THE DIELS-ALDER-REACTION WITH INVERSE-ELECTRON-DEMAND – A REVIEW OF AN EFFICIENT & ATTRACTIVE CLICK-REACTION CONCEPT

THE DIELS-ALDER-REACTION WITH INVERSE-ELECTRON-DEMAND - A REVIEW OF AN EFFICIENT & ATTRACTIVE CLICK-REACTION CONCEPT

Manfred Wiessler ¹, Waldemar Waldeck ², Ruediger Pipkorn ³, Peter Lorenz ¹, Jürgen Debus ¹, Hans-Hermann Schrenk ¹, and Klaus Braun*¹

Department of Imaging and Radio-oncology ¹, Division of Biophysics of Macromolecules ², Central Peptide Synthesis Unit ³,  German Cancer Research Center, INF 280, D-69120 Heidelberg, Germany

Radiation Oncology, University of Heidelberg ⁴, Heidelberg, INF 110, D-69120 Heidelberg, Germany

ABSTRACT: Here, we review the development of prospective image processing systems in molecular diagnostics and of pharmacologically active ingredients for patient-specific therapeutic approaches. These projects require not only high demands on quality, safety, and specificity but also, rapid, efficient and irreversible ligation routes during the synthesis of future pharmaceuticals. The Diels-Alder ligation reaction with inverse electron demand (DAR_inv) is an eligible technology not restricted to medical applications, but valuable in selective modification by functionalization of polymers, organic and inorganic surfaces and micro arrays. Additionally, the DAR_inv technology is considered as an attractive strategy-platform for efficient syntheses of promising pharmacologically active components and derivatives of natural molecules with an optimized therapeutic index. We like to encourage scientists working with the brilliant concept of Sharpless’s “Click chemistry”, to intensify their research with this valuable DAR_inv technology able to open the door for regioselective, stereospecific, and bioorthogonal exigent syntheses of substances of highest quality inconceivable so far.

Bioorthogonal, Click-Chemistry, Ligation chemistry, inverse Diels Alder Reaction, Regioselectivity & Stereospecificity, Therapy & Diagnostics

INTRODUCTION:The pioneering work of Otto P.H. Diels and Kurt Alder started in 1926 in the field of chemical reactions between reaction partners with one ene- and diene- containing components to cyclohexene products. This cyclo-addition methodology was documented as “Diels-Alder-Reaction” (DAR) and started a meteoric rise in ligation chemistry. The Nobel Prize in Chemistry has been awarded in 1950 ¹.

The DAR principle and its chemical potential for the pharmaceutical research were well investigated ^2-8. Dependent on the chemical properties of the dienes and the dienophiles, the reaction led to different but preferred variants. One variant was documented as “Hetero-Diels-Alder-Reaction” and described the chemical reaction with a heteroatom substitution of the diene- and the dienophile-components ^{9, 10}.

The DAR also fulfils the criteria of the “Click-Chemistry” compiled by Sharpless (Scheme 1) ¹¹, comprising a cornucopia of qualified ligation reactions as shown in Huisgen’s work ¹². The Staudinger ligation ^{13, 14} and Bertozzi’s variant ^{15, 16} which were reviewed by Wiessler ¹⁷.

Under normal conditions the reaction rate was either low at room temperature or a catalyst was required. In the classical DAR, the dominating orbital interaction (corresponding to the lowest HOMO-LUMO energy separation) was between HOMO diene and LUMO dienophile ^18-20. In contrast, the Diels-Alder Reaction with inverse-electron demand (DAR_inv) was controlled mainly by the interaction of HOMO dienophile and LUMO diene and required an electron-rich dienophile and an electron-poor diene ^21-25.

Substituents with pushing electrons increased and, with pulling electrons reduced the electron density of the dienes as used in the DAR_inv. All ligation products were generated after a rapid and complete reaction at room temperature in organic solutions. The reaction of this ligation chemistry led to high purity, but with hurdles and difficulties on the way to the multifunctional drug-imaging-conjugates appropriate to multimodal “theranostic” approaches ²⁶. The synthesis of complex nature identical pharmacologically active molecules was mentioned here ¹⁷.

The wide spectrum of this universal and powerful DAR_inv methodology in the field of the complex macromolecular architecture is expounded here.

Scheme 1: The scheme points out that the DAR_inv fulfils the criteria of the “Click”-Reaction principle ^{3, 27, 28}. R₁ and R₂ represent different functional moieties harbouring –I and/or –M effects on the diene 1, which induces a decrease of the electron density of the tetrazine ring. In contrast, the R₃ features a +I effect resulting in a relatively high electron density in the dienophile compound 2. The stepwise reaction from 1 and 2 results in the stereoisomers 3 and 4, which can be attributed to the two different variants of intermediates (bracketed) after elimination of molecular nitrogen. As shown here, the reverse reaction is impossible (modified from Wiessler¹⁷).

The DAR_inv can help to overcome obstacles, like long reaction times, stringent conditions and the need of catalysts for the ligation reactions ^{12, 29, 30}. The first DAR_inv documentation by Carboni and Lindsey in 1959 ²⁷ describes the chemical reactions between tetrazines 1 and unsaturated compounds 5-8. These are dienes, or acetylenes 9-12 and are referred as the Carboni-Lindsey Reaction ^31-33 (Scheme 2). The scientific interest on this technology increased exponentially.

