Introduction

Background of the project Calcineurin and its physiological roles

Calcineurin (CaN) is a serine/threonine protein phosphatase^{[1-5], activated by calcium and the calmodulin-calcium complex. CaN was first purified from bovine brain, where it was found in high concentrations in neurons (over 1 % of total protein in brain). CaN is widely distributed in mammalian tissues and plants. There are two kinds of calcineurins: calcineurin A and calcineurin B.}

Calcineurin activity [6] is necessary for the synthesis of several cytokine genes through the dephosphorylation of a family of transcription factors known as NF-AT (nuclear factor of activated T cells). By inhibiting calcineurin activity, cyclosporin A (CsA) and FK506 prevent the nuclear translocation of NF-AT secondary to dephosphorylation, thereby suppressing T cell activation.

Calcineurin has numerous physiological roles in budding yeast including recovery from -factor-induced growth arrest, salt and temperature tolerance, Ca²⁺ homeostasis, and Mn²⁺ tolerance. In addition, calcineurin inhibits the activity of the vacuolar H⁺/Ca²⁺ exchanger and causes conversion of the K⁺ channel to the high-affinity state.

Inhibitors of calcineurin

Calcineurin inhibitors [3, 6-12], which specifically inhibit T-cell activation, are essential for T-cell activation and proliferation. They are very important for the activities of cells, metabolism and the health of humans. In order to cure a series of diseases (heart diseases, skin diseases, etc.) caused by lacking calcineurin inhibitors, many synthetic chemists and medicinal chemists are interested in developing new calcineurin inhibitors.

(1) Natural calcineurin inhibitors

A number of natural cyclic peptides have been isolated and demonstrated to be inhibitors of calcineurin and other serine/threonine protein phosphatases. The most potent, specific and well-known inhibitor of calcineurin is the immuno-suppressant drug, cyclosporin A. Other cyclic peptides, for example, microcystin LR, AKAP79 (A-kinase-anchoring-protein 79), and FKBP12, are also useful inhibitors of calcineurin.

A few non-peptide natural products also have inhibitory activities against calcineurins and other serine/threonine protein phosphatases, such as FK506, okadaic acid, and dibefurin. Some known natural calcineurin inhibitors are shown in Figure 1.1.

Figure 1.1 Natural product inhibitors of calcineurin

(2) Synthetic calcineurin inhibitors

Several synthetic compounds have been found to be reasonable inhibitors of calcineurin and other phosphatases. They are, exo-7-oxabicyclo[2.2.1]heptane-2,3-dicarboxylic^{acid (an endothal derivative), a variety of alkylphosphonic acid derivatives^{containing an additional thiol or carboxylate group, tyrphostin A8 and PD 144795 (a benzothiophene derivative). Some of the known synthetic inhibitors of calcineurin are shown in Figure 1.2.}}

Figure 1.2 Synthetic inhibitors of calcineurin

(3) Necessity to develop new non-peptidic calcineurine inhibitors

So far, the most important calcineurin inhibitors are from natural origin (natural peptidic calcineurin inhibitors).Peptidic drugs normally cause the problem of easy in vivo hydrolysis and short live time. The immunosuppressive currently used results in a number of unwanted side effects, such as neurotoxicity, nephrotoxicity and carcinogenity [1]. The known synthetic calcineurin inhibitors usually have poor inhibiting activities. Therefore, there is a high need to develop better calcineurine inhibitors, in particular to non-peptidic compounds.

Our research background

On the basis of previous work in our group [13-16], we try to develop a series of special calcineurin inhibitors, which have better inhibiting power or higher selectivity than the known calcineurin inhibitors. These inhibitors will represent a guidance structure for new immune suppressive drug with lower side effect.

For this purpose, an assembly of three aromatic systems and an aminoalkyl unbranched chain was developed as the guidance structure (Figure 1.3). The polar central heteroaromatic ring is hydrophilic, and always flanked by two typically hydrophobic aromatic rings and a saturated unbranched side chain. The side chain is terminated by a hydrophilic functional group. The results of this thesis will help to refine our structural model of calcineurin-inhibiting heterocycles.

Figure 1.3 The generic structural component of guiding structure

In this guidance structure, the polar hydrophilic π-systems are normally nitrogen containing heterocycles, the two hydrophobic π-systems are unsubstituted or substituted aromatic systems or arylvinyl systems, but not polar heteroaromatic systems. The terminal hydrophilic functional groups are unsubstituted and substituted amino groups, hydroxy group, etc.

