Abstract
By screening of a collection of 50 000 small-molecule compounds, we recently identified 4-arylazo-3,5-diamino-1H-pyrazoles as a novel group of anti-biofilm agents. Here, we report a SAR study based on 60 analogues by examining ways in which the pharmacophore can be further optimized, for example, via substitutions in the aryl ring. The SAR study revealed the very potent anti-biofilm compound 4-(2-(2-fluorophenyl)hydrazineylidene)-5-imino-4,5-dihydro-1H-pyrazol-3-amine (2).
By screening of a collection of 50 000 small-molecule compounds, we recently identified 4-arylazo-3,5-diamino-1H-pyrazoles as a novel group of anti-biofilm agents.
Introduction
Biofilms are aggregates of microorganisms embedded in a matrix of self-produced polymeric substances consisting of polysaccharides, proteins, nucleic acids and lipids1 and are formed by bacteria and fungi as part of their survival mechanism. Biofilm forming bacteria are involved in various infections, and in this mode of growth, the bacteria are protected against both the immune system and antibiotic treatment, making it nearly impossible to treat with the current antibiotics.2 This antibiotic resistance, provided by the biofilm life mode, has made biofilm infections a global challenge, especially when associated with medical implants.3
With the increased applications of modern medical technologies, biofilm-based infections have become a major socioeconomic burden, as they lead to increased hospitalization time and costs and reduced quality of life of the patient as well as loss of employment.4 Therefore, there is an urgent need to develop drugs with new modes of action, in particular, those capable of transcending the multiple layers of biofilm resistance, providing means to efficiently combat biofilm-based infections.5
Compelling evidence suggests that cyclic di-guanosine monophosphate (c-di-GMP) is a key second messenger for biofilm formation in several bacterial species.6,7 A high level of c-di-GMP in bacteria correlates with a higher amount of biofilm formation, while low intracellular levels of c-di-GMP lead to increased motility and biofilm dispersal. Diguanylate cyclase (DGC) enzymes catalyze the formation of c-di-GMP, whereas phosphodiesterase (PDE) enzymes catalyze c-di-GMP degradation. Several studies have been aiming at developing compounds that inhibit DGC, whereas studies on PDE activators are limited.8 Therefore, small molecules that efficiently activate PDEs, leading to a reduction of the c-di-GMP content in bacteria, may provide lead structures for the development of novel anti-biofilm drugs. We recently identified 4-arylazo-3,5-diamino-1H-pyrazoles as a novel group of such anti-biofilm agents that stimulate the activity of the PDE BifA present in Pseudomonas spp. including P. aeruginosa which is a common cause of hospital-acquired infections.9 In this article, we describe the synthesis and biological activity of 61 compounds, leading to a comprehensive understanding of the structure–activity relationship (SAR) of phenyl-azo-3,5-diamino-1H-pyrazole structures as novel activators of the P. aeruginosa PDE BifA.
Results and discussion
Biological activity
During the course of our ongoing biofilm inhibitor discovery and development program, 4-(3-fluorophenyl)azo-3,5-diamino-1H-pyrazole (3-F, 1, Fig. 1) was identified to reduce the c-di-GMP level by more than 70% (73% reduction according to c-di-GMP monitor measurements and 84% reduction according to MS quantification) through stimulation of the activity of the P. aeruginosa PDE, BifA.8 Cyclic di-GMP reductions presented in this work will be based on c-di-GMP monitoring measurements, unless otherwise noted. Aiming at elucidating the structure and activity relationship of the 4-arylazo-3,5-diamino-1H-pyrazole structure, 60 analogues were synthesized by varying the structure of the arylazo and pyrazole moieties, and their c-di-GMP reducing potency was evaluated using the c-di-GMP monitor strain MTR23510 as described by Andersen and co-workers.8 The c-di-GMP monitor strain is a GFP (green fluorescent protein)-based reporter established in the bacteria P. aeruginosa, where the intracellular level of c-di-GMP of the bacteria is directly reflected in the strength of the fluorescence signal, due to gene fusion between gfp and the c-di-GMP responsive promoter PcdrA.10 Briefly, using a 96 well platform, a 20 hour old culture of the c-di-GMP monitor strain was diluted 100 fold into 100 μL aliquots of fresh growth media supplemented with either 1% DMSO (control) or 100 μM test compound (also in 1% DMSO). The cultures in the resulting microtiter plate were propagated at 37 °C on a Tecan reader at 440 RPM, and the corresponding values of fluorescence (GFP fluorescence is equal to the c-di-GMP level) and cell densities (OD600) were measured for each culture every 20 minutes for 24 hours. Then, maximal GFP/OD values were calculated for each compound and compounds exhibiting c-di-GMP reducing potency were identified as compounds giving a maximal GFP/OD600 value lower than the maximal GFP/OD600 value of the DMSO control (see Andersen et al. 20218 for details).
