1. Project Title: Synthesis, Characterization, QSAR and Biological Evaluation of some novel DPP-4 inhibitors 2.

Introduction: Emerging as an epidemic of the 21st century, Type 2 diabetes has become a major health problem throughout the globe. Type 2 or non-insulin-dependent diabetes mellitus (NIDDM) is the most common form of diabetes and is primarily characterized by insulin resistance or reduced insulin sensitivity, combined with reduced insulin secretion and hyperglycaemia. Thus, the important contributing factors for Type 2 diabetes (T2D) include (i) body cell resistance to insulin, (ii) increased hepatic glucose production (e.g., from glycogen degradation), (iii) decreased insulin-mediated glucose transport into muscle and adipose tissues, and (iv) impaired beta-cell function leading to loss of early phase of insulin release in response to hyperglycaemic stimuli. Patients having T2D are at high risk of macro- and micro- vascular complications such as coronary artery disease, stroke, hypertension, nephropathy, peripheral vascular disease, neuropathy and retinopathy.6 The number of deaths attributable to diabetes reflects the insufficient glycaemic control achieved with the treatments used in recent past Origin of the Research Problem Enhancement of insulin secretion by pancreatic islet -cells has been a major goal for the treatment of T2D. The current therapeutic agents, although effective in increasing insulin secretion, are associated with undesirable side effects, including hypoglycaemias, weight gain, tolerability issues, complexity of regimens, abnormalities in cardiovascular responses and -cell apoptosis. Due to their adverse side effects, most of these treatments are considered to be unsatisfactory in terms of prevention of complications and preservation of quality of life.2 The following table compares some common anti-diabetic agents, generalizing classes although there may be substantial variation in individual drugs of each class:31 Agent Mechanism Site of action Main advantages Main side effects

Sulfonylureas

Metformin

Stimulating insulin production Pancreatic by beta cells inhibiting the KATP channel Decreases Liver insulin resistance

Effective Inexpensive May result in mild weight loss Does not cause

Hypoglycemia Weight gain GI symptoms, including diarrhea, nausea, abdominal pain

 hypoglycem ia Reduces intestinal glucose absorption

Lactic acidosis Metallic taste GI symptoms, including diarrhea, abdominal cramping, flatulence

Acarbose

GI tract

Low risk

Reduce insulin resistance Thiazolidinediones by activating PPAR-

Fat, muscle

Hepatoxicity

Also, none of the available treatments meet two important needs to be fulfilled for efficient diabetic control, namely:8 a) Suppression of glucagon secretion b) Progressive beta-cell apoptosis. DPP-4 inhibitors have been investigated as a new therapy with novel mechanisms of action and improved tolerability. DPP-4, a protease that specifically cleaves dipeptides from proteins and oligopeptides after a penultimate N-terminal proline or alanine, is involved in the degradation of a number of neuropeptides, peptide hormones and cytokines, including the incretins. Incretins are insulin secretagogues. The two main candidate molecules that fulfill criteria for being an incretin are: I. Glucagon-like peptide-1 (GLP-1) and II. Gastric inhibitory peptide (glucose-dependent insulinotropic peptide, GIP). Both GLP-1 and GIP are rapidly inactivated by the enzyme dipeptidyl peptidase-4 (DPP-4). Glucagon-like peptide analogs and agonists Glucagon-like peptide (GLP) agonists bind to a membrane GLP receptor. As a consequence, insulin release from the pancreatic beta cells is increased. As soon as released from the gut in response to food intake, GLP-1 and GIP exert a potent glucose-dependent insulinotropic action, thereby playing a key role in the maintenance of post-meal glycaemic control. Endogenous GLP has a half life of only a few minutes, thus an analogue of GLP would not be practical.31 Consequently, inhibiting DPP-4 prolongs the action of GLP-1 and GIP, which in turn improves glucose homeostasis with a low risk of hypoglycaemia and potential for increasing the beta-cell mass and maintaining beta-cell efficiency. As a result, DPP-4 inhibitors will not only provide better glycaemic control and insulinotropic effect but will also improve the quality of life of diabetic patients by preserving the beta-cell mass.6

