito pupa (50 per beaker) were put in a mosquito-rearing cage (90 × 90 × 90 cm3, plastic frames with chiffon walls) until adults appeared. Adult mosquitoes were continually given cotton wicks soaked in a 10% (w/v) glucose solution. Daily changes were made to the cotton, which was always maintained moist with the solution. Five days after discharge, females were provided with a blood meal via professional heated blood (lamb's blood), at a constant temperature of 38 °C and covered with a membrane of cow gut. After 30 min., the blood meal was removed and a fresh meal was introduced [42, 43].

Experimental conditions

The mosquitocidal activity of PTDz-Cs was evaluated against A. stephensi larvae according to the method used by Murugan et al. [44]. Different concentrations of PTDz-Cs derivative (5, 10, 15, 20, and 25 ppm) were tested toward 25 A. stephensi larvae (I, II, III, or IV instar) or pupae in 500-mL beakers for 24 h exposure in the presence of 0.5 mg larval food for each test. The mortality ratio (M) was determined using Eq. 1 as follows:

$$M\,(\%) = \frac{{{\text{Total}}\;{\text{dead}}\;{\text{individuals}}}}{{{\text{Total}}\;{\text{treated}}\;{\text{individuals}}}} \times 100$$
(1)

Statistical analysis

Three replicates were done, and all resulted values are the averages of three independent experiments. Data were analyzed using a one-way ANOVA model of analysis of variance (ANOVA) (α = 0.05) to determine the significance between groups. When a significant difference was detected by pairwise comparisons, multiple comparisons were performed using Tukey's test. The data obtained from the larvicidal and pupicidal tests were analyzed using Statistical Product and Service Solutions (SPSS), version 16.0, in terms of probit analysis. LC50 and LC90 were calculated as reported by Finney [45].

Results and discussion

Synthesis of PTDz-Cs derivative

PTDz-Cs was synthesized by grafting a 1,3,4-thiadiazol carboxylic acid derivative (PTDz-Acid) onto chitosan via a multi-step procedure as indicated in the Scheme 1. Firstly, the 5-amino-1,3,4-thiadiazole-2-thiol (TDz-amine) intermediate was prepared by the heterocyclization of hydrazine carbothioamide (TDz) with carbon disulfide in absolute ethanol containing anhydrous sodium carbonate as a base [15]. Furthermore, reaction of the TDz-amine moiety with propyl iodide in alcoholic potassium hydroxide at room temperature yields the target derivative (PTDz-amine) in an 86% yield. The formation of PTDz-amine is believed to occur through the initial formation of the nucleophilic substitution of the thiol group in TDz-amine to activate cite in an alkyl halide (propyl iodide), as shown in Scheme 2. PTDz-Acid was obtained in an 82% yield by exposing the primary amine derivative (PTDz-amine) in dry benzene to react with succinic anhydride with continuous stirring at 23–25 °C for 3 h. There is a plausible mechanistic pathway for the formation of an acid derivative via a nucleophilic attack of an amino group of PTDz-amine at one of the carbonyl groups of succinic anhydride to yield the corresponding non-isolable intermediate, which undergoes the shifting of the hydrogen atom followed by ring opening to yield the final product. The acid chloride intermediate (PTDz-AcylChloride) was prepared by the reaction of PTDz-Acid with SOCl2 in dry dichloromethane under reflux at 70–80 °C, followed by a reaction with chitosan through a nucleophilic substitution reaction of free amino groups to afford the secondary amide derivative (PTDz-Cs) as stated in Scheme 2. The microanalytical and spectral data of all the produced compounds and grafted chitosan supported all their structural claims.

Scheme 2
scheme 2

A plausible mechanistic pathway for the formation of grafting chitosan

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Analysis of Fourier transform infrared spectroscopy (FT-IR)

FTIR (KBr, υmax /cm−1) of PTDz-amine (Fig. 1a) confirmed the characteristic peaks that appeared at 3307 and 3268 (NH2 group), 2957 (C-H aliphatic), 1630 (C = C), 1509 (C = N), and 1431 (C-H, bending). Figure 1b displays the FTIR (KBr, υmax /cm−1) of PTDz-Acid, which indicated the formation of the acid derivative by the appearance of a broad peak including stretching vibrations of O–H, N–H, and C–H aliphatic groups. Another indication was the appearance of stretching vibrations of C=O (acid) at 1709 cm−1 and C=O (amide) at 1686 cm−1. Other significant peaks appeared at 1575 cm−1 (C=N), 1424 cm−1 (C–H bending), and 1333 cm−1 (O–H bending).

