Thiazoles in Peptides and Peptidomimetics

Abstract

The natural occurrence of the thiazole ring in chemistry and biology has inspired its widespread use in synthetic peptidomimetics as structural templates, biological probes, and pharmaceuticals. Thiazole can be viewed as a dehydrated cyclized derivative of cysteine, incorporated into peptide sequences through chemical synthesis or ribosomal biosynthesis. Thiazoles are planar heterocycles and valuable synthetic templates with a strong hydrogen bond accepting nitrogen, a sulfur atom with extended lone pair electron orbitals, and an aromatic π-cloud. These properties can influence molecular conformation and direct interactions with proteins, leading to development of thiazole-containing peptidomimetics as protein mimicking scaffolds, modulators of cell surface proteins like G protein-coupled receptors (GPCRs), inhibitors of enzymes, and agonists or antagonists of protein–protein interactions. The thiazole ring is the most common five-membered heterocycle present in pharmaceuticals. This perspective article describes important properties of thiazoles in synthetic peptidomimetics and highlights key examples, including some from the last 5 years.

1 From Cysteine to Thiazoles and Peptidomimetics

Thiazole (C₃H₃NS) is a five-membered heterocyclic aromatic compound found in many natural products [1–4] including vitamin B1 and has been widely incorporated into synthetic molecules including pharmaceuticals [5], catalysts [6], and dyes [7]. Indeed, thiazole is a more common component of FDA-approved pharmaceuticals than related five-membered heterocycles such as isothiazole, thiophene, furan, isoxazole, and oxazole (Fig. 1). This perspective briefly summarizes thiazole chemistry [8] and focuses on current knowledge of thiazole peptidomimetics and their interactions with proteins.

Fig. 1

Number of FDA-approved pharmaceuticals containing a single heterocyclic ring. The FDA1216 database [9] was searched for each heterocyclic ring using the ligand filtering module within Schrödinger software. Numbering convention shown for thiazole

From a peptidomimetic perspective, thiazole can be viewed as a derivative of cysteine, in much the same way as oxazole can be considered a derivative of serine or threonine (Scheme 1). Within a peptide sequence, a thiazole can be considered to represent a net condensation between the thiolate side chain of cysteine (e.g., in 1) and the C-terminus of an adjacent amino acid with elimination of water to produce a dipeptide surrogate (Scheme 1). This heterocyclization with dehydration forms a thiazoline (e.g., 3) that may undergo dehydrogenation to provide the thiazole (e.g., 5). This set of reactions occurs in both ribosomal and non-ribosomal biosynthesis. The consequences of this condensation include the following: (1) the C-terminal carboxylate is reoriented due to attachment to the sp² carbon of thiazole versus an sp³ carbon in cysteine; (2) replacement of the N-terminal amide and cysteine thiol by the thiazole reduces polarity and hydrogen bond donors; (3) the cysteine stereochemical center is removed; (4) overall, this cyclization rigidifies the peptide backbone and favors turn conformations that may facilitate peptide cyclization.

Scheme 1

Condensation of the cysteine (X = S, 1) and serine (X = O, 2) side chains to give, respectively, thiazoline 3 and oxazoline 4, followed by dehydrogenative aromatization to thiazole 5 and oxazole 6 dipeptide surrogates [10]

The thiazole ring in dipeptide surrogates (e.g., 5) may be reduced to the thiazoline (one double bond) or thiazolidine (no double bonds) to provide a range of conformational and stereochemical influences on the spatial disposition of the N- and C-terminus of the mimetic, to alter the capacity of the heterocycle to fit into hydrophobic binding sites in proteins, and to modify the hydrogen bonding properties of the heteroatoms. We shall return to peptidomimetics after briefly summarizing the chemistry of the thiazole heterocycle.

2 Chemistry of Thiazoles

2.1 Syntheses of Thiazoles

The reactivity and synthesis of thiazoles has been previously reviewed [8, 11–16], and only such chemistry relevant to their use as peptidomimetics is presented below. Thiazoles have been traditionally prepared from open-chained components by a number of methods, among which the Hantzsch synthesis is preferred (Scheme 2) [17]. Thioamides (e.g., 7) and α-halocarbonyls (e.g., 8) combine to form thiazoles (e.g., 11) via halide displacement (to give 9) and subsequent condensation (e.g., 10) [18]. All three carbons of the components may possess alkyl and aryl substituents, such that various substituted thiazoles may be synthesized [19]. For example, the thioamide component can be replaced by a thiourea to give the corresponding 2-aminothiazole [20].

Scheme 2

Mechanism of the Hantzsch thiazole synthesis

Racemization in the synthesis of thioamides derived from enantiomerically pure α-amino acids has been reported due to base-promoted epimerization [21]. Furthermore, application of α-amino acid-derived thioamides in the Hantzsch synthesis has been suggested to cause racemization from acid-catalyzed imine to enamine equilibrium during cyclization prior to aromatization [22, 23]. A modified Hantzsch synthesis protocol was developed to obtain enantiomerically pure α-amino acid-derived thiazoles (e.g., 15) [23, 24]. After alkylation of thioamide 12 with ethyl bromopyruvate gave 13, the hydroxyl group was trifluoroacetylated (to give 14) and then eliminated in one pot using trifluoroacetic anhydride and pyridine at low temperature (Scheme 3).

