Benzonitrile and diphenyltetrazine

Diphenyltetrazine (more exactly, 3,6-diphenyl-1,2,4,5-tetrazine) is a beautiful molecule both in structure and in real life. It has a nice pink color. Tetrazines have found wide application in the field of bioorthogonal/click chemistry because they can undergo an inverse electron-demand Diels-Alder reaction with dienophiles with loss of nitrogen to form pyridazines.^[6]

Step 1: Benzonitrile
A one-pot synthesis for the preparation of nitriles from a carboxylic acid has been disclosed in patent US3663589.^[1] The reaction has been reported here as a two step process via benzamide and ammonium sulphamate,^[2] and as a one-pot synthesis for the preparation of 2-chlorobenzonitrile.^[3] The one-pot reaction has been applied to benzoic acid itself as well, but chemplayer reported a violent runaway on heating.^[4] When I attempted it, I used a large flask as a precaution and ground all reagents separately and on heating did not observe a runaway. The yield of the two preparations was similar. Because of the high recovery during the distillation step I think even the initial product is fairly pure. The overall yield was 64-67 % based on benzoic acid, which is about the same as the two step process previously reported here,^[2] although somewhat higher (85 %) yields are claimed in the literature for the two step process.^[5] Then again I believe the yields of this procedure here could probably be optimized further.



Synthesis #1:
11.7 g (96 mmol, 1 eq.) benzoic acid (prepared from sodium benzoate and hydrochloric acid, recrystallized once from boiling water and air dried), 17.4 g (179 mmol, 1.9 eq.) sulfamic acid and 8.1 g (135 mmol, 1.4 eq.) urea (prills) were all ground separately with a mortar and pestle and placed in a 500 mL round bottom flask. The flask was stoppered and shaken around to mix the powders. The flask was then equipped with a Liebig condenser and lowered into an oilbath preheated to 250 °C and kept at around 230-250 °C (oilbath) for 1 hour. The mass initially expands into a foamy mass. The flask was then removed from the oil bath, cooled to below 100 °C and 200 mL of water were added. A simple distillation was arranged and the contents in the flask were then steam-distilled until around 150 mL of distillate were obtained. The distillate was saturated with around 30 g of NaCl, the upper organic layer was removed and dried with Na₂SO₄.

Yield: 7.1 g (69 mmol, 72 %) of colorless, highly refractive benzonitrile with a smell somewhere between benzaldehyde and nitrobenzene.

Synthesis #2:
23.4 g (192 mmol, 1 eq.) benzoic acid (commercial), 34.8 g (358 mmol, 1.9 eq.) sulfamic acid and 16.2 g (270 mmol, 1.4 eq.) urea (prills) were all ground separately with a mortar and pestle, placed in a 500 mL round bottom flask and shaken around to mix the powders. It was then lowered into an oil bath and heating was started. Around 150 °C some foamy bubbling was observed with some sublimation and local melting around the edges. After about 30 minutes the oil bath had reached 250 °C. Some slight reflux was noted. The temperature was maintained for 1 hour and then the flask was removed and left to cool to below 70 °C. 300 mL of water were added and the mixture was steam distilled, while more water was added dropwise to keep the volume constant. After around 400-450 mL of distillate had collected the distillation was stopped. About 100 g of NaCl were dissolved in the steam distillate and the solution was extracted with 50 mL MTBE (Note 1), the organic layer was washed with a solution containing 16 g NaOH, 50 mL of water and 100 mL of saturated NaCl solution in two portions, then washed with 100 mL of saturated sodium chloride solution and dried over Na₂SO₄. All the MTBE was distilled off until the residue reached 110 °C.

Yield: 13.28 g (129 mmol, 67 %) of slightly turbid, highly refractive liquid benzonitrile.


Fig 1: Progression of the flasks appearance during the synthesis.


Fig 2: Left: The cake in the flask. Right: The steam distillate.

