Azim Ziyaei Halimehjani1, Faezeh Dehghan1, Vida Tafakori2, Elaheh Amini3, Seyyed Emad Hooshmand4, Yazdanbkhsh Lotfi Nosood1. 1. Faculty of Chemistry, Kharazmi University, 49 Mofateh St., 15719-14911, Tehran, Iran. 2. Department of Cell and Molecular Biology, Faculty of Biological Sciences, Kharazmi University, Tehran, Iran. 3. Department of Animal Biology, Faculty of Biological Sciences, Kharazmi University, Tehran, Iran. 4. Department of Medical Nanotechnology, Faculty of Advanced Technologies in Medicine, Iran University of Medical Sciences, Tehran, Iran.
Abstract
A metal-free multicomponent synthetic route for the diverse preparation of dithiocarbamate-containing piperazine derivatives was developed through the C-N bond cleavage of DABCO ring. This multicomponent re-engineering approach proceeds via the reaction of amines, CS2 and DABCO salts in one pot. Various DABCO salts and secondary amines are tolerated well in this protocol to afford a broad spectrum of dithiocarbamate-containing piperazines in good to high yields. Then, the selected compounds have been deployed against some critical types of bacteria and fungi. A certain number of synthesized compounds revealed not only appropriate antibacterial activity as investigated by disc fusion and minimum inhibitory concentration methods against bacteria (Gram-positive and Gram-negative), but also depicted good to excellent antifungal activity.
A metal-free multicomponent synthetic route for the diverse preparation of dithiocarbamate-containing piperazine derivatives was developed through the C-N bond cleavage of DABCO ring. This multicomponent re-engineering approach proceeds via the reaction of amines, CS2 and DABCO salts in one pot. Various DABCO salts and secondary amines are tolerated well in this protocol to afford a broad spectrum of dithiocarbamate-containing piperazines in good to high yields. Then, the selected compounds have been deployed against some critical types of bacteria and fungi. A certain number of synthesized compounds revealed not only appropriate antibacterial activity as investigated by disc fusion and minimum inhibitory concentration methods against bacteria (Gram-positive and Gram-negative), but also depicted good to excellent antifungal activity.
Nowadays, the world is encountering the coronavirus disease 2019 (COVID-19) pandemic. It has been amply clear that this is a viral disease. Nevertheless, it could still weaken patients' immune systems, hence leading to secondary bacterial or fungal infections taking hold [1, 2]. For instance, the COVID-19 delta variant in some patients led to apparent of mucormycosis, previously called zygomycosis and also known as black fungus [3, 4]. Considering the extensive repercussion of this pandemic, scientists must come up with novel and effective antiviral drugs [5, 6], and need to find new antibacterial and antifungal compounds [7, 8]. Indisputably, discovering highly efficient remedies for these infectious diseases has exponentially emerged as an area of focus [9, 10, 11]. In this context, the synthesis of various dithiocarbamates (DTCs), which revealed remarkable properties and diverse applications in bioorganic and medicinal chemistry such as fungicides and pesticides, crop protection agents and anticancer agents, is thoroughly crucial [12, 13, 14, 15, 16, 17, 18]. For example, take Zineb, Maneb and disulfiram as imperative dithiocarbamate-based biologically active molecules (Figure 1) [19,20]. Further, piperazine is a six-membered heterocyclic compound encompassing two nitrogen atoms in the positions 1 and 4 [21]. On account of the importance of the substituted piperazines in many different biomedical applications, a swift approach to the direct synthesis of novel piperazines has been tremendously beneficial. Among various methods reported for the synthesis of piperazines, DABCO bond cleavage found widespread applications for the direct preparation of valuable piperazines scaffolds. A broad spectrum of nucleophiles such as carboxylic acids, amines, thiols, phenolates, indoles, azide, and alcohols were successfully deployed for the cleavage of C–N bond in DABCO [22]. Piperazine-based drugs such as Indinavir, an antiretroviral drug used to treat HIV, Ciprofloxacin, Buspirone, and Prazosin are available in the market (Figure 1). In addition, Vestipitant, a NK-1 receptor antagonist is in clinical trials to treat anxiety and tinnitus [23, 24]. Until now, MDL@Drug Data Report (MDDR) database includes approximately 11800 scaffolds bearing the piperazine heterocycles. Since piperazine compounds are normally applied as a linker between two portions of a bioactive molecules, combining piperazine along with dithiocarbamate moieties in a single structure may have synergistic effect for designing of novel and promising pharmacologically active compounds.