Scheme 3

Modified Hantzsch thiazole synthesis for production of enantiomerically pure α-amino acid-derived thiazoles

The practicality of the Hantzsch synthesis was demonstrated by the preparation of orthogonally protected thiazole diamino acids 21. β-Alanines 16 were converted to β-keto esters 18 by acylation with Meldrum’s acid (17) and solvolytic decarboxylation. α-Chlorination of 19 with sulfuryl chloride followed by condensation with thiourea gave 2-aminothiazoles 20, which were suitably N-protected and saponified to give 21 (Scheme 4) [25].

Scheme 4

Synthesis of orthogonally protected thiazole diamino acids 21

The Hantzsch reaction has also demonstrated utility for peptide macrocyclization (Fig. 2). In the case of macrocycle 22, the Hantzsch synthesis installed a cysteine mimetic in the form of a thiazole ring, possessing a 4-position alkyl chloride, which was displaced by the thiol of a cysteine residue in the macrocyclization step [26].

Fig. 2

Hantzsch strategy for the synthesis of peptide macrocycle 22

The Hantzsch synthesis has been conducted on solid phase to synthesize thiazole-containing peptide macrocycles [27]. The N-terminus of peptide 23 on solid support was reacted with N,N′-di-Boc-thiourea and Mukaiyama’s reagent to give thiourea 24. Subsequent treatment of 24 with α-bromopyruvic acid gave thiazole 25 en route to macrocycle 26 (Scheme 5).

Scheme 5

Hantzsch thiazole formation in solid phase macrocycle synthesis

Alternatively, N-Fmoc-isothiocyanate has been used to prepare thioureas 28 for solid phase Hantzsch syntheses of thiazoles 29 (Scheme 6) [28].

Scheme 6

Solid phase Hantzsch thiazole synthesis with N-Fmoc-NCS

A one-pot formation of 1,3-thiazoles from nitrostyrenes 30 (Scheme 7) was developed based on a Hantzsch strategy [29]. Oxidation of nitrostyrenes 30 gave α-nitro-epoxides 31, which serves as the α-halocarbonyl equivalent in condensations with thioamides to give the thiazoles 32, albeit the reaction scope was limited to the formation of 2,5-diaryl thiazoles.

Scheme 7

One-pot synthesis of 2,5-diaryl thiazoles from nitrostyrenes 30

The Cook–Heilbron synthesis gives access to 5-aminothiazoles (e.g., 36) with 2-position substituents [30–32]. Thioamidation of α-aminonitrile 34 with thiocarbonyl electrophiles, such as carbon disulfide, dithioesters, dithioacids (as exemplified by 33), and isothiocyanates, provides thioamide intermediate 35, which cyclizes to 5-aminothiazole 36 (Scheme 8).

Scheme 8

Thiazole formation from dithioacid 33 and α-aminonitrile 34 in the Cook–Heilbron synthesis

Employment of activated methylene isocyanide 38, instead of the aminonitrile, in the condensation with dithioester 37 gave access to 4,5-substituted thiazoles 40 by way of thioketone intermediate 39 (Scheme 9) [33].

Scheme 9

Modified Cook–Heilbron synthesis of 4,5-disubstituted thiazoles 40 using activated methylene isocyanides 38

The Gabriel synthesis features conversion of α-acylamino ketones 41 into the corresponding thioketones 42, which undergo intramolecular condensation to thiazole 43 [34, 35]. Substitution of Lawesson’s reagent in place of phosphorus pentasulfide as thionating agent has expanded the approach to α-amido esters to make 5-alkoxythiazoles (Scheme 10) [36].

Scheme 10

Simplified mechanism of the Gabriel synthesis of thiazoles

2,4-Disubstituted thiazoles 46 have also been synthesized from cysteine. For example, cysteine ethyl ester (44) has been condensed with aldehydes to give thiazolidines 45, which were oxidized with manganese dioxide to give the corresponding substituted thiazoles 46 on multi-gram scale (Scheme 11) [37].

Scheme 11

Synthesis of 2,4-disubstituted thiazoles from cysteine ethyl ester

2.2 Stereoelectronic Properties of Thiazoles

Thiazoles possess a number of stereoelectronic properties that may impact on their peptidomimetic derivatives. Thiazoles are planar aromatic heterocycles in which the relative connectivity of the heteroatoms, the bond lengths, and the bond angles all compare favorably with the planar amide bond in peptides, except that the C–S bond is considerably longer than the carbonyl C=O bond (Fig. 3) [38]. Replacement of a cysteine residue by a thiazole ring forces three additional atoms into the same plane as the amide (Fig. 3, shown in bold). Furthermore, a 5-position substituent (R₃) may be introduced, occupying space inaccessible by the cysteine residue.