Fig. 1. (A) The hit compound from the library screen. All percentages reported are based on the reduction of the c-di-GMP level, unless otherwise noted. (B) A hybrid of tautomeric forms of structure 1.
Chemical synthesis
The phenyl-azo-3,5-diamino-1H-pyrazole structure has a large number of contributing tautomeric forms, which combine electrons from both the phenyl-azo and pyrazole systems in the same hybrid (Fig. 1B). Therefore, alternation of one of the two moieties separately is expected to modify the overall electronic properties of the structure.
A simple two-step synthetic procedure to assemble the 4-arylazo-3,5-diamino-1H-pyrazole system, as reported by Kryštof et al.,11 prompted us to initiate SAR studies by synthesizing 3,5-diamino-1H-pyrazole derivatives that vary in the phenyl substitution pattern. The first synthetic step is a diazotization reaction of aniline (A) to form the corresponding diazonium ion (see Fig. 2). The diazonium ion is a high energy molecule and therefore, it is important to keep the reaction temperature around 0 °C. The diazo intermediate is quenched with malonate in the presence of a base to give the N-phenylcarbonohydrazonoyl dicyanide intermediate (B). The second and last step is a cyclization reaction with hydrazine, resulting in the corresponding pyrazole derivative (C).
Fig. 2. Synthetic route for the preparation of phenyl-3,5-diamino-1H-pyrazole derivatives. Reagents and conditions: (a) NaNO2 (1 eq.), conc. HCl, water, 0 °C, 30 min; (b) H2C(CN)2 (1.5 eq.), NaOAc, water, 0 °C → rt; (c) NH2NH2·H2O (50%, 1.15 eq.), EtOH, rt, overnight.
SAR studies
Starting from different aniline derivatives (A), a library consisting of 32 phenyl-3,5-diamino-1H-pyrazole derivatives (C), with different phenyl substitution patterns, was synthesized, see Table 1. First, the position of the fluorine substituent in the phenyl ring, was investigated (entries 2 and 3), showing the highest activity to be achieved when flourine was located ortho to the hydrazine moiety (2-F, entry 2), resulting in 83% reduction of the c-di-GMP level (90% reduction according to MS quantification), compared to 73% for the hit compound 3-F (entry 1) and 44% for 4-F (entry 3). In comparison, the analogue without substituents in the phenyl ring (4), decreased the activity to 71%, while other halogen substituents (5–7) resulted in a complete loss of activity, see Fig. 3A. Also, addition of more fluorine substituents was investigated (entries 8–15), which resulted in a decrease of activity, Fig. 3B. Next, the presence of additional substituents on the 2-F analogue was investigated. This included a methyl group as well as halogens (16–19), see Table 1; however, for all these analogues, no activity was observed. These results suggest a narrow pocket in the binding site of the target protein. We included six more analogues with different halogens and number of substituents and their positions (20–24); however, none of these analogues showed any biological activity. As control experiments, the reagents involved in the synthesis of the hit compound 2-F (entry 2) were also tested. This included the starting material 2-flouroaniline and the N-(phenyl)carbonohydrazonoyl dicyanide intermediate 34 (see Fig. 5); however, none of these showed any effect on the c-di-GMP level.