Fig. 1
figure 1

FTIR spectra of a PTDz-amine, b PTDz-Acid, c chitosan, and d PTDz-Cs. (FTIR conditions: KBr plates, scanning range 4000–400 cm−1, and resolution 16)

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FTIR (KBr, υmax/cm−1) of chitosan (Fig. 1c) was in an agreement with that reported previously [20, 46, 47] showing peaks at 3331 (O–H group), 3291 (N–H), 2921 and 2877 (C-H aliphatic), 1645 (C=O (amide I) residual N-acetyl groups), 1589 (N–H bending), 1262 (CH3 in N-COCH3), 1325 (C-N (amide III)), 1154 (C-O in CH2OH), and 1066, 1028, and 896 (pyranose ring). A significant change in the FTIR of PTDz-Cs (Fig. 1d) was noted by comparing it with that of chitosan, which confirmed the grafting reaction between the chitosan and the 1,3,4-thiadiazol-carboxylic acid derivative. The carbonyl group (C=O) intensity was significantly increased and shifted to 1722 cm−1, and the N–H bending peak disappeared due to NH2 consumption during the reaction with the acid derivative (PTDz-Acid). Additionally, the appearance of new peaks at 1574 cm−1 and 629 cm−1 ʋ which were attributed to (C=N thiadiazole) and (C-S-C), respectively, evidenced the introduction of the thiadiazole derivative to the chitosan structure. All these indications confirmed the synthesis of PTDz-Cs.

Analysis of 1H /13C-nuclear magnetic resonance and mass spectrometry

PTDz-amine. (Fig. 2a): 1H-NMR(500 MHz,chloroform-d) [48]: δ/ppm = 7.28 (s, 2H, NH2,canceled by D2O, Fig.S1), 3.11 (t, J = 7.2 Hz, 2H, SCH2CH2CH3), 1.80–1.71 (m, 2H, SCH2CH2CH3), 1.01 (t, J = 7.5 Hz, 3H, SCH2CH2CH3). 13C-NMR (126 MHz, chloroform-d) (Fig. 2b): δppm = 168.36(carbon of thiadiazole ring ((S–C=N)), 155.29 (carbon of thiadiazole ring (N=C–S)), 36.88(SCH2CH2CH3), 22.89(SCH2CH2CH3), 13.31 (SCH2 CH2CH3). MS (m/z) (Fig.S2): δ = 175.27 [%]:[M+, (0.15%)], Anal. Calcd. for C5H9N3S2 (175.27). PTDz-Acid. (Fig. 3a): 1H-NMR (500 MHz, DMSO-d6):δ/ppm = 12.60 (s, 1H, COOH, canceled by D2O), 12.14 (s, 1H, CONH, canceled by D2O, Fig.S3), 3.14 (t, J = 7.2 Hz, 2H, SCH2CH2CH3), 2.65 (t, J = 6.5 Hz, 2H, CH2-acidic), 2.52 (t, J = 6.5 Hz, 2H, CH2-amidic), 1.64 (tq, J = 14.2, 7.2 Hz, 2H, SCH2CH2CH3), 0.93 (t, J = 7.4 Hz, 3H, SCH2CH2CH3). 13C NMR (126 MHz, DMSO-D6) (Fig. 3b): δppm = 174.15(carbon of carboxylic group), 174.00 (carbon of amidic), 171.27 (carbon of thiadiazole ring ((S–C=N)), 159.10 (carbon of thiadiazole ring (N=C–S)), 36.04 (SCH2CH2CH3), 30.33 (carbon of CH2-acidic), 28.81 (carbon of CH2-amidic), 22.95 (SCH2CH2CH3), 13.45(SCH2CH2CH3). MS (m/z) (Fig. S4 & Fig. 4)δ): δ = 275.34 [%]: [M+, (0.42%)], Anal. Calcd. for C9H13N3O3S2 (275.34). 1H-NMR (500 MHz, DMSO-D6) of PTDz-Cs (Fig. 5) showed the characteristic signals of chitosan combined with additional new signals due to the modification reaction. The signal at 1.91 ppm corresponds to CH3 of –NHCOCH3, the signals at 3.06 ppm and 4.76 ppm to H2 and H1 in the glucosamine moiety, respectively [49], and the multiplets at 3.50–3.70 ppm to H3 and H6 protons on Cs Skelton [50]. Compared to the Cs, some new proton signals were noted in the PTDz-Cs polymer, such as the signals at 0.93 ppm, 1.64 ppm, 2.51 ppm, 2.65 ppm, and 3.14 ppm, which were related to the protons of the aliphatic hydrogen atoms of (-SCH2CH2CH3), (-SCH2CH2 CH3), (-CH2-amidic), (-CH2-acidic), and (-SCH2CH2CH3), respectively. The singlet proton of CONH in the acid derivative shifted from 12.14 ppm to 12.3 ppm. Another strong indication to confirm the successful synthesis of PTDz-Cs is the disappearance of the singlet proton of the -COOH group (appeared at 12.60 ppm, Fig. 3a) and the appearance of a new singlet proton at 9.87 ppm, which is due to the chitosan-NH-CO amide linkage.