Fig. 3

Selected bond lengths and angles of amide 47 and thiazole 48 with coplanar atoms in bold. Bond angles and lengths correspond to unsubstituted thiazole [38]

The thiazole ring structure satisfies Hückel’s rule (n = 1) courtesy of the delocalization of the lone pair of the sulfur atom. Relative to oxazole, which has little aromatic character, thiazole is considered to be aromatic, due in part to π-electron delocalization of the lone pair on the sulfur atom [39]. Calculations of the electron density distribution in thiazole by many different methods indicate the sulfur and nitrogen atoms have, respectively, net positive and negative charges, and C-2 and C-5 are, respectively, slightly positive, but close to neutral, and slightly negative [40]. The computational results are consistent with experiments indicating nucleophiles attack preferentially at C-2 and electrophiles react with C-5 (Fig. 4).

Fig. 4

The 2- and 5-positions of thiazole react preferentially with nucleophiles and electrophiles, respectively

The thiazole sulfur has been suggested to form non-covalent interactions with electron-rich regions of molecules [41]. For example, replacement of a thiazole for the pyrazole ring in Janus kinase 2 inhibitor 49 gave analogue 50, which exhibited similar potency (Fig. 5). Based on the predicted binding mode, a co-planar electrostatic interaction between the nitrogen (δ−) of the pyrazine and the sulfur (δ+) of the thiazole of inhibitor 50 was presumed to replace the hydrogen bond between the pyrazine and 5-position proton of the pyrazole in 49 [41].

Fig. 5

The predicted N–HC hydrogen bond in pyrazole 49 was successfully replaced by a N–S electrostatic interaction in thiazole 50

The thiazole sp² nitrogen is basic. With lone pair electrons sitting outside of the aromatic π-system, the thiazole nitrogen may act as a moderately strong hydrogen bond acceptor, similar to the oxygen of an amide. With a pK _a of 2.5 for the protonated form of unsubstituted thiazole, the nitrogen is typically not protonated under physiological conditions; however, thiazole basicity is affected by ring substitution [42]. Typically, 2-, 4-, and 5-position alkyl groups increase basicity with decreasing potency. For example, the pK _a values of protonated 2-, 4-, and 5-methylthiazoles are 3.4, 3.2, and 3.1, respectively. Protonated 2-aminothiazole has a pK _a of 5.4, due to resonance between the exocyclic amine and the conjugated π system. Protonated benzothiazole and electron-deficient thiazoles, such as nitrothiazole, are relatively more acidic than thiazole.

2.3 Chemical Reactivity of Thiazoles

Unlike their structurally related oxazole counterparts, thiazoles do not usually react in cycloaddition reactions, due to their greater aromaticity [43–45]. Unless activated by electron donating groups (e.g., amino or hydroxyl groups) [46], similar to π-deficient pyridines, thiazoles typically resist electrophilic attack, due in part to deactivation by the nitrogen [40]. For example, thiazoles are resistant to nitration, sulfonation, and halogenation in strongly acidic media, because nitrogen protonation depletes ring electron density. The C-2 proton possesses greater kinetic and thermodynamic acidity [40], relative to those at C-4 and C-5, and may be deprotonated with alkyl lithium bases [47] and lithium diisopropylamide [48]. Together with thiazole Grignard reagents [47], C-lithiated species have been reacted with various carbon electrophiles. The C-2 position is prone to nucleophilic attack and becomes even more reactive to nucleophiles upon alkylation of the thiazole nitrogen to give the corresponding thiazolium salts [19].

Appropriately functionalized thiazole derivatives have been substrates in palladium-catalyzed reactions, such as Suzuki, Stille, Negishi, Sonogashira, as well as Heck coupling reactions [49, 50], albeit few examples of the latter have been reported [19]. The thiazole tin and zinc reagents serve, respectively, as nucleophilic partners in Stille and Negishi cross-coupling reactions. In Suzuki cross-couplings, however, halothiazoles act typically as the electrophilic component, because of the instability of thiazole boronates, especially those with boron at C-2 [19, 51]. Scalable syntheses of 2-bromothiazole, as well as 2,4- and 2,5-dibromothiazoles from inexpensive commercially available starting materials, have facilitated the use of metallation and palladium-catalyzed reactions [52].

The Ugi reaction has been exploited for the synthesis of thiazole-containing peptides, albeit without stereochemical control [53, 54]. After the Ugi reaction and ester hydrolysis afforded thioamide 53, treatment with triflic anhydride in CH₂Cl₂ gave thiazole triflate 54, which was employed in a series of palladium-catalyzed cross-coupling reactions to prepare thiazoles possessing proton, allyl, acetylenyl and aryl 5-position substituents (e.g., 55, 56, and 57, Scheme 12) [55].

Scheme 12

Synthesis of 5-substituted thiazole peptides via the Ugi reaction

3 Thiazoles as Peptide Surrogates and Synthetic Building Blocks

3.1 Thiazoles as Synthetic Building Blocks

There are many useful thiazole synthetic blocks that are commercially available (e.g., 58–66, Fig. 6). Thiazoles have found wide application within peptidomimetics, due in part to effective methods for installing substituents at each of the ring carbons [6, 56–58].