Scheme 2: illustrates the variability of synthetic routes of substituted tetrazines 1 with unsaturated compounds (dienes, acetylenes) 5-8 to the formation of 3,6-disubstituted pyridazines 9-12. R₁ and R₂ represent different functional moieties harbouring –I and/or –M effects on the diene 1. (R₃ – R₆ = H; Me; alkyl) (modified from Carboni and Lindsay²⁷).

It is obvious that in this rapidly increasing field of the syntheses of pharmacologically active substances, the DAR_inv technology offers a tremendous potential for the development of pharmaceutically interesting active nature identical molecules.

The almost infinite variability of the DAR_inv-products is supported by the huge reservoir of different diene and dienophile compounds as reaction partners which are referred in detail as follows.

Diene components: Heterocyclic molecules have a prominent role in the DAR_inv and were exemplarily reported elsewhere ^34-36.

Synthesis of tetrazine compounds: The most frequently used diene in the DAR_inv is the easily available 1,2,4,5-tetrazine-3,6-di-carbonic acid17, which can be produced in three-steps starting with diazo etylacetate 13 as shown in Scheme 3. The carboxylic acids 14 and 15 were synthesized first by Curtius ³⁷. The diazo ester and the hydrazine molecule 16 were isolated and described by Sauer ³⁶. Almost all these molecules underwent a quantitative reaction at room temperature within minutes as proposed by Nenitzescu. The research groups of Boger and Largeron comprehensively described regioselective properties of substituted 1,2,4,5-tetrazines in the DAR_inv ³⁸. In 2012, derivatized tetrazines were lodged as an US Patent application for bio-orthogonal coupling agents ³⁹.

The electro negativity was crucial for maintaining the high diene-activity. The stability of functionalized tetrazine esters, however, proved to be insufficient, whereas functionalized tetrazine amides provided the ability for the synthesis of many functionalized derivatives. These could be obtained via the dihydrotetrazine-amides followed by an oxidation step. Nevertheless the use of these tetrazine derivatives as described in Scheme 3 turned out to be problematic for two reasons:

Their insufficient stability and

Their poor solubility in aqueous solutions obviates applications in living systems.

The sensitivity against nucleophiles also seemed to be the cause of the low stability of these tetrazine derivatives in aqueous solutions. Because of these chemical decomposition processes, tetrazines modified with the dicarboxylic acid 15 were not qualified for ligation under conditions for the solid phase peptide synthesis (SPPS) and use under physiological conditions as proven in our experiments.

Considerations to circumvent these limitations the development gave rise to the synthesis of aryl substituted tetrazines featuring –I attributes. Following the reaction, the colour change during DAR_inv from a tetrazine (magenta) to a diazine (yellow) under degassing nitrogen occurred rapidly ⁴⁰.

Scheme 3: Illustrates the chemical route of the synthesis of the diamide of the tetrazine dicarbonic acid 17. The reaction steps were initiated and carried out in i) 13, a) 50% NaOH, b) H₂SO₄; ii) NaNO₂ in glacial acetic acid; iii) SOCl₂, MeOH; iv) NaNO₂ (modified from Wiessler ¹⁷).

Synthesis of triazine components: Wu and Gomez-Galeno documented studies to investigations of the DAR_inv-based synthesis of purine and pyrimidine analogues 22 with 1,3,5-triazine derivatives 19 as diene compounds as reaction partners ^{23, 41, 42} (Scheme 4). De Rosa and Arnold investigated the electronic and steric effects of the DAR_inv‘s reaction mechanism with 1, 3, 5-triazines 19⁴³.

Scheme 4: Illustrates, strongly simplified, the chemical route of the reaction of 1,3,5-triazine derivative 19 and 2-aminopyrrole 18 to the final aromatic cycloadduct 22 (modified from De Rosa ⁴³).

Yu and co-workers published in 2001 a detailed theoretical study of a DAR_inv-based reaction mechanism with 1,3,5-triazine and 2-amino pyrrole as diene and dienophile reaction partners for the synthesis of purine analogues ²³.

The huge potential of triazines was substantiated by the work of Branowska who documented a direct chemical synthesis route with 5,5'-bi-1,2,4-triazines with bicyclo[2.2.1]hepta-2,5-dienes to disubstituted-2, 2'-bipyridine derivatives ^44-46.

Whereas the Synder group showed the importance of the triazine as a diene reaction partner in the DAR_inv in 1,2,4-triazine studies ^47-49, the Boger group systematically investigated the 1,2,3-triazines ^{50, 51}; earlier, the group exhibited expertise in the research of the DAR_inv azadiene chemistry in the field of the total synthesis of nature identical molecules ^{4, 5, 52}.

Diazines as diene reaction partners: Recently, in 2012, the DAR_inv of 1,2-diazines as azadienes and siloxy alkynes, which were silver-catalyzed, was published by the Rawal group. An example for a silver-catalyzed reaction phthalazines 23and siloxy alkynes 24, reacting under loss of nitrogen to silyl-protected 2-naphthols 26⁵³ is shown in Scheme 5.

Scheme 5: illustrates the chemical route of the DAR_inv of 1,2-diazine 23 and siloxy alkyn 24 to the silyl-protected 2-naphthol (modified from Turkmen ⁵³).

