Educational guide
Peptide Coupling Reagent - an overview
Chapters and Articles You might find these chapters and articles relevant to this topic. V Peptide Coupling Peptide coupling reagents are more than fancy dehydrating reagents [82] , and there is a wide range of costs. Some commercially available reagents shown
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Chapters and Articles
You might find these chapters and articles relevant to this topic.
V Peptide Coupling
Peptide coupling reagents are more than fancy dehydrating reagents [82], and there is a wide range of costs. Some commercially available reagents shown in Table 4.5 are ranked by the cost per mol relative to thionyl chloride, along with some exemplary references. (Thionyl chloride was selected as the least expensive reagent, for the formation of acid chlorides. Acid chlorides are rarely used for amide couplings of amino acids, and acid fluorides have some advantages over acid chlorides [83].) HBTU and HATU highly active and may be used for more difficult amide bond formations, as with resins or hindered carboxylic acids [84]. Byproducts from HBTU and BOP-Cl are water-soluble. An amine and a carboxylic acid can be mixed with either carbodiimides or T3P for coupling without prior activation of the carboxylic acid. The urea byproduct from the water-soluble carbodiimides can be readily removed by extractions, due to the basic amine functionality. Dicyclohexylurea will crystallize from DMF and can be removed by filtration. The phenolic byproduct from CDMT couplings can be removed by extractions into a basic aqueous phase. SAFETY precautions should be taken with all these reagents, as they can be severe irritants.
TABLE 4.5. Relative Costs of Peptide Coupling Reagents
| Reagent | Relative cost/mol | Reference |
|---|---|---|
| O-(Benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate (HBTU) | 1048 | (1) |
| Bis(2-oxo-3-oxazolidinyl)phosphinic chloride (BOP-Cl) | 273 | |
| 2-Chloro-4,6-dimethoxy-1,3,5-triazine (CDMT) | 250 | (2, 3) |
| (Chloromethylene)dimethyliminium chloride (Vilsmeier reagent) | 82 | (4) |
| N-(Dimethylaminopropyl)-N′-ethyl-carbodiimide (as the liquid free base or solid hydrochloride: EDAC, EDC, WSC) | 70 | (5) |
| Carbonyl diimidazole (CDI) | 53 | (See text) |
| Propylphosphonic anhydride (cyclic trimer, T3P) | 44 | (6) |
| Diethyl chlorophosphate | 19 | (7, 8) |
| Dicyclohexylcarbodiimide (DCC) | 10 | |
| Isobutyl chloroformate | 5 | (9) |
| Pivaloyl chloride | 2 | (10) |
| Thionyl chloride | 1 | (11) |
The selection of peptide coupling reagents may change as the development of an NCE progresses. The water-soluble carbodiimides have been used for initial studies for convenience, and then processes have been developed to use reagents that are more atom-economical. On large scale the mixed anhydrides from chloroformates, pivaloyl chloride, or benzoyl chloride are often used. More recently carbonyl diimidazole (CDI) has frequently been employed on a large scale, even for forming amides of hindered carboxylic acids (Figure 4.16) [85–92]. The benefits of CDI are that the byproducts are innocuous and water-soluble, reactions can be monitored readily by the evolution of CO2, the imidazolides often are fairly stable thermally, and CDI is inexpensive when purchased in bulk. In general imidazolides are less reactive than the corresponding acid chloride. Imidazolium chloride has been found to catalyst CDI couplings [93], and a small amount of CO2 has also been shown to be effective [89] (see Figure 17.1).
FIGURE 4.16. Some compounds prepared on scale by CDI couplings.