DPP-4 cleaves two amino acids from the N-terminal end of peptides, such as GLP-1. DPP-4 selectively cleaves two amino acids from peptides, such as GLP-1 and GIP which have proline or alanine in the second position. At the active site of the protease there is a characteristic motif of three amino acids; Asp-His-Ser. DPP-4 is widely distributed in human organs and tissue. Tissues which strongly express DPP-4 include the exocrine pancreas, sweat glands, salivary and mammary glands, thymus, lymph nodes, biliary tract, kidney, liver, placenta, uterus, prostate, skin and the capillary bed of the gut mucosa where most GLP-1 is inactivated locally. Review of Research and Development in the subject: Since its discovery in 1967, serine protease DPP-4 has been a popular subject of research. Inhibitors of DPP-4 have long been sought as tools to elucidate the functional significance of the enzyme. Since DPP-4 is a protease, it is not unexpected that inhibitors would likely have a peptide nature and this theme has carried through to contemporary research. Structure X-ray structures of DPP-4 that have been published since 2003 give rather detailed information about the structural characteristics of the binding site. Many structurally diverse DPP-4 inhibitors have been discovered and it is not that surprising considering the properties of the binding site: 1. A deep lipophilic pocket combined with several exposed aromatic side chains for achieving high affinity small molecule binding. 2. A significant solvent access which makes it possible to tune the physicochemical properties of the inhibitors that leads to better pharmacokinetic behavior. DPP-4 is a 766 amino acid transmembrane glycoprotein which belongs to the prolyloligopeptidase family. It consists of three parts; a cytoplasmic tail, a transmembrane region and an extracellular part. The extracellular part is divided into a catalytic domain and an eight-bladed -propeller domain. The latter contributes to the inhibitor binding site. The catalytic domain shows an /hydrolase fold and contains the catalytic triad Ser630 - Asp708 - His740. The S1pocket is very hydrophobic and is composed of the side chains: Tyr631, Val656, Trp662, Tyr666 and Val711. Existing X-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 for proline residues.31 Binding site

The key interactions between the ligand and DPP-4 complex. The ligand's basic amine forms a hydrogen bonding network. The nitrile reacts with the catalytic active serine and forms an imidate adduct DPP-4 inhibitors usually have an electrophilic group that can interact with the hydroxyl of the catalytic serine in the active binding site. Frequently that group is a nitrile group but can also be boronic acid or diphenyl phosphonate. This electrophilic group can bind to the imidate complex with covalent bonds and slow, tight-binding kinetics but this group is also responsible for stability issues due to reactions with the free amino group of the P2-amino acid. Therefore inhibitors without the electrophilic group have also been developed, but these molecules have shown toxicity due to affinity to other dipeptidyl peptidases, e.g. DPP-2, DPP-8 and DPP-9. DPP-4 inhibitors span diverse structural types. In 2007 few of the most potent compounds contain a proline mimetic cyanopyrrolidine P1 group. This group enhances the potency, probably due to a transient covalent trapping of the nitrile 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 the nitrile group was investigated. Amino acids with aryl or polar side chains did not show appreciable DPP-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 the nitrile group. The first inhibitors were characterized in the late 1980s and 1990s. Each inhibitor was important to establish an early structure activity relationship (SAR) for subsequent investigation. It should be noted that the inhibitors fall into two main classes- those that interact covalent with DPP-4 and those that do not. DPP-4 is a dipeptidase that selectively binds substrates that contain proline at the P1-position, thus many DPP-4 inhibitors have 5-membered heterocyclic rings that mimic proline, e.g. pyrrolidine, cyanopyrrolidine, thiazolidine and cyanothiazolidine. These compounds commonly form covalent bonds to the catalytic residue Ser630. It is important to find a fast and accurate system to discover new DPP-4 inhibitors with ideal therapeutic profiles. One of the first reported DPP-4 inhibitor was P32/98 from Merck. It used thiazolidide as the P1-substitute and was the first DPP-4 inhibitor that showed effects in both animals and humans but it was not developed to a market drug due to side effects.10

Fig 1: P32/98

Another old inhibitor is DPP-728 from Novartis, 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 either substrate-like or non-substrate-like.