The van der Plas group investigated intramolecular DAR_inv–based cycloadditions and documented the synthesis of 7,7-dicyano-6,7dihydro-5h-1-pyridines from the intermediacy of cycloadducts 2-(1,1-dixyanopent-4-yn-1-yl)pyrimidine and 2-(1,1-dicyanohex-5-yn-1-yn)pyrimidine after reaction of 2-chloro- or 2-methylsulfonyl-pyrimidines and the sodium salt of 5,5-dicyanopent-1-yne and 6,6-dicyanohex-1-yne ⁵⁴.

Dienamine intermediates as diene reaction partners: In 2012, Albrecht and co-workers described an asymmetric variant of the DAR_inv using metal catalysis and organocatalysis facilitating the synthesis of optical active dihydropyran derivatives 27. This was achieved by DAR_inv using dienamines intermediates as reaction partners (Scheme 6). The high stereo- and regiocontrol was realized by use of a bifunctional H-bond aminocatalyst ⁵⁵.

Scheme 6: Illustrates the catalyst facilitated hetero DAR_inv (modified from Albrecht ⁵⁵). Further, aminocatalytic DAR and DAR_inv via HOMO activation were documented using dienamine species from α, β-unsaturated aldehydes which act either as electron-rich dienes in normal-electron-demand DAR or as dienophiles in DAR_inv. All these reactions occur with high chemo-, regio-, and stereoselectivity as mentioned above ⁵⁶.

Functionalization of dienes with imaging components

Dienes can be considered as coupling molecules for diagnostic as well as for therapeutic use: The Devaraj group intensively investigated benzylamino-tetrazines functionalized with different fluorescent dyes in imaging studies ^57-60. Tetrazines derivatized with functional groups like 3,6-diaryl-s-tetrazines, suitable for labeling with biomolecules were demonstrated by the Fox group. They developed an a_nb₅ integrin targeted PET tracer by DAR_inv. reaction ⁶¹

For optical imaging studies the Wiessler group developed a_nb₃ and a_nb₅ integrin addressed to cyclic RGD-BioShuttle carrier molecules. The chemical reaction to the tetrazine diene, exemplarily functionalized with the polymethine-based Cyanine dye Cy7 30as an imaging component suitable for NIR imaging, is illustrated in Scheme 7.

Scheme 7: Illustrates the chemical reaction of 28 and 29. The Cy7 reaction product 30 acts as a diene reaction partner for the DAR_inv.

The conjugate 30 is composed of the diene N-(2-aminopropyl)-4-(6-(pyrimidine-2-yl)-1, 2, 4, 5-tetra zine-3-yl)benzamide 29 and the indotricarbo cyanine fluorescent dye Cy7 28 ²⁶. The synthesis of further diene building blocks improved the variability of the “Click Chemistry” for fluorescence imaging as exemplarily documented here by the coupling of 5-(dimethylamino)-naphthalene-1-sulfonyl (dansyl) 28 and bis2,6-[5-carboxylic acid-pyrid-2-yl]-tetrazine 29 ^{62, 63}.

Hilderbrand et al, designed an asymmetric aryl-tetrazine derivative, functionalized with the chelators 1,4,7,10-tetraazacyclo-dodecane 1,4,7,10-tetraacetic acid (DOTA) or desferrioxamine (DFO) bearing the positron emitting radioisotopes ⁶⁴Cu or ⁸⁹Zr. These functionalized components react in turn under the DAR_inv route with trastuzumab (Herceptin^®), an antibody against the human epidermal growth factor receptor HER2/neu norbonene-modified ⁶⁴.

Functionalization of tetrazines with biologically active molecules: In future, dienes, functionalized with pharmacologically active molecules are definitely conceivable. The functionalization of a diaryl-tetrazine with “old fashioned” drugs like temozolomide (TMZ) and the effects of the DAR_inv. reaction product were first documented by the Wiessler group as an important example of reformulation of established drugs ⁶⁵.

The synthesis of dienes used in our TMZ-BioShuttle studies was highly efficient but the synthesis of functionalized tetrazines suitable for the DAR_inv posed an experimental challenge ⁶⁶. As illustrated in Scheme 8, the nitriles 2-cyanopyrimidine 31 and 4-cyanobenzoic acid 32 reacted with hydrazine 33 to the dihydro-intermediate 34. The oxidation and the conversion to the acid chloride which in turn reacted with the Boc-mono-protected 1, 3-propylenediamine are the next steps to product 35. After deprotection, the amino group reacted with the acid chloride derivative of the TMZ 36 to the final product TMZ-diaryl-tetrazine 37 acting as diene reaction partner for the DAR_inv–mediated ligation ¹⁷.

Scheme 8: Illustrates the synthesis route of the TMZ tetrazine derivative 37 which reacts as a diene partner for DAR_inv. The 1,3-diaminopropyl modified 4-diaryl-3, 8-dihydro-1,2,4,5-tetrazine 35 is reacted with the acyl chloride derivative of the TMZ 36 (modified from Wiessler ¹⁷).

Functionalization of the peptide nucleic acid-backbone (PNA) with dienophile compounds: Our group also developed the following prototype of a theranostic agent (Scheme 9).

Scheme 9: The simplified illustration shows the reaction route to the ligation product 41 after the DAR_inv of the Reppe anhydride double functionalized peptide nucleic acid (PNA) pentamer 38. This is completely loaded with four diaryl-1,2,4,5-tetrazine-3,6 functionalized with two dansyl chloride 40 and the N-(2-aminopropyl)-4-(6-(pyrimidine-2-yl)-1,2,4,5-tetrazine-3-yl)benzamide. It is functionalized with 4-methyl-5-oxo-2,3,4,6,8-pentazabicyclo[4.3.0]nona-2,7,9-triene-9-carboxamide (temozolomide) 39 (modified from Wiessler⁶⁷).