So far, only a few structural types of this kind are known. The ring-chain-transformation-synthesis concept, developed by us [16a] represents an efficient entrance to such structures and provided access to pyrazolo[1,5-a]pyrimidines and other heterocyclic derivatives with aminoalkyl substituents in 7-position. ( Scheme 1.1)

Scheme 1.1 Synthesis of pyrazolo[1,5-a]pyrimidines by ring-chain-transformation

Some structural examples with high activity of calcineurin inhibitor are successfully synthesized by our group [16], using ring-chain-transformation and other synthetic routes. They are either bicyclic heterocycles (Figure 1.4) or monocyclic heterocycles (Figure 1.5).

Bicyclic heterocycles:

Figure 1.4 Potent bicyclic heterocyclic inhibitors

Monocyclic heterocycles:

Figure 1.5 Potent monocyclic heterocyclic inhibitors

These known examples are limited to pyrazolo[1,5-a]triazines, pyrazolo[1,5-a]pyrimidines, pyrimidines, triazines and thiazoles as the core heterocycles.

Target molecules and synthetic strategies Synthetic target molecules

According to the analysis and discussion above, a synthetic target molecular model of non-peptide calcineurin inhibitors are designed as below (Figure 1.6). The general structure 8 will be followed up in the present work.

Figure 1.6 Synthetic target molecules

The following variation of structural parameters is envisaged:

Ar1, Ar2: phenyl, substituted phenyl, pyridyl, and other aromatic group.

Y: CH₂, CH=CH, C≡C, C=O, CH₂NH, CH(OH), NH, NR, O, S.

Z: NH₂, NHR, NR₁R₂, OH, OR, CO₂R,CN, CONH₂, CONR₂, etc.

Aliphatic-chain: unbranched chain (saturated or unsaturated chain) with 2 to 5 carbon atoms.

Heterocyclic cores: mono-, or bicyclic nitrogen-containing heterocycles, such as pyrazolo[1,5-a]pyrimidine, purine, pyrido[2,3-b]pyrazine, imidazo[1,2-a]pyridine, pyrimidine, pyridine, pyrazine, oxazole, pyrazole, imidazole.

Disconnection of target molecules

The desired target molecules can be retrosynthetically disconnected as follow:

Scheme 1.2 Disconnection of target molecules

Synthetic strategies for target molecules

The chemo-, regio-, stereoselective formation of new carbon-carbon (carbon-oxygen or carbon-nitrogen) bonds is our major goal.

At first, we synthesized a series of heterocyclic ring cores, and introduced one or more leaving groups X (I, Br, Cl) into the heterocyclic cores, then one or two aryl groups were introduced. In the last and most important step, the side chains were introduced into the heterocycles.

Introducing the Y-aliphatic-chain-Z chain into diarylheterocycleis the key step of our project. In order to introduce the Y-aliphatic-chain-Z into the heterocyclic cores, there are two main methods. One way is using nucleophlic substitution, and the other one is using Pd-catalyzed cross-coupling reactions.

Classic nucleophilic substitution is used for active electron deficient halo-heterocycles. For example, the nucleophilic substitution of 4-chloro-2,6-diphenylpyrimidine [17] is shown in Scheme 1.3:

Scheme 1.3 Introducing branch chains by nucleophilic substitution

The transition metal catalyzed cross-coupling reactions, which were developed starting from the 1970s, are extensively used for the formation of C-C bonds, C-N bonds, C-O bonds and C-P bonds. Among them, the most important are Pd-catalyzed cross-coupling reactions. This is a good way to introduce aryl group as well as aliphatic groups into carbocyclic arenes or heteroarenes.