Fig. 2
figure 2

a 1H-NMR spectrum, b 13C-NMR spectrum of PTDz-amine

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Fig. 3
figure 3

a 1H-NMR spectrum and b 13C-NMR spectrum of PTDz-Acid

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Fig. 4
figure 4

Mass fraction of PTDz-Acid

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Fig. 5
figure 5

1H-NMR spectrum of PTDz-Cs

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Elemental analysis and degree of substitution (DS)

Qualitative presence of C, H, N, and S elements was determined in the chitosan and the prepared derivative (PTDz-Cs) (Table 2), and the data were used to calculate the DS (Eq. 2) [51].

$$DS\,\left( \% \right) = \frac{{A\left( {{\text{C}}/{\text{N}}} \right)_{{{\text{Cs}}\;{\text{derivative}}}} - \left( {{\text{C}}/{\text{N}}} \right)_{{{\text{Cs}}}} }}{{\text{B}}} \times 100$$
(2)

where (C/N)Cs-derivative and (C/N)Cs are the carbon/nitrogen ratio of PTDz-Cs and Cs, respectively; A and B are the number of nitrogen and carbon atoms, respectively, added on Cs after the modification reaction. The DS value was found to be 78.90%. This value indicated that the chitosan derivative was successfully prepared.

Table 2 Elemental analysis of Cs and PTDz-Cs
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XRD results

XRD of Cs (Fig. 6a) showed two sharp crystalline peaks at 2θ = 10° and 2θ = 20° in agreement with the previous reports indicating the high degree of crystallinity of chitosan [20, 52]. The crystallinity of Cs has clearly decreased after the modification reaction with the PTDz moiety, which is indicated by the broadening of the peaks (Fig. 6b) as compared with Cs, pointing to the fact that the PTDz-Cs derivative has a less crystalline structure than chitosan. The loss in the Cs crystallinity after PTDz modification could be assigned to the breaking of the intermolecular and intramolecular hydrogen bonding systems that exist between the hydroxyl and amino groups [53].

Fig. 6
figure 6

XRD of chitosan (Cs, a) and the modified chitosan (PTDz-Cs, b)

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Thermal properties

The decomposition pattern for Cs and PTDz-Cs in response to a programed heating (10 °C min−1) was recorded and is tabulated in Table 3. The mass loss data presented in Table 3 showed an obvious change in the thermal profiles of both polymers, which indicated a change in the structure of chitosan, confirming the modification reaction. Moreover, the modification of the chitosan structure with the prepared heterocyclic compound resulted in a loss in the thermal stability of the chitosan derivative, as expected, because of the breaking of the hydrogen bonding system, which leads to a decrease in the crystallinity. As the crystallinity decreases, the thermal stability also decreases. Broido’s method [54] was used to calculate the activation energy of decomposition, which is 317.33 kJ/mol for chitosan and significantly decreased after chitosan modification to 62.22 kJ/mol for PTDz-Cs, indicating lower stability when compared with chitosan.