Fig 2: DPP-728 Substrate-like inhibitors Substrate-like inhibitors (Figure 5) are more common than the non-substrate-likes. They bind either covalently 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 a proline mimetic that occupies the S1-pocket. Large substituents on the 2-cyanopyrrolidine ring are normally not tolerated since the S1pocket is quite small. Since DPP-4 is identical with the T-cell activation marker CD26 and DPP-4 inhibitors are known to inhibit T-cell proliferation, these compounds were initially thought to be potential immunomodulators. When the function against type 2 diabetes was discovered, the cyanopyrrolidines became a highly popular research material. A little later vildagliptin and saxagliptin, which are the most developed cyanopyrrolidine DPP-4 inhibitors to date, were discovered.6

Fig.5: A generic structure of a substratelike inhibitor. Cyanopyrrolidines Cyanopyrrolidines have two key interactions to the DPP-4 complex: 1. Nitrile in the position of the scissile bond of the peptidic substrate which is important for high potency. The nitrile group forms reversible covalent bonds with the catalytically active serine hydroxyl (Ser630), i.e. cyanopyrrolidines are competitive inhibitors with slow dissociation kinetics. 2. Hydrogen bonding network between the protonated amino group and a negatively charged region of the protein surface, Glu205, Glu206 and Tyr662. All

cyanopyrrolidines have basic, primary or secondary amine 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 a hydrazine, but it is claimed that these compounds do not only act via DPP-4 inhibition but also prevent diabetic vascular complications by acting as a radical scavenger.22 Structure-activity relationship (SAR) 1. Strict steric constraint exists around the pyrrolidine ring of cyanopyrrolidine-based inhibitors, with only hydrogen, fluoro, acetylene, nitrile or methano substitution permitted. 2. Presence of a nitrile moiety on the pyrrolidine ring is critical to achieving potent activity Also, systematic SAR investigation has shown that the ring size and stereochemistry 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 the pyrrolidine ring can be tolerated since the good fit of the ring with the hydrophobic S1 pocket is very important for high affinity. Some trials have been made, e.g. by replacing the pyrrolidine with a thiazoline. 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 with DPP-8 and DPP-9. These interactions have been connected with increased toxicity and mortality 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 that biaryl-based side chains can also give highly active inhibitors. Originally it was believed that only lipophilic substitution would be tolerated, now its stated that also the substitution of polar negatively charged side chains as well as hydrophilic substitution can lead to excellent inhibitory activity.31 Chemical stability

Fig.6: Trans-rotamers are more stable then cis-rotamers. Cis-rotamers undergo intramolecular cyclisation. DPP-4 inhibitors generally 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 a steric bulk. The two cyanopyrrolidines that have been most pronounced, vildagliptin and saxagliptin, were created in this manner. One major problem in DPP-4 inhibitor stability is intramolecular cyclisation. The precondition for the intramolecular cyclisation is the conversion of the trans-rotamer, which is the DPP-4 binding rotamer (Figure 6). 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 didnt undergo the intramolecular cyclisation and was even more selective over different DPP enzymes. It has also been reported that a cyanoazetidine in the P1 position and a -amino acid in the P2 position increased stability. Vildagliptin Vildagliptin (Galvus)(Figure 7) was first synthesized in May 1998 and was named after Edwin B. Villhauer. It was discovered when researchers at Novartis examined adamantyl derivatives that had proven to be very potent. The adamantyl group worked as a steric bulk and slowed intramolecular cyclization while increasing chemical stability. Furthermore, the primary metabolites were highly active. To avoid additional chiral center a hydroxylation at the adamantyl ring was carried out (Figure 5). The product, vildagliptin, was even more stable, undergoing intramolecular cyclisation 30-times slower, and having high DPP-4 inhibitory activity and longerlasting pharmacodynamic effect. 31 Saxagliptin