Pd-catalyzed cross-coupling of heterocycles
Palladium was discovered by W. H. Wollaston in 1803. It is known for its ability to absorb large amounts of hydrogen gas (up to 900 times of its own volume of H₂ at room temperature), which led to one of its earliest chemical uses, as a hydrogenation catalyst. In the last few decades, palladium compounds have been used as catalyst to develop many new synthetic transformations, such as carbon-carbon and carbon-heteroatom coupling reactions (e.g., by Buchwald-Hartwig, Heck, Suzuki-Miyaura, Kumada, Negishi, Nozaki-Hiyama, Sonogashira, Stille, and Tsuji-Trost) [18-23]. The Pd-catalyzed cross-coupling reactions gained increasing popularity amongst pharmaceutical chemists as they are generally tolerant of a wide-range of functional groups and therefore can be used for the synthesis of complicated molecules.
Overview of relevant Pd-catalyzed cross-coupling Cross-coupling reactions with organometallic reagents
Palladium-catalyzed cross-coupling reactions of organohalides (organotriflates, etc.) with organometallic reagents follow a general mechanistic cycle. The L₂Pd(0) 17, as a 14-electron structure [the active catalyst PdL is 12 electron when P(o-tol)₃ is used as the ligand] is sometimes reduced from a Pd(II) species 14 by an organometallic reagent R₁M 15. The transmetalation product 16 from 14 and 15 undergoes a reductive elimination step, giving rise to the Pd(0) species 17, along with the homocoupling product R₁-R₁. This is one of the reasons why the organometallic coupling partners are often used in a slight excess relative to the electrophilic partners. When the Pd(0) catalyst 17 is generated, the catalytic cycle goes through a three-step sequence. (a) Electrophile R₂-X 18 undergoes an oxidative addition step to Pd(0) to afford a 16-electron Pd(II) intermediate 19. (b) Subsequently, 19 undergoes a transmetalation and isomerisation step with the organometallic reagent R₁M 15 to produce the intermediate 21. When there is more than one group attached to the metal M, such as with Sn, the order of transmetalation for different substituents is:

alkynyl > vinyl > aryl > allyl ~ benzyl >> alkyl

The transmetalation step, often rate-limiting, is the step to which attention should be directed if the reaction goes awry. (c) Finally, with appropriate syn geometry, intermediate 21 undergoes a reductive elimination step to produce the coupling product R₂—R₁ 22, regenerating the palladium (0) catalyst 17 to close the catalytic cycle (Scheme 1.4).

Scheme 1.4 Catalytic cycle of coupling reactions of organometallic reagents

(1) Negishi coupling

The Negishi reaction is the Pd-catalyzed cross-coupling between organozinc reagents and organohalides (or triflates) [24-26], for an example [27] see Scheme 1.5. It is compatible with many functional groups including ketones, esters, amine and nitriles. Organozinc reagents are usually generated and used in situ by transmetalation of Grignard or organolithium reagents with ZnCl₂.In addition, some organo halides can be oxidatively added to Zn (0) to give the corresponding organozinc reagents. The Negishi coupling is often advantageous over other cross-coupling, because organozinc reagents have a high tolerance of functional groups.

Scheme 1.5 Example of the Negishi coupling reaction

(2) The Stille coupling

The Stille coupling is the Pd-catalyzed cross-coupling between an organostannane and an electrophile to form a new C-C single bond [28-30], for an example [31] see Scheme 1.6. This is regarded as one the most versatile methods in Pd-catalyzed cross-coupling reactions with organometallic reagents for two reasons. First, the organostannanes are readily prepared, purified and stored. Second, the conditions of the Stille reaction tolerate a wide variety of functional groups. In contrast to the Suzuki, Kumada, Heck, and Sonogashira reactions which are run under basic conditions, the Stille reaction can be run under neutral conditions. The pitfall of the Stille reaction is the toxicity of stannanes, making it not suitable for large-scale synthesis or the synthesis of pharmaceutical products.

Scheme 1.6 Examples of the Stille coupling reactions

(3) The Suzuki coupling

The Suzuki reaction is the Pd-catalyzed cross-coupling between organoboron reagents and organohalides (or triflates) [32-34], some examples [35, 36] are shown in Scheme 1.7 .

Scheme 1.7 Example of the Suzuki coupling reactions

In comparison to the abundance of heterarylstannanes, heteroarylboron reagents are not as prevalent. There are major reasons why one should consider the Suzuki coupling when designing a Pd-catalyzed reaction in heteroaryl synthesis. First, a growing number of heteroarylboron reagents are known now. Second, judiciously designing the coupling partners will enable the use of a heteroaryl halide to couple with a known organoboron reagent for the use of certain molecules. Third, there is no toxicity issue involved in organoboron reagents. Therefore, Suzuki reaction is a more attractive choice in carbon-carbon bond formation reactions.

(4). The Kumada coupling

The Kumada coupling represents the Pd-catalyzed cross-copling of a Grignard reagent with an electrophile such as an alkenyl-, aryl-, and heteroaryl halide or triflate [37-39], for an example [40] see Scheme 1.8. The advantage of this reaction is that numerous Grignard reagents are commercially available. Those that are not commercially available may be readily prepared from the corresponding halides. Another advantage is that the reaction can often be run at room temperature or lower. A drawback of this method is the intolerance of many functional groups (such as –OH, -NH₂, -C=O, etc.) by the Grignard reagents.