During peptide coupling, racemization α- to the carbonyl of what was formerly the carboxylic acid group is a concern, usually through oxazolone formation (see discussion with Figure 7.1). N-Hydroxybenzotriazole (HOBt) has classically been used as a catalyst for peptide coupling to minimize racemization and O- to N-acyl transfer (Figure 9.6) through formation of the active ester. Unfortunately HOBt is known to react violently, especially when dry, and the shipping of HOBt is restricted. The nucleus of HOBt is incorporated into some peptide coupling reagents, such as HBTU, HATU, and PYBOP (see Figure 4.17), and these reagents should be treated with care appropriate to HOBt. Pfizer researchers and others have examined substitutes for HOBt [85,86,94]. For the coupling of a hindered imidazolide with the relatively poor nucleophile aniline the activities of the catalysts were found to be (in descending order) HOAt, HOBt·H2O, 5-nitro-2-hydroxypyridine, and 2-hydroxypyridine. For SAFETY the ranking was (in descending order) 2-hydroxypyridine, 5-nitro-2-hydroxypyridine, HOBt·H2O, then HOAt. The Pfizer researchers concluded that 5-nitro-2-hydroxypyridine was the preferred catalyst. The inexpensive 2-mercaptobenzothiazole has been shown to be an effective substitute for HOBt in the preparation of peptides by solid-phase peptide synthesis [95], and should be suitable for solution phase syntheses (although it has smelled like rubber tires). Other active esters have been generated or isolated for peptide couplings, such as the pentafluorophenyl ester or the N-hydroxysuccinimide (HOSu) ester. The latter has been shown to undergo a Lossen rearrangement to produce β-alanine derivatives. A lower reaction temperature and less basic conditions during coupling may minimize this type of side product [96,97]. 4-Dimethylaminopyridine (DMAP) is a highly active acylation catalyst [98] that might promote racemization through ketene formation from the acyl pyridinium intermediate. Pyridine has also been used as an acylation catalyst. If a catalyst is needed in place of HOBt, perhaps 2-hydroxypyridine will be effective and economical.
FIGURE 4.17. Alternatives to HOBt.
URL: https://www.sciencedirect.com/science/article/pii/B9780123865373000046
Uses
Benzotriazole-based peptide coupling reagents and additives, as activators or additives (Figure 1), are used in peptide bond formation reactions both in solution and solid-phase synthesis. There are many different types of peptide coupling reagents (e.g., carbodiimides, aminium/uranium salts, and phosphonium salts). The choice of method(s) and reagent(s) depends on a variety of factors, including the specific sequence of peptide to be synthesized, the preferred method of deprotection, the preferred solvents, and the type of active intermediate desired.
Figure 1. Representative benzotriazole-based peptide coupling reagents and additives.
URL: https://www.sciencedirect.com/science/article/pii/B9780123864543006424
5.06.1.2.7.(iv) Iminium salts
The use of iminium salts as peptide coupling reagents has been extensively explored, and the CIP reagent 12 is useful for couplings involving hindered amino acids, especially in the presence of the additive HOAt <1996JOC3350>. More recently, a range of new iminium coupling reagents has been developed by Li and Xu <2000T4437, 2000T8119, 2000MI456>, and the comparison of these and other coupling reagents in the synthesis of cyclosporine O <2000JOC2951> provides an opportunity to compare the practical merits of the different coupling reagents. They report the synthesis and properties of several other iminium-type salts <1999TL8301, 2000TL721, 2000MI110, 2000T9949>, but it has yet to be seen whether these reagents will be considered by the general community to be more efficient than the alternatives that are currently available. Iminium salts related to the Vilsmeier reagent have also been developed <2002TL7597> (e.g., CMPA 13), and ones based on thiazolium salts <2003TL4393> are also receiving attention (e.g., BMTB 14).
URL: https://www.sciencedirect.com/science/article/pii/B0080446558000969
Uses
HOAt, as additives, and HOAt-based peptide coupling reagents, as activators, are used in peptide bond formation reactions both in solution and solid phase synthesis.
There are many different types of peptide coupling reagents (e.g., carbodiimides, aminium/uranium salts, and phosphonium salts). The choice of method(s) and reagent(s) depend on a variety of factors, including the specific sequence of peptide to be synthesized, the preferred method of deprotection, the preferred solvents, and the type of active intermediate desired.