Fig. 6

Useful thiazole-containing synthetic building blocks

3.2 Thiazoles as Capping Groups in Peptidomimetics

Thiazoles have been used to cap the N- and C-termini of peptides and peptidomimetics, such as in the anticancer agent dolastatin-10 (68, Fig. 7) [59, 60]. In the PAR2 agonist 2at-LIGRL-NH₂ (67, Fig. 7 2at=2-aminothiazol-4-oyl), the aminothiazoyl group was used as a metabolically stable N-terminus cap [61], which may confer additional chemical and biological stability to the peptide [62, 63]. Frequently, the thiazole fits neatly into an indentation in the surface of a protein target, offering interactions with its π cloud or its hydrogen bond accepting nitrogen (see Sect. 5).

Fig. 7

PAR2 agonist 2at-LIGRL-NH₂ (67, 2at=2-aminothiazol-4-oyl) and dolastatin-10 (68)

2-Aminothiazole was used as a heterocyclic cysteine surrogate in the development of Ras protein C-terminal CAAX tetrapeptide mimetics for the inhibition of the zinc metalloenzyme farnesyltransferase (FTase, Fig. 8) [64]. In previous studies, a pyridine group had been successfully employed as a metabolically stable cysteine mimetic. The 2-aminothiazole was tested as an alternative, because of its comparable basicity to pyridine, and pronounced properties for forming metal complexes. Thiazole peptidomimetic 69 inhibited FTase activity (IC₅₀ 49 nM) and rat smooth muscle cell proliferation (IC₅₀ 107 μM). Described as a potentially promiscuous scaffold [65], 2-aminothiazole is a component of some drugs and has gained significant consideration for employment in drug discovery.

Fig. 8

Based on the C-terminal Ras protein tetrapeptide CAAX sequence, 2-aminothiazole served as a cysteine mimetic in FTase inhibitor 69

3.3 Thiazole Influences Conformation in Peptidomimetics

Thiazoles exert stereoelectronic effects on adjacent carboxamides that dictate their conformation [66, 67]. The solid-state structures of six thiazole-5-carboxamide analogues have been deposited in the Cambridge crystallographic database. The four thiazole-5-carboxamides 71 possessing a 1,4-relationship between the sulfur and carbonyl oxygen atom exhibited a S–C–C–O dihedral angle value between 11° and 37° and S–O distances that were less than the sum of the van der Waal radii, suggesting an attractive interaction (Fig. 9). On the other hand, the two thiazole-5-carboxamides 70 with a 1,4-relationship between the thiazole nitrogen and the amide carbonyl oxygen had N–C–C–O torsion angle values of 165° and 173°. On switching locations of the nitrogen and sulfur heteroatoms in thiazole-5-carboxamides 70 and 71 (Fig. 9), the altered dipole and orbital alignments result in opposite amide orientations with different three-dimensional electrostatic surfaces. These differences translated into opposing agonist versus antagonist activity in activating a G protein-coupled receptor (GPCR, i.e., the complement C-3a receptor) on human macrophages [66]. The influence of the heteroatom was confirmed by locking the heterocyclic amide conformation using fused bicyclic rings.

Fig. 9

The dipole moment of the thiazole ring can dictate the conformation of the neighboring amide

Toward the development of potential anticancer agents, peptidomimetics of the cytotoxic cyclic pentapeptide sansalvamide A (72) were prepared employing triazole, oxazole, and thiazole rings as amide bond isosteres (Fig. 10) [68]. Replacement of the Leu-Val residue with thiazole (73) gave an analogue with equal cytotoxicity to the parent sansalvamide A (72). The relative cytotoxicity of the heterocycle analogues was rationalized on their ability to adopt backbone conformation and side chain presentations similar to the native peptide [68].

Fig. 10

Sansalvamide A (72) and its thiazole analogue 73

4-Amino(methyl)-1,3-thiazole-5-carboxylic acids have served as turn-inducing constraints in peptides such as an analogue of gramicidin S that maintained strong antibacterial activity with reduced hemolytic activity [69]. Oligomers of the 4-amino(methyl)-1,3-thiazole-5-carboxylic acids in γ-peptides adopted a right-handed 9-helix structure [70]. Potent examples of thiazole GPCR ligands include cholecystokinin-1 (CCK1) agonist SR146131 (74) and antagonist SR27897 (75, IC₅₀ > 0.58 nM CCK; 489 nM CCK2), orally active corticotropin-releasing factor (CRF) antagonist SSR-125543A (76, K _i = 2 nM, CRF1), and orally active and brain permeable neuropeptide Y antagonist J-104870 (77, Y1 < 1 nM; Y2,Y4 > 10 μM; Y5 6 μM) (Fig. 11) [71].

Fig. 11

Thiazole-containing GPCR modulators (74–77) and enzyme inhibitors (78–82)

Thiazoles have served as linkers and capping groups in peptide-mimicking ligands that inhibit enzymes, for example, in tumor necrosis factor-α converting enzyme inhibitor 78 (IC₅₀ 0.08 μM), cyclin-dependent kinase inhibitor 79 (IC₅₀ 40 nM, CDK2), cathepsin K inhibitor 79 (K _i 10 nM), ERK inhibitor 81 (K _d 16 μM, ERK2), and the HIV protease inhibitor ritonavir (80, IC₅₀ ~ 1 nM, HIV-1 protease) [72].