Scheme 1.8 Example of the Kumada coupling reaction

(5) The Hiyama coupling

The Hiyama coupling is the Pd-catalyzed cross-copling of an organosilicon reagent (activated by F or alkyloxy) with organohalides (or triflates) [41-42], for an example [43] see Scheme 1.9. One of the advantages of the Hiyama coupling is that organosilicon reagents are innocuous. Another advantage is the better tolerance of functional groups in comparison to other strong nucleophilic organometallic reagents.

Scheme 1.9 Example of the Hiyama coupling reaction
The Sonogashira reaction
The Sonogashira reaction is the palladium-catalyzed cross-coupling reaction between terminal alkynes with aryl and vinyl halides in the presence of an aliphatic amine or inorganic base under mild conditions [44-46]. The proposed catalytic cycle is shown in Scheme 1.10:

Scheme 1.10 Catalytic cycle of the Sonogashira coupling

Some examples [47, 48], which are also interesting with respect to this thesis are shown in Scheme 1.11:

Scheme 1.11 Examples of the Sonogashira coupling reactions

The Heck reaction
The Heck reaction is the palladium-catalyzed cross-coupling reaction of organohalides (or triflates) and olefins [49-51]. Nowadays it has become an indispensable tool for organic synthesis. The proposed catalytic cycle is shown in Scheme 1.10:

Scheme 1.12 Catalytic cycle of the Heck coupling

Some useful known examples [52, 53], where side chain with terminal N-atom were introduced are shown in Scheme 1.11:

Scheme 1.13 Examples of the Heck coupling reaction
The Buchwald-Hartwig C-N bond formation
The direct Pd-catalyzed C-N bond formations of aryl halides with amines were discovered by Buchwald and Hartwig independently in 1995 [54, 55]. Pd(OAc)₂ or Pd₂(dba)₃ was often chosen as catalyst, and t-Bu₃P, BINAP, or other bulky phosphorous compound was used as ligand. It is an effective way to introduce substituted amino groups into aromatic rings. The proposed catalytic cycle is shown in Scheme 1.14:

Scheme 1.14 Catalytic cycle of the Buchwald-Hartwig amination

Some useful examples [56, 57] are shown in Scheme 1.15:

Scheme 1.15 Examples of the Buchwald-Hartwig amination
Pd-catalyzed cross-coupling reactions of heterocycles The characteristics and importance of Pd-catalyzed cross-coupling reactions of heterocycles
The applications, in which the palladium chemistry is used for the synthesis of heterocycles, have increased exponentially. Several review articles summarize the development of palladium chemistry in the synthesis of heterocyclic products [58-62]. The importance of these reactions is shown below:

(1) A myriad of heterocycles are biologically active and therefore of paramount importance to medicinal and agricultural chemists. Many heterocycle-containing natural products have elicited great interest from both academic and industrial research groups. Today palladium-catalyzed cross-coupling reaction is the common method to the synthesis of a wide range of fine chemicals, pharmaceutical intermediates and active pharmaceutical ingredients.

In addition, palladium-mediated polymerisation of heterocycles is extensively used in material chemistry. Heterocycles are also important as ligands in coordination chemistry of palladium

(2) Palladium chemistry involving heterocycles has its unique characteristics stemming from the heterocycles’ inherently structural and electronic properties in comparison with the corresponding carbocyclic aryl compounds.

One example illustrating the striking difference in reactivity between a heteroarene and a carbocyclic arene is called “heteroaryl Heck reaction”, which is defined as an intermolecular or intramolecular Heck reaction occurring onto heteroaryl recipient. Intermolecular Heck reaction of carbocyclic arenes as the recipients are rare, whereas heterocycles including thiophenes, furans, thiazoles, oxozoles, imidazoles, pyrroles and indoles, etc. are excellent substrates. For instance, the heteroaryl Heck reaction of 2-chloro-3,6-diethylpyrazine and benzoxazole occurred at the C(2) position of benzoxazole to elaborate the pyrazinylbenzoxazole 54 [63] (Scheme1.16 ).