Combined purification:
Because some of the benzonitrile from the first run had already been used only 2.84 g of this product were used, it and all of the product from the second run were combined, the total weight was 16.12 g. This was placed in a 100 mL round bottom flask (although a smaller flask might be better) along with a stir bar and some ceramic boiling chips from a broken Büchner funnel. A simple distillation was set up and the flask was heated with an oil bath. Some small amount of forerun collected at 80-90 °C, which was discarded. The fraction boiling at 185-190 °C (literature 190 °C)^[6] was collected (oil bath around 210-220 °C). Some small amount of yellow liquid residue remained in the flask.

Yield: 15.28 g (95 % of starting amount recovered) of almost colorless, highly refractive benzonitrile.


Fig 3: Redistilled product.

Notes:
1. The choice of solvent is more or less arbitrary. MTBE can probably be replaced by a variety of organic solvents.

Discussion:
The exact mechanism for this procedure isn't clear as far as I know. Probably the benzoic acid and urea react together to form benzamide in situ, which is then dehydrated by the sulfamic acid probably similar to what has been reported for ammonium sulfamate (i.e. via an N-benzoyl sulfamate intermediate). It's also conceivable that the ammonia produced by the urea + benzoic acid reaction also forms the ammonium sulfamate in situ.

I opted for a steam distillation because the remaining cake in the flask is quite solid and would probably trap a lot of the benzonitrile.

Literature:
[1] - J. Luecke, Sandoz AG, "Process for the production of nitriles", US3663589, https://patents.google.com/patent/US3663589A/en
[2] - (a) Catalysed, "Synthesis of benzonitrile using ammonium sulphamate and benzamide", https://www.sciencemadness.org/whisper/viewthread.php?tid=15... (b) Catalysed, "Synthesis of Benzonitrile", YouTube 2021, https://www.youtube.com/watch?v=WTFM_UNeyTg
[3] - taffy, "one pot synthesis of a halobenzonitrile from a halobenzoic acid", https://www.sciencemadness.org/whisper/viewthread.php?tid=38...
[4] - chemplayer.., in "Benzonitrile in different ways", https://www.sciencemadness.org/whisper/viewthread.php?tid=65...
[5] - J. L. Boivin, "Synthesis of nitriles by fusion of amides with ammonium sulphamate", Can. J. Res. 1950, 28b, 11, 671-675, https://doi.org/10.1139/cjr50b-081
[6] - P. Jacobson et al, Beilsteins Handbuch der organischen Chemie, Band IX, Isocyclische Monocarbonsäuren und Polycarbonsäuren, Springer-Verlag, 4th edition Berlin 1926, p. 276, https://archive.org/details/BeilsteinsHandbuchDerOrganischen...

Step 2: Diphenyltetrazine
Now this is where I ran into trouble. The reaction is done with hydrazine hydrate and sulfur in ethanol. The intermediate dihydrotetrazine is then oxidized with sodium nitrite. The hydrazine hydrate was previously titrated with iodine and determined to be the pure monohydrate. All steps were performed in a fume hood.



Attempt #1 (Very low yield):
The synthesis followed a 2016 paper by Polezhaev et al.^[1]

In a 50 mL round bottom flask were placed 27.62 g absolute, undenatured ethanol dried over molecular sieves 3A, 4.26 g (41 mmol, 1 eq.) benzonitrile (from synthesis #1), 2.11 g (42 mmol, 1 eq.) of hydrazine monohydrate and 0.80 g (25 mmol as sulfur atoms, 0.61 eq.) of sulfur (recrystallized previously from toluene). Already a yellow coloration develops at room temperature. (also see Note 2) The flask was equipped with a reflux condenser, flushed with argon and covered loosly with a plastic stopper such to allow pressure release but limit air entry. The solution was then refluxed in an oil bath maintained initially at 130 °C, later at 110 °C for 4.5 hours. The solution turns dark orange-red and then fades to an light orange. The solution was filtered hot from some needle-like crystals (melting point 113 °C confirms this as sulfur) through a fluted filterpaper. The flask was left overnight. The next day it had turned a cherry red color, was filtered from some more precipitate by vacuum and then concentrated by vacuum distillation on a water bath. Initially only a light vacuum was applied to remove the alcohol, then a stronger vacuum was applied (water bath ~80 °C). The distillate smells of benzonitrile, in the flask some solid, partially pink-red residue remained.
This was suspended in 22 mL of glacial acetic acid + 8 mL water using an ultrasonic cleaner, which only partially dissolved it. Around 0.46 g (6.7 mmol, 0.16 eq.) of solid sodium nitrite was added with stirring slowly (some nitrogen dioxide is evolved), then the flask was left stirring for one hour. A pink solid in a pink solution resulted. Another portion of 0.55 g (8.0 mmol, 0.2 eq., in total 1.01 g, 14.6 mmol, 0.36 eq.) sodium nitrite was added, stirred for another hour and then filtered with vacuum over a Büchner funnel and washed with water.

