WO2013060860A1

WO2013060860A1 - N-acyl-n'-phenylpiperazine derivatives as srbp modulators for use in the treatment of diabetes and obesity

Info

Publication number: WO2013060860A1
Application number: PCT/EP2012/071286
Authority: WO
Inventors: John Stephens; John Findlay; Gemma KINSELLA; Darren Martin; Robert Devine; Trinidad VELASCO-TORRIJOS
Original assignee: National University Of Ireland, Maynooth
Priority date: 2011-10-27
Filing date: 2012-10-26
Publication date: 2013-05-02
Also published as: GB201118613D0

Abstract

The present invention relates to compounds having the general formula (I) as described herein; methods of preparing said compounds; and their use in treating Type 1 and/or Type 2 Diabetes Mellitus. The compounds were assayed for binding to serum retinol binding protein (sRBP) and for disruption of the sRBP:transthyretin (TTR) and sRBP:sRBP receptor interaction. The compounds were also assayed for their ability to induce glucose uptake into mouse muscle cells. Also described is a pharmaceutical composition comprising a compound according to the first aspect of the present invention, and use thereof in treating Type 1 and Type 2 Diabetes.

Description

N-ACYL-N' - PHENYLPIPERAZINE DERIVATIVES AS SRBP MODULATORS FOR USE IN THE TREATMENT OF DIABETES AND OBESITY

Field of the Invention This invention relates to compounds having the general formula (I) as described herein; methods of preparing said compounds; and their use in treating Type 1 and/or Type 2 Diabetes. The compounds were assayed for binding to serum retinol binding protein (sRBP) and for disruption of the sRBP:transthyretin (TTR) and sRBP:sRBP receptor interaction. The compounds were also assayed for their ability to induce glucose uptake in muscle cells.

Background to the Invention

Diabetes is a chronic disease, which occurs when the pancreas does not produce enough insulin, or when the body cannot effectively use the insulin it produces. This leads to an increased concentration of glucose in the blood (hyperglycaemia). Type 1 diabetes is characterized by a lack of insulin production. Type 2 diabetes is caused by the body's ineffective use of insulin.

The number of people suffering from Type-2 Diabetes (T2D) is rising from under 200 million in 2000 to estimates of about 400 million by 2030. Alarmingly, as many as 50% of the total number of people suffering, may continue to remain undiagnosed. The numbers of type 1 diabetes are smaller but still above 10% of sufferers. The oral anti-diabetic market is valued at $13.6Bn in the 7 largest markets. T2D is also associated with several complications, for example, (i) macrovascular complications, resulting from hyperlipidemia and hypertension, which can lead to end-stage renal disease, limb amputation, and accelerated atherosclerosis (cardiovascular disease); and (ii) chronic microvascular complications, such as retinopathy (blindness), nephropathy, and neuropathy.

Type 2 diabetes is a consequence of insulin resistance, which largely results from obesity. However, the causative link between obesity and insulin resistance remains to be validated. Treatment for typel diabetes involves taking insulin injections every day or using an insulin pump. The goal of insulin treatment is to control the amount of insulin in the bloodstream so that glucose levels are normal, or near normal. Investigational treatments include pancreas, islet cell, or stem cell transplants. Successful, extended control of T2D is not facile. Management of the pre-condition, insulin resistance, is not often attempted in practice. For some time, there has been great concern about the link between obesity, insulin resistance, T2D, and cardiovascular disease. The situation now is of global pandemic-like proportions and escalating. Clearly, therefore, therapeutic treatment is inevitable, but there is general agreement that the current arsenal of therapeutic drugs, and their respective targets, is inadequate; indeed some of the most widely used therapeutic drugs have been described as "dirty", having adverse and potentially dangerous side-effects. Hence, new, particularly preventative, therapies are required.

Conventionally, therapeutic drugs used in the treatment and management of T2D include: sulfonylureas, such as tolbutamide, which were the first widely used oral hypoglycaemic medications; and act by triggering insulin release by direct action on the K_ATP channel of the pancreatic beta cells; meglitinides, such as nateglinide and repaglinide, a class of therapeutic drug, which stimulate insulin release; and act by closing the potassium channels of the pancreatic beta cells, resulting in opening of the calcium channels to enhance insulin secretion; biguanides, which are a class of therapeutic drug that can function as oral anti-hyperglycaemic agents by reducing hepatic glucose output and increasing uptake of glucose by the periphery. Typically, biguanides improve hyperglycaemia primarily through suppression of hepatic glucose production, by activation of AMP-activated protein kinase (AMPK); thiazolinediones, such as rosiglitazone, which act by binding to peroxisome proliferator-activated receptor (PPAR)-gamma, a nuclear receptor involved in transcription of genes regulating glucose and lipid metabolism; and alpha-glucosidase inhibitors, which are oral anti-diabetic therapeutic drugs, which act by slowing the digestion of starch in the small intestine, so that the resultant glucose absorption into the bloodstream can be matched more effectively by an insulin response - these agents are effective therapy only in early stages of impaired glucose tolerance, and must be used in combination with other agents in treating T2D.

However, adverse effects are associated with the use of conventional therapeutic drugs used in the treatment and management of T2D. For example, sulfonylureas, thiazolodinediones, and insulin lead to weight gain, resulting in worsening glycaemic control with time. Other adverse effects include hypoglycaemia (associated with sulphonylureas, meglitinides, and insulin); gastrointestinal problems (associated with biguanides, and alpha-glucosidase inhibitors); and fluid retention and heart failure (associated with thiazolidinediones).

The mainstay of treatment in T2D is biguanides, and alpha-glucosidase inhibitors in impaired glucose tolerance. Patients can adapt to the adverse effects of biguanides, but it has been suggested that biguanides have only a small effect on reducing or postponing cardiovascular disease. In the long run, therefore, life expectancy and quality are still compromised. Unfortunately, clinical trials on thiazolidinediones have led to their abandonment. Moreover, thiazolidinediones have recently been associated with adverse cardiac effects and so are under re-examination by the FDA. Emerging therapies have focused on incretins such as: glucagon-like peptide (GLP) analogues (for example, exenatide and liraglutide, which stimulate insulin secretion); glucose-dependent insulinotropic peptide (GIP) antagonists, which affect plasma insulin; and dipeptidyl peptidase IV inhibitors to delay the breakdown of endogenous GLP-1. However, the prospects are unclear. Several of these agents are associated with nausea and vomiting, which may limit their use clinically. Administration of the most tolerated of this class of therapies is by injection and there have recently been concerns about pancreatitis (associated with exenatide). Furthermore, all of these therapies address only the full-blown disease rather than its development. Pramlintide acetate (Symlin) is an analogue of amylin, a small peptide hormone that is released into the bloodstream after a meal by the beta-cells of the pancreas along with insulin. Symlin has been approved by the FDA for type 1 and type 2 diabetics who use insulin.

Summary of the Invention

According to a first aspect of the present invention, there is provided a compound having the general formula (I):

(I)

wherein,

X is an atom selected from carbon and nitrogen;

Y is selected from:

0) a halide selected from fluoride, chloride, bromide, iodide, and astatide; and

(ii) a hydrogen atom;

Z is selected from:

(i) a short-chain alkylene, alkenylene, or alkynylene, which can be linear or branched, and which can be substituted or unsubstituted; and (ii) a short-chain thio-alkylene, thio-alkenylene, or thio-alkynylene, which can be linear or branched, and which can be substituted or unsubstituted;

R and R1 are each independently selected from:

(i) a hydrogen atom;

(ii) an oxygen atom; and

(iii) a hydroxyl group;

R2 is selected from:

(i) a five-membered heterocyclic moiety;

(ii) a six-membered hydrocarbon moiety, optionally which can be substituted or unsubstituted;

(iii) a carbonyl group, optionally which can be substituted or unsubstituted;

and

(iv) a hydrogen atom; and

R3 is an atom selected from carbon and nitrogen; or a pharmaceutically acceptable salt thereof.

For the purposes of this specification, in the case of a polyatomic molecule represented by text, a single bond extending between any two atoms is represented by a solid dashed line (-), a double bond extending between any two atoms is represented by a double solid dashed line (=), and a triple bond extending between any two atoms is represented by a triple solid dashed line (≡), unless otherwise stated.

By "short chain" is meant a polyatomic molecule comprising at least one carbon atom. Optionally, the polyatomic molecule comprises 1-7 carbon atoms. Further optionally, the polyatomic molecule comprises 3-6 carbon atoms. Still Further optionally, the polyatomic molecule comprises one carbon atom.

By the term "linear" is meant a molecule comprising at least two atoms, any of which can be the same or different, wherein each atom of the molecule is bonded to an adjacent atom in a substantially straight series. Each atom can be bonded to an adjacent carbon atom by a single-, double-, triple-, or higher order-bond. Non-limiting examples include n-methane, n-ethane, n- propane, n-butane, and n-pentane.