3.4 Thiazoles as Amide Bond Replacements

Thiazoles have been used as amide bond isosteres. For example, in the development of inhibitors of apoptosis protein (IAP) antagonists, the proline amide bond of lead peptide 83 did not make any specific interactions with the target protein (Fig. 12) [73]. Thus, the amide bond was replaced with thiazole and benzothiazole surrogates to fine-tune the physicochemical properties. The most potent of these thiazole analogues (e.g., 84 and 85) had K _i values of 20–60 nM against targeted IAPs.

Fig. 12

Thiazoles as amide replacements in IAP antagonists

Thiazoles were used as amide bond replacements in the development of peptidomimetic oligomers that modulate the activity of human p-glycoprotein [74]. Linear and cyclic trimer oligomers of (S)-valine-derived thiazole units 5 (R = i-Pr, Scheme 1) exhibited low micromolar inhibition of human p-glycoprotein. Consistent with being effective amide bond replacements, thiazole-based amino acids have also been used to construct peptide secondary structures analogous to those of natural amino acids, such as helical oligomers [70, 75], β-strand mimics [72], and turn mimics [69, 71].

Bi-thiazoles (Fig. 13) may be considered peptide mimics and have served in enzyme inhibitors, protein-binding motifs, and natural products. For example, bi-thiazole-2,2′-diamines (86 and 87, Fig. 13) compete against peptide substrates that bind cJun N-terminal kinases (JNKs) [76]. When incorporated into peptide sequences, these scaffolds can restrict conformation. The bi-thiazole natural product antibiotic cystothiazole A (88) kills human colon cancer and leukemia cells (Fig. 13) [77, 78]. Bi-thiazoles occur in other natural products such as micrococcinic acid (89) and macrocyclic peptides (see section below).

Fig. 13

Bithiazole-2,2′-diamines 86 and 87 and bithiazole natural products cystothiazole A (88) and micrococcinic acid (89)

4 Thiazoles in Peptidomimetics and Cyclic Peptides

Thiazoles are present in numerous classes of natural products, particularly alkaloids and cyclic peptides [1, 3, 79–81]. Only a few thiazole macrocycles can be accommodated within the size constraints of this article. The first to be considered here is largazole (90, Fig. 14), a metabolite from a cyanobacterium that potently inhibits the growth of triple-negative human breast cancer cells (GI₅₀ = 7.7 nM) [82–86]. Largazole features a macrocycle containing a thiazole fused to a 4-methylthiazoline. An octanoyl moiety is attached to the macrocycle by way of a thioester to the 3-hydroxy-7-mercaptohept-4-enoic acid subunit and serves to produce a prodrug, which on thioester hydrolysis releases the thiolate that potently inhibits histone deacetylase enzymes. The thiazole–thiazoline bicyclic unit serves primarily as a scaffold that orients the other components of the molecule for protein binding.

Fig. 14

Largazole (90), a thiazole-containing cyclic peptide natural product

Bistratamides (e.g., 91–93, Fig. 15) are a family of thiazole-containing hexapeptide analogues, which were isolated from the aplouso-branch ascidian Lissoclinum bistratum [87]. Bistratamides have exhibited antitumour activity against human colon cancer cells [87]. Their cytotoxicity and that of related macrocycles are contingent on the variety of l-amino acid, oxazole, oxazoline, and thiazole components, which leads to different conformational restrictions [88].

Fig. 15

Bistratamides C (91), E (92), and J (93)

Sanguinamides A and B (94 and 96, Fig. 16) are thiazole-containing cyclic hepta- and octa-peptide analogues. Sanguinamide A has inspired analogues exhibiting membrane permeation and oral absorption [89–91]. Sanguinamide B analogues have been the subject of synthetic efforts, due in part to their cytostatic activity [92, 93]. Originally isolated from the sea slug H. sanguineus [91], sanguinamide A (94) features an isoleucine-thiazole dipeptide surrogate in a 21-member cyclic heptapeptide, cyclo-[Ile(Thz)AlaPheProIlePro]. The total synthesis of 94 enabled NMR studies, which revealed two strong intramolecular hydrogen bonds between Ala² and Leu⁵ rigidify the cyclic peptide, which adopts a predominant conformation possessing a prolyl amide cis-isomer between Phe³ and Pro⁴ [90]. Replacement of the thiazole constraint by Ala gave a more flexible cyclic peptide with conformers due to cis–trans isomerization about the Ile⁵-Pro⁶ peptide bond, both retaining the amide cis-isomer between Phe³ and Pro⁴ [89]. Guided by amide H–D exchange rates and NMR structures, Ala² was replaced by the bulkier tBuGly (i.e., 95, Fig. 16) to shield solvent-exposed polar atoms, substantially increasing oral bioavailability in rats (7–50%) [89]. Sanguinamide B (96, cyclo-[Pro-Val-(Ala-Thz)Ile-(ProThzOx)]) was also isolated from H. sanguineus. Featuring two prolyl amide trans-isomers and an unusual 4,2-oxazole-thiazole unit in a 24-member cyclic octapeptide [92], 96 has exhibited antibacterial (P. aeruginosa) and anticancer activity [92–94].