Yield: 0.082 g (1 %) of a pink solid.


Fig 4: Progression of the flask during the synthesis.


Fig 5: Left: Flask after solvent removal. Right: Oxidation with nitrite.


Fig 6: Final product.

Analysis:
Thin layer chromatography on silica 60 F₂₅₄ using DCM/n-hexane, 1/1 as eluent^[2] revealed a pink spot with an R_f of around 0.69 (reported^[2] with heptane as 0.50) and some minor impurities at R_f 0.10 and 0.45 (visualized using 250 nm UV). The initial sulfur precipitate was also spotted and showed a faint spot running with the solvent front, R_f ~ 1.00.


Fig 7: TLC plate in visible light and under 250 nm UV. From left to right: sulfur precipitate, co-spot, final product.

Attempt #2 (Failure):
This attempt was based on a 2006 paper by Robins et al.^[3]

In this attempt, a solution of hydrazine in ethanol was prepared from the sulfate and more hydrazine was used. 2.4 g (60 mmol, 3.0 eq.) of NaOH, ground to a powder, was suspended in 10 mL of absolute, undenatured ethanol. There was a substantial exotherm. The beaker was placed in an ice bath and in portions 3.90 g (30 mmol, 1.5 eq) of hydrazine sulfate was added slowly with manual stirring. The entire addition took about five minutes. Manual stirring of the slurry was continued for 15 minutes, the solution was then vacuum filtered through a coarse glass frit and the solid residue washed with 10 mL of absolute ethanol. The filtrate was slightly cloudy.
Into a 25 mL round bottom flask were added 2.06 g (20 mmol, 1 eq.) benzonitrile (distilled), 0.110 g (3.4 mmol as sulfur atoms, 0.17 eq.) of sulfur (recrystallized from toluene) and the entire hydrazine-ethanol filtrate. The flask was refluxed with stirring in an oil bath for 9 hours, but already after 2 hours stirring became practically impossible because the flask was full of yellow precipitate. The flask was then cooled for 1.5 hours on ice, filtered and washed with 10-15 mL of cold ethanol.
The pale yellow solid was then transferred to a beaker containing 27 g of glacial acetic acid and stirred at room temperature for 10 minutes, during which almost everything dissolves. The solution was filtered from some insoluble residue (probably sulfur) and cooled in an ice bath before 2.1 g (30 mmol, 1.5 eq.) NaNO₂ dissolved in 12 mL water was added dropwise. No nitrogen dioxide was visible in this case, but also no pink color. Even after an hour of stirring and with more solid sodium nitrite no pink color was obtained. The filtrate of the crystals, when diluted with some water and acetic acid, also showed no pink coloration on treatment with nitrite.


Fig 8: The flasks appearance after 2 hours of heating.

Thus, this second reaction was a complete failure. The initially precipitated product was apparently not that soluble in ethanol, but was quite soluble in glacial acetic acid, which does exactly match what has been reported for the dihydrodiphenyltetrazine,^[4] but since the oxidation failed it must have been something else.

Could the presence of free NaOH have caused this? Did heating for too long destroy the product?