Figure 1
Representative piperazine- and dithiocarbamate-centered drugs and biologically active compounds.
Representative piperazine- and dithiocarbamate-centered drugs and biologically active compounds.Since the conventional synthesis encompassing the reaction of two reagents have limitations for preparing diverse and complex products, multicomponent reactions (MCRs) or one-pot three or more reactants procedures, making it viable to generate ensued molecules with more complexity and efficiency [25]. Besides, the re-engineering approach makes it possible to adopt specific nonelementary two-component reactions into higher-order MCRs. Implementing the re-engineering approach as a rational design culminated in increasing the dimensionality of MCRs and achieving diverse skeletal molecules [26]. In addition, in terms of the green chemistry criteria, MCRs enjoy excellent green properties such as pot, atom, and step economy and appropriate environmental impact factor (E-factor), as the lower amount of waste products were produced [27]. Consequently, in this project, dithiocarbamate-containing piperazine derivatives which were recently prepared with us via a two-component reaction [28], effectively were synthesized through a novel re-engineering multicomponent approach and subsequently, their antifungal and antibacterial properties were investigated (Scheme 1).
Scheme 1
Single-pot three-component approach for the synthesis of dithiocarbamate-containing piperazines (4) using secondary amines (1), CS2 (2) and DABCO salts (3).
Single-pot three-component approach for the synthesis of dithiocarbamate-containing piperazines (4) using secondary amines (1), CS2 (2) and DABCO salts (3).
Experimental
General
Starting materials and solvents were purchased from Merck and Fluka and were applied as received. All reactions were carried out in sealed vessel at high temperature for overnight. 1H and 13C NMR spectra were recorded on a Bruker AMX 300 MHz device with tetramethylsilane (TMS) as internal standard for NMR solvents. Purity of products and progress of the reactions were checked by thin-layer chromatography using TLC silica gel 60 F254 plates and visualization was carried out using iodine or KMnO4 solution. Melting points were measured by an electrothermal digital apparatus. HRMS (High Resolution Mass Spectra) was measured on a THERMO SCIENTIFIC Advantage and a THERMO SCIENTIFIC Exactive instrument equipped with an APCI source in the positive-ion mode.
General procedure for preparation of quaternary ammonium salts from DABCO
DABCO (10 mmol, 1.12 g) was dissolved in THF (20 mL) and then an alkyl halide such as isopropyl, isobutyl, allyl, benzyl, isopentyl halides (10 mmol) was added and the reaction mixture was heated at 70 °C for 24 h to afford a precipitate. Filtration of the precipitate, washing with diethyl ether (3 × 15 mL), and drying under vacuum afforded the corresponding product. In the case of bis ammonium salt of DABCO, 20 mmol of DABCO reacted with 10 mmol of the corresponding 1,3-dibromopropane.
General procedure for preparation of functionalized piperazines
In a round bottom flask, a DABCO salt (1 mmol), a secondary amine (1 mmol), carbon disulfide (1.5 mmol), potassium carbonate (1 mmol), and THF (5 mL) were added and the mixture was stirred at 90 °C for 16 h. At the end, the solvent was evaporated and the crude mixture was subjected to column chromatography (SiO2, EtOAc/petroleum ether; 70/30) to afford pure products. The isolated compounds were well characterized by NMR spectroscopy and HRMS analysis.
For antibacterial assays Gram-positive bacteria, Bacillus subtilis (ATTC 6633), Methicillin Resistance Staphylococcus aureus (ATTC 33593), and Gram-negative bacteria, Escherichia coli (ATTC 25922), Klebsiella pneumonia (ATTC 10031) were used. For antifungal assays molds, Aspergillus niger (DSM 1957) and Fusarium oxysporum (DSM 62338) and yeasts, Candida albicans (ATTC 10231), Trichosporon asahii (CBC 8904) were used. Bacterial strains were cultured in nutrient agar and fungi were cultured in Sabouraud dextrose agar.