Table 3 Mass loss data extracted from the thermal profile curve for Cs and the synthesized derivatives as well as the activation energy
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X-ray photoelectron spectroscopy (XPS)

XPS measurements are a valuable tool for surface investigation due to their capacity to distinguish between distinct chemical forms of certain atoms depending on their oxidation states [55]. The full XPS spectra of the chitosan and its derivative PTDz-Cs are shown in Fig. 7.

Fig. 7
figure 7

Full scans of XPS spectra of chitosan and its derivative (PTDz-Cs)

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It is obvious that the sulfur signals at around 226 eV, and 166 eV, which are attributed to S 2s and S 2p, respectively, occur in the spectrum of the chitosan derivative, PTDz-Cs, in contrast to the chitosan spectrum, which corresponds to the existence of sulfur atoms in chitosan’s derivatives [56]. However, the carbon (C 1s), nitrogen (N 1s), and oxygen (O 1s) signals of the PTDz-Cs were slightly displaced and combined with increased intensity compared to the Cs peaks, which shows the rising quantity of deacetylation of chitosan and the formation of the new derivative [57].

Further, in the range of 162–168 eV of the bending energy, the S 2p peaks at 163.9 and 165.4 eV (Fig. 8a) were correlated with S-C in the aliphatic and aromatic groups, respectively [58]. The XPS spectrum of Cs for C S1 presents three deconvoluted peaks at 284.7, 286.1, and 287.1 eV, which are attributed to C–C, C–N/C–O, and N–C=O, respectively [59], as shown in Fig. 8b. As expected, there is a significant shift in the peak at 284.7 eV for PTDz-Cs towards higher binding energy as a result of the substituted acetyl group with a different terminal chain of the polymers. However, Fig. 8c shows the corresponding peaks of N1s of the chitosan compound at 399 and 400.5 eV, which are attributed to NH2 and NH, respectively. New peaks at ~ 402 eV related to –N=C occurred with a gradual shift after substitution for PTDz-Cs. Noteworthy differences were found in the spectra of O 1S for chitosan or PTDz-Cs. Overall, the results show a significant relationship between the formation of PTDz-Cs polymers.

Fig. 8
figure 8

XPS spectra of chitosan and its derivative PTDz-Cs and bending energy of sulfur S 2p (a); carbon C 1s (b); and nitrogen N 1s (c)

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Antifungal activity

Due to increasing use of AMB in the treatment of fungal infections, it has led to the antifungal resistant of mucorales to AMB. Therefore, in this study, a new chitosan derivative, PTDz-Cs, was prepared for use as an antifungal agent. Antifungal activity of PTDz-Cs and Cs was evaluated against fungi causing mucromycosis, such as R. microspores, M. racemosus, L. corymbifera, and S. racemosum, as illustrated in Fig. 9 and Table 4. Results in Table 4 show that AMB exhibited weak antifungal activity against L. corymbifera only, but R. microspores, M. racemosus and S. racemosum were resistant to AMB as a traditional antifungal agent. Moreover, results revealed that PTDz-Cs exhibited promising antifungal activity against all tested fungal strains compared to AMB. Inhibition zones of PTDz-Cs were 17.76, 20.1, 38.2, and 18.3 mm toward R. microspores, M. racemosus, L. corymbifera,, and S. racemosum, respectively. Also, the results showed that the highest efficacy of PTDz-Cs was toward L. corymbifera where MIC was 3.9 µg/ml, but both R. microsporus and S. racemosum were least affected by PTDz-Cs, where the MIC was 125 for both of them. On the other hand, Cs only exhibited weak antifungal activity against some tested fungal strains, where inhibition zones were 14.3, 9.1, and 11.0 mm against M. racemosus, L. corymbifera, and S. racemosum, respectively. This indicates the promising antifungal activity of PTDz-Cs is attributed to the addition of the PTDz compound to the original chitosan. Previous studies have reported that thiadiazole derivatives have antifungal activity against pathogenic fungi [60,61,62]. Pham et al. [63] synthesized a series of novel thiadiazole derivatives and found these derivatives have antifungal activity against unicellular and multicellular fungi. Another study reported that thiadiazole derivatives based on pyrimidin exhibited antifungal activity against A. flavus and Fusarium solani. Mohamed et al. [64] illustrated that a novel 1,3,4-thiadiazole modified chitosan has antifungal activity against C. albicans. Li et al. [65] synthesized novel chitosan derivatives containing thiadiazole groups and found these derivatives have promising antifungal activity against Colletotrichum lagenarium and Monilinia fructicola. The mechanism of thiadiazole derivatives may be attributed to their contact with fungal cell which leads to a change in cell membrane permeability and affects the growth of the hyphae [66] and the inhabitation of ergosterol biosynthesis [62].

