Introduction

The anthraquinone derivatives Reactive Blue 2 (RB-2, 1a, Fig. 1) and Cibacron Blue 3GA (1b, Fig. 1) have been identified as new lead compounds in drug discovery1,2,3,4. They have been found to interact with a variety of nucleotide-binding proteins in the human body, including a number of different P2 receptor subtypes and ectonucleotidases5,6, as well as with certain RNA sequences7. Furthermore, (many) anthraquinone derivatives are brightly colored and are used for coloring natural and synthetic fibers (e.g., cotton, silk, wool, polyamide and polyester)8,9. As a result of their significant potential as therapeutics and as coloring agents, interest has grown in the development of methods for the efficient and rapid synthesis of derivatives of anthraquinones, especially because the available methods have significant limitations.

Figure 1
figure 1

Structures of Reactive Blue 2 (RB-2) and Cibacron Blue 3GA.

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Recent studies have shown that the substituent in the 4-position of the anthraquinone core has a crucial role with respect to affinity and selectivity for certain targets10,11,12,13,14,15,16,17. Consequently, our group and others have been interested in the preparation of a series of anthraquinone derivatives related to the lead structures 1a and 1b (Fig. 1) for pharmacological evaluation as antagonists of purine P2 receptors4,6,10,11,12,18,19 and as potential ectonucleotidase inhibitors17,20,21 to study their structure–activity relationships.

The classical and most widely used strategy for the synthesis of 4-anilino-substituted anthraquinone derivatives utilizes the Ullmann coupling reaction22,23,24, which involves the treatment of sodium 1-amino-4-bromo-9,10-dioxo-9,10-dihydroanthracene-2-sulfonate (bromaminic acid sodium salt, 2a) with an amine in the presence of a copper catalyst (Fig. 2)10,11,12,25. The reaction typically requires harsh conditions, e.g., high temperatures and long reaction times; it suffers from mostly poor yields (typically clearly below 50%; in many cases no reaction at all)10,11,12,13 and the formation of side-products, such as 4 and 5.

Figure 2
figure 2

Classical syntheses of anilinoanthraquinone derivatives.

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Development of the protocol

Initially we investigated two different classical procedures for reacting bromaminic acid sodium salt (2a) with a test set of aniline derivatives as outlined in Figure 2. The reaction conditions were as follows:

  1. A

    Cu(I) method10,11: reaction in the presence of CuCl, Na2CO3 and Na2SO3 in H2O at room temperature (25 °C) for 8–24 h, or under reflux at 120 °C for 8–10 h;

  2. B

    Cu(II) method25: reaction in the presence of CuSO4 and Na2CO3 in H2O at 120 °C for 12–48 h.

In many cases, no products or only poor yields could be obtained (see Fig. 3 for Cu(II) method). The main undesirable product identified in all reactions was 1-amino-4-hydroxy-9,10-dioxo-9,10-dihydroanthracene-2-sulfonate (4, Fig. 2) formed by attack of the competing nucleophile hydroxide. In addition, the des-bromo compound (5) was detected.

Figure 3
figure 3

Comparison of yields in Ullmann coupling reactions of bromaminic acid with aniline derivatives using various conditions (Cu(II) method: CuSO4, Na2CO3, H2O, 120 °C, 12–48 h; Cu(II)(μW) method: μW, CuSO4, Na2CO3, H2O, 120 °C, 30 min; Cu(0) method: Cu(0), sodium phosphate buffer, pH 6–7, 120 °C, 2–15 h; and Cu(0)(μW) method: μW, Cu(0), sodium phosphate buffer, pH 6–7, 80–120 °C, 2–30 min).

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As a next step we investigated a procedure reported in the patent literature suggesting the use of Cu(0) as a catalyst26. Thus, model reactions using a set of aniline derivatives were performed in the presence of Cu(0) in sodium phosphate buffer, pH 6–7, at 120 °C for 2–15 h (method Cu(0)). In comparison to the classical methods using Cu(I) or Cu(II), the application of Cu(0) appeared to be superior for the majority of products. Nevertheless, the method was still unsatisfactory (Fig. 3).