Functionalization on the benzene.
| ||||||
|---|---|---|---|---|---|---|
| Entry | R 1 | R 2 | R 3 | R 4 | R 5 | Percentage reduction of c-di-GMP |
| 4 | H | H | H | H | H | 71% |
| Fluorinated | ||||||
| 2 | F | H | H | H | H | 83% |
| 8 | F | H | H | H | F | 77% |
| 1 | H | F | H | H | H | 73% |
| 9 | F | H | H | F | H | 65% |
| 10 | F | H | H | F | F | 61% |
| 11 | F | F | H | H | H | 48% |
| 3 | H | H | F | H | H | 44% |
| 12 | F | H | F | H | H | 41% |
| 13 | H | F | H | F | H | 35% |
| 14 | F | H | F | H | F | 30% |
| 15 | F | F | F | H | H | 0% |
| Oxygen | ||||||
| 27 | OH | H | H | H | H | 55% |
| 30 | H | OH | H | H | H | 0% |
| 26 | OMe | H | H | H | H | 0% |
| 31 | H | H | OMe | H | H | 0% |
| 29 | OH | H | H | H | OH | 15% |
| Amine | ||||||
| 25 | NH2 | H | H | H | H | 0% |
| Halogens | ||||||
| 5 | Cl | H | H | H | H | 0% |
| 6 | Br | H | H | H | H | 0% |
| 7 | I | H | H | H | H | 0% |
| 20 | Br | H | H | F | H | 0% |
| 18 | F | H | H | Br | H | 0% |
| 21 | H | Br | H | Br | H | 0% |
| 22 | Br | H | H | Br | H | 0% |
| 23 | Br | H | H | H | Br | 0% |
| 19 | F | H | H | H | Br | 0% |
| 24 | CF3 | H | H | H | H | 0% |
| Other | ||||||
| 28 | F | H | H | H | OH | 75% |
| 16 | F | H | Me | H | H | 0% |
| 17 | F | H | H | H | Me | 0% |
Fig. 3. The c-di-GMP reduction of 4-arylazo-3,5-diamino-1H-pyrazole with a variation in the phenyl substitution pattern. All percentages reported are based on the reduction of the c-di-GMP level, unless otherwise noted. (A) Varying the halogen at the 2-position; (B) varying the quantity and position of fluorides; (C) varying the position and quantity of the hydrogen bond acceptor/donor groups.
Fig. 5. Functionalization of the pyrazole nitrogen in the 4-arylazo-3,5-diamino-1H-pyrazole. Reagents and conditions: (a) NH2NHR (1.15 eq.), EtOH, rt or 50 °C (see the ESI†), overnight; (b) NaOAc (0.56 mol%), Ac2O (22 eq.), 100 °C.
It was speculated if the importance of fluorine was not solely due to an electronic inductive effect on the aromatic system, but whether it could also be involved in a hydrogen bond acceptor/donor interaction. To investigate this, we synthesized and studied a series of aryl-3,5-diamino-1H-pyrazole derivatives containing a substituent in the phenyl 2-position with hydrogen bond donor properties, see Fig. 3C. While the amino- and methoxy-functionalized analogues (25 and 26, respectively) showed no activity, the hydroxyl-functionalized analogue (27) showed a 55% reduction of the c-di-GMP level. To further investigate the effect of the hydroxyl substituent, we synthesized the 2-F,6-OH (28, Fig. 3B) and 2,6-OH (29) analogues which showed activities at 75% and 15%, respectively. Importantly, the hydroxyl-functionalized derivatives showed better water solubility compared to analogues containing fluorine substituents. Further investigation of the position of the hydroxyl (30) or methoxy (31) group, again resulted in no biological activity.
To summarize, out of all the phenyl analogues synthesized and tested, 2-F (2) proved to have the best c-di-GMP lowering effect with 83% reduction, Fig. 3 and 4. Additionally, 2,6-F (8), 2-F,6-OH (28) and the non-substituted phenyl derivative (4) showed a c-di-GMP reduction of more than 70%, Fig. 4.
Fig. 4. The 4 most promising 3,5-diamino-1H-pyrazole derivatives with a variation in the phenyl substitution pattern. All percentages reported are based on the reduction of the c-di-GMP level, unless otherwise noted.