Scheme 1.16 Intermolecular heteroaryl Heck reaction

The second salient feature of heterocycles is the marked activationat position α- and γ- to the heteroatom. For N-containing 6-membered heterocycles, the presence of N-atom polarizes the aromatic ring, thereby activating α and γ positions, making them more prone to nucleophilic attack. The order of S_NAr displacement of heteroaryl halides with EtO^- is:

4-chloropyrimidine

> 2-chloroquinoline

> 2-chloropyridine

>> chlorobenzene

7×10⁵

3×10²

1

There is certain similarity in the order of the reactivities between S_NAr displacement reactions and oxidative additions in palladium chemistry. Therefore, the ease with which the oxidative addition occurs for these heteroaryl chlorides has a comparable trend. Even α- and γ-chloro-N-heterocycles are sufficiently activated for Pd-catalyzed reactions, whereas chlorobenzene requires sterically hindered, electron-rich phosphine ligands.

As a consequence of α and γ activation of di- or trihaloheterocycle, Pd-catalyzed chemistry may take place regioselectively at the more activated position. This phenomenon is rarely seen in carbocyclic analogues.
Regioselective Pd-catalyzed cross-coupling of heterocycles
Regioselectivity of reactions are very interesting and also very important in organic synthesis, especially in the synthesis of heterocycles. In this way, a functional group can be introduced to the desired position of a substrate. There are a lot of regioselective reactions involving Pd-catalyzed cross-coupling of heterocycles.

In polyhalo-pyrimidines, the 4-position is more active than 2-position, allowing regiospecific Pd-catalyzed coupling at 4-position. The reaction of 2,4-dichloropyrimidine and styrylstannane first preceeded regiospecifically at C(4), giving rise to 55,which was subsequently coupled with phenylstannane at C(2), under more forcing conditions to afford disubstituted pyrimidine 56 [64].

Scheme 1.17 Synthesis of 2,4-disubstituted pyrimidine

In Pd-catalyzed cross-coupling of polyhalopyridines, the 2-position is more active than 4-position and 3-position. For example, the Suzuki reactionof 2,4-dichloropyridine [65] and the carbonylation reaction of 2,3-dichloro-5-methoxypyridine occurred regioselectively at 2-position [66].

Scheme 1.18 Regioselective Pd-catalyzed cross-couplings of polyhalopyridine

The positional preference can be overridden by choosing different leaving groups. Thus, the iodo-substituted position is more active than the chloro-substituted position.When 2-chloro-3-iodopyridine reacts with 4-methylaniline, catalyzed by Pd(OAc)₂, the amino group was introduced to 3-position [67]. Similarly, the reaction of 2-chloro-5-iodopyridine with an olefin took place at 5-position [68, 69].

Scheme 1.19 Regioselective Pd-catalyzed cross-coupling of halopyridines

Facing the vast variety of heterocyclic compounds, the aspect of reactivity and regioselectivity of Pd-catalyzed cross-coupling reactions of heterocycles is still a weakly explored and important field of organic synthesis. We tried to employ these reactions in the synthesis of new calcineurin inhibitors with the general structure 8. Either the aryl groups or the fuctionalized side chains can be introduced into the central heterocycles in this way.
Prospected application of Pd-catalyzed cross-coupling to the synthesis of new calcineurin inhibitors Introducing aryl groups to the heterocycles
There are a lot of ways to introduce aryl groups to the heterocycles, for example, Suzuki coupling, Negishi coupling, Kumada coupling, Hiyama coupling, etc. The most important and most effective reaction is the Suzuki reaction, using aromatic halides cross-coupling with aryl boronic acid. Some examples [70, 71]^{which are interesting to our project are shown in Scheme 1.20:}

Scheme 1.20 Introducing aryl groups to heterocycles by Suzuki couplings
Introducing the functionalized side chains
In the establishment of calcineurin inhibiting assemblages of the general structure 8, the introduction of the functionalized side chains is often the key step to the target molecules. The application of Pd-catalyzed cross-coupling reactions envisaged for these synthetic transformations is shown by the following protocols. When the side chain contains π-bond, it should be possible to transform it into saturated side chain by reduction or hydrogenation.

(1) Using Heck cross-coupling

Designed synthetic strategy:

Scheme 1.21 Introducing side chains by the Heck coupling

(2) Using Sonogashira cross-coupling

Designed synthetic strategy:

Scheme 1.22 Introducing side chains by the Sonogashira coupling

(3) Using Suzuki cross-coupling

Designed synthetic strategy:

Scheme 1.23 Introducing side chains by the Suzuki coupling

(4) Using Negishi cross-coupling

Designed synthetic strategy:

Scheme 1.24 Introducing side chains by the Negishi coupling

(5) Using Buchwald-Hartwig amination

Designed synthetic strategy:

Scheme 1.25 Introducing side chains by the Buchwald-Hartwig amination