Experimental design

In recent years, microwave (μW) irradiation has emerged as an efficient tool in organic synthesis, and its benefits have been well documented. In a number of studies it has been shown that microwave irradiation can circumvent the need for prolonged heating and it generally accelerates the rate of chemical reactions, often with increased yields and cleaner reaction profiles, which allow for more rapid reaction optimization and library synthesis. Higher reaction temperatures can be obtained by combining rapid microwave heating with sealed-vessel (autoclave) technology. Using the dedicated microwave synthesizer, simplified online control of temperature and pressure profiles is possible, which leads to highly reproducible reaction conditions27,28,29,30,31,32,33.

Figure 3 shows the results from reactions of the model compounds subjected to microwave irradiation, typically for only 2–30 min at 80–120 °C (Fig. 4). All other conditions (catalyst, salts, solvent) were the same as in the classical methods using either Cu(I) or Cu(II), and the improved method applying Cu(0) (Figs. 2 and 4). Microwave irradiation (Fig. 4) dramatically increased yields, particularly for the Cu(0) method, and especially of those compounds that were only poorly accessible without microwaves.

Figure 4
figure 4

New, general synthesis of 4-substituted anthraquinones.

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Reactions were typically performed with 0.3 mmol (121.3 mg) of 2a, but the reaction can be upscaled to multigram amounts with similar high yields13. It is also possible to scale it down to 0.1 mmol (40.4 mg) of 2a. The described reaction conditions can be generally used, and only the reaction time needs to be adjusted according to the amine derivatives, usually within a time range of 2–30 min13,14,15,16,17. However, this protocol failed in the coupling of 2a with heteroaromatic amines, such as 4-aminopyridine, 2-aminopyrimidine, 4-amino-2-methylquinoline and 3-amino-1,2,4-triazole, probably because of the low nucleophilicity of such compounds.

Comparison with other methods. The classical method using copper(I) chloride as a catalyst performed in the presence of sodium carbonate and sodium sulfite (Fig. 2) did not lead to detectable conversion in most cases (monitoring by RP-TLC). Subsequent rise of the temperature from room temperature to 40, 60, 80, 100 up to 120 °C did not alter the yields. As conventional heating did not significantly affect the yields, it was also not expected that microwave heating would have a dramatic effect. Nevertheless we applied microwave heating for one sample (employing 4-hydroxyaniline) without any success (0% yield)13.

A comparison of yields obtained by various methods (Cu(II)/Cu(0) with or without microwaves) is shown in Figure 3. For example, 3-amino-5-carboxy- and 4-carboxy-anilino-substituted derivatives that could be obtained by all other methods investigated in only <10% yield, were obtained in 70% yield by the new microwave-assisted method Cu(0) (μW). Also 3-amino- and 3-carboxy-anilino-substituted derivatives were synthesized in 70% and 76%, respectively, using this new protocol, whereas these two compounds were obtained in only <20% by all other described methods.

Applications of the method. This new protocol was discovered in 2007 (ref. 13), and it was subsequently shown that this protocol could easily be used for the coupling of a highly diverse range of amines (aliphatic or aromatic) with various 4-bromo-substituted anthraquinone derivatives (bromaminic acid (2a): R1 = SO3Na; 1-amino-4-bromo-2-carboxyanthraquinone (2b): R1 = CO2H; 1-amino-4-bromo-2-methylanthraquinone (2c): R1 = CH3) yielding the target compounds in good to excellent isolated yields (Fig. 4). Since then, the method has been used for the development of very potent and selective P2 receptor subtype antagonists as well as ectonucleotidase inhibitors.

The P2Y12 receptor expressed on blood platelets is an ADP-activated G protein-coupled receptor that mediates platelet aggregation34,35,36. P2Y12-antagonists are efficient antithrombotic drugs34,35,36. In radio ligand-binding studies at P2Y12 receptors of human thrombocytes, compounds 6, 7 (PSB0739) and 8 (PSB0702) were found to be the most potent ligands with Ki value in the low nanomolar range (Fig. 5)15. In functional cAMP studies, 7 exhibited antagonistic activity with subnanomolar potency37.