Fig.7: The basic structure of cyanopyrrolidines compared with vildagliptin, saxagliptin and denagliptin

Researchers at Bristol-Myers Squibb found that increased steric bulk of the Nterminal amino acid side chain led to increased stability. To additionally increase stability they stabilized the trans-rotamer with a cis-4,5-methano substitution of the pyrrolidine ring, resulting in an intramolecular van-der-Waals interaction, thus preventing intramolecular cyclisation. Because of that increased stability, the researchers continued their investigation on cis-4,5-methano cyanopyrrolidines and came across with a new adamantyl derivative which showed extraordinary ex vivo DPP-4 inhibition in rat plasma. Also noted, high microsomal turnover rate which indicated that the derivative was quickly converted to an active metabolite. After hydroxylation on the adamantyl group they had a product with better microsomal stability and improved chemical stability. That product was named saxagliptin (Onglyza) (Figure 7). In June 2008 AstraZeneca and Bristol-Myers Squibb submitted a new drug application for Onglyza in the United States and a marketing authorization application in Europe. Approval is still pending. Denagliptin Denagliptin (Figure 7) is an advanced compound with a branched side chain at the P2 position, but also has (4S)-fluoro substitution on the cyanopyrrolidine ring. It is a well known DPP-4 inhibitor developed by GlaxoSmithKline (GSK). Biological evaluations have shown that the S-configuration of the amino acid portion is essential for the inhibitory activity since the R-configuration showed reluctantly inhibition. These findings will be useful in future designs and synthesis of DPP-4 inhibitors. GSK suspended phase-III clinical trials in October 2008. Azetidine based compounds Informations for this group of inhibitors are quite restircted. Azetidine-based DPP-4 inhibitors can roughly be grouped into three main subcategories; 2-cyanoazetidines, 3-fluoroazetidines and 2-ketoazetidines. The most potent ketoazetidines and cyanoazetidines have large hydrophobic amino acid groups bound to the azetidine nitrogen and are active below 100nM.

Fig 8: Azetidine-based DPP-4 inhibitors Non-substrate-like inhibitors Non-substrate-like inhibitors do not take after dipeptidic nature of DPP-4 substrates. They are non-covalent inhibitors and usually have an aromatic ring that occupies the S1-pocket, instead of the proline mimetic. In 1999, Merck started a drug development program on DPP-4 inhibitors. When they started internal screening and medicinal chemistry program, two DPP-4

inhibitors were already in clinical trials, isoleucyl thiazolidide- P32/38 (Fig. 1) and NVP-DPP728 (Fig. 2)from Novartis. Merck in-licensed L-threo-isoleucyl thiazolidide and its allo stereoisomer. In animal studies, they found that both isomers had similar affinity for DPP-4, similar in vivo efficacy, similar pharmacokinetic and metabolic profiles. Nevertheless, the allo isomer was 10-fold more toxic. The researchers found out that this difference in toxicity was due to the allo isomer's greater inhibition of DPP-8 and DPP-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 and toxicity 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 used positional scanning libraries. From scanning these libraries, the researchers discovered that both DPP-4 and DPP-8 showed a strong preference for breaking down peptides with a proline 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 over DPP-8. Merck kept up doing even more research and screening. They stopped working on compounds from the -amino acid series related to isoleucyl thiazolidide due to lack of selectivity but instead they discovered a very selective -amino acid piperazine series through SAR studies on two screening leads. When trying to stabilize the piperazine moiety, a group of bicyclic derivatives were made, which led to the identification of a potent and selective triazolopiperazine series. Most of these analogs showed excellent pharmacokinetic properties in preclinical species. Optimization of these compounds finally led to the discovery of sitagliptin.32 Sitagliptin

Fig. 9: The structure of sitagliptin

Sitagliptin (Januvia) has a novel structure with -amino amide derivatives (Figure 9). Since sitagliptin has shown excellent selectivity and in vivo efficacy it urged researches to inspect the new structure of DPP-4 inhibitors with appended amino acid moiety. Further studies are being developed to optimize these compounds for the treatment of diabetes. In October 2006 Sitagliptin became the first DPP-4 inhibitor that got FDA approval for the treatment of type 2 diabetes. It has been shown with an X-ray crystallography how sitagliptin binds to the DPP4 complex. 1. The trifluorophenyl group occupies the S1-pocket 2. The trifluoromethyl group interacts with the side chains of residues Arg358 and Ser209. 3. The amino group forms a salt bridge with Tyr662 and the carboxylated groups of the two glutamate residues, Glu205 and Glu206.