By the term "branched" is meant a molecule comprising at least three atoms, any of which can be the same or different, bonded in a substantially straight series, wherein the molecule further comprises at least one other atom, which is not bonded to either of the terminal atoms of the substantially straight series. Each atom can be bonded to an adjacent atom by a single-, double-, triple-, or higher order-bond. Non-limiting examples include isopentane (2-methylbutane), and neopentane (2,2- dimethylpropane).

By the term "five-membered heterocyclic moiety" is meant a molecule comprising five atoms, bonded to form at least one loop or ring, wherein at least one of the atoms is selected from nitrogen, oxygen, and sulphur.

Optionally, Y is selected from fluoride, chloride, bromide, iodide, and astatide. Further optionally, Y is selected from chloride and bromide. Still further optionally, Y is chloride.

Optionally, Z is a short-chain thio-alkylene, thio-alkenylene, or thio-alkynylene, which can be linear or branched, and which can be substituted or unsubstituted. Further optionally, Z is a thioalkylene, optionally thioethylene (C-C-S), which can be substituted or unsubstituted. Optionally, Z is a short-chain alkylene, alkenylene, or alkynylene, which can be linear or branched, and which can be substituted or unsubstituted.

Further optionally, Z is a substituted short-chain alkylene, alkenylene, or alkynylene. Optionally, Z is a short-chain alkylene, alkenylene, or alkynylene, which is substituted by an oxygen atom.

Optionally or additionally, R is an oxygen atom. Alternatively, R is a hydrogen atom. Further alternatively, R is a hydroxyl group.

Optionally or additionally, R1 is a hydrogen atom.

Optionally, the five-membered heterocyclic moiety is an organic five-membered heterocyclic moiety. Further optionally, five-membered heterocyclic moiety comprises carbon atoms and at least one atom selected from nitrogen, oxygen, and sulphur, bonded to form at least one loop or ring.

Optionally, R2 is a saturated or unsaturated five-membered heterocyclic moiety. Further optionally, R2 is an unsaturated five-membered heterocyclic moiety. Optionally, R2 is a five-membered heterocyclic moiety selected from thiophene, furan, imidazole, and pyrazole.

Optionally, R2 is a carboxyl group (-COOH), which can be substituted or unsubstituted. Further optionally, R2 is an unsubstituted carboxyl group. It is understood that a carbonyl group, which is substituted, is also referred to as a substituted carbonyl group; and that a carbonyl group substituted with at least one oxygen atom can also be referred to as an ester group, optionally represented by the general structure RCOOR', wherein R is any substitutent; R' is any substitutent, which can be the same as or different to R; C is a carbon atom; and O is an oxygen atom.

Optionally, R2 is a substituted carbonyl group. Further optionally, R2 is an alkoxyl substituted carbonyl group. Still further optionally, R2 is a methoxyl substituted carbonyl group. Still further optionally, R2 is an ester group. Still further optionally, R2 is a methyl alkanoate group.

Optionally, R2 is an amide group, which can be substituted or unsubstituted. Further optionally, R2 is a carboxamide group (-CONH₂), which can be substituted or unsubstituted. Further optionally, R2 is a substituted carboxamide group. Further optionally, R2 is a carboxamide group, substituted with an ether group. Further optionally, R2 is a carboxamide group, substituted with an ether group comprising at least one a short-chain alkylene, alkenylene, or alkynylene, which can be linear or branched, and which can be substituted or unsubstituted. Still further optionally, R2 is a carboxamide group, substituted with a methoxyethanyl (methyl ethyl ether; ethyl methyl ether) group.

Optionally, the six-membered hydrocarbon moiety can be substituted or unsubstituted. Further optionally, the six-membered hydrocarbon moiety is unsubstituted.

Optionally, R2 is a saturated or unsaturated six-membered hydrocarbon moiety. Further optionally, R2 is an unsaturated six-membered hydrocarbon moiety.

Optionally or additionally, R2 is a saturated or unsaturated six-membered cyclic hydrocarbon moiety. Further optionally, R2 is an unsaturated six-membered cyclic hydrocarbon moiety.

Optionally or additionally, the six-membered hydrocarbon moiety is an organic hydrocarbon moiety. Further optionally, the six-membered hydrocarbon moiety comprises six carbon atoms bonded to form at least one loop or ring. Still further optionally, the six-membered hydrocarbon moiety is a phenyl group.

Optionally, the compound has the general formula (I); X is a nitrogen atom; Y is chloride, optionally attached to the carbon atom at position 3 of the pyridinyl moiety; Z is thioethylene; R and R1 are each a hydrogen atom; R2 is thiophene; and R3 is nitrogen. Further optionally, the compound has the general formula (I); X is a nitrogen atom; Y is chloride attached to the carbon atom at position 3 of the pyridinyl moiety; Z is thioethylene; R and R1 are each a hydrogen atom; R2 is thiophene; and R3 is a nitrogen. Still further optionally, the compound is 1-(4-(3-Chloro-5-(trifluoromethyl)pyridin-2- yl)piperazin-1-yl)-3-(thiophen-2-ylthio)propan-1-one.

Optionally, the compound has the general formula (I); X is a carbon atom; Y is a hydrogen atom; Z is propylene; R and R1 are each a hydrogen atom; R2 is thiophene; and R3 is nitrogen. Further optionally, the compound is the compound is 4-(thiophen-2-yl)-1-{4-[4- (trifluoromethyl)phenyl]piperazin-1-yl}butan-1-one.

Optionally, the compound has the general formula (I); X is a carbon atom; Y is a hydrogen atom; Z is a short-chain alkylene, alkenylene, or alkynylene, which is substituted by an oxygen atom; R2 is a thiophene; and R3 is nitrogen. Further optionally, the compound has the general formula (I); X is a carbon atom; Y is a hydrogen atom; Z is an alkylene, which is substituted by an oxygen atom; R2 is a thiophene; and R3 is nitrogen. Still further optionally, the compound has the general formula (I); X is a carbon atom; Y is a hydrogen atom; Z is propylene, which is substituted by an oxygen atom; R2 is a thiophene; and R3 is nitrogen. Still further optionally, the compound has the general formula (I); X is a carbon atom; Y is a hydrogen atom; Z is propylene, wherein one R is an oxygen atom; R1 is a hydrogen atom; R2 is a thiophene; and R3 is nitrogen. Still further optionally, the compound is 1- (thiophen-2-yl)-4-{4-[4-(trifluoromethyl)phenyl]piperazin-1-yl}butane-1 ,4-dione. Optionally, the compound has the general formula (I); X is a nitrogen atom; Y is a hydrogen atom; Z is propylene; R is an oxygen atom; R1 is a hydrogen atom; R2 is thiophene; and R3 is nitrogen. Further optionally, the compound is 1-(thiophen-2-yl)-4-{4-[5-(trifluoromethyl)pyridin-2-yl]piperazin-1- yl}butane-1 ,4-dione. Optionally, the compound has the general formula (I); X is a nitrogen atom; Y is chloride, optionally attached to the carbon atom at position 3 of the pyridinyl moiety; Z is propylene; R is an oxygen atom; R1 is a hydrogen atom; R2 is thiophene; and R3 is nitrogen. Further optionally, the compound has the general formula (I); X is a nitrogen atom; Y is chloride attached to the carbon atom at position 3 of the pyridinyl moiety; Z is propylene; R is an oxygen atom; R1 is a hydrogen atom; R2 is thiophene; and R3 is nitrogen. Still further optionally, the compound is 1-{4-[3-chloro-5- (trifluoromethyl)pyridin-2-yl]piperazin-1-yl}-4-(thiophen-2-yl)butane-1 ,4-dione.

Optionally, the compound has the general formula (I); X is a nitrogen atom; Y is a hydrogen atom; Z is propylene; R and R1 are each a hydrogen atom; R2 is thiophene; and R3 is nitrogen. Further optionally, the compound is 4-(thiophen-2-yl)-1-{4-[5-(trifluoromethyl)pyridin-2-yl]piperazin-1-yl}butan- 1-one.