Antimicrobial Tests
Antibacterial and antifungal activities were evaluated with CLSI [29, 30] and EUCAST [31] standard methods by the agar well diffusion and the microbroth dilution techniques. Microbroth dilution method was applied for minimum inhibitory concentration (MIC) determination. All experiments were performed in three biological replicates. A stock solution was prepared by dissolving 10 mg of the hydrochloride salt of each compound (10 mg) in 1 mL of distilled water or related culture media as solvent and then sterilized by microbial filter with 0.22 μm pore size.
Agar well diffusion method for antibacterial activity assay
Bacterial homogenous suspensions were prepared by transferring the fresh grown bacteria from the plates into sterile normal saline solution and vortexing. In continue, the turbidity was adjusted to 0.5 McFarland standard units, containing 1–2 ∗ 108 cell/ml, and was spread over the entire Mueller Hinton agar (MHA) surface plates with sterile swab. Next, by the aid of a sterile tip, a hole of 6–8 mm diameter is punched aseptically, and 100 μL of the prepared stock of each compound was introduced into the well. A well was poured with Refampicin as positive control. Eventually, the plates were incubated at 37 °C for 24 h. Diffusion of the antimicrobial agent in the agar medium inhibits the growth of the tested microbial strain. The inhibition zones were measured in millimeter [32].
Agar well diffusion method for antifungal activity assay
The fungal strains were cultured on Sabouraud dextrose agar and incubated at 35 °C (for yeasts) and 28 °C (for molds). A microbial suspension of yeast was prepared in sterile normal saline and adjusted to 0.5 McFarland standard units, containing 1–5 ×106 cell/ml. For the mold, a sterile tissue paper putted on the surface of the Sabouraud dextrose agar containing each mold, then sterile normal saline (1 mL) supplemented with 0.1% Tween 20 was implemented to cover them. The spores (without any hypha) were collected through tissue paper with sterile tip. After that, by applying a hemocytometer, the suspension was adjusted to 1–5×106 conidia/mL and was spread over the entire Mueller Hinton agar containing %2 glucose surface plates by the aid of a sterile swab. Then, a hole of 6–8 mm diameter is punched aseptically with a sterile tip, and 100 μL of the tested compounds (10 mg/mL dissolved in water) is introduced into the well. Nystatin was used as positive control standard antifungal drug and incubated at 35 °C and 28 °C for 48 h.
Determination of minimum inhibitor concentration of synthetic compounds
The MIC is the minimum concentration of antimicrobial compound that completely prevents the growth of the microorganism in culture medium as detected by the unaided eye [33]. The procedure involved preparing two-fold dilutions of the antimicrobial agent in MHB or MHB containing %2 glucose dispensed in 96-well microtitration plate (microdilution). Therefore, the compounds that could impact on microorganisms (4f for bacteria and 4v for fungi) in agar well diffusion method were selected and diluted. Therefore, each well was poured with 50 μl of e.g. 10, 5, 2.5, 1.25, 0.625, 0.312, 0.156, 0.078, 0.039 and 0.019 mg/mL of proper medium of effective compounds. Then, each well is inoculated with 50 μl of microbial inoculum prepared in MHB or and MHB+ %2 glucose after dilution with culture media, 1:150 v:v of standardized microbial suspension adjusted to 0.5 McFarland scale. After complete mixing, the inoculated 96-well microtitration plates were incubated in 37 °C for 24–48 h [29,30,33].Optical density measuring was used for MIC determination. Thus, by using an ELISA reader, the absorbance of microtiter plates at 570 nm and 530 nm was evaluated for bacteria and yeasts, respectively. The drug free wells were considered as positive control and the lowest concentration with optical density of less than 0.1 was considered as MIC. For molds, MIC was determined visually.
