Dipeptidyl peptidase-4 inhibitors (DPP-4 inhibitors) areenzyme inhibitors that inhibit theenzymedipeptidyl peptidase-4 (DPP-4). They are used in the treatment oftype 2 diabetes mellitus. Inhibition of the DPP-4 enzyme prolongs and enhances the activity ofincretins that play an important role ininsulin secretion andblood glucose control regulation.^[1]Type 2 diabetes mellitus is a chronic metabolic disease that results from inability of theβ-cells in the pancreas to secrete sufficient amounts ofinsulin to meet the body's needs. Insulin resistance and increasedhepatic glucose production can also play a role by increasing the body's demand for insulin. Currenttreatments, other than insulin supplementation, are sometimes not sufficient to achieve control and may cause undesirableside effects, such asweight gain andhypoglycemia. In recent years, new drugs have been developed, based on continuing research into the mechanism of insulin production and regulation of the metabolism of sugar in the body. The enzyme DPP-4 has been found to play a significant role.^[2]

Since its discovery in 1967, serine protease DPP-4 has been a popular subject of research.^[2] Inhibitors of DPP-4 have long been sought as tools to elucidate the functional significance of theenzyme. The first inhibitors were characterized in the late 1980s and 1990s. Eachinhibitor was important to establish an earlystructure activity relationship (SAR) for subsequent investigation. The inhibitors fall into two main classes, those that interact covalently with DPP-4 and those that do not.^[3]DPP-4 is adipeptidase that selectively bindssubstrates that containproline at the P1-position, thus manyDPP-4 inhibitors have 5-memberedheterocyclic rings thatmimicproline, e.g.pyrrolidine, cyanopyrrolidine,thiazolidine and cyanothiazolidine.^[4][5] These compounds commonly form covalent bonds to thecatalytic residue Ser630.^[5]

In 1994, researchers from Zeria Pharmaceuticals unveiled cyanopyrrolidines with anitrilefunction group that was assumed to form animidate with thecatalyticserine. Concurrently other DPP-4 inhibitors without anitrile group were published but they contained other serine-interacting motifs, e.g.boronic acids,phosphonates or diacylhydroxylamines. Thesecompounds were not as potent because of the similarity ofDPP-4 andprolyl oligopeptidase (PEP) and also suffered from chemicalinstability.Ferring Pharmaceuticals filed for patent on two cyanopyrrolidine DPP-4 inhibitors, which they published in 1995. These compounds had excellentpotency and improved chemical stability.

In 1995, Edwin B. Villhauer atNovartis started to explore N-substituted glycinyl-cyanopyrrolidines based on the fact that DPP-4 identifies N-methylglycine as an N-terminalamino acid. This group of new cyanopyrrolidines became extremely popular field of research in the following years. Some trials with dual inhibitors of DPP-4 and vasopeptidase have been represented, since vasopeptidase inhibition is believed to enhance the antidiabetic effect ofDPP-4 inhibition by stimulatinginsulin secretion. Vasopeptidase-inhibiting motif is connected to theDPP-4 inhibitor at the N-substituent.^[3][6]

Fig.1: During ameal, theincretinsglucagon-like peptide 1 (GLP-1) and glucose-dependentgastric inhibitory polypeptide (GIP) are released by thesmall intestine into the blood stream. Thesehormones regulate insulin secretion in a glucose-dependent manner. (GLP-1 has many roles in thehuman body. It stimulates insulin biosynthesis, inhibits glucagon secretion, slows gastric emptying, reduces appetite and stimulates regeneration of isletβ-cells.)

GLP-1 and GIP have extremely short plasmahalf-lives due to very rapid inactivation, catalyzed by theenzyme DPP-4. Inhibition of DPP-4 slows their inactivation, thereby potentiating their action, leading to lower plasma glucose levels, hence its utility in the treatment of type 2 diabetes. (Figure 1).^[2][7]

DPP-4 is attached to theplasma membrane of the endothelium of almost every organ in thebody.Tissues which strongly express DPP-4 include theexocrine pancreas,sweat glands,salivary andmammary glands,thymus,lymph nodes,biliary tract,kidney,liver,placenta,uterus,prostate,skin, and thecapillary bed of thegutmucosa (where most GLP-1 is inactivated locally). It is also present, in soluble form, inbody fluids, such asblood plasma andcerebrospinal fluid. (It also happens that DPP-4 is the CD26T-cell activatingantigen.)