Attempt #3 (Success):
This attempt was based on a 1968 procedure by Abdel-Rahman, Kira and Tolba for the formation of the dihydrotetrazine.^[5] The oxidation followed as in the first attempt the procedure by Polezhaev et al.^[1]

In a 50 mL round bottom flask with 29/32 joint (Note 1) and a reflux condenser were added, in the following order, 1.5 g (14.6 mmol, 1 eq.) benzonitrile (redistilled), 3.0 g (60 mmol, 4.1 eq.) hydrazine monohydrate, 3.7 g anhydrous ethanol and 0.3 g (9.4 mmol as sulfur atoms, 0.6 eq.) sulfur (recrystallized from toluene). The solution immediately starts turning orange when the sulfur is added. (Note 2) No stirbar was used. The mixture was refluxed on a hot water bath for 1 hour, during which time the solution first turns orange, then red, the sulfur dissolves and some H₂S is evolved, then the entire mass turns into an orange paste. It was held for another hour in the now cooling water bath, then the flask was briefly placed on ice and filtered by suction and the solid washed with 10 mL cold anhydrous ethanol. The mass of the moist solid was about 2.12 g. (See below for TLC.) (Note 3)
The orange more or less crystalline solid was suspended in 30 mL of 30 % acetic acid in a 250 mL beaker and slowly treated with 5.0 g (72 mmol, 5 eq.) solid NaNO₂, the solution turns pink and foamy. Some nitrogen dioxide is released, but the foam also traps some of the nitrogen dioxide. The solution was extracted with 100 mL of DCM (Note 4) and gave a clear red-pink organic phase, which was washed twice with 50 mL of water, once with 100 mL of half-saturated sodium bicarbonate solution and once with 100 mL of saturated NaCl. The DCM was then dried over anhydrous sodium sulfate and distilled to dryness on a water bath. The last traces of solvent were stripped off at 300 mbar.

Yield: 0.982 g (4.2 mmol, 58 %) of pink-purple crystalline solid with faint smell of benzonitrile.

The product was recrystallized from 180 mL of boiling 95 % EtOH, the hot beaker was placed in a ~75 °C water bath as thermal mass to slow cooling. Soon long needles started to grow in the solution. The solution was placed in the fridge after coming to room temperature and then in the freezer at -18 °C for half an hour. The crystals were filtered using vacuum on a glass frit and washed twice with 20 mL of cold 95 % ethanol.

Yield: 0.611 g (2.6 mmol, 36 %) of long, pink-purple odorless needles.


Fig 9: Progression of the flask during the first step.


Fig 10: Left: Oxidation with nitrite. Right: DCM extraction.


Fig 11: Left: Product after removal of DCM. Middle: Recrystallization from ethanol. Right: Recrystallized diphenyltetrazine.

Notes:
1. The mixture forms a thick paste and the easier a spoon or spatula can reach into the flask, the better.
2. This has been mentioned in the literature.^[11]
3. After a day about 0.15 g of further solid yellow precipitate had collected from this filtrate, which wasn't used for the following step, but it probably could have.
4. At first, extraction with 100 mL of MTBE was attempted, however, the solubility of the diphenyltetrazine was too low, and while the solid tetrazine seemed to float on top of the aqueous phase, the formation of a complex bubbly phase boundary made complete separation impossible. After two partial washes with water the entire suspension was distilled on a hot water bath to remove the MTBE and what remained was partioned between 100 mL of DCM and 100 mL of water, and processed as described.

Analysis:
TLC was performed on silica 60 F₂₅₄ plates using DCM/n-hexane, 1/1, as the eluent.^[2] The samples were spotted in acetone. The plate was visualized with 270 nm UV. (It seems like the acetone wasn't fully evaporated from the plate, so the spots spotted last skew upwards and have a higher R_f.)

A - Diphenyldihydrotetrazine intermediate
B - Final diphenyltetrazine crystals
C - Second yellow precipitate (see note 2)

From left to right: A, A+B, B, B+C, C, A+C, where the plus indicates a cospot.


Fig 12: TLC of the intermediate and product.