Figure 5
figure 5

Structures of potent human P2Y12 receptor antagonists.

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To our knowledge, 7 (PSB-0739) is the most potent competitive antagonist acting at the human P2Y12 receptor described to date, and it shows high selectivity versus other P2 receptor subtypes and ecto-nucleotidases37. Hence, 7 serves as a valuable experimental tool for the pharmacological characterization of P2Y12 receptors. Compound 7 also has potential as a lead structure for antithrombotic drugs. In contrast to P2Y12 antagonists that are currently in clinical development38,39,40,41,42, 7 is non-nucleotide/nucleoside-derived, which may have advantages with respect to stability, ease of synthetic access and side-effect profile. Its main drawback is the presence of two sulfonate functions, which prevent peroral bioavailability owing to their high acidity. Therefore, we bioisosterically replaced the sulfonates with more weakly acidic function, i.e., carboxylate groups. Carboxylates are known be perorally bioavailable, if not directly, they may be converted to methyl or ethyl esters that can act as lipophilic prodrugs43. Compound 8 has already been described in a recent publication to be a potent P2Y12 antagonist15, showing that a carboxylate group is well tolerated as a bioisosteric replacement of carboxylate. In this protocol we describe the preparation of the new compound 9, in which both sulfonate groups of the lead structure 7 are bioisosterically replaced by carboxylate (Fig. 5). We describe not only the Ullmann coupling reaction step but also the preparation of the required aniline derivative (5-amino-2-(phenylamino)benzoic acid, 20), as this compound is obtained in situ without isolation and directly employed in the final coupling reaction.

Furthermore, the newly developed Ullmann coupling protocol has been used to access a library of diverse anthraquinone derivatives, which allowed the analysis of their structure–activity relationships as antagonists for P2Y2 receptors14 and as inhibitors of ectonucleotidases: ecto-nucleoside triphosphate diphosphohydrolases (NTPDase) inhibitors16 and ecto-5′-nucleotidase (ecto-5′-NT) inhibitors17. The following lead compounds (Fig. 6) have been identified and some of them have already been optimized to obtain biological and pharmacological tools13.

Figure 6
figure 6

Structures of selected potent P2Y2 receptor antagonists.

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Potent NTPDase inhibitors have been identified. The most potent derivatives are depicted below (Fig. 7)16. These compounds are appropriate starting points for further optimization.

Figure 7
figure 7

Structures of selected potent inhibitors of rat NTPDases1,2,3.

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Furthermore, the newly developed protocol led to the discovery of potent ecto-5′-NT inhibitors. In fact compound 18 (PSB-0963) is the most potent inhibitor of ecto-5′-NT described to date (Fig. 8)17. ecto-5′-NT inhibitors have potential as new drugs for cancer therapy17.

Figure 8
figure 8

Structures of the most potent and selective competitive inhibitors of rat ecto-5′-nucleotidase.

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These results demonstrate that the anthraquinone scaffold appears to behave as a privileged structure in medicinal chemistry, and the substitution pattern, especially in the 4-position, can direct its interaction with specific targets. The developed Cu(0)-catalyzed Ullmann reaction might even be more generally applicable to amination reactions of aryl halogenides structurally unrelated to bromaminic acid.

Materials

REAGENTS

Caution

Organic materials must be handled with care: wear gloves, safety glasses/goggles and a laboratory coat, and all operations have to be done under a fume hood (for more information about safety in the chemistry laboratory visit the following website: http://delloyd.50megs.com/hazard/labsafety.html).

  • Bromaminic acid sodium salt (90%, Aldrich, cat. no. 456063)

    Irritant.

  • 1-Amino-4-bromo-2-methylanthraquinone (95%, Aldrich, cat. no. 246700)

    Irritant.

  • 1-Amino-4-bromo-2-carboxyanthraquinone (96%, Enamine, cat. no. T5799846)

  • 3,5-Diaminobenzoic acid dihydrochloride (puriss p.a., Fluka, cat. no. 32780)

    Irritant.