Results and discussion
Chemistry
Here, a straightforward and three-component procedure for the synthesis of dithiocarbamate-containing piperazines 4 is reported via the reaction of secondary amines 1, carbon disulfide 2, and quaternized derivatives of DABCO 3. Normally, design and discovery of a direct step-economy synthetic route to generate substituted piperazine rings is partly complicated; as a result, it has been garnered widespread attention in recent years. In this regard, 1,4-diazabicyclo [2.2.2]octane (DABCO) which has universally known as a base in organic reactions [28, 34], has been deployed as a reagent in C–N bond cleavage synthetic routes for the straightforward preparation of unsymmetrical piperazine compounds [22].To optimize the reaction conditions, a model reaction was considered (Scheme 2). Initially, we observed that the reaction of DABCO salt 3b (0.5 mmol) with diethylamine (0.5 mmol) and CS2 (0.75 mmol) in THF for 24 h at 120 ᵒC without a base provided the product 4f in 40% isolated yield (Table 1, entry 1). Under these conditions, various bases such as NaOH, KOH, K3PO4, Na2CO3 and K2CO3 were used in the model reaction (Table 1, entries 2–6) and the elevated yield (89 %) was achieved using K2CO (Table 1, entry 6). By screening the model reaction in various protic and aprotic solvents in the presence of K2CO3, we observed that higher yields were obtained in MeOH and EtOH (Table 1, entries 7–8) compare to DMF, n-hexane and CHCl3 (Table 1, entries 9–11). Using aqueous media gave inferior results (Table 1, entry 14). No desired product was obtained in CH2Cl2 and DMSO (Table 1, entry 12–13). By decreasing the reaction temperature to 50, 70 and 90 °C, the corresponding product was obtained in 25, 37 and 87 %, respectively (Table 1, entries 15–17). Besides that, we observed that by decreasing the reaction time to 16 h, the yield was improved to 95% (Table 1, entry 19). At the end, deploying THF as a solvent and heating the reaction mixture to 90 °C for 16 h in the presence of K2CO3 as a base was used as optimal conditions for the direct synthesis of a wide range of piperazine derivatives.
Scheme 2
Model reaction for optimization of the reaction conditions for the synthesis of dithiocarbamate-containing piperazine (4f) using amine (1a), CS2 (2) and DABCO salt (3b).
Table 1
Selected conditions for optimization of the model reaction of diethylamine (1a), CS2 (2) and DABCO salt (3b) for the synthesis piprazine derivative (4f).
Entry
Base
Solvent
T (ᵒC)
Time (h)
Yield (%)a,b
1
-
THF
120
24
40
2
NaOH
THF
120
24
37
3
KOH
THF
120
24
32
4
K3PO4
THF
120
24
66
5
Na2CO3
THF
120
24
54
6
K2CO3
THF
120
24
89
7
K2CO3
MeOH
120
24
83
8
K2CO3
EtOH
120
24
80
9
K2CO3
n-hexane
120
24
33
10
K2CO3
DMF
120
24
37
11
K2CO3
CHCl3
120
24
26
12
K2CO3
CH2Cl2
120
24
NRc
13
K2CO3
DMSO
120
24
NRc
14
K2CO3
H2O
120
24
8
15
K2CO3
THF
50
24
28
16
K2CO3
THF
70
24
35
17
K2CO3
THF
90
24
87
18
K2CO3
THF
90
12
82
19
K2CO3
THF
90
16
95
Isolated yield.
Reaction conditions: DABCO salt 3b (0.5 mmol), diethylamine (0.5 mmol), carbon disulfide (0.75 mmol), base (0.5 mmol), solvent (3 mL).
NR = no reaction.