Optionally, the compound has the general formula (I); X is a nitrogen atom; Y is chloride, optionally attached to the carbon atom at position 3 of the pyridinyl moiety; Z is a propylene; R and R1 are each a hydrogen atom; R2 is thiophene; and R3 is nitrogen. Further optionally, the compound has the general formula (I); X is a nitrogen atom; Y is chloride attached to the carbon atom at position 3 of the pyridinyl moiety; Z is a propylene; R and R1 are each a hydrogen atom; R2 is thiophene; and R3 is nitrogen. Still further optionally, the compound is 1-{4-[3-chloro-5-(trifluoromethyl)pyridin-2- yl]piperazin-1-yl}-4-(thiophen-2-yl)butan-1-one. Optionally, the compound has the general formula (I); X is a carbon; Y is a hydrogen atom; Z is thioethylene; R and R1 are each a hydrogen atom; R2 is thiophene; and R3 is nitrogen. Further optionally, the compound is 3-(Thiophen-2-ylthio)-1-(4-(4-(trifluoromethyl)phenyl)piperazin-1- yl)propan-1-one. Optionally, the compound has the general formula (I); X is a carbon; Y is a hydrogen atom; Z is heptylene; R and R1 are each a hydrogen atom; R2 is an unsubstituted carboxyl group; and R3 is nitrogen. Further optionally, the compound is 9-oxo-9-(4-(4-(trifluoromethyl)phenyl)piperazin-1- yl)nonanoic acid. Optionally, the compound has the general formula (I); X is a carbon; Y is a hydrogen atom; Z is heptylene; R and R1 are each a hydrogen atom; R2 is a carboxamide group, substituted with an ether group; and R3 is nitrogen. Further optionally, the compound has the general formula (I); X is a carbon; Y is a hydrogen atom; Z is heptylene; R and R1 are each a hydrogen atom; R2 is a carboxamide group, substituted with a methoxyethanyl group; and R3 is nitrogen. Still further optionally, the compound is N-(2-methoxyethyl)-9-oxo-9-(4-(4-(trifluoromethyl)phenyl)piperazin-1- yl)nonanamide.

Optionally, the compound has the general formula (I); X is a carbon; Y is a hydrogen atom; Z is propylene; R and R1 are each a hydrogen atom; R2 is pyrazole; and R3 is nitrogen. Further optionally, the compound is 4-(1 H-Pyrazol-4-yl)-1-(4-(4-(trifluoromethyl)phenyl)piperazin-1-yl)butan- 1-one.

Optionally, the compound has the general formula (I); X is a carbon; Y is a hydrogen atom; Z is butylene; R and R1 are each a hydrogen atom; R2 is a hydrogen atom; and R3 is nitrogen. Further optionally, the compound is 1-{4-[4-(trifluoromethyl)phenyl]piperazin-1-yl}pentan-1-one.

Optionally, the compound has the general formula (I); X is a carbon; Y is a hydrogen atom; Z is butylene; R and R1 are each a hydrogen atom; R2 is thiophene; and R3 is nitrogen. Further optionally, the compound is 5-(thiophen-2-yl)-1-{4-[4-(trifluoromethyl)phenyl]piperazin-1-yl}pentan-1- one.

Optionally, the compound has the general formula (I); X is a carbon; Y is a hydrogen atom; Z is propylene; R and R1 are each a hydrogen atom; R2 is thiophene; and R3 is carbon. Further optionally, the compound is 4-(thiophen-2-yl)-1-{4-[4-(trifluoromethyl)phenyl]piperidin-1-yl}butan-1- one. Optionally, the compound has the general formula (I); X is a carbon; Y is a hydrogen atom; Z is ethylene; R and R1 are each a hydrogen atom; R2 is thiophene; and R3 is nitrogen. Further optionally, the compound is 3-(thiophen-2-yl)-1-{4-[4-(trifluoromethyl)phenyl]piperazin-1-yl}propan-1- one.

Optionally, the compound has the general formula (I); X is a carbon; Y is a hydrogen atom; Z is heptylene; R and R1 are each a hydrogen atom; R2 is a hydrogen atom; and R3 is nitrogen. Further optionally, the compound is 1-{4-[4-(trifluoromethyl)phenyl]piperazin-1-yl}octan-1-one. Optionally, the compound has the general formula (I); X is a carbon; Y is a hydrogen atom; Z is ethylene; R and R1 are each a hydrogen atom; R2 is furan; and R3 is nitrogen. Further optionally, the compound is 3-(furan-2-yl)-1-{4-[4-(trifluoromethyl)phenyl]piperazin-1-yl}propan-1-one.

Optionally, the compound has the general formula (I); X is a carbon; Y is a hydrogen atom; Z is methylene; R and R1 are each a hydrogen atom; R2 is thiophene; and R3 is nitrogen. Further optionally, the compound is 2-(thiophen-2-yl)-1-{4-[4-(trifluoromethyl)phenyl]piperazin-1-yl}ethan-1- one.

Optionally, the compound has the general formula (I); X is a carbon; Y is a hydrogen atom; Z is pentylene; R and R1 are each a hydrogen atom; R2 is thiophene; and R3 is nitrogen. Further optionally, the compound is 6-(thiophen-2-yl)-1-{4-[4-(trifluoromethyl)phenyl]piperazin-1-yl}hexan-1- one.

Optionally, the compound has the general formula (I); X is a carbon; Y is a chloride, optionally attached to the carbon atom at position 2 of the benzene moiety; Z is propylene; R and R1 are each a hydrogen atom; R2 is thiophene; and R3 is nitrogen. Further optionally, the compound has the general formula (I); X is a carbon; Y is a chloride attached to the carbon atom at position 2 of the benzene moiety; Z is propylene; R and R1 are each a hydrogen atom; R2 is thiophene; and R3 is nitrogen. Still further optionally, the compound is 1-(4-(2-chloro-4-(trifluoromethyl)phenyl)piperazin-1- yl)-4-(thiophen-2-yl)butan-1-one.

Optionally, the compound has the general formula (I); X is a carbon; Y is hydrogen; Z is methylene; R and R1 are each a hydrogen atom; R2 is hydrogen; and R3 is nitrogen. Further optionally, the compound is 1-(4-(4-(trifluoromethyl)phenyl)piperazin-1-yl)ethanone.

Optionally, the compound has the general formula (I); X is a carbon atom; Y is a hydrogen atom; Z is a short-chain alkylene, alkenylene, or alkynylene, which is substituted by an oxygen atom; R2 is a thiophene; and R3 is carbon. Further optionally, the compound has the general formula (I); X is a carbon atom; Y is a hydrogen atom; Z is an alkylene, which is substituted by an oxygen atom; R2 is a thiophene; and R3 is carbon. Still further optionally, the compound has the general formula (I); X is a carbon atom; Y is a hydrogen atom; Z is propylene, which is substituted by an oxygen atom; R2 is a thiophene; and R3 is carbon. Still further optionally, the compound has the general formula (I); X is a carbon atom; Y is a hydrogen atom; Z is propylene, wherein one R is an oxygen atom; R1 is a hydrogen atom; R2 is a thiophene; and R3 is carbon. Still further optionally, the compound is 1- (thiophen-2-yl)-4-(4-(4-(trifluoromethyl)phenyl)piperidin-1-yl)butane-1 ,4-dione.

Optionally, the compound has the general formula (I); X is a nitrogen atom; Y is a chloride, optionally attached to the carbon atom at position 2 of the pyridinyl moiety; Z is propylene; R and R1 are each a hydrogen atom; R2 is a thiophene; and R3 is nitrogen. Further optionally, the compound has the general formula (I); X is a nitrogen atom; Y is a chloride attached to the carbon atom at position 2 of the pyridinyl moiety; Z is propylene; R and R1 are each a hydrogen atom; R2 is a thiophene; and R3 is nitrogen. Still further optionally, the compound is 1-(4-(6-chloro-5-(trifluoromethyl)pyridin-2- yl)piperazin-1-yl)-4-(thiophen-2-yl)butan-1-one.

Optionally, the compound has the general formula (I); X is a carbon atom; Y is hydrogen; Z is heptylene; R and R1 are each a hydrogen atom; R2 is a substituted carbonyl group; and R3 is nitrogen. Further optionally, the compound has the general formula (I); X is a carbon atom; Y is hydrogen; Z is heptylene; R and R1 are each a hydrogen atom; R2 is an ether substituted carbonyl group; and R3 is nitrogen. Still further optionally, the compound has the general formula (I); X is a carbon atom; Y is hydrogen; Z is heptylene; R and R1 are each a hydrogen atom; R2 is a methoxyl- substituted carbonyl group; and R3 is nitrogen. Optionally, the compound is methyl 9-oxo-9-(4-(4- (trifluoromethyl)phenyl)piperazin-1-yl)nonanoate.

Optionally, the compound has the general formula (I); X is a carbon atom; Y is a bromide, optionally attached to the carbon atom at position 2 of the benzene moiety; Z is propylene; R and R1 are each a hydrogen atom; R2 is a thiophene; and R3 is nitrogen. Further optionally, the compound has the general formula (I); X is a carbon atom; Y is a bromide attached to the carbon atom at position 2 of the benzene moiety; Z is propylene; R and R1 are each a hydrogen atom; R2 is a thiophene; and R3 is nitrogen. Still further optionally, the compound is 1-(4-(2-bromo-4-(trifluoromethyl)phenyl)piperazin- 1-yl)-4-(thiophen-2-yl)butan-1-one.

Optionally, the compound has the general formula (I); X is a carbon atom; Y is hydrogen; Z is butylene; R and R1 are each a hydrogen atom; R2 is a six-membered hydrocarbon moiety, optionally which can be substituted or unsubstituted; and R3 is nitrogen. Further optionally, the compound has the general formula (I); X is a carbon atom; Y is hydrogen; Z is butylene; R and R1 are each a hydrogen atom; R2 is an unsubstituted six-membered hydrocarbon moiety; and R3 is nitrogen. Still further optionally, the compound is 5-phenyl-1-(4-(4-(trifluoromethyl)phenyl)piperazin-1-yl)pentan-1- one.