The diphenyltetrazine shows only a single spot at R_f ~ 0.72 that is visibly pink under normal light. The first diphenyldihydrotetrazine also contains some traces of the diphenyltetrazine from atmospheric oxidation.
It is also very clear that the initial diphenyldihydrotetrazine contains at least 5 different compounds. Apart from the diphenyltetrazine there are also spots at R_f 0.00, 0.11, 0.34 and 0.97 (taken from the leftmost lane). The spots at R_f ~ 0.34 showed blue fluorescence under UV (weak at 250 nm, stronger at 270 nm, weak at 310 nm and none at 365 nm). From attempt #1 I assume the sulfur might be the spot near the solvent front, but what spot corresponds to the diphenyldihydrotetrazine, I don't know.

Discussion:
There are many procedures that have been reported for the synthesis of tetrazines and extensive reviews have been compiled.^[6-8] The reaction used here has been described as sulfur catalyzed,^[6] sulfur-induced^[9] and sulfur-assisted.^[10] A suggested mechanism^[9-11] is shown below:



It is a difficult question whether the dihydrotetrazine produced is the 1,2-dihydro or the 1,4-dihydro isomer. On the one hand, the suggested mechanism produces the 1,2-dihydro isomer. On the other hand, according to X-ray analysis, the 1,4-dihydro isomer is preferred for all symmetrical dihydrotetrazines.^[12] Acylation studies of the ring nitrogens of "3,6-diphenyl-1,2-dihydrotetrazine" can produce products that correspond to either isomer.^[13] Depending on the literature source, either isomer is drawn.^{[14, 15]} I couldn't find data on the speed of the tautomerism.

As seen on TLC, the intermediate dihydrotetrazine is not pure. There are a variety of possible products. There could be present 4-amino-3,5-diphenyl-1,2,4,4H-triazole, 3,5-diphenyl-1,2,4-triazole,^[16] 2,5-diphenyl-1,3,4-thiadiazole,^[1] and possibly even more.^[4] Whether those explain some of the spots on the TLC, I don't know. And what the product of the second attempt was is also unclear to me.