  • 3-Aminobenzoic acid (>98%, Merck, cat. no. 800104)

    Harmful. Avoid skin or eye contact.

  • 4-Aminobenzoic acid (>99%, Merck, cat. no. 822312)

  • 2-Aminophenol (>99%, Merck, cat. no. 800419)

    Harmful. The substance is toxic to lungs, brain. Repeated or prolonged exposure to the substance can result in damage to the target organs.

  • 4-Aminophenol (>99%, Merck, cat. no. 800421)

    Harmful; dangerous for the environment.

  • 2-Amino-5-chlorobenzoic acid (98%, Aldrich, cat. no. A45475)

    Irritant.

  • 2-Amino-4-chlorobenzoic acid (98%, Aldrich, cat. no. A45467)

    Irritant.

  • 2-Chloro-5-nitrobenzoic acid (>99%, Acros Organics, cat. no. 154691000)

    Irritant.

  • o-Phenylenediamine (98%, Acros Organics, cat. no. 130552500)

    Toxic.

  • m-Phenylenediamine (>99%, Acros Organics, cat. no. 130560250)

    Toxic.

  • Aniline (≥99.5%, Merck, cat. no. 101261)

    Toxic.

  • 5-Amino-2-phenoxybenzenesulfonic acid sodium salt (purity and identity have not been verified, SALOR, cat. no. S628530)

  • 4-Aminodiphenylamine-2-sulfonic acid (purity and identity have not been verified, SALOR, S341118)

  • o-Anisidine (>98%, Fluka, cat. no. 10460)

    Toxic.

  • 4-Chloroaniline (98%, Aldrich, cat. no. C22415)

    Very toxic, possible carcinogen, absorbed through skin.

  • 1-Naphthylamine (purum, Fluka, cat. no. 70731)

    Toxic; dangerous for the environment.

  • 2-Naphthylamine (95%, Aldrich, cat. no. A66405)

    Toxic, known human carcinogenic, toxic for the environment, handle with extreme caution.

  • 2-Aminoanthracene (94%, Aldrich, cat. no. A38800)

    Harmful; causes photosensitivity.

  • 2-Amino-5-fluorobenzoic acid (≥97%, Fluka, cat. no. 07973)

    Irritant.

  • Deionized water

  • Dichloromethane (p.a., VWR, cat. no. 113460250)

    Harmful may be carcinogenic; do not breathe fumes.

  • Trifluoroacetic acid (99%, Alfa Aesar, cat. no. L06374)

    Corrosive.

  • Methanol (≥99.8%, Fluka, cat. no. 65543)

    Toxic.

  • Acetone (≥99.5%, Fluka, cat. no. 00570)

    Irritant.

  • Di-sodium hydrogen phosphate dodecahydrate (≥99%, AppliChem, cat. no. A2530)

  • Sodium di-hydrogen phosphate dihydrate (≥99%, AppliChem, cat. no. A3902)

  • Copper(I) chloride (≥97%, Merck, cat. no. 102739)

    Harmful; dangerous for the environment.

  • Copper(II) sulfate pentahydrate (≥99%, AppliChem, cat. no. A3880)

    Harmful; dangerous for the environment.

  • Copper (≥99.7%, Merck, cat. no. 102703)

  • Sodium carbonate (98%, Alfa Aesar, cat. no. L13098)

    Irritant.

  • Sodium sulfite (≥98%, Aldrich, cat. no. S0505)

  • Silica gel 60 RP-18 F254s (Merck, cat. no. 110167)

  • TLC silica gel 60 F254 aluminium sheets (Merck, cat. no. 105554)

  • RP-TLC silica gel 60 RP-18 F254s aluminium sheets (Merck, cat. no. 105559)

EQUIPMENT

  • Discover LabMate microwave synthesizer (CEM Corporation)

  • Microwave reaction vial 10 ml (CEM Corporation)

  • Microwave reaction vial 80 ml (CEM Corporation)

  • Teflon septum caps (CEM Corporation)

  • Magnetic stir bar

  • Erlenmeyer flask

  • Round bottom flask (50–1,000 ml)