We continued our SAR studies focusing on the 2-F analogue (2). In subsequent efforts, we investigated the functionalization of the 3,5-diamino-1H-pyrazole amino group, as summarized in Table 2. The pyrazole C3 and C5 amino groups were acetylated by reacting the 2-F analogue (2) with acetic anhydride, which provided a mixture of two products (32 and 33), see Fig. 5. The mixture of the acylated analogues (32 and 33) showed complete loss of activity. Also, selective functionalization of the N1-position was investigated. Synthesis of the N1-functionalized pyrazole derivatives was achieved by reacting the malonate intermediate (34) with different hydrazine or hydrazide derivatives, Fig. 5, step 1. Cyclization with hydrazine derivatives was carried out at room temperature overnight, whereas cyclization with hydrazides required heating of the reactions to 50 °C overnight (see details in the ESI†).
Functionalization on the pyrazole nitrogens.
| ||||
|---|---|---|---|---|
| Entry | R 1 | R 2 | R 3 | Percentage reduction of c-di-GMP |
| 44 | H |
|
H | 40% |
| 32 | Ac | H | Ac | 0% |
| 33 | H | Ac | Ac | 0% |
| 37 | H |
|
H | 0% |
| 40 | H |
|
H | 0% |
| 36 | H |
|
H | 0% |
| 41 | H |
|
H | 0% |
| 39 | H |
|
H | 0% |
| 38 | H |
|
H | 0% |
| 43 | H |
|
H | 0% |
| 42 | H |
|
H | 0% |
| 37 | H | Me | H | 0% |
A series of N1-alkylated pyrazole analogues were synthesized, with the alkyl group varying in both size and electronic properties, see Fig. 6 (35–40). The chosen substituents varied from a methyl group (35) to the bulkier benzyl (36) and para-fluorophenyl group (37) as well as an ethanol (38) and a para benzoic acid (39) substituent. However, all N1-alkylated pyrazole analogues showed a complete loss of activity. To further expand the SAR studies, the benzoylated variant (40) was also synthesized and tested in order to investigate the effect of changing the electronic character of the pyrazole ring. Once again, this analogue showed no activity.
Fig. 6. The c-di-GMP reduction of the N1-functionalized 3,5-diamino-1H-pyrazole analogues. All percentages reported are based on the reduction of the c-di-GMP level, unless otherwise noted.
Inspired by the sulfonamide antibiotics,12 a small series of 2-F analogues functionalized with a sulfonyl-containing group in the N1 position (41–43), see Fig. 7A, were synthesized and tested. Also, a fusion compound between isoniazid and the 2-F analogue was included. Isoniazid is a known antibiotic hydrazide used in the treatment of tuberculosis, where it disrupts the synthesis of mycolic acid.13 Reacting the malonate intermediate (34) with isoniazid gave the hydrazide N1-functionalized pyrazole 44, Fig. 7B.
Fig. 7. All percentages reported are based on the reduction of c-di-GMP level, unless otherwise noted. (A) Synthesized and tested sulfonyl-containing 3,5-diamino-1H-pyrazole derivatives. (B) Isoniazid and the corresponding synthesized 3,5-diamino-1H-pyrazole analogue.
While none of the sulfonyl-containing compounds had any effect on the c-di-GMP level, the isoniazid-fused analogue (44) reduced the c-di-GMP level by 40%. However, the observation that all other attempts to functionalize the N1-position resulted in a complete loss of activity suggests that the 40% c-di-GMP reduction of 44 results from another mechanism. This was not further evaluated, but we observed that isoniazid showed no reduction of the c-di-GMP level.
Next, we investigated the analogs where nitrogen atom(s) in the pyrazole ring were replaced by oxygen atom(s), as shown in Table 3 and Fig. 8 and 9. This was done for a series of the most active analogues from Table 1. For the synthesis of the isoxazole derivatives (45–49), the respective hydrazone derivative was reacted with hydroxylamine, Fig. 8. The mono- and di-carbonyl derivatives (50–54) were synthesized by first reacting the diazonium ion (cf.Fig. 2) with ethyl 2-cyanoacetate (55) or diethyl malonate (56) to give the intermediate 57 or 58 respectively, followed by ring closure with either hydrazine or hydroxylamine, see Fig. 9.