4. The triazolopiperazine group collides with the phenyl group of residue Phe357. Constrained phenylethylamine compounds

Fig.10: The structure of ABT-341

Researchers at Abbott Laboratories identified three novel series of DPP-4 inhibitors using HTS. After more research and optimization ABT-341 was discovered (Figure 10). It is a potent and selective DPP-4 inhibitor with a 2D-structure very similar to sitagliptin. However, the 3D-structure is quite different. ABT-341 also has a trifluorophenyl group that occupies the S1-pocket and the free amino 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 of steric hindrance between the cyclohexenyl ring and the tyrosine side chain. Pyrrolidine compounds The pyrrolidine type of DPP-4 inhibitors was first discovered after HTS. Research showed that the pyrrolidine rings were the part of the compounds that fit into the binding site. Further development has led to fluoro substituted pyrrolidines that show superior activity, as well as pyrrolidines with fused cyclopropylrings that are highly active. Xanthine-based compounds This is a different class of inhibitors that were identified with HTS. Aromatic heterocyclic-based DPP-4 inhibitors have gained increased attention recently. The first patents describing xanthines (Figure 11) as DPP-4 inhibitors came from Boehringer-Ingelheim(BI) and Novo Nordisk. When xanthine based DPP-4 inhibitors are compared with sitagliptin and vildagliptin it has shown a superior profile. Xanthines are believed to have higher potency, longer-lasting inhibition and longerlasting improvement of glucose tolerance. 17

Linagliptin

Fig.11: The structure of xanthine type inhibitors and linagliptin (known as BI-1356 in clinical trials) Researchers at BI discovered that using a buty-2-nyl group resulted in a potent candidate, called BI-1356 (Figure 11). In 2008 BI-1356 was undergoing phase III clinical trials; it was released as linagliptin in May 2011. X-ray crystallography has shown that xanthine type binds the DPP-4 complex in a different way than other inhibitors: 15 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 -stacking interaction with the Tyr547 residue 4. The quinazoline group undergoes a -stacking interaction with the Trp629 residue Alogliptin

Fig.12: Quinazolinone structure and alogliptin Alogliptin (Figure 12) is a novel DPP-4 inhibitor developed by the Takeda Pharmaceutical Company. Researchers hypothesized that a quinazolinone based structure (Figure 12) would have the necessary groups to interact with the active site on the DPP-4 complex. Quinazolinone based compounds interacted effectively with the DPP-4 complex, but suffered from low metabolic half-life. It was found that when replacing the quinazolinone with a pyrimidinedione, the metabolic stability was increased and the result was a potent, selective, bioavailable DPP-4 inhibitor named