DPP-4 selectively cleaves twoamino acids frompeptides, such as GLP-1 and GIP, which haveproline oralanine in the second position (Figure 2). At theactive site where DPP-4 has its effect, there is a characteristic arrangement of threeamino acids, Asp-His-Ser. Sincealanine andproline are crucial for thebiological activity of GPL-1 and GIP, they are inactivated by cleaving away theseamino acids. Thus, preventing the degradation of theincretinhormones GLP-1 and GIP by inhibition of DPP-4 has potential as atherapeutic strategy in the treatment of type 2 diabetes.^[1][8]

Since DPP-4 is aprotease, it is not unexpected that inhibitors would likely have apeptide nature and this theme has carried through to contemporary research.^[3]

X-ray structures ofDPP-4 that have been published since 2003 give rather detailed information about the structural characteristics of thebinding site. Many structurally diverseDPP-4 inhibitors have been discovered and it is not that surprising considering the properties of the binding site:^[9]

1. A deeplipophilic pocket combined with several exposedaromaticside chains for achieving high affinity small molecule binding.

2. A significantsolvent access that makes it possible to tune thephysico-chemical properties of the inhibitors that leads to betterpharmacokinetic behavior.

DPP-4 is a 766-amino acidtransmembrane glycoprotein that belongs to theprolyloligopeptidase family. It consists of three parts; acytoplasmic tail, a transmembrane region and anextracellular part. Theextracellular part is divided into acatalytic domain and an eight-bladed β-propeller domain. The latter contributes to the inhibitor binding site. The catalytic domain shows an α/β-hydrolase fold and contains the catalytic triad Ser630 - Asp708 - His740. The S1-pocket is veryhydrophobic and is composed of the side chains: Tyr631, Val656, Trp662, Tyr666 and Val711. ExistingX-ray structures show that there is not much difference in size and shape of the pocket that indicates that the S1-pocket has high specificity forproline residues^[9][8]

DPP-4 inhibitors usually have an electrophilic group that can interact with thehydroxyl of the catalytic serine in the active binding site (Figure 3). Frequently that group is anitrile group but can also beboronic acid or diphenylphosphonate. This electrophilic group can bind to theimidatecomplex withcovalent bonds and slow, tight-binding kinetics but this group is also responsible for stability issues due toreactions with the free amino group of the P2-amino acid. Therefore, inhibitors without the electrophilic group have also been developed, but thesemolecules have showntoxicity due to affinity to other dipeptidyl peptidases, e.g. DPP-2,DPP-8 andDPP-9.^[10]

DPP-4 inhibitors span diverse structural types. In 2007 few of the mostpotent compounds contain aprolinemimetic cyanopyrrolidine P1 group. This group enhances the potency, probably due to a transientcovalent trapping of thenitrile group by the active site Ser630 hydroxyl, leading to delayed dissociation and slow tight binding of certain inhibitors. When these potency enhancements were achieved, some chemical stability issues were noted and more advanced molecules had to be made. To avoid these stability issues, the possibility to exclude thenitrile group was investigated. Amino acids witharyl orpolarside chains did not show appreciableDPP-4 inhibition and in fact, all compounds without the nitrile group in this research suffered a 20 to 50-fold loss of potency corresponding to the compounds containing thenitrile group.^[11]