Modifications to the pyrazole ring.
| ||||||
|---|---|---|---|---|---|---|
| Entry | R | X 1 | X 2 | X 3 | X 4 | Percentage reduction of c-di-GMP |
| 46 | 2-F, 6-F | NH | O | N | NH2 | 0% |
| 47 | 2-F, 5-F | NH | O | N | NH2 | 0% |
| 48 | 2-F, 6-OH | NH | O | N | NH2 | 0% |
| 51 | 2-F | O | O | N | NH2 | 0% |
| 52 | 2-F | O | NH | O | NH | 0% |
| 54 | 2-F | O | O | N | OH | 0% |
| 49 | 2-F | NH | O | N | NH2 | 0% |
| 53 | 2-F | O | NH | N | OH | 0% |
| 50 | 2-F | O | NH | N | NH2 | 0% |
| 45 | 2-OH | NH | O | N | NH2 | 0% |
Fig. 8. Synthesis and the c-di-GMP lowering effect of 4-arylazo-3,5-diamino-isoxazoles (45–49). All percentages reported are based on the reduction of the c-di-GMP level, unless otherwise noted. Reagents and conditions: (a) NH2OH·HCl (1.15 eq.), 10% NaOtBu in methanol (0.5 mL mmol−1), MeOH, rt, overnight.
Fig. 9. A synthetic strategy for the preparation of the mono- and di-carbonyl derivatives of 4-arylazo-isoxazoles and 4-arylazo-1H-pyrazoles. All percentages reported are based on the reduction of the c-di-GMP level, unless otherwise noted. Reagents and conditions: (a) NaNO2 (1 eq.), conc. HCl, water, 0 °C, 30 min; (b1) ethyl 2-cyanoacetate (55, 1.5 eq.), NaOAc, water, 0 °C → rt; (c) or (b2) diethyl malonate (56, 1.5 eq.), NaOAc, water, 0 °C → rt; (c) NH2NH2·H20 (50%, 1.15 eq.), EtOH, rt, overnight; (d) NH2OH·HCl (1.15 eq.), 10% NaOtBu in methanol (0.5 mL mmol−1), MeOH, rt, overnight.
All attempts to substitute nitrogen atoms with oxygens led to a complete loss of activity (see Table 3). There is only a 1.08 difference between the atomic radius of nitrogen compared to that of oxygen; however, the difference in their chemical properties is more substantial. The ether bound oxygen can only function as a hydrogen acceptor whereas the nitrogen in the same position can function as both an acceptor and donor. Also, oxygen as the more electronegative element (on the Pauling scale, oxygen and nitrogen are, respectively, 3.5 and 3.0) holds more tightly to its lone pair than the nitrogen. The nitrogen lone pair, therefore, is more basic. The loss of activity for the group of oxygen analogues could be derived from one or more of these chemical differences.
Towards the end of the SAR study, a small series of fluorinated pyridine derivatives were synthesized and tested, as shown in Table 4 and Fig. 10A. The pyridine ring has full aromaticity similar to benzene with six π-electrons, but differs with an extra lone pair on the nitrogen perpendicular to the π-system, giving the molecule a higher basicity as compared to benzene.14 Hamada summarizes in his book from 201815 some of the most important characteristics of pyridine in drug scaffolds, which in addition to the basicity also include good water solubility and the ability to form hydrogen bonds. The pyridine derivatives synthesized in this study, differed in the position of the nitrogen in the fluorinated aryl ring (59–60). While the derivative having the pyridine nitrogen positioned meta to fluorine (59) showed no activity, the analogue with the pyridine nitrogen positioned ortho to fluorine (60) showed a 13% reduction of the c-di-GMP level.
Fluorinated pyridine analogues.
| Entry | Structure | Percentage reduction of c-di-GMP |
|---|---|---|
| 59 |
|
0% |
| 60 |
|
13% |
Fig. 10. All percentages reported are based on the reduction of the c-di-GMP level, unless otherwise noted. (A) Pyridine derivatives of 4-arylazo-3,5-diamino-1H-pyrazoles; (B) aryl functionalized 3,5-diamino-1H-pyrazole (61) and a substructure (62) of the 2-F analogue (2).
The final set of analogues to be synthesized and tested are shown in Table 5 and Fig. 10B. Compound 61 was synthesized as a more constrained analogue to limit the number of structures contributing to the resonance hybrid. However, this resulted in a complete loss of activity. We also tested a substructure (62) of the active 2-F analogue (2), but likewise this shut down all c-di-GMP lowering activity.