alogliptin. The quinazoline based compounds showed potent inhibition and excellent selectivity over related protease, DPP-8. However, short metabolic half-life due to oxidation of the A-ring phenyl group was problematic. At first, the researchers tried to make a fluorinated derivative. The derivative showed improved metabolic stability and excellent inhibition of the DPP-4 enzyme. However, it was also found to inhibit CYP 450 3A4 and block the hERG channel. The solution to this problem was to replace the quinazolinone with other heterocycles, but the quinazolinone could be replaced without any loss of DPP-4 inhibition. Alogliptin was discovered when quinazolinone was replaced with a pyrimidinedione. Alogliptin has shown excellent inhibition of DPP-4 and extraordinary selectivity, greater than 10.000 fold over the closely related serine proteases DPP-8 and DPP-9. Also, it does not inhibit the CYP 450 enzymes nor block the hERG channel at concentration up to 30M. Based on this data, alogliptin was chosen for preclinical evaluation. In January 2007 alogliptin was undergoing the phase III clinical trial and in October 2008 it was being reviewed by the U.S. Food and Drug Administration. Clinical trials involving diabetic patients have shown improved glucose control by administering DPP-4 inhibitors, thus demonstrating the benefit of this promising new class of antidiabetics. Long term effects of DPP-4 inhibitors therapy demonstrated in animals include suppression of glucagon secretion and increase in beta-cell mass, thus meeting the needs which were hitherto unmet with other hypoglycaemics. Intense research activities in this area, both in industries and academia, have resulted in the launch of some (sitagliptin, vildagliptin, saxagliptinand linagliptin) and the advancement of a few others into preregistration/phase 3. However, research in this field is still in the early stage and many challenges need to be met, nevertheless, treatment of T2D especially in the early stages via DPP-4 inhibition has been recognized as a validated principle. It is noteworthy that pyrrolidine derivatives have been most widely explored as DPP-4 inhibitors due to DPP-4s specificity for substrate having an amino-terminal proline at C-2. In the current project, we will try to synthesis some novel pyrrolidine based DPP-4 inhibitors.26 3. Objectives: i. ii. iii. iv. compounds. 4. Methodology: PROPOSED SYNTHESIS Step 1: Synthesis of a series of variously substituted thiovalyl amino acids. The amino acid portion of the molecule will be generated by starting with the commercially available D-penicillamine 1. A general synthetic route to afford the final desired compounds is described in (Scheme 1). Initially, the amino acid will be protected as the t-butyl carbamate with di-t-butyl-di-carbonate (BOC2O) under Synthesis of some novel pyrrolidine based DPP-4 inhibitors. Characterization of synthesised compounds. QSAR of synthesised compounds. Evaluation of DPP-4 inhibitory activity of the synthesised

basic conditions to generate acid 2. The acid will then be alkylated again under basic conditions with either the requisite alkyl bromide or chloride to give sulfide 3.

Scheme1: Reagents and Conditions: (a) THF, KOH, BOC2O (b) 1N NaOH, H2O, RBr/RCl Step2: Synthesis of a series of variously substituted 2-cyano pyrrolidines. Commercially available carboxylic acids 4 will be coupled with ammonia using 1-[3-(dimethylamino)- propyl]-3-ethylcarbodiimide hydrochloride (EDC) and 1hydroxybenztriazole (HOBt) as coupling reagents (Scheme 2). Acid-catalyzed cleavage of the N-Boc-protecting group in 5 will then yield the amines 6.

Scheme 2: (a) EDC, HOBt, NH3, DMF or CH3CN (b) 4M HCl/AcOEt or 4M HCl/1,4-dioxane Step 3: Standardised dipeptide coupling of compounds prepared in step 1 with those prepared in step 2 to give the final products. Compounds 6 will be coupled with amino acids 3 produced in step 1 using EDC/HOBt as coupling reagents to yield the dipeptides 7 quantitatively (scheme 3). Compounds 7 will then be dehydrated with cyanuric chloride in dimethylformamide to yield cyano derivatives 8. The N-Boc group in 8 will then be remoned with acids, in some cases producing salts with hydrochloric acid, yielding final compounds 9. Alternatively, in compounds 8 the sulfur will be oxidized to give either the sulfoxide (sodium periodate, MeOH) or the sulfone (m -chloro peroxy benzoic acid mCPBA, CH2Cl2) followed by deprotection yielding compounds 10 and 11.