It is important to find a fast and accurate system to discover newDPP-4 inhibitors with idealtherapeutic profiles.High throughput screening (HTS) usually gives low hit rates in identifying the inhibitors butvirtual screening (VS) can give higher rates.VS has for example been used to screen for small primaryaliphatic amines to identifyfragments that could be placed in S1 and S2 sites ofDPP-4. On the other hand, these fragments were not very potent and therefore identified as a starting point to design better ones.Three-dimensional models can provide a useful tool for designing novelDPP-4 inhibitors.Pharmacophore models have been made based on keychemical features ofcompounds withDPP-4 inhibitory activity. These models can provide a hypothetical picture of the primarychemical feature responsible for inhibitory activity.^[5]The first DPP-4 inhibitors were reversible inhibitors and came with badside effects because of low selectivity. Researchers suspected that inhibitors with shorthalf-lives would be preferred in order to minimize possibleside effects. However, sinceclinical trials showed the opposite, the latest DPP-4 inhibitors have a long-lasting effect. One of the first reported DPP-4 inhibitor was P32/98 fromMerck. It used thiazolidide as the P1-substitute and was the first DPP-4 inhibitor that showed effects in bothanimals andhumans but it was not developed to a marketdrug due toside effects. Another old inhibitor is DPP-728 fromNovartis, where 2-cyanopyrrolidine is used as the P1-substitute. The addition of the cyano group generally increases the potency. Therefore, researchers' attention was directed to those compounds. Usually, DPP-4 inhibitors are eithersubstrate-like or non-substrate-like.^[12]

Substrate-like inhibitors (Figure 4) are more common than the non-substrate-likes. They bind eithercovalently or non-covalently and have a basic structure where the P1-substituent occupies the S1-pocket and the P2-substituent occupies the S2-pocket. Usually they contain aprolinemimetic that occupies the S1-pocket. Large substituents on the 2-cyanopyrrolidine ring are normally not tolerated since the S1-pocket is quite small.^[12]SinceDPP-4 is identical with theT-cell activation markerCD26 andDPP-4 inhibitors are known to inhibit T-cellproliferation, these compounds were initially thought to be potentialimmunomodulators. When the function againsttype 2 diabetes was discovered, the cyanopyrrolidines became a highly popular research material. A little latervildagliptin andsaxagliptin, which are the most developed cyanopyrrolidineDPP-4 inhibitors to date, were discovered.^[6]

Cyanopyrrolidines have two key interactions to the DPP-4 complex:^[6]

1.Nitrile in the position of thescissile bond of the peptidic substrate that is important for high potency. The nitrile group forms reversiblecovalent bonds with the catalytically activeserine hydroxyl (Ser630), i.e. cyanopyrrolidines are competitive inhibitors with slow dissociation kinetics.

2.Hydrogen bonding network between the protonated amino group and a negativelycharged region of theprotein surface, Glu205, Glu206 and Tyr662. All cyanopyrrolidines have basic, primary or secondaryamine, which makes this network possible but these compounds usually drop in potency if these amines are changed. Nonetheless, two patent applications unveil that the amino group can be changed, i.e. replaced by ahydrazine, but it is claimed that these compounds do not only actvia DPP-4 inhibition but also prevent diabetic vascular complications by acting as aradical scavenger.

Importantstructure-activity relationship:^[11]

1. Strict steric constraint exists around thepyrrolidine ring of cyanopyrrolidine-based inhibitors, with onlyhydrogen,fluoro,acetylene,nitrile, or methano substitution permitted.

2. Presence of anitrile moiety on thepyrrolidine ring is critical to achieving potent activity

Also, systematic SAR investigation has shown that the ring size andstereochemistry for the P2 position is quite conditioned. A 5-membered ring and L-configuration has shown better results than a 4-membered or 6-membered ring with D-configuration. Only minor changes on thepyrrolidine ring can be tolerated, since the good fit of the ring with thehydrophobic S1 pocket is very important for high affinity. Some trials have been made, e.g. by replacing thepyrrolidine with athiazoline. That led to improved potency but also loss of chemical stability. Efforts to improve chemical stability often led to loss of specificity because of interactions withDPP-8 andDPP-9. Theseinteractions have been connected with increasedtoxicity andmortality in animals. There are strict limitations in the P1 position and hardly any changes are tolerated. On the other hand, a variety of changes can be made in the P2 position. In fact,substitution with quite big branched side chains, e.g.tert-butylglycin, normally increased activity and chemical stability, which could lead to longer-lasting inhibition of the DPP-4 enzyme. It has also been noted thatbiaryl-basedside chains can also give highly active inhibitors. It was originally believed that onlylipophilic substitution would be tolerated. Now it is stated that also the substitution ofpolar negatively charged side-chains as well ashydrophilic substitution can lead to excellent inhibitory activity.^[6]