Here, the synthesis of 41 was performed by a combined DAR_inv-mediated ligation of diene-functionalized pharmacologically active substances, like TMZ 39, with fluorescent dyes like 5-(dimethylamino)naphthalene-1-sulfonyl chloride (Dansyl chloride) 40. The Cyanine dyes C5 and C7 as further imaging components were also documented. A further possible reaction partner was a PNA-based polymer whose building blocks were functionalized with different dienophiles (instead of nucleobases) 42 (Scheme 10).

Scheme 10: Illustrates a PNA pentamer backbone which consists of momoners functionalized with dienophile structures offering different reactivity, like pentenoic acid and the Reppe anhydride 42 (modified from Wiessler ⁶⁷).

The variability of the charge of a polymer consisting of different molecules in desired ratios confers the key properties to the DAR_inv products. They reach optimal effects and minimal adverse reactions and are simultaneously able to monitor metabolic processes at the cellular level. This was feasible with the theranostic molecule in expressing cells and tissues after coupling to functional peptides like the cRGD for targeting to the a_nb₃ and a_nb₅ integrins ²⁶.

1. Synthesis of dienophile components: It should be mentioned that a lot of educts harbouring reactive terminal double bonds or double bonds in ring systems are commercially available or are easy to prepare. By this means a wide range of dienophilic compounds is available for certain DAR_inv reactions. In order to obtain reaction times in the range of minutes for the DAR_inv, the reactivity of the dienophile is decisive for the rapid reaction process besides the reactivity of the tetrazines as reactants. The allyl-group, a component of numerous chemical compounds which are easily accessible indeed may offer dienophile-activity, but nevertheless it is not qualified for a rapid chemical reaction. Therefore symmetric dienophiles with higher reactivity should be favoured.
2. Synthesis of the “Reppe-Anhydride”: Already in 1966, Sauer documented a very high dienophile activity of double bonds in cyclic ring systems adjoining to the terminal double bonds ⁶⁸. Incipient with strained rings like cyclopropene possessing very high dienophile reactivity, the reaction rate was reciprocally proportional to the ring’s size; the minimum was reached with the six-atom ring and in larger rings the reaction rate increased slightly.  In case of cyclobutene the derivatives disposed a sufficient stability as well as excellent dienophile reactivity.

A tetracyclic anhydride, synthesized and well described by Reppe (best-known under the name of Reppe-Anhydride), turned out to be the ideal compound for our purpose 45. It was easily available by the classic DAR of cyclooctatetraene 43 and maleic anhydride 44^69-71. (Scheme 11 exemplifies the chemical route). Other ring systems, dedicated for chemical reactions, as described above, are the cyclobutene-3,4-dicarboxylic acid anhydride ^{72, 73}.

Scheme 11: Illustrates the synthesis of a versatile building block for modification of peptides. The syntheses of the Reppe-Anhydride 44 and the corresponding (±) Boc-Lys derivative 46 were described ⁶⁵.

A valuable input to the fundamental research for the DAR_inv was given by Sauer’s group from 1966 to 2004 ^{28; 74-79}, An intensive research in the field of cycloaddition reactions of azabenzols 47 with different reaction partners ^80-85 was conducted by Neunhoeffer’s group (exemplified in Scheme 12).

Scheme 12: Describes the chemical reaction of diazobenzol derivatives 47 with ethylene-based vinylamines using as an example 1-methoxy-N,N-vinylamine [R = H; Me]; [X = OMe; Y = NMe₂] 48 to the structurally isomeric products 51 and 52 (modified from Neunhoeffer and Werner ⁸⁶).

Molecules with reactivity as dienophiles: The bioorthogonal reaction is characterized by rapid reaction rates and without need for catalysis. In 1962, the Sauer group first documented the cyclooctene as a dienophile ²⁸. The dienophile cyclooct-4-enol also was lodged in the US patent application US2011/0268654 A1 ⁸⁷. The Fox and the Robillard groups investigated the trans-cyclooctene 53as dienophile partner for DAR_inv with substituted s-tetrazines 54 as diene reaction partners with an excellent bioorthogonal reactivity ^{88, 89} (Scheme 13).

Scheme 13: Illustrates the DAR_inv.of 3,6-di-(2-pyridyl)-s-tetrazine 54 and trans-cyclooctene 53 to the (±)-4,5-dihydropyridizine 56 (modified from Blackman).

Schoch and co-workers first documented a bioorthogonal reaction established with DNA building blocks functionalized with cyclooctene-based dienophiles to rapidly synthesize oligonucleotides as reporter molecules for FRET studies ⁹⁰.

Alkenes, alkynes: The use of isolated olefinic double or triple bonds in hydrocarbon rings or linear systems as dienophile components 57 was comprehensively published by Wiessler ⁹¹.

Scheme 14: Shows the DAR_inv “Click”-Reaction of the diene 1 with the alkyne 57. R₁ and R₂ represent functional moieties harbouring –I and/or –M effects on the diene 1.

In 2012, Fox and co-workers published coupling reactions with 3-substituted cycopropenes in organic and aqueous solvents ³⁹ and documented, already in 2006, that chiral cyclopropenes are considered as eligible dienophile reaction partners for diastereoselective synthesis of methylene cyclopropanes ⁹².