Optionally, the compound has the general formula (I); X is a carbon atom; Y is a hydrogen atom; Z is a short-chain alkylene, alkenylene, or alkynylene, which is substituted by a hydroxyl group; R2 is a thiophene; and R3 is nitrogen. Further optionally, the compound has the general formula (I); X is a carbon atom; Y is a hydrogen atom; Z is an alkylene, which is substituted by a hydroxyl group; R2 is a thiophene; and R3 is nitrogen. Still further optionally, the compound has the general formula (I); X is a carbon atom; Y is a hydrogen atom; Z is propylene, which is substituted by a hydroxyl group; R2 is a thiophene; and R3 is nitrogen. Still further optionally, the compound has the general formula (I); X is a carbon atom; Y is a hydrogen atom; Z is propylene, wherein one R is a hydroxyl group; R1 is a hydrogen atom; R2 is a thiophene; and R3 is nitrogen. Still further optionally, the compound is 4- hydroxy-4-(thiophen-2-yl)-1-(4-(4-(trifluoromethyl)phenyl)piperazin-1-yl)butan-1-one.

Optionally, the compound has the general formula (I); X is a carbon atom; Y is hydrogen; Z is heptylene; R and R1 are each a hydrogen atom; R2 is a substituted carbonyl group; and R3 is carbon. Further optionally, the compound has the general formula (I); X is a carbon atom; Y is hydrogen; Z is heptylene; R and R1 are each a hydrogen atom; R2 is an alkoxyl substituted carbonyl group; and R3 is carbon. Still further optionally, the compound has the general formula (I); X is a carbon atom; Y is hydrogen; Z is heptylene; R and R1 are each a hydrogen atom; R2 is a methoxyl- substituted carbonyl group; and R3 is carbon. Still further optionally, the compound is Methyl 9-oxo- 9-(4-(4-(trifluoromethyl)phenyl)piperidin-1-yl)nonanoate.

Optionally, the compound has the general formula (I); X is a carbon atom; Y is hydrogen; Z is heptylene; R and R1 are each a hydrogen atom; R2 is a carboxyl group; and R3 is carbon. Further optionally, the compound has the general formula (I); X is a carbon atom; Y is hydrogen; Z is heptylene; R and R1 are each a hydrogen atom; R2 is an unsubstituted carboxyl group; and R3 is carbon. Still further optionally, the compound is 9-Oxo-9-(4-(4-(trifluoromethyl)phenyl)piperidin-1- yl)nonanoic acid. It is understood that, when Z is an alkylene, Z comprises a molecule comprising at least one carbon atom, wherein each atom of the molecule is bonded to an adjacent atom in a substantially straight series by a single bond, and which can be substituted or un-substituted. For example, the term "methylene" is intended to describe a molecule having one carbon atom having two hydrogen atoms attached thereto (-CH₂-), which can be substituted or unsubstituted. Optionally, the term "ethylene" is intended to describe a molecule having two carbon atoms, each carbon atom having two hydrogen atoms attached thereto (-C₂H₄-), which can be substituted or unsubstituted. Optionally, the term "propylene" is intended to describe a molecule having three carbon atoms, each carbon atom having two hydrogen atoms attached thereto (-C₃H₆-), which can be substituted or unsubstituted. Optionally, the term "butylene" is intended to describe a molecule having four carbon atoms, each carbon atom having two hydrogen atoms attached thereto (-C₄H₈-), which can be substituted or unsubstituted. Optionally, the term "pentylene" is intended to describe a molecule having five carbon atoms, each carbon atom having two hydrogen atoms attached thereto (-C₅H₁₀-), which can be substituted or unsubstituted. Optionally, the term "hexylene" is intended to describe a molecule having six carbon atoms, each carbon atom having two hydrogen atoms attached thereto (-C₆H₁₂-), which can be substituted or unsubstituted. Optionally, the term "heptylene" is intended to describe a molecule having seven carbon atoms, each carbon atom having two hydrogen atoms attached thereto (-C₇H₁₄- ), which can be substituted or unsubstituted.

Optionally, the pharmaceutically acceptable salt is selected from hydrochloride, hydrobromide, sulphate, methane-sulfonate, nitrate, maleate, acetate, citrate, fumarate, tartrate, succinate, benzoate, and salts with amino acids.

According to a second aspect of the present invention, there is provided a pharmaceutical composition comprising a compound according to the first aspect of the present invention; and, optionally a pharmaceutically acceptable excipient.

Optionally, the excipient is at least one non-ionic surfactant, or a mixture thereof, or a solution thereof. Further optionally, the non-ionic surfactant is selected from Polysorbate 80 (Tween 80), polyethoxylated castor oil, polyethylene glycol, polyethylene glycol-400, polyethylenglycol 660-12- hydroxystearate, and 2-pyrrolidone; or a solution each thereof. Preferably, the excipient is 2- pyrrolidone.

Alternatively, the excipient is dimethyl sulfoxide (DMSO), or a solution thereof. Optionally, the excipient is dimethyl sulfoxide (DMSO), or an aqueous solution thereof. Further optionally, the excipient is an aqueous solution of DMSO. Still further optionally, the excipient is a 0.2% aqueous solution of DMSO.

Optionally or additionally, the excipient is a cyclodextrin, optionally hydroxypropyl beta cyclodextrin (ΗΡβΟϋ).

Optionally, the excipient is 20: 1 solution of hydroxypropyl beta cyclodextrin and a 2% aqueous solution of DMSO.

According to a third aspect of the present invention, there is provided a compound according to the first aspect of the present invention, or a pharmaceutical composition according to the second aspect of the present invention, for use in treating a disorder caused by or associated with retinol-binding protein.

Optionally, the disorder is caused by or associated with retinol-binding protein function. Retinol- binding protein function may refer to one or more alteration in retinol-binding protein that result in a non-normal (disease-free) phenotype, and can include, but is not limited to, retinol-binding protein expression including translation, retinol-binding protein activity, retinol-binding protein signalling, presence, absence, or quantitative level of retinol-binding protein, and/or retinol-binding protein localisation.

Optionally, the disorder is caused by or associated with dysfunctional retinol-binding protein function. Optionally, the disorder is diabetes.

Optionally, the disorder is diabetes selected from typel or type2 diabetes mellitus. Optionally, the disorder is type-ll diabetes mellitus.

Optionally, the disorder is obesity.

Examples

Embodiments of the present invention will now be described, with reference to the accompanying, non-limiting examples and drawings, in which:

Figure 1A is a sensorgram showing interactions of His-sRBP with TTR in the presence of compounds of the present invention, reflecting the interaction of TTR with immobilized sRBP, in which responses were recorded as a function of time and are expressed in resonance units (RU); Figure 1 B is SDS-PAGE analysis, using silver staining, of the capacity of compounds to disrupt the interaction between sRBP and TTR, examined by a pull-down assay;

Figure 2 is a sensorgram showing interactions of His-sRBP with solubilized HEK293 cell membranes in the presence of compounds of the present invention, reflecting the interaction of HEK293 cell solubilized membrane preparations passed over immobilized sRBP, in which responses were recorded as a function of time and are expressed in resonance units (RU);

Figure 3 is a graph illustrating glucose (3A) and insulin (3B) tolerance tests of diabetic mice treated with 1-(4-(3-Chloro-5-(trifluoromethyl)pyridin-2-yl)piperazin-1-yl)-3-(thiophen-2-ylthio)propan-1-^ Figure 4 is a graph illustrating the animal weights across the animal trial of high fat diet induced obese mice treated with (A) 1-(4-(3-Chloro-5-(trifluoromethyl)pyridin-2-yl)piperazin-1-yl)-3-(thiophen- 2-ylthio)propan-1-one and (B) 4-(thiophen-2-yl)-1-{4-[4-(trifluoromethyl)phenyl]piperazin-1-yl}butan-1- one

Figure 5 is a graph illustrating glucose (5A) and insulin (5B) tolerance tests of high fat diet induced obese mice treated with 4-(thiophen-2-yl)-1-{4-[4-(trifluoromethyl)phenyl]piperazin-1-yl}butan-1-one Figure 6 is a graph illustrating glucose (6A) and insulin (6B) tolerance tests of high fat diet induced obese mice treated with 4-(thiophen-2-yl)-1-{4-[4-(trifluoromethyl)phenyl]piperazin-1-yl}butan-1-one Figure 7A is a graph depicting glucose uptake in C2C12 muscle cells pre-incubated with compounds of the present invention (10μΜ);

Figure 7B is a graph illustrating glucose uptake of C2C12 cells treated with 10μΜ of 4-(thiophen-2- yl)-1-{4-[4-(trifluoromethyl)phenyl]piperazin-1-yl}butan-1-one; Cytochalasin B (10μΜ), an inhibitor of glucose uptake, inhibits this increase.