URL: https://www.sciencedirect.com/science/article/pii/B0123694000010401
2.4.2 Biological activity
Some patents and a few literature references describe the use of 1-hydroxy-1H-indazole derivatives as peptide-coupling reagents,61 dye additives62 or inhibitors of β-catenin/T-cell factor (Tcf) protein–protein interactions.59, 63 This last application is very interesting for outlining the concept of this hydroxyazole as a bioisostere of the carboxylic functionality. The selective inhibition of the β-catenin/Tcf protein–protein interaction (PPi) represents an appealing therapeutic target for many cancers. Three hot spots on β-catenin are critical for binding to Tcf and one of them has been explored as an available pocket for small-molecule binding (schematic representation shown in Fig. 12). This involves the positively charged side chains of Lys435 and Lys508 of β-catenin where the carboxylic acid groups of Asp16 and Glu17 of human Tcf bind. With the aim of disrupting charge–charge and H–bond interactions between the two proteins, Hu et al.59 generated bioisosteric fragments of the carboxylic acid functionality and then linked two bioisosteres together with the aid of an electron-rich aromatic ring, generating series 2.4.4 and series 2.4.5 In particular, the 1-hydroxyindazole series (compounds 2.4.5) was shown to be more potent than the corresponding 1-hydroxybenzotriazole series (compounds 2.4.4), thanks to a more favorable cation–π interaction with the positively charged guanidino group of an arginine residue (Arg469) present in the binding pocket. In fact, the N3 atom of hydroxybenzotriazole is postulated to have more electron-withdrawing effect than CH on the fused ring, rendering indazole a more electron-rich heterocycle than the benzotriazole ring.59 The most active compound belongs to series 2.4.5 (compound 2.4.5.A) and incorporates a two-carbon linker (Y = –CH2CH2–) between the two carboxyl bioisostere fragments. The authors predicted a binding conformation of 2.4.5.A with β-catenin: the hydroxyl group of 1-hydroxyindazole interacts with Lys508, mimicking Glu17 carboxylic group, while the tetrazole ring interacts with residue Lys435, mimicking the charge–charge and H–bond interactions with Asp16 of Tcf.59
Fig. 12. Representative scheme of one of the three hot spots on β-catenin critical for binding to Tcf. This spot is considered available for small-molecule binding, in particular side chain carboxylic acids of Asp16 and Glu17 were mimicked to anchor acidic inhibitors to Lys435 and Lys508 of β-catenin.59
URL: https://www.sciencedirect.com/science/article/pii/S0065272520300647
2.2.3 Reaction Discovery
The Biginelli reaction is a classic reaction providing the easy access to multifunctionalized dihydropyrimidinones, which in principle is a convenient precursor of the corresponding multifunctionalized pyrimidines. However, although the Biginelli reaction has been known for more than 100 years and the multifunctionalized pyrimidines are highly useful building blocks in medicinal chemistry, this particular transformation has remained largely undiscovered.
In 2004–2005, we reported the study of direct conversion of the Biginelli pyrimidinones to pyrimidines under mild conditions via the in situ activation and coupling reaction of this heterocycle using the well-known peptide coupling reagents, such as the carbodiimides, phosphonium salts, and guanidinium/uronium salts. Interestingly, we discovered that, while carbodiimides (DCC, EDC) and guanidinium/uronium salts (HATU, HBTU) were not effective, phosphonium salts (PyBroP, PyBOP) exhibited extraordinary reactivity to enable the in situ activation and coupling to afford the desired pyrimidine products in high yields (04AP702, 05JOC1957).
| Reagent | DCC | EDC | HATU | HBTU | PyBOP | PyBroP |
|---|---|---|---|---|---|---|
| Yield (%) | 0 | 0 | 0 | 0 | 90 | 94 |
URL: https://www.sciencedirect.com/science/article/pii/B9780081000243000027
1.2.2.1.3 From Diacid Monoesters
Particularly for the synthesis of N-phthalimido derivatives of amino acids, reaction with monomethyl phthalate provides an efficient and mild method. The reaction of 50 with α-amino acid amides 51 in the presence of the BOP peptide coupling reagent and Hünig’s base initially gives the coupled amide products 52 and these are readily cyclized with base to afford the imido acid amides 53 (Scheme 1.13).50 This method is also effective for α-amino esters and dipeptide amides and esters.50 Alternatively, ultrasonication of 50 and 51 with BOP reagent, Hünig’s base, and zinc chloride affords the products 53 directly.51 In a related method, the reaction of methyl 2-(succinimidoyloxycarbonyl)benzoate 54 with amino acids and other amines gives the corresponding phthalimides 55 in good yield.52
Scheme 1.13. Synthesis of imides from diacid monoesters.