Alkenes and alkynes also could act as typical dienophile counterparts for chemical reactions with dienes like dicyanocyclohexa-1,3-dienes and substituted phthalonitriles resulting in the synthesis of a broad class of heterocyclic derivatives like dicyano-indoles, and -carbazoles ⁹³.

The use of 2(1H)quinolones acting as dienophile components was not restricted to the synthesis of 5(6H)-Phenanthridone derivatives, pharma-cologically active as inhibitors of poly (ADP-ribose)-polymerase (PARP) ⁹⁴.

Mendez and co-workers investigated ethylene as a dienophile partner with oxazole under a DAR_inv whose reaction was facilitated by addition of Brønsted or Lewis acids ⁹⁵.

The Largeron group first documented a multistep electrochemical synthesis of polyfunctional 1,4-benzoxazine derivatized by alkylamino substituents via a DAR_inv-based reaction route ^{96, 97}. Due to their pharmacological activity 1,4-benzoxazine derivatives could play a role as neuroprotective agents ⁹⁸.

During the cycloaddition reaction via the regiospecific and distereospecific DAR_inv, the reactions of o-iminoquinone with secondary alkylenamines were generated in situ and produced aryl-2H-3,4-dihydro-1,4-benzoxazine intermediates which were unstable and chemically not accessible ⁹⁹.

Enamines: Bodwell et al, comprehensively described the synthesis of (±)-6H-dibenzo[b,d]pyran-6-one derivatives using the DAR_inv. Coumarin-fused electron-deficient 1,3-dienes ¹⁰⁰ were synthesized and reacted with a series of electron-rich enamines derived from acyclic carbonyl compounds, cyclic ketones and pyrrolidines. They led to the functionalized products dibenzopyranones 61 after aromatization by oxidation of the nondehydrogenated precursor intermediates which were due to be produced (Scheme 15). It is important to note, that the tested enamines 60 were chemically accessible by their generation before or in situ ¹⁰¹.

The key importance of dibenzopyranones 59 as educts and intermediates in the synthesis of molecules with multi-faceted pharmacologically activities is clearly documented. We mention here a few examples of steroid hormone analogues, receptor agonists, growth factor inhibitors and flavonoid derivatives with protective properties against cardiovascular diseases and cancer ^101-105.

Scheme 15: Illustrates in a simple way the DAR_inv-based step of the chemical route for the synthesis of (±)-6H-dibenzo[b,d]pyran-6-one 61 with the enamine dienophile 60 (produced by reacting cyclopentenone and pyrrolidine) with the coumarin diene compound 59 as reaction partner (modified from Bodwell ¹⁰⁰).

An additional attractive DAR_inv-strategy, documented by the Dujardin group ¹⁰⁶, is based on the reaction groups of electron-rich chiral enamines N-vinyl-2-oxazolidonones with the b,g-unsaturated a-ketoesters to the heterocycloadducts N-2-deoxyglykosyl-oxazolidinones. The distinctive feature was the oxazolidine compound, which acted not only as a chirally inducing agent but also as a monomer which allowed a stereocontrolled de novo synthesis of N-2-deoxyglykosides or sugar-a-amino acid hybrid derivatives.

Total synthesis of pharmacologically active nature identical products: The old fashioned total synthesis of naturally occurring molecules was representative for undesired adverse reactions, which hampered the therapeutic success. The DAR_inv-methodology could help to use specific modifications of the molecular structure during synthesis of the active molecules realizing an optimization of their therapeutic index.

Synthesis of purine analogues with antibiotic properties: DAR_inv-based cycloaddition reactions with different p-electron rich heteroaromatic “inverse” dienophiles like furanes, pyrroles, thiophenes, and N-methylimidazoles 62 and a negatively substituted tetrazine derivative 1 were described by the Seitz group ^107-110 Scheme 16). They provided a promising reaction for purine analogues which were pharmacologically highly active as repellents against microorganisms.

Scheme 16: Describes the reaction steps of the [4+2] cycloaddition of the tetrazine derivative 1 with N-methylimidazole 62 to the corresponding not isolatable cycloaddition intermediate (bracketed) 63 [64 and 65 represent tautomeric structures after nitrogen elimination] which are oxidized under recovery of the aromatic imidazole ring to the resulting reaction product 66 (modified from Seitz and Kämpchen ¹¹¹).

Synthesis of macrolides: The macrolide structural element is found in several pharmacologically active molecules like alkaloids and antibiotics. Despite the efforts in the development of synthetic lactones a challenge remained for the chemical synthesis. The Wang group presented the first approach of the macrolide synthesis. They used the asymmetric hetero DAR_inv to construct 69 after chemical reaction of cyclic ketones 68 with enones 67, thereby affording densely functionalized bicyclic skeletons in (±)-macrolides 69, 70 ¹¹² as shown in Scheme 17.

Scheme 17: Illustrates the catalytic asymmetric hetero DAR_inv during the synthesis of chiral (±)-macrolides 69 (modified from Jiang¹¹²).

Synthesis of antibiotics with antitumor properties: The Boger and Snyder groups succeeded in the total synthesis of multifaceted substances identical to nature: as an example, the complex synthesis of streptonigrin 76¹¹³. It is an antibiotic with antitumoral properties. A sequential implementation of the steps a and b of the DAR_inv methodology is illustrated in the highly simplified Scheme 18 ^{114; 115}.