Figure 7C is a graph illustrating the glucose uptake response of various concentrations of 4- (thiophen-2-yl)-1-{4-[4-(trifluoromethyl)phenyl]piperazin-1-yl}butan-1-one;

Figure 7D is a graph illustrating glucose uptake in C2C12 muscle cells pre-incubated with compounds of the present invention (10μΜ); Figure 7E is a graph illustrating that the 4-(thiophen-2-yl)-1-{4-[4-(trifluoromethyl)phenyl]piperazin-1- yl}butan-1 -one-induced uptake of ³H deoxy-2-glucose is inhibited by addition of 5mM 'cold' glucose; Figure 7F is a graph illustrating that 4-(thiophen-2-yl)-1-{4-[4-(trifluoromethyl)phenyl]piperazin-1- yl}butan-1-one does not induce glucose uptake in erthrocytes;

Figures 8-20 are each a graph illustrating glucose uptake in C2C12 muscle cells pre-incubated with compounds of the present invention (10μΜ); and

Figure 21 is a graph illustrating glucose (21 A) and insulin (21 B) tolerance tests performed after 1 and 2 weeks of 4-(thiophen-2-yl)-1-{4-[4-(trifluoromethyl)phenyl]piperazin-1-yl}butan-1-one administration.

Example 1

It was confirmed that the compounds of the present invention had the capacity to disrupt the interaction between serum Retinol binding protein 4 (sRBP), and transthyretin homotetramer (TTR) or membranes containing the stimulated by retinoic acid gene 6 (STRA6) receptor as examined through a surface plasmon resonance (SPR) assay before proceeding to animal studies on the compound 4-(thiophen-2-yl)-1-{4-[4-(trifluoromethyl)phenyl]piperazin-1-yl}butan-1-one.

The ability of the compound 4-(thiophen-2-yl)-1-{4-[4-(trifluoromethyl)phenyl]piperazin-1-yl}butan-1- one to disrupt the interaction between sRBP and TTR in real time was assessed using a surface plasmon resonance (SPR) assay. Retinol (ROH) strengthens the interaction between sRBP and TTR. As shown in Figure 1A, 4-(thiophen-2-yl)-1-{4-[4-(trifluoromethyl)phenyl]piperazin-1-yl}butan-1- one did not strengthen the interaction with TTR, and was more disruptive than fenretinide (FEN) itself.

The ability of the compound 4-(thiophen-2-yl)-1-{4-[4-(trifluoromethyl)phenyl]piperazin-1-yl}butan-1- one to disrupt the interaction between sRBP and TTR was also assessed using a pull down assay. Retinol (ROH) strengthens the interaction between sRBP and TTR and therefore less TTR is observed in the flow-through. As shown in Figure 1 B, 4-(thiophen-2-yl)-1-{4-[4- (trifluoromethyl)phenyl]piperazin-1-yl}butan-1-one weakened the interaction with TTR, and was similar to fenretinide (FEN) itself. 1-{4-[3-chloro-5-(trifluoromethyl)pyridin-2-yl]piperazin-1-yl}-3- (thiophen-2-ylsulfanyl)propan-1-one also weakened the interaction, whereas N-(2-methoxyethyl)-9- oxo-9-{4-[4-(trifluoromethyl)phenyl]piperazin-1-yl}nonanamide was similar to ROH. As shown in Figure 2, the ability of the compound 4-(thiophen-2-yl)-1-{4-[4- (trifluoromethyl)phenyl]piperazin-1-yl}butan-1-one to disrupt the interaction between sRBP and solubilised HEK293 cell membranes in real time was assessed using a surface plasmon resonance (SPR) assay. 4-(thiophen-2-yl)-1-{4-[4-(trifluoromethyl)phenyl]piperazin-1-yl}butan-1-one was more disruptive than fenretinide (FEN) and retinol (ROH). Example 2

In a prevention study, wildtype C57BI/6 background male mice (purchased from Charles River Laboratories, UK) were maintained in standard laboratory conditions. Mice were housed (4 mice per cage) in a conventional animal facility with a 12: 12-h light: dark cycle, with ad libitum access to water and an obesogenic diet (HFD - Diet F3282; 5450 kcal/kg; Bio-serv; Frenchtown, NJ - see http://www.bioserv.com/newcatalog/eeprod/rodent/highfat.html). Each group comprised 8 mice, with the high fat diet for Group A containing a compound according to a first aspect of the present invention at 0.04% w/w relative to the amount of diet. Usually, 1 kg of batch of HFD was prepared by thoroughly mixing 0.4g of compound (dissolved in 10ml of DMSO). Group B received high fat diet with DMSO only (the solvent), for example, 10 ml of DMSO thoroughly mixed with 1 kg HFD. All animals were acclimatised to high fat diet for one week before compounds and controls were introduced. Glucose (GTT) and insulin tolerance testing (ITT) was performed after 8 weeks of high- fat diet feeding. Following a period of fasting (12 hours and 4 hours for GTT and ITT, respectively), repeated whole blood sampling was performed on conscious mice at 30-minute intervals after an initial intra-peritoneal injection of either glucose or insulin (for GTTs or ITTs, respectively) (Figures 3A and 3B).

Animals treated with 1-(4-(3-Chloro-5-(trifluoromethyl)pyridin-2-yl)piperazin-1-yl)-3-(thiophen-2- ylthio)propan-1-one were able to clear glucose more rapidly than the HFD-fed group (control) and were also more sensitive to exogenous insulin, suggesting that mice fed with a compound of the present invention included in their HFD have improved glucose homeostasis compared to the control (HFD only) littermates. Compared to controls fed the diet +/- the solvent (DMSO), no evidence of an adverse reaction and no deaths have been obtained with any compound tested up to a 16-week period. There was no discernible difference in the characteristic "oily" appearance of the mice or in their activity or behaviour, nor was there any evidence of diarrhoea. The glucose tolerance tests show that the animals treated with compounds of the present invention exhibited significantly improved glucose handling compared to controls (Figure 3A). The insulin tolerance test showed increased sensitivity to insulin (Figure 3B), so much so in one case that animals rapidly became hypoglycaemic, requiring a bolus of glucose for recovery. Type-2 diabetes is known to be associated with weight, with over 90% of newly diagnosed patients suffering from type-2 diabetes above their ideal weight. Weight gain is a common problem with insulin therapy - this is often distressing and potentially harmful. The animals were weighed weekly and then before each test due to the number of hours fasting (either o/n for GTTs or 4h for ITTs). Food preference trials indicated that there was no adverse taste reaction or reduced food intake. As can be seen from Figures 4A and 4B, from week 2, there is significant difference in body weight between those kept on HFD and those that also received 1-(4-(3-Chloro-5-(trifluoromethyl)pyridin-2- yl)piperazin-1-yl)-3-(thiophen-2-ylthio)propan-1-one.

Both groups fed on HFD, with or without compound, gained more weight during the 16 weeks of feeding than the CHOW fed group, From week 29, there are significantly lower body weights between those kept on HFD and those that also received 4-(thiophen-2-yl)-1-{4-[4- (trifluoromethyl)phenyl]piperazin-1-yl}butan-1-one.

Regarding the causative link between obesity and insulin resistance, and without being bound by theory, a viable extant hypothesis concerns serum retinol-binding protein 4 (sRBP), elevated serum levels of which are thought to arise from the excess adipose tissue in obesity. This elevated sRBP in turn may be acting via one or both of two proposed mechanisms. The first acting through its plasma membrane receptor, appears to stimulate an intracellular pathway, which results in the attenuation of the insulin response by inhibiting the activation of components in the signalling pathway. This inhibition prevents the recruitment of glucose transporters to the plasma membrane and hence the insulin-stimulated uptake of glucose from serum does not occur. In the second proposed mechanism, sRBP activates macrophage cells to produce proinflammatory cytokines such as TNFa and IL-6. These, in turn, act to inhibit the insulin-signalling pathway via serine phosphorylation of IRS-1.

It is important to distinguish this mechanism from that which occurs in type 1 diabetes. In this condition, there is no sRBP effect. The defect lies either in the levels of insulin or in failure of any of the protein components of the signalling pathway. Thus, any drug which impedes the behaviour of sRBP would have a very limited sensitizing effect, if any. It would not be obvious, therefore, to believe an sRBP-directed therapy would be beneficial in the case of typel . Even more remote is any expectation of an effect on glucose uptake since here one would be expecting the compound to be a substitute for insulin, stimulating the normal pathways. In the event, the primary target appears to be quite different. Example 3

4-(Thiophen-2-yl)-1-{4-[4-(trifluoromethyl)phenyl]piperazin-1-yl}butan-1-one was tested in mice using a similar methodology to that described in Example 2, with similar positive effects against insulin resistance (Figures 5A and 5B). Wildtype mice (C57BI/6 background) were maintained in standard laboratory conditions as described in Example 1 , with ad libitum access to water and either chow (BK001 (E) rodent breeder and grower) comprising wheat, dehulled extracted toasted soya, barley, wheatfeed, macro minerals, vitamins, trace elements and anti-oxidents and soya oil; or an obesogenic HFD diet (HFD- high-fat diet: Diet F3282, 5450 kcal/kg; Bio-serv, Frenchtown NJ; for details of diet composition, see http://www.bioserv.com/ newcatalog/eeprod/rodent/highfat.html.]). Each group fed on HFD comprised 12 mice, with the high fat diet for Group A containing a compound of the present invention at 0.04% w/w relative to the diet and Group B received HFD with DMSO only (the solvent). A third group of 8 mice (control) received CHOW diet. All animals were acclimatised to the diet for one week before the compound was introduced. Glucose and insulin tolerance testing was performed after 16 weeks of high-fat diet feeding as described in Example 2. Following a period of fasting (12 hours and 4 hours for GTT and ITT respectively), repeated whole blood sampling was performed on conscious mice at 30-minute intervals after an initial intraperitoneal injection of either glucose or insulin (for GTTs or ITTs, respectively) (Figures 5A and 5B).