In general,DPP-4 inhibitors are not very stable compounds. Therefore, many researchers focus on enhancing the stability for cyanopyrrolidines. The most widespread technique to improve chemical stability is to incorporate asteric bulk. The two cyanopyrrolidines that have been most pronounced,vildagliptin andsaxagliptin, were created in this manner. K579 is aDPP-4 inhibitor discovered by researchers at Kyowa Hakko Kyogo. It had improved not only chemical stability but also a longer-lasting action. That long-lasting action was most likely due to slow dissociation of the enzyme-inhibitor complex and an activeoxidemetabolite that undergoes enterohepatic circulation. The discovery of the activeoxide was in fact a big breakthrough as it led to the development ofvildagliptin andsaxagliptin. One major problem inDPP-4 inhibitor stability isintramolecular cyclization. The precondition for the intramolecular cyclization is the conversion of thetrans-rotamer, which is the DPP-4 bindingrotamer (Figure 5). Thus, preventing this conversion will increase stability. This prevention was successful when incorporating an amide group into a ring, creating a compound that kept the DPP-4 inhibitory activity that, did not undergo the intramolecular cyclization and was even more selective over different DPP enzymes. It has also been reported that a cyanoazetidine in the P1 position and a β-amino acid in the P2 position increased stability.^[6]

Vildagliptin (Galvus)(Figure 6) was first synthesized in May 1998 and was named after Edwin B. Villhauer. It was discovered when researchers atNovartis examined adamantylderivatives that had proven to be verypotent. Theadamantyl group worked as asteric bulk and slowed intramolecular cyclization while increasing chemical stability. Furthermore, the primarymetabolites were highly active. To avoid additionalchiral center ahydroxylation at theadamantyl ring was carried out (Figure 6). The product,vildagliptin, was even more stable, undergoing intramolecular cyclization 30-times slower, and having highDPP-4 inhibitory activity and longer-lastingpharmacodynamic effect.^[6]

Researchers atBristol-Myers Squibb found that increasedsteric bulk of theN-terminalamino acidside-chain led to increased stability. To additionally increase stability thetrans-rotamer was stabilized with acis-4,5-methano substitution of thepyrrolidine ring, resulting in anintramolecularvan-der-Waals interaction, thus preventingintramolecular cyclisation. Because of that increased stability, the researchers continued their investigation oncis-4,5-methano cyanopyrrolidines and came across with a newadamantylderivative, which showed extraordinaryex vivo DPP-4 inhibition in rat plasma. Also noted, highmicrosomal turnover rate which indicated that the derivative was quickly converted to an activemetabolite. Afterhydroxylation on theadamantyl group they had a product with bettermicrosomal stability and improved chemical stability. That product was namedsaxagliptin (Onglyza) (Figure 6).^[6] In June 2008AstraZeneca andBristol-Myers Squibb submitted a new drug application for Onglyza in theUnited States and a marketing authorization application inEurope.^[13] Approval was granted in theUnited States by the FDA in July 2009 for Onglyza 5 mg and Onglyza 2.5 mg. This was later combined with extended-releasemetformin (taken once daily) and approved by the FDA in January 2011 under the trade name Kombiglyze XR.

Denagliptin (Figure 6) is an advancedcompound with a branchedside-chain at the P2 position, but also has (4S)-fluoro substitution on the cyanopyrrolidine ring.^[6] It is a well-knownDPP-4 inhibitor developed byGlaxoSmithKline (GSK). Biological evaluations have shown that theS-configuration of theamino acid portion is essential for the inhibitory activity since theR-configuration showed reluctantly inhibition. These findings will be useful in future designs and synthesis ofDPP-4 inhibitors.^[14] GSK suspended Phase III clinical trials in October 2008.^[15]