Scheme 18       exemplifies the key chemical reactions of: The cycloaddition of tetrazine derivative dimethyl 1,2,4,5-tetrazine-3,6-dicarboxylate 1 with the heterodienophile S-methyl 6-methoxy-5-nitro-2-quinolinethio-imidate 71 for the formation of the 1,2,4-triazine streptonigrin ABC ring system 72.

a)      The [4+2] cycloaddition of the dimethyl 5-(6-methoxy-5-nitro-2-quinolyl)-l,2,4-tri azine-3,6-dicarboxylate 73 with the morpholino enamine derivative 2-(benzyl oxy)-3,4-dimethoxypropiophenone 74 to the formation of the CD biaryl ring system in a mixture which contains the Diels-Alder adducts 75 and 76. The adduct 76 is composed of streptonigrin’s framework (modified from Boger ¹¹⁴).

Total synthesis of the antimitogenic (­)-Reveromycin A: El Sous in the Rizzacasa group reported the total synthesis of (-)-Reveromycin A 80 using the DAR_inv in a “hetero strategy” to construct the critical spiroketal moiety of the molecule ^{116, 117}. The central step, the Lewis acid catalyzing the hetero-DAR_inv, is visualized in Scheme 19.

Scheme 19: illustrates the key step of the Lewis acid [Eu(fod)₃]. Hexane as solvent at O°C promoted the cycloaddition of the diene 77 with methylene pyran 78 to the spiroketal fragment 79. The intermediate to the final product (-)-Reveromycin A is 80. The hetero DAR_inv was catalyzed by Eu(fod)₃. without solvent (modified from El Sous ¹¹⁷). The natural product (-)-Reveromycin A 80 (Scheme 19) derived from a member of the soil actinomycete family Streptomyces sp. acts as an inhibitor of the mitogenic activity of the epidermal growth factor (EGF). It inhibited the human tumor cell lines KB ¹¹⁸ and K-562 ¹¹⁹ by blocking of the isoleucyl-tRNA-synthetase ^120-122.

Synthesis of antiinsectans: In the year 1996, Snider and Lu described the first total synthesis of the antiinsectans (±)-Leporin A 123, isolated from the  sclerotia of Aspergillus-Leporis by the Gloer group in 1991 ¹²⁴. The speciality of this synthesis was the “tandem Knoevenagel condensation” and the intramolecular DARinv. It was first documented by the Tietze group in 1995 ¹²⁵ and qualified to construct the unstable tricyclic intermediate o-quinone methide 83 from 4-hydroxy-5-phenyl-2-pyridone 81 and the acyclic 2-methyl-6E,8E-decadienal 82. This molecule underwent the intramolecular DARinv followed by hydroxylation and methylation steps resulting in the hydroxypyridone product (±)-Leporin A 84 as shown in Scheme 20.

Scheme 20: Shows the chemical tandem Knoevenagel-intramolecular DAR_inv reaction steps of the (±)-Leporin A synthesis 84 (modified from Snider ¹²³).

Total synthesis of alkaloids: The multi-facetted pharmacological potential of alkaloids and the increasing demand for therapeutic approaches are in contrast to their availableness because of their extensive isolation and purification procedures. The complex chemical synthesis hampered their clinical breakthrough, unfortunately.

Therefore the DAR_inv increasingly comes into the pharmaceutical research’s field of vision, as demonstrated exemplarily by the following syntheses.

Synthesis of Zarzissine: Here the synthesis of the cytotoxic guanidine alkaloid, an imidazole pyridazine derivative is documented. It was originally extracted from the Mediterranean sponge Anchinoe paupertas.

Zarsissine was first isolated and characterized by the Puel group ¹²⁶. The synthesis route by DAR_inv was described by Snyder ¹¹⁵ (Scheme 21).

Scheme 21: Shows the simplified chemical reaction of the cycloaddition of 2-aminoimidazole 85 with 1,2,4,5-tetrazine derivative 1 to the final product 2-amino-1H-imidazo[4,5-d]pyridazine derivative (zarzissine: R₁ = R₂ = H) 86 (modified from Synder ¹¹⁵).

The DAR_inv bases on the reaction of dienophilic heteroaromatic compounds 1 and imidazoles 85 as documented by Seitz ¹⁰⁹. Due to the pharmacological activity the imidazo[4,5-d]pyridazines ¹²⁷ as purine analogues were in the focus of the pharmaceutical research of heart diseases ^{128; 129}.

Synthesis of (±)-Epibatidine: The synthesis of the pyridazine analogue (±)-Epibatidine 90 is representative for the fact that the DAR_inv technology is not limited to the synthesis of derivatives offering structural similarity to naturally occurring molecules. The DAR_inv was also critical in the synthesis of nicotinic acetylcholine receptor agonists like the pyridazine analogue (±)-Epibatidine 90 which was exemplified by the Methfessel group ¹³⁰. Their synthetic route to 90 started with the commercially available 3-tropanone 87.

After the conversion to the corresponding racemic ester and transformation to the electron-rich dienophile enol ether 88 reacted with the electron-deficient diazadiene 1,2,4,5-tetrazine 1. It was carried out in a LUMO_diene/HOMO_dienophile-regulated DAR_inv after inverse [4+2]-cycloaddition and elimination of nitrogen via the racemic intermediates 89a/89b. This leads to the pyridazine analogon product (±)-Epibatidine 90 (Scheme 22).