After 16 weeks of feeding, both groups fed on HFD with or without compound were less glucose tolerant than the CHOW fed group, but animals treated with 4-(thiophen-2-yl)-1-{4-[4- (trifluoromethyl)phenyl]piperazin-1-yl}butan-1-one were able to clear glucose more rapidly than the HFD-only. Both groups fed on HFD, with or without administration of compound, gained more weight during the 16 weeks of feeding than the CHOW fed group, but the compound-treated animals were more sensitive to exogenous insulin than the HFD-only.

Example 4

An intervention study was conducted, in which 4-(thiophen-2-yl)-1-{4-[4- (trifluoromethyl)phenyl]piperazin-1-yl}butan-1-one (0.04% w/w) was administered not at the start of the HFD treatment but at 17 weeks when insulin resistance had become established. The results indicate that 4-(thiophen-2-yl)-1-{4-[4-(trifluoromethyl)phenyl]piperazin-1-yl}butan-1-one restored glucose and insulin sensitivity to the normal levels seen with CHOW fed animals (Figures 6A&B). 4- (thiophen-2-yl)-1-{4-[4-(trifluoromethyl)phenyl]piperazin-1-yl}butan-1-one has a statistically significant anti-diabetic effect in high fat fed mice relative to controls. All animals remained alive, alert, active and free from any discomfort or diarrhoea.

Example 5

Those compounds of the present invention that pass Lipinski's guidelines (< 5 hydrogen bond donors; < 10 hydrogen bond acceptors; molecular weight < 500; logP < 5) have potential use as bioavailable and orally administered drugs. The exceptions are 1-(4-(4- (trifluoromethyl)phenyl)piperazin-1-yl)octan-1-one with a predicted logP of 5.099, which falls just above the cut-off, methyl 9-oxo-9-(4-(4-(trifluoromethyl)phenyl)piperidin-1-yl)nonanoate with a predicted logP of 5.075, and 5-phenyl-1-(4-(4-(trifluoromethyl)phenyl)piperazin-1-yl)pentan-1-one at 5.219. The early consideration of Absorption, Distribution, Metabolism, and Excretion (ADME) was used to assist selection of the compounds of the present invention. ADME Descriptors (Discovery Studio, Accelrys, advanced computer modeling software) was used to examine and eliminate compounds with unfavourable ADME characteristics early in the discovery process and evaluate proposed structural refinements prior to synthesis. For example, one ADME computer model employed, predicts human intestinal absorption (HIA) after oral administration. This computer model was developed at Accelrys using 182 compounds in the training set with descriptors including AlogP98 and 2D polar surface area (PSA_2D). Well-absorbed compounds have at least 90% absorption into the human bloodstream. Compounds of the present invention are illustrated in Tables 1 and 2, and are predicted to have good absorption potential and good solubility. According to Discovery Studios ADME descriptors, some of the compounds of the present invention may pass the blood brain barrier (BBB), with 1-(thiophen-2-yl)-4-{4-[5-(trifluoromethyl)pyridin-2- yl]piperazin-1-yl}butane-1 ,4-dione and the acid half of 4-(thiophen-2-yl)-1-{4-[4- (trifluoromethyl)phenyl]piperazin-1-yl}butan-1-one being less likely to pass. According to the Discovery Studios ADME descriptors, the compounds range from low to optimal solubility.

Table 1 : ADME Predictions

Compound BBB Absorption Solubility AlogP

RTB-70 1 0 2 4.346

RTC-1 0 0 2 4.067

RTC-1 acid half 2 0 4 1.71 1

RTC-1 amine half 1 0 3 2.355

RTC-2 1 0 2 3.497

RTC-3 2 0 3 2.885

RTC-4 1 0 3 3.549

RTC-5 1 0 2 3.455

RTC-6 1 0 2 4.12

RTC-8 0 0 2 4.731

RTC-7 0 0 2 4.293

RTC-12 1 0 3 2.151

RTC-15 1 0 2 4.277

RTC-16 0 0 2 5.219

RTC-18 0 0 3 3.725

RTC-29 1 0 2 3.496

RTC-39 1 0 2 4.068

RTC-44 1 0 2 4.329

RTC-56 1 0 2 4.503

RTC-62 0 0 2 4.815

RTC-78 1 0 2 5.075

RTC-79 1 0 2 4.849

RTC-193 1 0 2 3.206 RTC-194 1 0 2 3.73

RTC-195 0 0 2 4.523

RTC-196 0 0 2 4.639

RTC-532 1 0 2 3.61 1

RTC-533 0 0 2 5.099

RTC-535 1 0 2 3.28

RTC-536 1 0 2 3.576

RTC-537 0 0 2 4.979

For BBB 0 and 1 are very high and high respectively, while 2 is medium; Absorption - level 0 indicates good absorption; Solubility - 3 indicates good and 2 indicates low solubility. Most of the compounds of the present invention are not expected to be hepatotoxic (liver toxicity).

The compounds are not predicted to inhibit Cytochrome P450 2D6 (CYP2D6), a member of the cytochrome P450 mixed-function oxidase system with the exception of the 4-(thiophen-2-yl)-1-{4-[4- (trifluoromethyl)phenyl]piperazin-1-yl}butan-1-one - amine half. Moreover, the compounds of the present invention are predicted to have a higher unbound portion, with 90% or greater binding to plasma proteins, indicating high drug efficiency owing to the more efficiently the compound can traverse cell membranes or diffuse.

Table 2: ADME Predictions

Compound Hepatoxicity CYP2D6 PPB

RTB-70 F F T

RTC-1 F F T

RTC-1 acid half T F T

RTC-1 amine half T T T

RTC-2 T F T

RTC-3 F F T

RTC-4 F F T

RTC-5 F F T

RTC-6 F F T

RTC-7 F F T

RTC-8 F F T

RTC-12 F F T

RTC-15 F F T

RTC-16 F F T

RTC-18 F F T

RTC-29 T F T

RTC-39 T F T RTC-44 F F T

RTC-56 F F T

RTC-62 F F T

RTC-78 F F T

RTC-79 F F T

RTC-193 F F T

RTC-194 F F T

RTC-195 F F T

RTC-196 T F T

RTC-532 T F T

RTC-533 F F T

RTC-535 F F T

RTC-536 T F T

RTC-537 F F T

Hepatoxicity - F for False indicates non-toxic. CYP2D6 - F for False indicates a non-inhibitor. PPB T for true indicates > 90% binding. Example 6

A hallmark of type-2 diabetes is decreased sensitivity of muscle and adipose cells to insulin. Upon insulin treatment, the insulin receptor is phosphorylated, which activates a signal transduction pathway leading to increased glucose uptake by glucose transporter 4 (GLUT4) in fat or muscle. Therefore, measuring glucose uptake provides a relevant end point assay. Compounds that increase glucose uptake may be useful in the treatment of diabetes and/or associated complications. Therefore, glucose uptake was selected as the most relevant end point assay.

Figure 7 illustrates the results obtained with C2C12 muscle cells when monitoring the uptake of glucose. The tested compound, 4-(thiophen-2-yl)-1-{4-[4-(trifluoromethyl)phenyl]piperazin-1-yl}butan- 1-one (10μΜ), increased glucose uptake to a level comparable with insulin. It is important to note that there is no sRBP present. The effect is seen following overnight incubation; and is not seen with 1-{4-[3-chloro-5-(trifluoromethyl)pyridin-2-yl]piperazin-1-yl}-3-(thiophen-2-ylsulfanyl)propan-1-one (RTB-70) or another of its derivatives, RTB-95 (2-[(4-chlorophenyl)thio]-1-{4-[3- (trifluoromethyl)pyridin-2-yl]piperazino}ethan-1-one), with fenretinide (an sRBP ligand which also protects against the development of insulin resistance) or with A1 120 (a ligand for sRBP which does NOT prevent insulin resistance or type-2 diabetes (Motani, A.; Wang, Z.; Conn, M.; Siegler, K.; Zhang, Y.; Liu, Q.; Johnstone, S.; Xu, H.; Thibault, S.; Wang, Y.; Fan, P.; Connors, R.; Le, H.; Xu, G.; Walker, N.; Shan, B.; Coward, P. Identification and characterization of a non-retinoid ligand for retinol-binding protein 4, which lowers serum retinol-binding protein 4 levels in vivo. J Biol Chem 2009. 284, 7673-7680.) Referring to Figure 7B, C2C12 cells were treated overnight with 10μΜ 4-(thiophen-2-yl)-1-{4-[4-5 (trifluoromethyl)phenyl]piperazin-1-yl}butan-1-one, Cytochalasin-B or both and glucose uptake monitored. 4-(thiophen-2-yl)-1-{4-[4-(trifluoromethyl)phenyl]piperazin-1-yl}butan-1-one induced a significant increase in glucose uptake, which was inhibited by Cytochalasin-B co-treatment.