Informations for this group of inhibitors are quite restricted.Azetidine-basedDPP-4 inhibitors can roughly be grouped into three main subcategories: 2-cyanoazetidines, 3-fluoroazetidines, and 2-ketoazetidines. The most potent ketoazetidines and cyanoazetidines have largehydrophobicamino acid groups bound to theazetidinenitrogen and are active below 100nM.^[16]

Non-substrate-likeinhibitors do not take after dipeptidic nature of DPP-4 substrates. They arenon-covalent inhibitors and usually have anaromatic ring that occupies the S1-pocket, instead of the proline mimetic.^[12]

In 1999,Merck started adrug development program on DPP-4 inhibitors. When they started internal screening andmedicinal chemistry program, twoDPP-4 inhibitors were already inclinical trials, isoleucyl thiazolidide (P32/38) and NVP-DPP728 fromNovartis.Merck in-licensed L-threo-isoleucylthiazolidide and itsallostereoisomer. In animal studies, they found that bothisomers had similar affinity for DPP-4, similarin vivo efficacy, similarpharmacokinetic and metabolic profiles. Nevertheless, theallo isomer was 10-fold moretoxic. The researchers found out that this difference intoxicity was due to theallo isomer's greater inhibition ofDPP-8 andDPP-9 but not because of selective DPP-4 inhibition. More research also supported that DPP-4 inhibition would not cause compromised immune function. Once this link between affinity for DPP-8/DPP-9 andtoxicity was discovered,Merck decided on identifying an inhibitor with more than a thousandfold affinity for DPP-4 over the other dipeptidases. For this purpose, they usedpositional scanning libraries. From scanning these libraries, the researchers discovered that both DPP-4 andDPP-8 showed a strong preference for breaking downpeptides with aproline at the P1 position but they found a great difference at the P2 site; i.e., they found that acidic functionality at the P2 position could provide a greater affinity for DPP-4 overDPP-8.Merck kept up doing even more research and screening. They stopped working on compounds from the α-amino acid series related to isoleucylthiazolidide due to lack of selectivity but instead they discovered a very selective β-amino acidpiperazine series throughSAR studies on two screening leads. When trying to stabilize thepiperazinemoiety, a group ofbicyclic derivatives were made, which led to the identification of apotent and selective triazolopiperazine series. Most of theseanalogs showed excellentpharmacokinetic properties inpreclinical species. Optimization of these compounds finally led to the discovery ofsitagliptin.^[17]

Sitagliptin (Januvia) has a novel structure with β-amino amidederivatives (Figure 7). Sincesitagliptin has shown excellent selectivity andin vivo efficacy it urged researchers to inspect the new structure ofDPP-4 inhibitors with appended β-amino acid moiety. Further studies are being developed to optimize these compounds for the treatment ofdiabetes.^[4]In October 2006 sitagliptin became the first DPP-4 inhibitor that got FDA approval for the treatment oftype 2 diabetes.^[18]Crystallographic structure ofsitagliptin along withmolecular modeling has been used to continue the search for structurally diverse inhibitors. A newpotent, selective and orallybioavailable DPP-4 inhibitor was discovered by replacing the centralcyclohexylamine insitagliptin with 3-aminopiperidine. A 2-pyridyl substitution was the initial SAR breakthrough since that group plays a significant role in potency and selectivity for DPP-4.^[2]

It has been shown with anX-raycrystallography howsitagliptin binds to the DPP-4 complex:^[12]

1. The trifluorophenyl group occupies the S1-pocket

2. The trifluoromethyl group interacts with the side chains of residues Arg358 and Ser209.

3. Theamino group forms asalt bridge with Tyr662 and the carboxylated groups of the two glutamate residues, Glu205 and Glu206.

4. The triazolopiperazine group collides with thephenyl group of residue Phe357

Researchers atAbbott Laboratories identified three novel series of DPP-4 inhibitors using HTS. After more research and optimization ABT-341 was discovered (Figure 8). It is a potent and selective DPP-4 inhibitor with a 2D-structure very similar tositagliptin. However, the 3D-structure is quite different. ABT-341 also has a trifluorophenyl group that occupies the S1-pocket and the freeamino group, but the two carbonyl groups are orientated 180° away from each other. ABT-341 is also believed to interact with the Tyr547, probably because ofsteric hindrance between the cyclohexenyl ring and thetyrosine side-chain.^[12]Omarigliptin is one of such compound which is in Phase-III development byMerck & Co.