Simpler and more constrained analogues tested.
| Entry | Structure | Percentage reduction of c-di-GMP |
|---|---|---|
| 61 |
|
0% |
| 62 |
|
0% |
Among all the 60 tested aryl-azo-3,5-diamino-1H-pyrazole compounds, 16 successfully reduced the c-di-GMP level, with only five analogues resulting in more than 70% reduction, see Fig. 11. These results suggest a narrow binding pocket for the target protein, allowing only a limited variation in size and electronic effects of the pyrazole compounds.
Fig. 11. Overview of the 16 4-arylazo-3,5-diamino-1H-pyrazoles showing a c-di-GMP lowering effect. All percentages reported are based on the reduction of the c-di-GMP level, unless otherwise noted.
Biological studies
The most potent compound, the analogue 2-F (2), was investigated for further in vitro and in vivo studies,8 showing a significant reduction in the biofilm mass in mice that harbored infected implants. 2-F (2) is capable of dispersing bacteria from already formed biofilms. For medical applications, it is therefore possible to combine 2-F induced biofilm dispersal with the synergistic antibiotic treatments of tobramycin or ciprofloxacin (standard of care antibiotics). As a result, biofilm infections can be dismantled and subsequently eradicated. All in vitro and in vivo studies are described in a recent paper, discussing the identification of small molecules that interfere with c-di-GMP signaling and induce dispersal of P. aeruginosa.8
Conclusion
Aiming at developing a drug that is effective against recalcitrant P. aeruginosa biofilms, a broad range of arylazo-3,5-diamino-1H-pyrazole derivatives were synthesized and tested for their c-di-GMP lowering activity in a comprehensive SAR study.
Initially, the phenyl substitution pattern was investigated, and 14 compounds were synthesized and tested to reveal activities ranging from 14% to 83% reduction of the c-di-GMP level. It was evident that fluorine in the phenyl 2-position was an important substituent.
To investigate whether fluorine in the 2-position was important for hydrogen donor/acceptor bonding interactions, phenylazo-3,5-diamino-1H-pyrazole derivatives with various hydrogen bond acceptors in the phenyl 2-position were synthesized and tested. Here, complete loss of activity was observed except for the analogue with a hydroxyl group in the ortho position (27) showing 55% reduction.
Further SAR investigations studied the modifications of the 3,5-diamino-1H-pyrazole moiety. However, both alkylation and acylation of the nitrogen atoms in general led to loss of activity, while also analogues where one or more nitrogen atoms in the 3,5-diamino-1H-pyrazole moiety were replaced with oxygen atoms showed no reduction of the c-di-GMP level. Our studies led to the identification of the highly active 4-(2-fluorophenyl)azo-3,5-diamino-1H-pyrazole (2-F, 2), which reduced the c-di-GMP level by 83%. Rewardingly, the 2-F analogue (2) showed both high chemical and mechanical stability, and no weight loss/gain was observed upon exposure of the compound to mechanical stress, while also no degradation was observed over more than six days when dissolved in a variety of solvents, including basic and acidic solutions.
The bacteria that are involved in hospital-acquired infections are primarily Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa and Enterobacter spp., which are denoted by the acronym ESKAPE, and declared by the WHO and other agencies to be a top global threat.16 Among the ESKAPE pathogens, the anti-biofilm agent (2) is specific for P. aeruginosa and might serve as a lead compound for the development of drugs that can be used in combination with antibiotics for the treatment of a number of P. aeruginosa biofilm infections, including cystic fibrosis-associated lung infections, chronic wound infections, catheter-associated urinary tract infections, and ventilator-associated pneumonia.
Experimental section
The procedures were carried out as reported by Kryštof et al.11 with minor modifications. All experimental and analytical data can be found in the ESI.†
General procedure A1 – diazotization with malononitrile
In a 250 mL conical flask, a solution of aniline (0.01 mol, 1 eq.) in a suitable amount of H2O/ice (50 mL) and conc. HCl (3 mL) was cooled to 0 °C. Then, a cold solution of sodium nitrite (0.01 mol, 1 eq.) in 10 mL H2O was dropwise added with stirring. The mixture was allowed to stir for 30 min, to form the diazonium salt, before the slow addition of a cold aqueous solution of malononitrile (0.015 mol, 1.5 eq.) and sodium acetate (25 g) in 85 mL H2O. After stirring of the reaction mixture at 0 °C for 1 h, the formed solid product was collected by filtration and washed with ice-cold water. For those compounds that do not precipitate, the water was removed and then the compound was extracted from the salt with EtOAc. The product was then dried under high vacuum overnight.