  • Büchner funnel

  • Glass column 26/460 cpl (Büchi, cat. no. 044037)

  • Preparative flash column chromatography (Büchi)

  • LCMS (Applied Biosystems; API 2000 LCMS/MS, HPLC Agilent 1100)

  • HPLC column (Phenomenex Luna 3 μ C18, 50 × 2.00 mm, Phenomenex)

  • Freeze dryer (CHRIST ALPHA 1-4 LSC, SciQuip)

  • UV lamp

  • Separatory funnel (250 ml, 1,000 ml)

  • Rotary evaporator (IKA)

  • Hydrogen generator (HOGEN GC)

  • Hydrogenation reaction vial 10 ml (KONTES Kimble Chase LLC)

EQUIPMENT SETUP

Microwave synthesizer

  • CEM Discover LabMate microwave synthesizer (LabMate) features the IntelliVent Pressure Control System. IntelliVent ensures operator safety by offering an automated over pressure venting capability. Reactions that exceed 300 psi (20 bar) are automatically and safely vented in a controlled manner before the operator can access the vial.

10 ml Reaction vessel

  • The 10 ml reaction vessel can be used to perform small-scale (0.1–0.4 mmol) reactions in volumes between 5 and 7 ml.

80 ml Reaction vessel

  • The 80 ml reaction vessel can be used for larger-scale reactions (1–2 mmol) in a volume of up to 50 ml. The 80 ml vessel can be equipped with an optional fiber optic probe for rapid temperature monitoring.

Flash column chromatography

  • The separation is carried out at room temperature using silica gel RP-18 and applying 5, 10, 20, 40, 60 and 80%, successively, of either methanol/water or acetone/water as eluent. The products are blue colored, whereas starting material (orange and red) and by-products (dark red or violet) show a different color. This means that the product can be easily separated by visual detection.

Phosphate buffer preparation:

  • solution A: Dissolve 7.16 g of di-sodium hydrogenphosphate dodecahydrate in 100 ml of distilled water to give 0.2 mol of pH 9.67.

Phosphate buffer preparation:

  • solution B: Dissolve 1.872 g of sodium di-hydrogenphosphate dihydrate in 100 ml of distilled water to give 0.12 mol of pH 4.17. Both solutions (A and B) are refrigerated at 4 °C, the two solutions (A and B) are mixed just before using it in the reactions.

LC-MS

  • The purities of isolated products can be determined by HPLC-UV. We used an LC-MS instrument applying the following procedure: the compounds were dissolved at a concentration of 0.5 mg ml−1 in H2O:MeOH = 1:1, containing 2 mM NH4CH3COO. Then, 10 μl of the sample was injected into an HPLC column. Elution was performed with a gradient of water:methanol (containing 2 mM NH4CH3COO) from 90:10 to 0:100 for 30 min at a flow rate of 250 μl min−1, starting the gradient after 10 min. UV absorption was detected from 200 to 950 nm using a diode array detector. The purity of the compounds was determined at 254 nm and proved to be ≥ 95%.

Procedure

Preparation of 5-nitro-2-(phenylamino)benzoic acid (compound 19, Fig. 9)

Timing 25 min

  1. 1

    Place a magnetic stir bar in an 80 ml microwave reaction vessel. (Fig. 9)

    Figure 9
    figure 9

    Preparation of 1-amino-4-(3-carboxy-4-(phenylamino)phenylamino)-9,10-dioxo-9,10-dihydroanthracene-2-carboxylic acid 9 (PSB-0704).

    Full size image
  2. 2

    Add 2.520 g (12.5 mmol) of 5-nitro-2-chlorobenzoic acid and 4.5 ml (40 mmol, 3.2 equivalent) of aniline to the microwave vial.

  3. 3

    Put the microwave vial in the proper position in the microwave instrument.

  4. 4

    Screw the cap into its place on the top of the microwave and close it very well.

    Critical Step

    The cap on the microwave oven must be even and tight; otherwise leakage of the reagents or solvent can occur under microwave irradiation.