Referring to Figure 7C, various concentrations of 4-(thiophen-2-yl)-1-{4-[4- (trifluoromethyl)phenyl]piperazin-1-yl}butan-1-one were tested in the glucose uptake assay, and the resultant EC₅₀ was shown to be 12.99μΜ. Figure 7D illustrates glucose uptake in C2C12 muscle cells pre-incubated with compounds of the present invention (10μΜ).

4-(Thiophen-2-yl)-1-{4-[4-(trifluoromethyl)phenyl]piperazin-1-yl}butan-1-one has accordingly been examined in a cellular assay using C2C12 muscle cell cultures in the absence of sRBP (Figures 7A- 7D). These data demonstrate that glucose uptake is stimulated. The effect of 4-(thiophen-2-yl)-1-{4- [4-(trifluoromethyl)phenyl]piperazin-1-yl}butan-1-one is dose-dependent. In an embodiment, the effect of the present compounds, such as 4-(thiophen-2-yl)-1-{4-[4-(trifluoromethyl)phenyl]piperazin-

1- yl}butan-1-one, is shown to occur via the glucose transporter (given that the effect is inhibited by cytochalasin B (a specific inhibitor; Figure 7B) and, competitively, by unlabelled glucose; Figure 7E). It has also been shown that the effect of, for example, 4-(thiophen-2-yl)-1-{4-[4- (trifluoromethyl)phenyl]piperazin-1-yl}butan-1-one does not involve direct binding to the transporter (given there is no effect in erythrocytes; Figure 7F).

These observations demonstrate that the compounds of the present invention provide an insulin independent therapeutic approach for use in the treatment of either type-1 or type-2 diabetes, potentially circumventing or markedly reducing the necessity for injectable agents for treatment.

Example 7 Following the methods of Example 6, the mouse muscle cell line (C2C12) was used to test the effect of the compounds of the present invention on glucose uptake by measuring the amount of ³H deoxy-

2- glucose taken up by the cell (see Example 6). As seen in Figure 8, for 4-(thiophen-2-yl)-1-{4-[4- (trifluoromethyl)phenyl]piperazin-1-yl}butan-1-one, an increase in glucose uptake is observed; but not for the amine and acid halves (designated RTC-1 Amine and RTC-1 Acid, respectively) from which 4-(thiophen-2-yl)-1-{4-[4-(trifluoromethyl)phenyl]piperazin-1-yl}butan-1-one is derived.

As seen in Figure 9, for 4-(thiophen-2-yl)-1-{4-[4-(trifluoromethyl)phenyl]piperazin-1-yl}butan-1-one, an increase in glucose uptake is observed; but not for the amine half (designated RTC-1 Amine) from which 4-(thiophen-2-yl)-1-{4-[4-(trifluoromethyl)phenyl]piperazin-1-yl}butan-1-one is derived; and not for 4-(thiophen-2-yl)-1-{4-[5-(trifluoromethyl)pyridin-2-yl]piperazin-1-yl}butan-1-one; and not for 1-{4-[3-chloro-5-(trifluoromethyl)pyridin-2-yl]piperazin-1-yl}-4-(thiophen-2-yl)butan-1-one. As seen in Figure 10, for 4-(thiophen-2-yl)-1-{4-[4-(trifluoromethyl)phenyl]piperazin-1-yl}butan-1-one, an increase in glucose uptake is observed; also for 1-(thiophen-2-yl)-4-{4-[4- (trifluoromethyl)phenyl]piperidin-1-yl}butane-1 ,4-dione, an increase in glucose uptake is observed; also for 4-(thiophen-2-yl)-1-{4 4-(trifluoromethyl)phenyl]piperidin-1-yl}butan-1-one, increase in glucose uptake is observed.

As seen in Figure 1 1 , for 4-(thiophen-2-yl)-1-{4-[4-(trifluoromethyl)phenyl]piperazin-1-yl}butan-1-one, an increase in glucose uptake is observed; but not for 1-{4-[6-chloro-5-(trifluoromethyl)pyridin-2- yl]piperazin-1-yl}-4-(thiophen-2-yl)butan-1-one.

As seen in Figure 12, for 4-(thiophen-2-yl)-1-{4-[4-(trifluoromethyl)phenyl]piperazin-1-yl}butan-1-one, an increase in glucose uptake is observed; but not for the acid half (designated RTC-1 Acid) from which 4-(thiophen-2-yl)-1-{4-[4-(trifluoromethyl)phenyl]piperazin-1-yl}butan-1-one is derived; but also for 1-{4-[4-(trifluoromethyl)phenyl]piperazin-1-yl}ethan-1-one, an increase in glucose uptake is observed.

As seen in Figure 13, for 4-(thiophen-2-yl)-1-{4-[4-(trifluoromethyl)phenyl]piperazin-1-yl}butan-1-one, an increase in glucose uptake is observed; but not for 1-(thiophen-2-yl)-4-{4-[4- (trifluoromethyl)phenyl]piperazin-1-yl}butane-1 ,4-dione; but also for 3-(Thiophen-2-ylthio)-1-(4-(4- (trifluoromethyl)phenyl)piperazin-1-yl)propan-1-one, an increase in glucose uptake is observed; and also for 4-hydroxy-4-(thiophen-2-yl)-1-{4 4-(trifluoromethyl)phenyl]piperazin-1-yl}butan-1-one, an increase in glucose uptake is observed. As seen in Figure 14, for 4-(thiophen-2-yl)-1-{4-[4-(trifluoromethyl)phenyl]piperazin-1-yl}butan-1-one, an increase in glucose uptake is observed; and also for 5-phenyl-1-{4-[4- (trifluoromethyl)phenyl]piperazin-1-yl}pentan-1-one, an increase in glucose uptake is observed; and also for 4-(1 H-Pyrazol-4-yl)-1-(4-(4-(trifluoromethyl)phenyl)piperazin-1-yl)butan-1-one, an increase in glucose uptake is observed; and also for 1-{4-[4-(trifluoromethyl)phenyl]piperazin-1-yl}pentan-1-one, an increase in glucose uptake is observed, and also for 1-{4-[4-(trifluoromethyl)phenyl]piperazin-1- yl}octan-1-one, an increase in glucose uptake is observed, but not for 3-(furan-2-yl)-1-{4-[4- (trifluoromethyl)phenyl]piperazin-1-yl}propan-1-one.

As seen in Figure 15, for 4-(thiophen-2-yl)-1-{4-[4-(trifluoromethyl)phenyl]piperazin-1-yl}butan-1-one, an increase in glucose uptake is observed; and also for 5-(thiophen-2-yl)-1-{4-[4- (trifluoromethyl)phenyl]piperazin-1-yl}pentan-1-one, an increase in glucose uptake is observed; but not for 3-(thiophen-2-yl)-1-{4-[4-(trifluoromethyl)phenyl]piperazin-1-yl}propan-1-one; but also for 2- (thiophen-2-yl)-1-{4-[4-(trifluoromethyl)phenyl]piperazin-1-yl}ethan-1-one, an increase in glucose uptake is observed, and also for 6-(thiophen-2-yl)-1-{4-[4-(trifluoromethyl)phenyl]piperazin-1- yl}hexan-1-one, an increase in glucose uptake is observed. As seen in Figure 16, for 4-(thiophen-2-yl)-1-{4-[4-(trifluoromethyl)phenyl]piperazin-1-yl}butan-1-one, an increase in glucose uptake is observed; and also for 9-oxo-9-{4-[4- (trifluoromethyl)phenyl]piperazin-1-yl}nonanoic acid, an increase in glucose uptake is observed; and also for N-(2-methoxyethyl)-9-oxo-9-{4-[4-(trifluoromethyl)phenyl]piperazin-1-yl}nonanamide, an increase in glucose uptake is observed.

As seen in Figure 17, for 9-oxo-9-{4-[4-(trifluoromethyl)phenyl]piperazin-1-yl}nonanoic acid, an increase in glucose uptake is observed; and also for methyl 9-oxo-9-{4-[4- (trifluoromethyl)phenyl]piperazin-1-yl}nonanoate, an increase in glucose uptake is observed.