Thepyrrolidine type ofDPP-4 inhibitors was first discovered afterHTS.^[19] Research showed that thepyrrolidine rings were the part of the compounds that fit into thebinding site. Further development has led tofluoro substituted pyrrolidines that show superior activity, as well aspyrrolidines with fused cyclopropylrings that are highly active.^[20]

This is a different class of inhibitors that was identified with HTS.^[12]Aromaticheterocyclic-basedDPP-4 inhibitors have gained increased attention recently. The first patents describingxanthines (Figure 10) asDPP-4 inhibitors came fromBoehringer-Ingelheim(BI) andNovo Nordisk.^[21]Whenxanthine based DPP-4 inhibitors are compared withsitagliptin andvildagliptin it has shown a superior profile.Xanthines are believed to have higherpotency, longer-lasting inhibition and longer-lasting improvement ofglucose tolerance.^[22]

Alogliptin (Figure 9) is a novelDPP-4 inhibitor developed by theTakeda Pharmaceutical Company.^[22] Researchers hypothesized that aquinazolinone based structure (Figure 9) would have the necessary groups to interact with theactive site on the DPP-4complex.Quinazolinone based compounds interacted effectively with the DPP-4 complex, but suffered from low metabolichalf-life. It was found that when replacing thequinazolinone with a pyrimidinedione, the metabolic stability was increased and the result was apotent, selective,bioavailableDPP-4 inhibitor namedalogliptin. The quinazoline based compounds showed potent inhibition and excellent selectivity over relatedprotease,DPP-8. However, shortmetabolichalf-life due tooxidation of the A-ringphenyl group was problematic. At first, the researchers tried to make afluorinated derivative. The derivative showed improved metabolic stability and excellent inhibition of the DPP-4 enzyme. However, it was also found to inhibitCYP 450 3A4 and block thehERG channel. The solution to this problem was to replace thequinazolinone with otherheterocycles, but thequinazolinone could be replaced without any loss of DPP-4 inhibition.Alogliptin was discovered when quinazolinone was replaced with apyrimidinedione.Alogliptin has shown excellent inhibition of DPP-4 and extraordinary selectivity, greater than 10.000 fold over the closely related serine proteasesDPP-8 andDPP-9. Also, it does not inhibit theCYP 450 enzymes nor block thehERG channel at concentration up to 30 μM. Based on this data,alogliptin was chosen for preclinical evaluation.^[23] In January 2007alogliptin was undergoing the phase IIIclinical trial and in October 2008 it was being reviewed by theU.S. Food and Drug Administration.^[24]

Researchers at BI discovered that using a buty-2-nyl group resulted in a potent candidate, called BI-1356 (Figure 10). In 2008 BI-1356 was undergoingphase III clinical trials; it was released aslinagliptin in May 2011.X-ray crystallography has shown that thatxanthine type binds the DPP-4 complex in a different way than otherinhibitors:^[12]

1. The amino group also interacts with the Glu205, Glu206 and Tyr662

2. The buty-2-nyl group occupies the S1-pocket

3. The uracil group undergoes a π-stacking interaction with the Tyr547 residue

4. The quinazoline group undergoes a π-stacking interaction with the Trp629 residue

Thepharmacokinetic properties of sitagliptin and vildagliptin appear unaffected by age, sex orBMI.^[18]Clinical researches have shown thatsitagliptin andvildagliptin do not have theside effects that tend to followtype 2 diabetes treatment, e.g.weight gain andhyperglycemia, but however, other side effects have been observed, includingupper respiratory tractinfections,sore throat anddiarrhea.^[5]

• Dipeptidyl peptidase-4
• Dipeptidyl peptidase-4 inhibitors
• Linagliptin
• Vildagliptin
• Sitagliptin
• Saxagliptin
• Berberine
• teneligliptin
• gosogliptin

• Dipeptidyl peptidase-4 inhibitors
• Drug discovery

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