Scheme 22: Shows the chemical DAR_inv reaction steps of the synthesis of (±)-Epibatidine products 90 (modified from Methfessel in a simple form ¹³⁰).

Epibatidine derivatives were used as highly effective non-opioid analgesic molecules, but due to their toxicity the therapeutical application had to to be restrained ^{131; 132}.

The synthesis of Epibatidine homologues may produce relief for approaches to potential nicotinic acetylcholine receptor ligands ¹³³.

Synthesis of isoquinoline derivatives: The Haider lab scrutinized the [4+2] cycloaddition potential of the DAR_inv on condensed pyridazines for the construction of higher order annealed ring systems ^134-138.

Here, the key synthesis of the isoquinoline derivative 96by DAR_inv of pyrido[3,4-d]pyridazine 91with 1-pyrrolidino-1-cyclopentene 60¹³⁹ was illustrated (Scheme 23).

Scheme 23: Shows the reaction of the biphenyl-substituted pyrido[3,4-d]pyridazine 91 with 1-pyrrolidino-1-cyclopentene 60 to the two not-isolatable isomeric dihydro intermediate products 92 and 93 (in brackets) reacting to the two corresponding cycloadducts 94, 95. The elimination of pyrrolidine and the following aromatization resulted in the formation of the single final isoquinoline derivative product 96 (modified from Haider ¹³⁹). It is worth to note, that the isomeric cyclopentane-fused dihydroiso quinolines 94 and 95 react to the single final product 96.

Tetrahydroquinoline synthesis: Quinolines were of great interest for the pharmaceutical and chemical research as basic drug molecules and for the synthesis of herbicides and fungicides. The synthetic access by isolation from the gas tar exclusively was documented by Masson ¹⁴⁰. The synthesis route of 2,3,4-trisubstituted tetrahydro quinolines 100 using the DAR_inv modified as a three-component aza-DAR_inv of aldehydes (R2) 99, anilines 98 and isoeugenol 97 ¹⁴⁰ is shown in Scheme 24.

Scheme 24: Illustrates the synthesis step of the three-component aza-DAR_inv of the synthesis of 2,3,4-trisubstituted tetrahydroquinolines 100. [R2= -CHO] (modified from Masson ¹⁴⁰) .

The synthesis route is complex but the reaction mechanism was well investigated and described as a nucleophilic attack of isoeugenol to the N-arylimine under cyclization and formation of intermediates ¹⁴⁰.

Total synthesis of cannabinol and derivatives: The molecular class of cannabinoids consists of 70 natural products isolated from Cannabis sativa.

This alkaloid’s synthesis is extensive and requires several steps. The Bodwell group clearly documented an efficient methodology (yields up to 79%) employed in a concise total synthesis of cannabinol 104 whose intermediate 6H-dibenzo[b,d]pyranone (DBP) 103 was formed by an asymmetric DAR_inv of the compounds 6-methoxy-3-pentyl-salicylaldehyde 101 and dimethyl glutaconate 102 ¹⁴¹ (Scheme 25). The generation of the enamine 60 acting as an electron-rich dienophile was documented by Bodwell and co-workers as herein mentioned before ¹⁰⁰. The process was already shown in Scheme 15.

Scheme 25: Describes the key steps and the DAR_inv-based reaction of 6-methoxy-3-pentyl-salicylaldehyde 101 and dimethyl glutaconate 102 and the enamine 1-pyrrolidino-1-cyclopentene 60 to 6H-dibenzo[b,d]pyranone (DBP) 103. After elimination of pyrrolidine and the following aromatization step (not shown), it shows the condensed route to the final cannabinol derivative product 104 (modified from Nandaluru ¹⁴¹).

Total synthesis of analogues of natural hormones: The Posner group depicted the first DAR_inv-based synthesis step of dihydroxyvitamin D3 analogues by [4+2]-cycloaddition between the commercial 3-methoxycarbonyl-2-pyrone 105 as a diene and the difluorinated electron-rich vinyl ether 106 as a dienophile. The reaction product was the racemic (±)-lactone cycloadduct intermediate 107^{142; 143} which reacted under a nucleophilic opening of the lactone ring with the Li-allyloxide under formation of the polyfunctionalized racemic (±)-cyclohexene 108.

This happened without inter- or intramolecular displacement of fluoride ions and the coupling of the enantiomerically pure ketone 109. Additional reaction steps resulted in the final reaction product 1a, 25-dihydroxyvitamin D3 110 (shown in the condensed Scheme 26).

Scheme 26: Simply shows the DAR_inv reaction steps of the synthesis of the 1a, 25-dihydroxyvitamin D3 product 110 (modified from Posner ¹⁴³).

The pharmacologic properties of dihydroxyvitamin D3 analogues were well investigated ¹⁴⁴. Currently, the analogues 1a, 25-dihydoxy-19-nor-vitamin D2 and calcipotriol are in clinical use for therapeutic intervention for hyperparathyroidism and psoriasis ¹⁴⁵.

Functionalization of Polymer Surfaces:

Glass carbohydrate-microarrays: The Wittmann group developed a promising carbohydrate microarray platform ¹⁴⁶ with inorganic SiO₂-surfaces functionalized with tetrazine derivatives as diene molecules 113. Molecules, like norbonenes or terminal alkenes which were ligated with carbohydrates 114 act as dienophile reaction partners for DAR_inv chemistry whose reaction kinetics realized an immobilization of the carbohydrates in a high homogeneity. This fulfilled the requirement criteria of glycomix high-throughput screening studies of carbohydrate-protein interactions ^147-150.