URL: https://www.sciencedirect.com/science/article/pii/B9780128156759000011
6.11.5 Onium and Pyridinium Peptide Coupling Agents
A bewildering range of coupling agents have been developed to facilitate efficient peptide coupling reactions and address problems associated with poor yields and racemization for the synthesis of ‘difficult’ peptide sequences. One of the most popular range of peptide coupling reagents in current use are those known as ‘onium’ salts,161 which have proven to be particularly effective for the formation of active ester species for peptide bond coupling in situ. A number of different phosphonium, phosphate, and phosphinium162-derived reagents have been developed for amide bond-forming applications (Figure 37). For example, bromo-tris-pyrrolidino-phosphonium hexafluorophosphate (PyBroP) has been reported to be an excellent reagent for incorporating challenging protected N-methyl-amino acid residues.163 3-(Diethoxy-phosphoryloxy)-3H-benzo[d][1,2,3]triazin-4-one (DEPBT) is adept at preventing racemization during peptide bond formation,164 whereas (7-azabenzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate (PyAOP) is particularly useful for carrying out solid-phase peptide synthesis.165 A mixture of n-propanephosphonic anhydride (T3P) and pyridine has recently been reported as a safe, relatively inexpensive coupling agent for racemization-prone peptide bond syntheses, which affords water-soluble byproducts that makes it suitable for use on a large scale.166 Uronium species (Figure 38) such as O-benzotriazole-N,N,N′,N′-tetramethyluronium hexafluorophosphate (HBTU) react in a similar manner as phosphonium salts to afford well-behaved activated N-acyl oxime coupling species. The salts HBTU, O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetra-methyluronium hexafluorophosphate (HATU), bromotri(pyrrolidino)phosphonium hexafluorophosphate (PyBop),167 benzotriazoloxy-bis(pyrrolidino)carbonium hexafluorophosphate (BBC),168 bromotris(dimethylamino)-phosphonium hexafluorophosphate (BroP), and O-benzotriazol-1-yl-1,1,3,3-tetramethyluronium tetrafluoroborate (TBTU) have all been shown to be effective coupling reagents for carrying out sterically demanding peptide bond formation.154 2-Chloro-1,3-dimethylimidazolidinium hexafluorophosphate (CIP) is reported to be extremely useful for the coupling of sterically hindered α,α-dimethyl amino acid and N-methyl-amino acid residues.169 1-[(1-(Cyano-2-ethoxy-2-oxoethylideneaminooxy)dimethylamino-morpholinomethylene)]methanaminium hexafluorophosphate (COMU) has recently been introduced as a reagent that demonstrates improved peptide-coupling properties and greater stability than other ‘onium’ reagents.170 Immonium salts such as 5-(1H-benzotriazol-1-yloxy)-3,4-dihydro-1-methyl-2H-pyrrolium hexachloroantimonate (BDMP) are considered to be more reactive coupling agents than phosphonium/uronium reagents that mediate racemization-free peptide bond-forming reactions in good yield (Figure 39).171 Triazine-based coupling reagents (Figure 39) such as 4-(4,6-dimethoxy[1,3,5]triazin-2-yl)-4-methylmorpholinium chloride (DMTMM)172 and polymer-supported variants173 have been used to prepare highly reactive triazine esters in situ that are reported to have improved performance over coupling reagents such as TBTU or HATU.174 Pyridinium-based coupling reagents such as 2-bromo-1-ethylpyridinium tetrafluoroborate (BEP)175 or Mukaiyama's reagent176 also display good activity for peptide bond-forming reactions of sterically hindered nonproteinogenic α-amino acids (Figure 39).
Figure 37. Common phosphorus reagents employed for peptide bond formation. CloP, chlorotris(dimethylamino)phosphonium hexafluorophosphate; BroP, bromotris(dimethylamino)phosphonium hexafluorophosphate; BOP, benzotriazolyl-N-oxytrisdimethylaminophosphonium hexafluorophosphate; AOP, (7-Azabenzotriazol-1-yl)oxytris(dimethylamino) phosphonium hexafluorophosphate; PyClop, chlorotri(pyrrolidino)phosphonium hexafluorophosphate; PyBroP, bromotri(pyrrolidino)phosphonium hexafluorophosphate; PyBOP, (benzotriazol-1-yl)-oxy-tripyrrolidin ophosphonium hexafluorophosphate; PyAOP, (7-azabenzotriazole-1-yl)-oxy-tripyrrolidinophosphonium hexafluorophosphate; DEPBT, (Diethoxyphosphoryloxy)-1,2,3-benzotriazin-4(3H)-one; FDPP, pentafluorophenyl diphenyl phosphonate; T3P, n-Propanephosphonic anhydride.