Model reaction for optimization of the reaction conditions for the synthesis of dithiocarbamate-containing piperazine (4f) using amine (1a), CS2 (2) and DABCO salt (3b).Selected conditions for optimization of the model reaction of diethylamine (1a), CS2 (2) and DABCO salt (3b) for the synthesis piprazine derivative (4f).Isolated yield.Reaction conditions: DABCO salt 3b (0.5 mmol), diethylamine (0.5 mmol), carbon disulfide (0.75 mmol), base (0.5 mmol), solvent (3 mL).NR = no reaction.The generality of the reaction was investigated using various DABCO salts and secondary amines and the results are shown in Scheme 3. Various linear and cyclic secondary amines encompassing diethylamine, dimethylamine, morpholine, pyrrolidine, and piperidine were successfully deployed in this synthetic route to give the desired 1,4-disubstituted piperazines 4a-u in good to excellent yields. In addition, DABCO salts with primary and secondary alkyl groups are compatible with this protocol. By using bis-ammonium salt of DABCO, the corresponding N, N′-bis piperazine 4v containing two dithiocarbamate groups was obtained in moderate yield. In this MCRs, the regioselectivity seems to be related to the alkyl moiety attached to the nitrogen in DABCO salts. In the case of allyl and benzyl salts of DABCO, due to the competition between the carbon of the bridge and the alkyl group for nucleophilic attack, the corresponding alkyl dithiocabamates were obtained as main byproduct in the reaction mixture. The structures of all products were confirmed by 1H NMR, 13C NMR and HRMS analyses (Figures S1–S44 in supplementary material).
Scheme 3
Diversity in the synthesis of piperazines containing dithiocarbamate motif a,b (4) from secondary amines (1), CS2 (2) and DABCO salts (3). a Isolated yield. b Reaction conditions: DABCO salt (1 mmol), secondary amine (1 mmol), carbon disulfide(1.5 mmol), K2CO3 (1 mmol), solvent (5 mL), 90 ᵒC and 16 h.
Diversity in the synthesis of piperazines containing dithiocarbamate motif a,b (4) from secondary amines (1), CS2 (2) and DABCO salts (3). a Isolated yield. b Reaction conditions: DABCO salt (1 mmol), secondary amine (1 mmol), carbon disulfide(1.5 mmol), K2CO3 (1 mmol), solvent (5 mL), 90 ᵒC and 16 h.
Biological studies
Based on the results, the selected synthetic compounds were effective antifungal compounds because they could inhibit yeasts and molds.
Antimicrobial effects of the synthetic compounds with the disk diffusion test
As shown in Figure 2, 4f, 4j and 4v were affected on different bacteria. But, based on zone inhibition diameter, only 4f was affected on B.subtillis and E.coli with zone inhibition diameter of 20 ± 1 and 22 ± 0.6 mm, respectively and had no effect on MRSA and K.pneumoniae.
Figure 2
Antibacterial effects of synthetic compounds on bacteria: (a) B.subtilis was sensitive to 4f (20 ± 1 mm inhibition zone) and intermediate to 4j (18 mm ± 1 inhibition zone), (b) MRSA was resistant to all of synthetic compounds, (c) K.pneumoniae was resistant to all of synthetic compounds, and (d) E.coli was sensitive to 4f (22 ± 0.6 mm inhibition zone) and intermediate to 4j (15 ± 0.7 mm inhibition zone).
Antibacterial effects of synthetic compounds on bacteria: (a) B.subtilis was sensitive to 4f (20 ± 1 mm inhibition zone) and intermediate to 4j (18 mm ± 1 inhibition zone), (b) MRSA was resistant to all of synthetic compounds, (c) K.pneumoniae was resistant to all of synthetic compounds, and (d) E.coli was sensitive to 4f (22 ± 0.6 mm inhibition zone) and intermediate to 4j (15 ± 0.7 mm inhibition zone).Addition antifungal effects of these synthetic compounds were shown in Figure 3. 4d, 4f and 4v had antifungal activity. 4d and 4v were affected on C.albicans with zone inhibition of 23 ± 1 and 22 ± 0.5 mm, respectively, 4f and 4v had 19 ± 1 and 22 ± 1 mm inhibition zone for T.asahii.
4d, 4f and 4v with 19 ± 0.7, 19 ± 0.5 and 25 ± 1 mm inhibition zone were effected on A.niger, respectively. The results of zone inhibition diameter were summarized in Table 2.
Figure 3
Antifungal activity of synthetic compounds: (a) 4d and 4v were affected on C.albicans with zone inhibition of 23 ± 1 and 22 ± 0.5 mm respectively, (b) 4f and 4v had 19 ± 1 and 22 ± 1 mm inhibition zone on T.asahii. (c) 4d, 4f and 4v with 19 ± 0.7, 19 ± 0.5 and 25 ± 1 mm inhibition zone were effect on A.niger respectively. (d) The selected compounds had no effect on F.oxysporum.