Scheme 3: (a) EDC, HOBt, N,N-diisopropylethylamine, THFDMF (b) cyanuric chloride, DMF (c) 2M HCl (d) sodium periodate, MeOH (e) mCPBA, CH2Cl2 CHARACTERIZATION OF THE SYNTHESISED COMPOUNDS AND QSAR STUDIES The synthesised compounds will be characterized by TLC, UV, IR, NMR, and Mass spectroscopy. The characterization will be followed by QSAR studies. EVALUATION OF DPP-4 ACTIVITY Evaluation of the synthesised compounds will be done by DPP-IV drug discovery kit - AK-499 (A QuantiZyme Assay System) The DPPIV Drug Discovery Kit is a complete assay system designed to screen DPPIV inhibitors, providing enough material to perform at least 96 assays. The kit contains both a chromogenic substrate (H-Gly-Pro-pNA; Km=114 M) and a fluorogenic substrate (H-Gly-Pro-AMC; Km=50 M). Cleavage of the pnitroaniline (pNA) from the colorimetric substrate increases absorbance at 405 nm. The fluorimetric assay is based on the cleavage of 7-amino-4-methylcoumarin (AMC) moiety from the C-terminus of the peptide substrate, which increases its fluorescence intensity at 460 nm. The kit is useful to screen inhibitors of DPPIV, a potential therapeutic target. A DPPIV inhibitor, P32/98 (KI=130 nM12), is included for use as a control. Antidiabetic study of the potential DPP-4 inhibitors will be done on animal model [Streptozotocin induced animal model( neonatal Wistar albino mice)]

Toxicity studies will be carried out for the synthesized compounds following OECD guideline 423.

The acute toxic class method set out in this Guideline is a stepwise procedure with the use of 3 animals of a single sex per step. Depending on the mortality and/or the moribund status of the animals, on average 2-4 steps may be necessary to allow judgment on the acute toxicity of the test substance. The method uses pre-defined doses and the results allow a substance to be ranked and classified according to the Globally Harmonized System for the classification of chemicals which cause acute toxicity. In principle, the method is not intended to allow the calculation of a precise LD50, but does allow for the determination of defined exposure ranges where lethality is expected since death of a proportion of the animals is still the major endpoint of this test. The preferred rodent species is the rat, although other rodent species may be used. Normally females are used. This is because literature surveys of conventional LD50 tests show that, although there is little difference in sensitivity between the sexes, in those cases where differences are observed females are generally slightly more sensitive. The test substance is administered in a single dose by gavage using a stomach tube or a suitable intubation canula. In the unusual circumstance that a single dose is not possible, the dose may be given in smaller fractions over a period not exceeding 24 hours. Animals should be fasted prior to dosing (e.g. with the rat, food but not water should be withheld over-night, with the mouse, food but not water should be withheld for 3-4 hours). Following the period of fasting, the animals should be weighed and the test substance administered. After the substance has been administered, food may be withheld for a further 3-4 hours in rats or 1-2 hours in mice. Where a dose is administered in fractions over a period it may be necessary to provide the animals with food and water depending on the length of the period. Three animals are used for each step. The dose level to be used as the starting dose is selected from one of four fixed levels, 5, 50, 300 and 2000 mg/kg body weight. The starting dose level should be that which is most likely to produce mortality in some of the dosed animals. The flow charts of Annex 2 describe the procedure that should be followed for each of the starting doses. When available information suggests that mortality is unlikely at the highest starting dose level (2000 mg/kg body weight), then a limit test should be conducted. When there is no information on a substance to be tested, for animal welfare reasons it is recommended to use the starting dose of 300 mg/kg body weight. The time interval between treatment groups is determined by the onset, duration, and severity of toxic signs. Treatment of animals at the next dose, should be delayed until one is confident of survival of the previously dosed animals. Exceptionally, and only when justified by specific regulatory needs, the use of additional upper dose level of 5000 mg/kg body weight may be considered (see Annex 3). For reasons of animal welfare concern, testing of animals in GHS Category 5 ranges (2000-5000mg/kg) is discouraged and should only be considered when there is a strong likelihood that results of such a test have a direct relevance for protecting human or animal health or the environment.

5.

Streptozotocin induced neonatal rat model for NIDDM: NIDDM maybe induced by administering streptozotocin (90 mg/kg i.p.) in two-dayold neonatal rats. 8 animals maybe used for teasting each compound. After six weeks of streptozotocin injection, animals showing the fasting blood glucose level more than 140 mg/dl maybe considered as NIDDM positive. Changes in blood glucose levels of diabetic animals after administration of the suitable dose of selected novel DPP 4 inhibitor maybe done using a glucometer.

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