Scheme 27: Describes the preparation of carbohydrate microarrays 115. The amine-coated glass slides 111 were diene-functionalized with 112 in DMSO/pyridine solution. Then follows the DAR_inv based ligation of 113 and the dienophile 114. This is connected via a linker to a carbohydrate (modified from Wittmann ¹⁴⁶).

Biocompatible silicon surfaces: The Beck-Sickinger group investigated the potential of inorganic SiO₂-surfaces as possible linkers and coupling sites for functional peptides in biomaterials. The Scheme 28 highlights the SiO₂-surface 116 functionalized with different linkers realizing independently ligation reactions.

The 1,4-diaryl-1,2,4,5-tetrazine reacts with dienophiles like the Reppe anhydride in a DAR_inv clickreaction. The well-established copper(I) catalyzed Huisgen azide-alkyne [3+2] cyclo-addition (CuAAC) is possible using the cyclic azides.

Scheme 28: Describes schematically the structure of the inorganic SiO₂ material whose surface is functionalized with silica binding peptides which in turn are connected to different linkers like the diaryl tetrazine as diene component for DAR_inv (upper part) and the cyclic azide for the copper (I) catalyzed Huisgen azide-alkyne cycloaddition (CuAAC) (lower part). (Modified from Beck-Sickinger).

The functionalization was exemplarily carried out with the well investigated cRGD ^151-153, a small molecule which acts as a ligand for specific binding to the a_nb₅ integrin receptor, involved in neoangiogenic and metastasis processes ^154-156. The characteristics of the used DAR_inv-allowed a bioorthogonal click reaction strategy ^157-159 for a bridge formation between inorganic surfaces and biological active molecules ¹⁶⁰.

CONCLUSION:The DAR_inv chemistry is an increasing and expanding technology for rapid, efficient, irreversible ligations of user-defined functional molecules or genetic materials. These properties fulfil not only the requirements for the “Click Chemistry”, but also for chemical reactions under conditions for bioorthogonal chemistry. These topics were introduced and developed further by Bertozzi in 2000 ^{15; 161; 162},and are currently in the focus of the scientific interest.

Additionally, these reaction products are dedicated as a combination of cargo to carrier and as biological address-molecules to realize high local concentrations of active substances in living cells. These chemical reaction products are able to overcome cellular membrane barriers during a safe and efficient transfer of therapeutic and / or diagnostics cargos into target cells and tissues. It is important to point out that this DAR_inv technology is not restricted to application and easier handling of our developed BioShuttle delivery platform ^{67, 163} and other related carrier molecules.

An enhancement towards functionalization of surfaces and polymers by a proper ligation at surfaces like biotechnical arrays for genome or transcriptome analysis can be realized by this technique. Depending on the scientific subject and formulation of the scientific project, the coupling of dienes and dienophiles at a defined surface could be demonstrated.

The following points support this technology:

I)             The presented kinetic data with high reaction rates demonstrate the potential of the Diels-Alder-Reaction with inverse electron demand (DAR_inv) as a method of choice for ligation of molecules.

II)          The reaction process could be easily monitored by use of photometrical methods with a decreasing absorption maximum at 520 nm, which is typical for tetrazines.

III)       We could demonstrate that the DAR_inv features all the conditions for the successful “Click”-Chemistry and as a consequence, it turns out to be a dedicated tool for ligation reactions important but not restricted to the medical and pharmaceutical science.

The great advantage of DAR_inv is based on

A)      Compounds’ accessibility¹⁶⁴,

B)      The high and quantitative reaction rate,

C)      Bioorthogonal reactions,

D)      The potential for selective multiple reactions at the identical molecule,

E)       High regioselectivity,

F)       The ease of monitoring of the chemical reaction and

G)      The feasibility of the reaction on surfaces.

With this report we like to emphasize the great potential of the DAR_inv technology in many attractive fields. It was exemplarily documented in the field of drug-re-formulation, which dramatically increased the therapeutic potential of classic drugs as pointed out here with the alkylating agent temozolomide (TMZ). It was also able to enhance the therapeutic spectrum in malignant gliomas or in hormone-refractory prostate cancer ^{65, 66, 164}. Additionally this DAR_inv technology attracts increasing notice to further medical applications, especially in oncological diagnostics and therapy at the cellular and molecular level.

As illustrated above impressively, theDAR_inv technology is considered as an attractive strategy-platform for efficient syntheses of promising pharmacologically active components and derivatives of natural molecules (Scheme 16 – Scheme 26) with an optimized therapeutic index. The DAR_inv methodology contributed to the enhancement of the quality of already established syntheses of natural materials which are difficult to isolate and enrich ¹⁶⁵. The technical superiority like chemoselectivity, regio- and stereo-selectivity allow the synthetic access to increasingly more and more complex target structures ¹⁶⁶.

ACKNOWLEDGEMENTS: This work was supported in part by grant from the Deutsche Krebshilfe Foundation (Project No. 106335).

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     Wiessler M, Waldeck W, Pipkorn R, Lorenz P, Debus J, Hans Schrenk H, and Braun K: The Diels-Alder-reaction with Inverse-Electron-Demand - A review of an efficient & attractive Click-reaction concept. Int J Pharm Sci Res 2013: 4(10); 3678-3698. doi: 10.13040/IJPSR. 0975-8232.4(10).3678-98

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