Table 2
Numerical results of inhibition diameter zones for synthetic compounds.
Microorganism
B.subtillis
E. coli
C.albicans
T.asahii
A.niger
inhibition diameter zones in millimeters (mm)
(4f) 20 ± 1
(4f) 22 ± 0.6
(4d) 23 ± 1(4v) 22 ± 0.5
(4f) 19 ± 1(4v) 22 ± 1
(4d)19 ± 0.7(4f) 19 ± 0.5(4v) 25 ± 1
Antifungal activity of synthetic compounds: (a) 4d and 4v were affected on C.albicans with zone inhibition of 23 ± 1 and 22 ± 0.5 mm respectively, (b) 4f and 4v had 19 ± 1 and 22 ± 1 mm inhibition zone on T.asahii. (c) 4d, 4f and 4v with 19 ± 0.7, 19 ± 0.5 and 25 ± 1 mm inhibition zone were effect on A.niger respectively. (d) The selected compounds had no effect on F.oxysporum.Numerical results of inhibition diameter zones for synthetic compounds.
Minimum inhibitory concentration (MIC) results of the selected synthetic molecules
For determination of MIC, we selected 4f (effect on both bacteria) and 4v (effect on three fungi). Figure 4 shows the MIC results of the 4f and 4v on sensitive microorganisms. The results of MIC were summarized in Table 3. As shown in Figure 4, compound 4f illustrated antibacterial activity with MIC values of 2.5 mg/mL against E.coli and B.sutilis and also compound 4v showed antifungal activity with MIC values of 2.5, 0.625 and 5 mg/mL against C.albicans, T.asahii and A.niger, respectively.
Figure 4
The results of MIC test for 4f and 4v. All experiments had three replicates. (a) 4f was tested for bacteria and (b) 4v was tested for fungi.
Table 3
Numerical results of MIC for synthetic compounds.
Microorganism
E.coli (4f)
B.subtilis (4f)
C.albicans (4v)
T.asahii (4v)
A.niger (4v)
MIC mg/mL (Average)
2.5
2.5
2.5
0.625
5
The results of MIC test for 4f and 4v. All experiments had three replicates. (a) 4f was tested for bacteria and (b) 4v was tested for fungi.Numerical results of MIC for synthetic compounds.
Conclusion
In conclusion, expansion of modern biologically active molecules inspired the organic chemists to render DABCO bond cleavage technique. In this project, a one-pot three-component reaction based on re-engineering approach has been devised for the synthesis of dithiocarbamate-containing piperazine scaffolds by in situ reaction of amines, CS2 and DABCO salts. The reaction proceeds via C–N bond cleavage of DABCO ring. A certain number of synthesized compounds revealed appropriate antibacterial activity against both Gram-positive as well as Gram-negative bacteria and depicted good to excellent antifungal activity. Finally, this synthetic route enjoys various merits namely easy workup procedure, operator friendliness, economical use of reagents, provided satisfactory yields of the small molecules.
Declarations
Author contribution statement
Azim Ziyaei Halimehjani: Conceived and designed the experiments; Analyzed and interpreted the data; Contributed reagents, materials, analysis tools or data; Wrote the paper.Faezeh Dehghan, Yazdanbkhsh Lotfi Nosood: Performed the experiments; Analyzed and interpreted the data.Vida Tafakori, Elaheh Amini: Performed the experiments; Analyzed and interpreted the data; Contributed reagents, materials, analysis tools or data; Wrote the paper.Seyyed Emad Hooshmand: Conceived and designed the experiments; Wrote the paper.
Funding statement
This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.
Data availability statement
Data included in article/supplementary material/referenced in article.
Declaration of interests statement
The authors declare no conflict of interest.
Additional information
No additional information is available for this paper.
Authors: Marwa Ayman; Shahenda M El-Messery; Elsayed E Habib; Sara T Al-Rashood; Abdulrahman A Almehizia; Hamad M Alkahtani; Ghada S Hassan Journal: Bioorg Chem Date: 2019-01-03 Impact factor: 5.275
Figure 1
Scheme 1
Scheme 2
Scheme 3
Figure 2
Figure 3
Figure 4