Fig. 9
figure 9

Antifungal activity of PTDz-Cs and Cs against R. microspores (a), M. racemosus (b), L. corymbifera (c) and S. racemosum (d) compared to Amphotericin B (AMB) as standard antifungal agent

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Table 4 Inhibition zones and MICs of PTDz-Cs and Cs against tested fungal strains
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Larvicidal and pupicidal activity

The toxicity of PTDz-Cs derivative on A. stephensi larvae and pupae was tested in a laboratory setting. The LC50 value (lethal concentration that kills 50% of the targeted organisms) and LC90 value (lethal concentration that kills 90% of the targeted organisms) for PTDz-Cs are recorded in Table 5 as LC50 (LC90): 5.432 (10.398) ppm (larva I), 5.975 (11.556) ppm (II), 6.543 (12.888) ppm (III), 7.511 (13.092) ppm (IV), and 8.641 (14.703) ppm (pupa). We discovered that early instar mosquito larvae were more sensitive to PTDz-Cs derivative exposure than later instar mosquito larvae, however, pupae were not significantly affected by the chitosan derivative. These findings are consistent with previous research on green pesticides on mosquitoes [67], which demonstrated the poisonous effects of methanolic extracts of Sargassum wightii in combination with the microbial pesticide Bacillus thuringiensis var. israelensis against A. sundaicus, with LC50 values of 0.88 for I larval instar. In terms of chitosan toxicity on arthropod pests, Sahab et al. [68] studied the effect of nano-chitosan on Aphis gossypii eggs under laboratory and semi-field conditions, in addition to our previous research on mosquito vector control [69]. Furthermore, Badawy and El-Aswad [70] investigated the poisonousness of chitosan compounds on larval mortality, growth inhibition, and antifeedant activity in S. littoralis III larval instar. Zhang et al. [71] discovered that chitosan has insecticidal activity against a variety of aphids at concentrations ranging from 600 to 6,000 mg/l. Furthermore, chitosan had 70–80% pesticidal activity against the aphid pests Rhopalosiphum padi, Metopolophium dirhodum, and A. gossypii. There are few studies that look at the biological activities of benzothiazole derivatives against insects, even though they exhibit a wide range of biological activities. Previous research by Zhao demonstrated the fumigant toxicity of benzothiazole against the stored grain pest insect Tribolium castaneum (Coleoptera: Tenebrionidae) [72]. The repellency and moderate toxicity of the fungicide 2-(thiocyanomethylthio) benzothiazole (TCMTB) on termites were demonstrated [73]. These compounds are slightly lipophilic and thus have expected biological activity due to their effects on the structure of biological membranes, on transport and distribution processes, on binding receptor interactions, or on modulating cellular processes [74]. Other benzothiazole derivatives influence the nervous system and exhibit acetylcholinesterase (AChE) inhibitory potential [75]. There is currently little information available on the precise mechanisms of action of the chemicals examined in this study in Anopheles. It would be useful to carry out further studies aimed at elucidating the mechanism of action of promising larvicidal and repellent compounds.

Table 5 PTDz-Cs toxicity against larvae and pupa of Anopheles stephensi
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Conclusions

This study reported the successful development of a new chitosan derivative from the reaction of chitosan with a new propyl thiadiazol carboxylic acid compound, which was confirmed by FT-IR, 1H/13C-NMR, GC–MS, elemental analysis, XRD, TGA, and XPS. The modified chitosan (PTDz-Cs) showed various activities, such as antifungal action and larvicidal action, greater than chitosan itself. These results may orient medicinal researchers to apply this new derivative in the biomedical field as well as anti-mosquito finished products.