Fig. 16

Sanguinamide A (94), tBuGly analogue 95, and sanguinamide B (96)

Other thiazole-containing 24-member cyclic octapeptide derivatives include urukthapelstatin A (97), patellamide D (98), and ascidiacyclamide (99) (Fig. 17). Urukthapelstatin A (97) was isolated from Mechercharimyces asporophorigenens YM11-542 and was characterized by crystallography. The 24-member cycle of 97 contains a sequential oxazole–thiazole moiety that has also been observed in mechercharstatin and telomestatin [95]. Urukthapelstatin A is a potent inhibitor of human cancer cell growth (e.g., IC₅₀ 12 nM, A549 lung fibroblasts).

Fig. 17

Thiazole-containing cyclic octapeptide derivatives urukthapelstatin A (97), patellamide D (98), and ascidiacyclamide (99)

The patellamides are a diverse class of cyclic peptides that exhibit cancer cell cytotoxicity. Biosynthetically produced by enzymes, they possess various oxazoline, oxazole, thiazoline, and thiazole moieties [96–98]. For example, patellamide D (98) and ascidiacyclamide (99) were isolated from Lissoclinum patella, and both have two thiazole and two oxazoline components that create a saddle-shaped conformation that bestows metal binding capability [99]. Each thiazole nitrogen in the cyclic peptide can coordinate to copper, zinc, calcium, and potassium to form one or two metal ion complexes [100]. In complexes with two copper ions, the cyclic peptide can trap carbon dioxide from solution to form a carbonate bridge between the two metal ions [101]. These cleft-forming thiazole peptides can also trap small organic molecules in their cavities [2]. Removal of two of the heterocycles from an ascidiacyclamide analogue completely altered the cleft structure [102] and inspired the use of thiazoles, oxazoles, and their reduced analogues to generate other shapes, including cylindrical and conical structures [103], as well as the preparation of very large macrocycles containing as many as 19 thiazoles and 76 amino acids [104].

There are many larger macrocycles of varying sizes containing thiazoles (Figs. 18 and 19) that are ribosomally derived from complex gene clusters. Most notable are the thiopeptide antibiotics, which typically have antibacterial activity against Gram-positive bacteria, and sometimes anticancer, antimalarial, immunosuppressive, and cytotoxic activities at low micromolar concentrations [105]. For example, micrococcin P1 (100, Fig. 18) incorporates the four-thiazole unit micrococcinic acid (89, Fig. 13), in a 26-member ring that exhibits antimalarial, antiprotozoal, antibacterial, and cytotoxic activities [106]. The natural product GE2270A (101) contains five thiazoles and exhibits potent antibacterial activity [107]. The antibiotic GE37468A (102) inhibits bacterial protein synthesis. Containing three thiazoles, a thiazoline, an oxazole, and a proline in a 29-member ring, 102 inhibits the growth of a number of bacteria including Clostridium difficile. An analogue LFF571 [108] is in clinical trials for treating bacterial infections in the GI tract. Micrococcin P1 (100), GE2270A (101), and GE37468A (102) all have a central pyridine ring. Thiostrepton A (103, Fig. 19) is a bis-macrocycle possessing a dehydropiperidine core linked to one macrocycle comprising of three thiazoles and a thiazoline, a second cycle containing a quinaldic acid macrocycle, and a bis-dehydroalanine tail. Biosynthesis of 103 involves 21 separate genes. Thiostrepton A has also been made by chemical synthesis [109]. Demonstrated to be active against breast cancer cells and Gram-negative bacteria, 103 is an inhibitor of 50S ribosome and 20S proteasome [110], as well as a valuable tool in molecular biology for gene selection in nucleotide metabolism. Although 103 had limited solubility, thiostrepton A is marketed as a drug for treating mastitis and skin infections in domestic animals. The related bis-macrocycle, nosiheptide (104, Fig. 19), is a thiopeptide antibiotic from a marine actinomycete strain that has exhibited potency (MIC 0.25 mg/L) against methicillin-resistant Staphylococcus aureus (MRSA), drug-resistant clinical isolates, and a virulent strain of Clostridium difficile, but was inactive against most Gram-negative strains tested [111]. With negligible cytotoxicity against mammalian cells and in vivo activity in a murine model of MRSA infection, nosiheptide is marketed for veterinary use as an antibiotic, as well as a food preservative.

Fig. 18

Micrococcin P1 (thiocillin, 100), GE2270A (101), and GE37468A (102)

Fig. 19

Thiostrepton (103) and nosiheptide (104)

5 Protein-Binding Thiazoles and Thiazole Drugs

The thiazole ring is present in biologically active molecules and pharmaceuticals that interact with various proteins [112–114], in part due to the hydrogen bond acceptor ability and aromatic character of the heterocycle. A search of the protein data bank found over 200 crystal structures that feature thiazole–protein interactions exhibiting aromatic π–π, π-cation and π-halogen attractive forces. To exemplify key molecular interactions that underpin biological properties of thiazole peptidomimetics, eight specific thiazole–protein crystal structures are discussed below including six inhibitors of kinases, a protease inhibitor, and one example of a thiazole bound to DNA: 2GQG [115], 3G5D [116], 2Y6O [117], 3BX5 [118], 3IW8 [119], 3QXP [120], 1A61 [121], and 2R2U [122].