  5. 5

    Program the microwave reactor (see table below).

    Table 2
    Full size table
  6. 6

    After the program has completed, the reactor will cool automatically to ca. 50 °C by gas jet cooling (compressed air, 30 psi); unscrew the pressure sensor cap from the microwave reactor, lift the reaction vessel and open it.

Purification of the product

Timing 12 h + 10 min

  1. 7

    Transfer the reaction mixture from the vial into a 1,000 ml separatory funnel containing 150 ml of DCM.

  2. 8

    Wash the vial with ca. 100 ml of DCM to collect any remaining material.

  3. 9

    Add 500 ml of 0.1 N of NaOH aqueous solution.

  4. 10

    Extract the organic soluble materials with 250 ml of DCM (three times).

  5. 11

    Transfer the water-soluble material into a 1,000 ml round-bottom flask, and wash the separatory funnel with ca. 50 ml of distilled water.

  6. 12

    Acidify the aqueous solution by the dropwise addition of concentrated HCl with cooling in an ice bath, control this step using pH indicator paper (pH ≤3).

  7. 13

    Collect the yellow precipitate by filtration using Büchner funnel, wash with distilled water (3 × 100 ml) and dry it in oven at 70 °C, yielding 2.6 g (81%) of a pure yellow product 19, Figure 9 (ref. 44).

    Pause point

    Product 19 can be left to dry in oven at 70 °C overnight.

Preparation of 5-amino-2-(phenylamino)benzoic acid (compound 20)

Timing 2 h + 13 min

  1. 14

    Place a magnetic stir bar in a 10 ml hydrogenation vial.

  2. 15

    Add 160 mg (0.6 mmol) of 19, 10 mg of 10% Pd/C and 5 ml phosphate buffer (consists of 4.5 ml of solution A and 0.5 ml of solution B) to the hydrogenation vial.

  3. 16

    Apply H2 pressure of 60 psi (using a HOGENGC hydrogen generator) and stir the reaction at room temperature for 2 h, then remove the palladium by suction filtration.

    Caution

    Use the exactly calculated amount of 10% Pd/C to avoid the necessity to add extra Pd/C during the reaction, which might lead to fire or even explosion. In case the added amount of Pd/C was not enough, transfer the reaction vial under the fume hood and let it stir there for a while to allow the removal of remaining H2 gas. Then Pd/C can be added portion-wise.

  4. 17

    Place a magnetic stir bar in a 10 ml microwave reaction vessel.

  5. 18

    Add 140 mg (0.4 mmol) of 2b, 10 mg (5 mol%) Cu(0) and the filtrate of the reaction mixture from Step 16 to the microwave vial.

  6. 19

    Fit the Teflon septum containing cap into the microwave vial and close it very well.

    Critical Step

    The cap on the microwave vial must be even and tight; otherwise leakage of the reagents or solvent can occur under microwave irradiation.

  7. 20

    Put the microwave vial in the proper position in the microwave instrument.

  8. 21

    Place the pressure sensor over the microwave vial.

  9. 22

    Program the microwave reactor (see table below).

    Table 3
    Full size table
  10. 23

    After cooling to ca. 50 °C via gas jet cooling (compressed air, 30 psi), remove the pressure sensor from the microwave reactor, lift the reaction vessel and open it.

Purification of the product

Timing 2 h + 45 min + 24 h

  1. 24

    Transfer the reaction mixture from the vial into a 250 ml separatory funnel.

  2. 25

    Wash the vial with ca. 100 ml of distilled water.

  3. 26

    Extract the organic soluble materials with 100 ml of DCM.

  4. 27

    Repeat Step 26 till the DCM layer becomes almost colorless (two to three times).

  5. 28

    Transfer the water-soluble material into a 500 ml round-bottom flask, wash the separatory funnel with ca. 50 ml of distilled water.

  6. 29

    Evaporate the water using a rotavap until ca. 10 ml of water remains.

    Troubleshooting

  7. 30

    Pack a chromatography column (26/460 cpl) with 125 g of silica gel 60 RP-18 material using 5% acetone/water.