General procedure A2 – diazotization with ethyl-2-cyanoacetate
In a 250 mL conical flask, a solution of aniline (0.01 mol, 1 eq.) in a suitable amount of H2O/ice (50 mL) and conc. HCl (3 mL) was cooled to 0 °C. Then, a cold solution of sodium nitrite (0.01 mol, 1 eq.) in 10 mL H2O was dropwise added with stirring. The mixture was allowed to stir for 30 min, to form the diazonium salt, before the slow addition of a cold aqueous solution of ethyl-2-cyanoacetate (0.015 mol, 1.5 eq.) and sodium acetate (25 g) in 85 mL H2O. After stirring of the reaction mixture at 0 °C for 1 h, the formed solid product was collected by filtration and washed with ice-cold water. For those compounds that do not precipitate, the water was removed and then the compound was extracted from the salt with EtOAc. The product was then dried under high vacuum overnight.
General procedure A3 – diazotization with diethyl malonate
In a 250 mL conical flask, a solution of aniline (0.01 mol, 1 eq.) in a suitable amount of H2O/ice (50 mL) and conc. HCl (3 mL) was cooled to 0 °C. Then, a cold solution of sodium nitrite (0.01 mol, 1 eq.) in 10 mL H2O was dropwise added with stirring. The mixture was allowed to stir for 30 min, to form the diazonium salt, before the slow addition of a cold aqueous solution of diethyl malonate (0.015 mol, 1.5 eq.) and sodium acetate (25 g) in 85 mL H2O. After stirring of the reaction mixture at 0 °C for 1 h, the formed solid product was collected by filtration and washed with ice-cold water. For those compounds that do not precipitate, the water was removed and then the compound was extracted from the salt with EtOAc. The product was then dried under high vacuum overnight.
General procedure B1 – cyclization with hydrazine/hydrazide
The product from procedures A1–3 (1 eq.) was dissolved in EtOH (2.9 mL mmol−1) and then hydrazine/hydrazide (1.2 eq.) was added. The reaction was monitored with LCMS. Upon completion, the mixture was filtered (if the product precipitated) to give the product. If the product did not precipitate, the solvent and hydrazine were evaporated off to give the product. If purification was needed, it was done with either flash chromatography or preparative HPLC.
General procedure B2 – cyclization with heat and hydrazine/hydrazide
The product from procedures A1–3 (1 eq.) was dissolved in EtOH (2.9 mL mmol−1) and then the appropriate hydrazine/hydrazide (1.2 eq.) was added. The reaction was refluxed until LCMS showed full completion. Afterwards, the mixture was cooled to room temperature, and if the product precipitated, it was filtered. If the product did not precipitate, the solvent and hydrazide were evaporated off to give the product. If purification was needed, it was done with either flash chromatography or preparative HPLC.
General procedure B3 – cyclization with hydroxylamine
The product from procedures A1–3 (1 eq.) was dissolved in methanol (6.5 mL mmol−1) and a solution of 10% NaOtBu in methanol (0.5 mL mmol−1) and hydroxylamine hydrochloride (1.15 eq.) was added. The mixture was refluxed overnight. The solvent was removed in vacuo and afterwards the residue was purified by flash chromatography or preparative HPLC.
Abbreviations
- c-di-GMP
Cyclic di-guanosine monophosphate
- DGC
Diguanylate cyclase
- EPS
Extracellular polymeric substances
- PDE
Phosphodiesterase
- SAR
Structure–activity relationships
Conflicts of interest
There is no conflict of interest to declare.
Supplementary Material
Acknowledgments
This work was supported by a grant to KQ from the Carlsberg Foundation Young Researcher Fellowship (grant No. CF18-0631), as well as grants to MG and TTN from the Danish Council for Independent Research, the Lundbeck Foundation, the Novo Nordisk Foundation, and the Danish Ministry of Higher Education and Science (the DK-Openscreen program).
Electronic supplementary information (ESI) available: General methodologies; experimental and analytical Data. See DOI: 10.1039/d1md00275a
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