5.1 Kinase Inhibition by Dasatinib

Dasatinib (105, Fig. 20a–c) is a kinase inhibitor used to treat chronic myelogenous leukemia (CML) and other cancers. It features a central 2-amido thiazole-5-carboxamide component derived from amino acid 111 (Fig. 21).

Fig. 20

Interactions between thiazole-containing molecules and their targets in crystal structures: (a–c) dasatinib (105) bound to BCR-ABL kinase (PDB: 2GQG), the human kinase domain of cSrc (PDB: 3G5D), and human EphA4 kinase (PDB: 2Y6O); (d) inhibitor 106 bound to p38α MAP kinase (PDB: 3BX5); (e) inhibitor 107 bound to p38α MAP kinase (PDB: 3IW8); (f) inhibitor 108 bound to Cdk2 kinase (PDB: 3QXP); (g) inhibitor 109 bound to thrombin (PDB: 1A61); (h) intercalating agent 110 bound to DNA (PDB: 2R2U)

Fig. 21

The central thiazole amino acid of dasatinib

Dasatinib (105) has been crystallized with several human kinases including BCR-ABL kinase, the human kinase domain of cSrc, erythropoietin-producing hepatocellular EphA4 kinase, Lyn protein kinase [123], and Bruton’s tyrosine kinase [124]. A common feature of most dasatinib structures is that the thiazole nitrogen acts as a hydrogen bond acceptor that binds to a protein backbone amide NH, often from a methionine (Fig. 20).

BCR-ABL kinase is activated in chronic myeloid leukemia [115] and inhibited by the drug imatinib (Gleevec); however, kinase mutation leads to resistance. Dasatinib (105) is 325-fold more potent than imatinib against wild-type and most mutant forms of BCR-ABL and interacts in the ATP binding site (Fig. 21a, PDB: 2GQG) flanked by the two classical kinase sub-lobes. The 2-amino thiazole component occupies a site normally occupied by the adenine group of ATP. The 2-chloro-6-methyl phenyl moiety binds within a hydrophobic site formed by M290, V299, T315, and K217. The thiazole nitrogen and the proton of its 2-amido nitrogen form respectively hydrogen bonds with the amide and carbonyl oxygen of Met318. A hydrogen bond is also made between the hydroxyl oxygen of Thr315 and the thiazole 5-carboxamide NH of dasatinib. The terminal hydroxyethyl group contacts the backbone carbonyl oxygen of Tyr320 via a hydrogen bond. Comparison of the crystal structures of dasatinib and imatinib bound, respectively, to BCR-ABL led to the suggestion that the higher kinase affinity of the former was due to its ability to recognize multiple states of the enzyme [115].

The kinase cSrc is overexpressed in certain tumors such as glioblastoma, gastrointestinal, and prostate cancers [125–127]. The duration of action of cSrc inhibitors is compromised by the development of drug resistance due to the buildup of substrate pressure on the kinase. Crystallographic analysis of dasatinib (105) bound to the human kinase domain of cSrc facilitated development of more effective kinase inhibitors by showing that a T388M mutation, involving a change from a hydrogen bond acceptor side chain to a larger hydrophobic side chain, abolished an important hydrogen bond and caused a steric clash with the known inhibitors (Fig. 21b, PDB: 3G5D) [116].

Erythropoietin-producing hepatocellular kinase EphA4 is associated with axon growth and angiogenesis [128, 129]. Among fourteen known human Eph receptors, EphA4 is overexpressed in a range of malignant carcinomas, including gastric cancer [130], prostate cancer [131], and cutaneous lymphomas [132]. Crystallographic analysis of the broad-spectrum kinase inhibitor dasatinib (105) bound to EphA4 revealed a network of hydrogen bonds between the thiazole ring nitrogen and Met702 amide NH, between the thiazole 2-amido NH with the carbonyl oxygen of Met702, and between the thiazole 5-carboxamide NH and the side chain hydroxyl oxygen of Thr699 (Fig. 21c, PDB: 2Y6O) [117]. Similar to the binding modes of the kinases described above, the 2-chloro-6-methyl phenyl ring is accommodated in a hydrophobic pocket, and the hydroxyethyl piperazine is mainly solvent exposed.

5.2 p38α Kinase Inhibitors

Mitogen-activated protein kinase p38α (MAPK14) plays an important role in inflammatory stress and conditions such as rheumatoid arthritis, by causing the up-regulation of pro-inflammatory cytokines, such as TNFα and IL-1β [133]. Iterative structure-activity relationship studies have led to the development of a potent and selective MAPK14 inhibitor BMS-640994 (106, IC₅₀ 3.5 nM, [118]) [134]. Crystallographic analysis of the inhibitor-kinase structure has revealed the importance of the amido thiazole carboxamide moiety for the binding of 106 to MAPK14. Stationed in a small hydrophobic pocket lined by Y35 and L167, the thiazole engages in a hydrogen bond with its ring nitrogen to the backbone NH of Met109, and with the amide at its 2-position with the carbonyl of Met109. The linker amide H forms a hydrogen bond with side chain hydroxyl oxygen of T106. In addition, the C-terminal carboxamide of 106 interacts in hydrogen bonds with Glu71 and Asp168 (Fig. 21d, PDB: 3BX5). From such information, orally bioavailable inhibitors were designed exhibiting anti-inflammatory activity in rodent models of inflammatory disease.