  8. 31

    Load the 10 ml reaction mixture into the column through an injector valve.

  9. 32

    Elute the column using 10% acetone/water by pump (approximate flow rate: 50 ml min−1; Fig. 10a).

    Figure 10: Purification of anthraquinone derivatives by reverse phase flash column chromatography.
    figure 10

    (a) Initial separation of orange, dark red and blue compounds. (b) Washing out of the orange and the dark red compounds (side products). (c) Collection of the blue product (for R1 and R2 see Fig. 4)19.

    Full size image
  10. 33

    Increase gradually (20% and 40%) acetone concentration to wash out the orange and the dark red compounds (impurities and side products; Fig. 10b).

  11. 34

    Increase gradually (60% and 80%) acetone concentration and collect the blue product in a round-bottom flask (1,000 ml) (Fig. 10c).

    Troubleshooting

  12. 35

    Evaporate the solvent using a rotavap until ca. 50 ml of water and product remain.

  13. 36

    Transfer the mixture into a smaller round-bottom flask (100 ml).

  14. 37

    Freeze the material in liquid nitrogen.

  15. 38

    Attach the flask containing frozen mixture to a freeze dryer (ca. 24 h).

    Troubleshooting

  16. 39

    Check the purity by LC-MS using the LC-MS method described in the LC-MS setup.

    Troubleshooting

Troubleshooting

Troubleshooting advice can be found in Table 1.

Table 1 Troubleshooting table.
Full size table

Timing

Steps 1–6, Preparation of 5-nitro-2-(phenylamino)benzoic acid: 25 min

Steps 7–13, Purification of the product: 12 h + 10 min

Steps 14–23, Preparation of 5-amino-2-(phenylamino)benzoic acid: 2 h + 13 min

Steps 24–39, Purification of the product: 2 h + 45 min + 24 h

Anticipated results

In general, the typical isolated yield of anthraquinone derivatives is 30–90% applying the purification method described above. This range of yields represents results from the synthesis of >150 anthraquinone derivatives. It strongly depends on the amine component: aliphatic amines usually result in high yields with fast reaction times (up to 5 min), whereas aromatic amines require longer reaction times (up to 30 min) and—depending on the substitution pattern—lower yields can be observed in some cases, in particular when substituted with electron withdrawing groups.

In the case of product 9 (see Figs. 5 and 9) the total isolated yield for three steps is 50%. A ≥95% purity was obtained employing the method described in the LC-MS setup (Fig. 11).

Figure 11: HPLC trace, MS and UV chromatograms of compound 9 (a–f).
figure 11

(a) HPLC total ion count chromatogram; (b) MS in positive mode; (c) MS in negative mode; (d) UV chromatogram, detection at 640 nm; (e) UV spectrum at 21 min; and (f) integration of the UV chromatogram, detection at 254 nm.

Full size image

Additional analytical data

mp >300 °C blue powder.

1H-NMR (500 MHz, DMSO-d6): δ 7.08 (dd, J = 7.3, 7.6 Hz, 1H, 4′′-H), 7.28, 7.38 (2m, each 3H, 6′-H, 5′-H, 2′′-H, 3′′-H, 5′′-H, 6′′-H), 7.80 (d, J = 2.5 Hz, 1H, 2′-H), 7.85 (m, 2H, 5-H, 8-H), 8.13 (s, 1H, 3-H), 8.26 (m, 2H, 6-H, 7-H), 9.60 (br, 2H, 1-NH2) and 11.65 (brs, 1H, 4-NH).

13C-NMR (125 MHz, DMSO-d6): δ 110.2, 113.6, 115.3, 121.5, 123.2, 126.0, 126.2, 127.2, 128.6, 129.0, 129.6, 131.0, 133.0, 133.4, 133.5, 134.1, 140.6, 140.9, 144.5, 147.6, 167.5, 169.4, 182.1 and 183.0.

LC-MS (m/z): 494 [M]+, 492 [M]−, 448 [M–CO2]− and 404 [M–2CO2]−.

Purity by HPLC-UV (254 nm)-ESI-MS: 98.3%.