Literature:
[1] - A. V. Polezhaev, N. A. Maciulis, C.-H. Chen, M. Pink, R. L. Lord, K. G. Caulton, "Tetrazine Assists Reduction of Water by Phosphines: Application in the Mitsunobu Reaction", Chem. Eur. J. 2016, 22, 13985, https://doi.org/10.1002/chem.201600913
[2] - C. D. Mboyi, C. Testa, S. Reeb, S. Genc, H. Cattey, P. Fleurat-Lessard, J. Roger, J.-C. Hierso, "Building Diversity in ortho-Substituted s-Aryltetrazines By Tuning N-Directed Palladium C–H Halogenation: Unsymmetrical Polyhalogenated and Biphenyl s-Aryltetrazines", ACS Catalysis 2017, 7, 12, 8493-8501, https://doi.org/10.1021/acscatal.7b03186 (procedure taken from the supporting information)
[3] - L. I. Robins, R. D. Carpenter, J. C. Fettinger, M. J. Haddadin, D. S. Tinti, M. J. Kurth, "Diazocinones: Synthesis and Conformational Analysis", J. Org. Chem. 2006, 71, 6, 2480-2485, https://doi.org/10.1021/jo052577a
[4] - A. Pinner, "Ueber die Einwirkung von Hydrazin auf Imidoäther", Justus Liebigs Ann. Chem. 1897, 297, 221-271, https://doi.org/10.1002/jlac.18972970302
[5] - N. O. Abdel-Rahman, M. A. Kira, M. N. Tolba, "A direct synthesis of dihydrotetrazines", Tetrahedron Lett. 1968, 9, 35, 3871-3872, https://doi.org/10.1016/S0040-4039(01)99123-3
[6] - L. M. Bickem, K. Gavriel, K. Neumann, "Accessing Functionalized Tetrazines as Click Chemistry Tools: A Synthesis Guide for Chemists and Chemical Biologists", Eur. J. Org. Chem. 2024, 27, e202301117, https://doi.org/10.1002/ejoc.202301117
[7] - H. Neunhoeffer, "1,2,4,5-Tetrazines", in E. Schaumann, Methoden der Organischen Chemie (Houben-Weyl), E 9c: Hetarenes IV, Georg-Thieme-Verlag, Stuttgart 1998, 870-890 https://doi.org/10.1055/b-0035-118247
[8] - M. Bohle, "Six-Membered Hetarenes with More Than Three Heteroatoms", in S. M. Weinreb, E. Schaumann, Science of Synthesis 17, Hetarenes and related ring systems, Six-Membered Hetarenes with Two Unlike or More than Two Heteroatoms and Fully Unsaturated Larger-Ring Heterocycles, Georg-Thieme-Verlag, Stuttgart 2004, 585-626, https://doi.org/10.1055/sos-SD-017-00928
[9] - C. Li, H. Ge, "Novel 3,6-unsymmetrically disubstituted-1,2,4,5-tetrazines: S-induced one-pot synthesis, properties and theoretical study", RSC Adv. 2015, 5, 12277-12286, https://doi.org/10.1039/C4RA10808F
[10] - G. Clavier, P. Audebert, "s-Tetrazines as Building Blocks for New Functional Molecules and Molecular Materials", Chem. Rev. 2010, 110, 6, 3299-3314, https://doi.org/10.1021/cr900357e
[11] - P. Audebert et al., "Synthesis of new substituted tetrazines: electrochemical and spectroscopic properties", New J. Chem. 2004, 28, 387-392, https://doi.org/10.1039/B310737J
[12] - B. Stanovnik, M. Tišler, A. R. Katritxky, O. V. Denisko, "The Tautomerism of Heterocycles. Six-Membered Heterocycles: Part 1, Annular Tautomerism", Adv. Heterocycl. Chem. 2001, 81, 253-303, https://doi.org/10.1016/S0065-2725(01)81013-8
[13] - (a) G.-W. Rao, W.-X. Hu, "Synthesis and Crystal Structure of Diethyl 3,6-Diphenyl-1,2-dihydro-1,2,4,5-tetrazine-1,2-dicarboxylate", Chinese Journal of Organic Chemistry 2004, 24, 12, 1622-1625, https://sioc-journal.cn/Jwkyjhx/EN/abstract/abstract349571.shtml, (b) - G.-W. Rao, W.-X. Hu, "Synthesis and crystal structure of 1-acetyl-3,6-diphenyl-1,4-dihydro-1,2,4,5-tetrazine", Journal of Chemical Crystallography_ 2004, 34, 207-210, https://doi.org/10.1023/B:JOCC.0000021567.28429.07, (c) - G.-W. Rao, W.-X. Hu, "Synthesis and crystal structure of 1-isobutyryl-3,6-diphenyl-1,4-dihydro-1,2,4,5-tetrazine", Journal of Chemical Research 2004, 2004, 6, 408-409, https://doi.org/10.3184/0308234041423763
[14] - W. Chen D. Wang C. Dai, D. Hamelberga, B. Wang, "Clicking 1,2,4,5-tetrazine and cyclooctynes with tunable reaction rates", Chem. Commun. 2012, 48, 1736-1738, https://doi.org/10.1039/C2CC16716F
[15] - G.-W. Rao, W.-X. Hu, "Synthesis, structure analysis, and antitumor activity of 3,6-disubstituted-1,4-dihydro-1,2,4,5-tetrazine derivatives", Bioorg. Med. Chem. Lett. 2006, 16, 14, 3702-3705, https://doi.org/10.1016/j.bmcl.2006.04.066
[16] - J. Allegretti, J. Hancock, R. S. Knutson, "Azo and Hydrazo Compounds. I. An Attempt to Prepare the N-Oxide of 3,6-Diphenyl-1,2,4,5-tetrazine", J. Org. Chem. 1962, 27, 4, 1463-1464, https://doi.org/10.1021/jo01051a521

[Edited on 18-6-2024 by Diachrynic]