Thiazole urea 107 binds an allosteric site of human p38α MAP kinase in a way that stabilizes an inactive conformation (Fig. 21e, PDB: 3IW8). In the crystal structure, the thiazole ring of 107 does not contribute to hydrogen bonding interactions with p38α MAP kinase, but forms a π–π contact with F169 [119]. Moreover, the thiazole serves as a pivot that orients the 3-chloro-4-fluoro-phenyl ring toward a hydrophobic pocket, the urea moiety to form two hydrogen bonds with backbone NH of Asp168 and Glu71, and the benzyl ether into a second pocket surrounded by I84, T106 and K53, which are proximal to the gatekeeper residues.

5.3 Cyclic Dependent Kinase Inhibitor

Cyclin-dependent kinases (CDK) are a group of Ser/Thr protein kinases that are cell cycle regulators and targets for drug design [135]. Diaminothiazole 108 (IC₅₀ 20 nM) proved to be a potent inhibitor of CDK2. In the crystal structure of 108 bound to CDK2, the diamino thiazole is anchored by hydrogen bonds to Glu81 and Leu83; places the ortho-nitro phenyl ketone into a hydrophobic pocket consisting of Val18, Ala31, and Lys33; and serves as a hinge-binding scaffold that extends the benzenesulfonamide residue outward to engage in polar interactions in a pocket composed of Gln85, Asp86, and Lys89 (Fig. 21f, PDB: 3QXP).

5.4 Thrombin Inhibitor

Thiazoles have been incorporated into inhibitors of many proteases. For example, thrombin is a serine protease involved in blood coagulation. In the crystal structure of thrombin bound to inhibitor 109, the thiazole occupies the S1′ pocket surrounded by side chains of Cys42, His57, Try60A, Trp60D, and Lys60F (Fig. 21g, PDB: 1A61) [121].

5.5 DNA-Binding Thiazole

A crystal structure for of 110 bound to the 5′-GT containing oligonucleotide d(ATTTAGTTAACTAAAT)₂, the bithiazole moiety intercalates between T7 and T9, interfering with stacking of DNA bases, and plays an essential role in DNA cleavage (Fig. 21h, PDB: 2R2U). Thiazoles are often components of DNA-intercalating dyes and other fluorescent dyes, especially when conjugated with aromatic rings that impart useful fluorescence properties [136].

5.6 Thiazole-Containing Drugs

Among FDA-approved drugs that contain thiazole (105, 112–133, Figs. 22 and 23 show twenty-three representative examples), about half are β-lactams that target a penicillin-binding protein, and the remainder target a variety of human and viral enzymes, GPCRs and DNA. Relatively stable from a biological perspective, thiazole drugs have been found to be prone to ring oxidation, as illustrated by the oxidative epoxidation across the C-4–C-5 double bond of 134 (Scheme 13) [137]. Metabolism proceeds by spontaneous rupture of epoxide 135 to form diol 136, which further ring opens to give dicarbonyl 137 and thioamide 138, both of which may be further metabolized [138–140].

Fig. 22

Lactam-based thiazole-containing antibiotics (112–124) that target penicillin-binding protein in bacteria cell walls

Fig. 23

Non-lactam thiazole-containing drugs (105, 125–133), their uses (in parentheses), and protein targets (in italics) gathered from http://www.drugbank.ca/

Scheme 13

Oxidative metabolism of thiazoles to give dicarbonyl and thioamide metabolites

Ring oxidation was retarded by the addition of substituents at the thiazole C-4 and C-5 positions [141, 142]. For example, the metabolism of the thiazole-containing drug sudoxicam (139, Fig. 24) caused oxidative ring opening in vivo in rat, dog, and monkey [143]. The addition of a methyl group to the thiazole 5-position of 139 gave meloxicam (128) and switched the oxidative metabolism to the methyl group without epoxidation of the heterocycle leading to little to no ring-opened metabolites [144, 145].

Fig. 24

Sudoxicam (139) and 5-methyl analogue, meloxicam (128)

6 Concluding Remarks

The thiazole ring is present in many peptidic natural products and peptidomimetics. This article has illustrated commonly used approaches to synthesize thiazole derivatives, described their key electronic properties, and highlighted their most important chemical reactivities. A particular focus has been on uses of thiazoles in synthetic peptidomimetics, for example, as a peptide capping group, an amide bond replacement, or a conformational constraint. Importantly, the thiazole ring is the most common five-membered heterocycle present in FDA-approved drugs, strongly supporting future development of additional thiazole-based peptidomimetics. To this end, this perspective assessment of currently known thiazole analogues provides a valuable platform for building new thiazole-based peptidomimetics for the future.