Figure 38. Common uronium and imidazolium reagents used for peptide bond formation. HBTU, O-(benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate; TBTU, O-(benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium tetrafluoroborate; HATU, O-(7-Azabenzotriazol-1-yl)-1,1,3,3-tetra-methyluronium hexafluorophosphate; TSTU, 2-Succinimido-1,1,3,3-tetramethyluronium tetrafluoroborate; TNTU, 2-(5-Norbornene-2,3-dicarboximido)-1,1,3,3-tetramethyluronium tetrafluoroborate; HPTU, 1-((Dimethylamino)(dimethyl iminio)methoxy)-2-hydroxypyridinium tetrafluoroborate; HDTU, O-(3,4-Dihydro-4-oxo-1,2,3-benzotriazin-3-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate; TDTU, 2-(3,4-Dihydro-4-oxo-1,2,3-benzotriazin-3-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate; HATU, O-(7-Azabenzotriazol-1-yl)-1,1,3,3-tetra-methyluronium hexafluorophosphate; TATU, O-(7-Azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate; BBC, benzotriazoloxy-bis(pyrrolidino)carbonium hexafluorophosphate; COMU, 1-[(1-(Cyano-2-ethoxy-2-oxoethylideneaminooxy)-dimethylamino-morpholinomethylene)]-methanaminium hexafluorophosphate; CIP, 2-Chloro-1,3-dimethylimidazolidinium hexafluorophosphate.
Figure 39. Common iminium, ammonium, and pyridinium coupling agents used for ‘in situ’ peptide bond formation.154 BOMI, benzotriazol-1-yloxy-N,N-dimethylmethaniminium hexachloroantimonate; BDMP, 5-(1H-Benzotriazol-1-yloxy)-3,4-dihydro-1-methyl-2H-pyrrolium hexachloroantimonate; DMTMM, 4-(4,6-Dimethoxy[1,3,5] triazin-2-yl)-4-methylmorpholinium chloride; BEP, 2-Bromo-1-ethylpyridinium tetrafluoroborate.
These advanced coupling agents have been developed for carrying out conventional peptide-coupling reactions between suitably protected proteinogenic α-amino acids, however, they have also proven to be extremely useful for carrying out challenging amide bond-forming reactions for the synthesis of advanced chiral intermediates for natural product synthesis (Figure 40). This is particularly true for the synthesis of advanced amide intermediates for the synthesis of nonribosomal cyclic peptides, whose synthesis requires construction of multiple amide bonds from a combination of structurally challenging natural and nonproteinogenic α-amino acids (Figure 41).
Figure 40. Selected ‘onium’-mediated peptide bond-forming reactions: (a) TBTU-mediated amide bond-forming reaction for the synthesis of vanchrobactin177; (b) BOP-mediated amide bond-forming reaction for the synthesis of antitumor depsipeptide FR-901,208178; (c) HATU-mediated amide bond-forming reaction for the synthesis of mirabazole C179; (d) CIP-mediated amide bond-forming reaction for the synthesis of microcystin-LA180; and (e) BEP-mediated amide bond-forming reaction for the synthesis of chloptosin.181
Figure 41. DEPTB-mediated amide bond-coupling reaction for the synthesis of ramoplanin A2.182
URL: https://www.sciencedirect.com/science/article/pii/B9780080977423006170
Macrolactonizations Applying Phosphorus-Based Reagents
Phosphates are prominent alternatives to established protocols. A variety of reagents are known such as phosphates of type 10069 and BOP-Cl 101,70 most widely used in the synthesis of esters and thiol esters.71 The phosphorus-based peptide-coupling reagents PyBrOP 10272 and PyBOP 10373 originally designed for peptide bond formation have also been successfully used in the synthesis of macrolactones (Fig. 3).
Fig. 3. Structures of frequently used phosphorus-based reagents for macrolactonization.
For the first two reagents, heat labile mixed carbon–phosphorus anhydrides (e.g., 104, Scheme 34), and for the latter two species 102 and 103, acyl-oxy-phosphonium intermediates74 were postulated as reactive intermediates. Since mixed carbon–phosphorus anhydrides (e.g., 104) tend to give symmetrical anhydrides (e.g., 105) under heating (Scheme 34), Masamune recognized the need to perform macrolactonizations at a temperature below 80 °C.75
Scheme 34. Scope and limitations of phosphorus-based reagents in macrolactonization reactions.
As most macrolactonizations are nontrivial, ambitious reactions and the best performance is often cumbersome as can exemplarily be seen in the total synthesis of (−)-chlorothricolide 108 (Scheme 35).76 Only after lengthy investigations, Roush and co-workers recognized that only by using Palomo–Coll's reagent BOP-Cl 10170 (1.9 equiv) in toluene at 100 °C, the pivotal macrolactonization proceeded in acceptable yield (50% of the desired macrocycle 107 and 31% of recovered starting material 106). On the other hand, other macrocyclization methods (Yamaguchi,32a Yamaguchi–Yonemitsu,36b Steglich,77 Boden–Keck,61 Colvin78 and Taub–Wendler79 methods) failed or gave unsatisfactory yields.
Scheme 35. BOP-Cl-mediated macrolactonization in the total synthesis of (−)-chlorothricolide, 108.
The enediyne-bridged tricyclic core rac-111 of dynemicin A 112 was obtained in 51% yield using a PyBrOP-mediated macrolactonization followed by a transannular Diels–Alder reaction at room temperature (Scheme 36).80 Although the Yamaguchi32a macrolactonization protocol had been employed in preliminary studies,81 improved yields were only observed when the reaction was effected with PyBrOP 102,72 which was also superior in large-scale cyclizations.
Scheme 36. PyBrOP-mediated macrolactonization followed by a transannular Diels–Alder reaction in the synthesis of the enediyne-bridged tricyclic core rac-111 of dynemicin A, 112.
Laborious investigations of the best macrolactonization strategy were imperative during synthetic studies on (−)-spinosin A 115 (Scheme 37).82 Initial attempts to effect the macrocyclization of 113 by using Mukaiyama63 conditions provided the desired lactone 114 in only 33% yield (Scheme 37, entry 1). Coste's peptide-coupling agent PyBrOP 10272 and the Yonemitsu variant36b of the Yamaguchi macrolactonization procedure provided 114 in equally low yield (37% and 38%) (Scheme 37, entries 2 and 3). A significant improvement (48–54% isolated yield of 114) was achieved when the Mitsunobu62 conditions were employed (Scheme 37, entries 4 and 5). Ultimately, the best results (70% yield) were obtained when the macrolactonization of 113 was performed by using Coste's peptide-coupling agent benzotriazol-1-yloxytripyrrolidinophosphonium hexafluorophosphate (PyBOP) (103)73 at ambient temperature (Scheme 37, entry 6).
Scheme 37. Various cyclization protocols used during synthetic studies on (−)-spinosin A, 115.
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10.17.9.2.3.2 Pyrido[3,2-d][1,2,3]triazines (Class 4)
In principle, the diazotization of 3-aminopicolinonitriles (3-amino-2-cyanopyridines) under conditions akin to those mentioned in Section 10.17.9.2.3.1 should provide pyrido[3,2-d][1,2,3]triazine (Class 4) derivatives but there are few reports of this approach (see Section 10.17.9.5.2 for an alternative). One important example of a closely related outcome has been the synthesis of the well-known peptide coupling reagent, 3-hydroxypyrido[3,2-d][1,2,3]triazin-4(3H)-one (HODhat) (Class 4), initially mentioned briefly by Harrison and Smith123 and then examined more fully by Carpino, Xia and El-Faham.124 The Carpino synthesis achieved the nitrosation of 3-amino-N-hydroxypicolinamide using sodium nitrite in hydrochloric acid as reagent.
The 4-chloropyrido[3,2-d]1,2,3]triazine (Class 4) expected from such a process and its hydrazine derivative have been reported.125 However, the reactant on this occasion was 2-amino-3-methyl-4-phenylnicotinonitrile, a substance used previously in studies by the reporting authors, but one that should have yielded isomeric 4-substituted pyrido[2,3-d][1,2,3]triazine (Class 1) heterocycles. It is therefore likely that the structures of the two reported product structures have been misreported.
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