As seen in Figure 18, for 4-(thiophen-2-yl)-1-{4-[4-(trifluoromethyl)phenyl]piperazin-1-yl}butan-1-one, an increase in glucose uptake is observed; but not for 1-{4-[2-chloro-4- (trifluoromethyl)phenyl]piperazin-1-yl}-4-(thiophen-2-yl)butan-1-one; and also not for 1-{4-[2-bromo-4- (trifluoromethyl)phenyl]piperazin-1-yl}-4-(thiophen-2-yl)butan-1-one and also not for 1-(4-(3-Chloro-5- (trifluoromethyl)pyridin-2-yl)piperazin-1-yl)-3-(thiophen-2-ylthio)propan-1-one..

As seen in Figure 19, for 4-(thiophen-2-yl)-1-{4-[4-(trifluoromethyl)phenyl]piperazin-1-yl}butan-1-one, an increase in glucose uptake is observed; but not for 1-(Thiophen-2-yl)-4-{4-[5- (trifluoromethyl)pyridin-2-yl]piperazin-1-yl}butane-1 ,4-dione; and also not for 1-{4-[3-Chloro-5- (trifluoromethyl)pyridin-2-yl]piperazin-1-yl}-4-(thiophen-2-yl)butane-1 ,4-dione.

As seen in Figure 20, for 4-(thiophen-2-yl)-1-{4-[4-(trifluoromethyl)phenyl]piperazin-1-yl}butan-1-one, an increase in glucose uptake is observed; but not for methyl 9-oxo-9-(4-(4- (trifluoromethyl)phenyl)piperidin-1-yl)nonanoate; but for 9-oxo-9-(4-(4- (trifluoromethyl)phenyl)piperidin-1-yl)nonanoic acid, an increase in glucose uptake is observed.

Example 8

A group of 4 mice were fed on a CHOW diet alone; and a group of 4 mice were fed on a CHOW diet containing a compound according to a first aspect of the present invention at 0.04% w/w relative to the amount of diet. Daily oral gavage at 1 mg/mouse/day in 25ul DMSO was performed for 2 weeks. GTT was performed after 1 week and ITT after 2 weeks as impaired glucose tolerance is often associated with insulin resistance. The GTTs were performed after 1 week of oral gavaging CHOW fed mice with 4-(thiophen-2-yl)-1-{4-[4-(trifluoromethyl)phenyl]piperazin-1-yl}butan-1-one. As seen in Figure 21 , values at point 0 and at 120 min are significantly different (<0.05) from the control indicating improved glucose tolerance. For the ITTs, no significant differences were seen at any of the points measured. There was no evidence of hypersensitivity and no observed toxic effects.

Figure 21 illustrates Glucose (21 A) and insulin (21 B) tolerance tests performed after 1 and 2 weeks of 4-(thiophen-2-yl)-1-{4-[4-(trifluoromethyl)phenyl]piperazin-1-yl}butan-1-one administration, respectively (n=3 to 4). The number of "mice fit for experiment on the day" at 1 week was: CHOW, n=3; CHOW+4-(thiophen-2-yl)-1-{4-[4-(trifluoromethyl)phenyl]piperazin^ n=4. The number of "mice fit for experiment on the day" at 2 weeks was: CHOW, n=3;CHOW+4-(thiophen-2- yl)-1-{4-[4-(trifluoromethyl)phenyl]piperazin-1-yl}butan-1-one, n=3. Values are the mean± STDEV. Significant differences between the two groups were observed for the GTT after 1 week and are indicated as * (TTEST performed and P<0.05 at 0 min and 120 min).

Table 3 - Nomenclature

4-(Thiophen-2-yl)-1-{4-[4-(trifluoromethyl)phenyl]piperazin-1- yl}butan-1-one

1-[4-(Trifluoromethyl)phenyl]piperazine

4-(Thiophen-2-yl)butanoic acid

1-(Thiophen-2-yl)-4-{4-[4-(trifluoromethyl)phenyl]piperazin-1- yl}butane-1 ,4-dione

1-(Thiophen-2-yl)-4-{4-[5-(trifluoromethyl)pyridin-2-yl]piperazin-1- yl}butane-1 ,4-dione

1-{4-[3-Chloro-5-(trifluoromethyl)pyridin-2-yl]piperazin-1-yl}-4- (thiophen-2-yl)butane-1 ,4-dione

4- (Thiophen-2-yl)-1-{4-[5-(trifluoromethyl)pyridin-2-yl]piperazin-1- yl}butan-1-one

1-{4-[3-Chloro-5-(trifluoromethyl)pyridin-2-yl]piperazin-1-yl}-4- (thiophen-2-yl)butan-1-one

3- (Thiophen-2-ylthio)-1-(4-(4-(trifluoromethyl)phenyl)piperazin-1- yl)propan-1-one

1- (4-(2-Chloro-4-(trifluoromethyl)phenyl)piperazin-1-yl)-4-(thiophen

2- yl)butan-1-one

1-(4-(4-(Trifluoromethyl)phenyl)piperazin-1-yl)ethanone

9-Oxo-9-{4-[4-(trifluoromethyl)phenyl]piperazin-1-yl}nonanoic acid

5- Phenyl-1-(4-(4-(trifluoromethyl)phenyl)piperazin-1-yl)pentan-1- one

N-(2-Methoxyethyl)-9-oxo-9-{4-[4-(trifluoromethyl)phenyl]piperazin- 1-yl}nonanamide

4- Hydroxy-4-(thiophen-2-yl)-1-(4-(4- (trifluoromethyl)phenyl)piperazin-1-yl)butan-1-one

1-(Thiophen-2-yl)-4-(4-(4-(trifluoromethyl)phenyl)piperidin-1- yl)butane-1 ,4-dione

1-(4-(6-Chloro-5-(trifluoromethyl)pyridin-2-yl)piperazin-1-yl)-4- (thiophen-2-yl)butan-1-one

9-Oxo-9-(4-(4-(trifluoromethyl)phenyl)piperazin-1-yl)nonanoate

1- (4-(2-Bromo-4-(trifluoromethyl)phenyl)piperazin-1-yl)-4-(thiophen

2- yl)butan-1-one

Methyl 9-oxo-9-(4-(4-(trifluoromethyl)phenyl)piperidin-1- yl)nonanoate

9-Oxo-9-(4-(4-(trifluoromethyl)phenyl)piperidin-1-yl)nonanoic acid

4- (1 H-Pyrazol-4-yl)-1-{4 4-(trifluoromethyl)phenyl]piperazin-1- yl}butan-1-one

1-{4-[4-(Trifluoromethyl)phenyl]piperazin-1-yl}pentan-1-one

5- (Thiophen-2-yl)-1-{4-[4-(trifluoromethyl)phenyl]piperazin-1- yl}pentan-1-one

4-(Thiophen-2-yl)-1-{4-[4-(trifluoromethyl)phenyl]piperidin-1- yl}butan-1-one

3-(Thiophen-2-yl)-1-{4-[4-(trifluoromethyl)phenyl]piperazin-1- yl}propan-1-one

1-{4-[4-(Trifluoromethyl)phenyl]piperazin-1-yl}octan-1-one

3-(Furan-2-yl)-1-{4-[4-(trifluoromethyl)phenyl]piperazin-1-yl}propan-

1- one

2- (Thiophen-2-yl)-1-{4-[4-(trifluoromethyl)phenyl]piperazin-1- yl}ethan-1-one

6- (Thiophen-2-yl)-1-{4-[4-(trifluoromethyl)phenyl]piperazin-1- yl}hexan-1-one

1-(4-(3-Chloro-5-(trifluoromethyl)pyridin-2-yl)piperazin-1-yl)-3- (thiophen-2-ylthio)propan-1-one

Materials and Methods

Materials

BL21 (DE3) Escherichia coli strain, Pichia pastoris strain KM71 H (aox1::ARG4, arg4), expression vector pPICZ-a A, Zero Blunt TOPO PCR cloning kit and europium-labeled anti-His-tag antibodies were purchased from Invitrogen. HEK293 cells (human embryonic kidney) were from the ATCC. SureLight streptavid in-conjugated allophycocyanin (APC) was from PerkinElmer. EZ-Link Sulfo- NHS-LC-Biotinylation kit was from Pierce. ROH, FEN, sRBP, native human TTR and synthetic oligonucleotide primers were from Sigma. PCR amplification of coding sequences was done with PfuTurbo Hotstart DNA polymerase from Stratagene. Ni-NTA superflow resin and cobalt affinity gel were purchased from Qiagen and Sigma, respectively. a-RBP and cr-rabbit IgG-HRP were purchased from Dako and GE Healthcare, respectively. Plasmid isolation and gel extraction kits were from Qiagen. Biacore materials were obtained from GE Healthcare. Other reagents used were molecular biology grade.

Mouse muscle cells (C2C12) were purchased from the European Collection of Cell Cultures. Dulbecco's Modified Eagle Medium (DMEM), Foetal Calf Serum (FCS), horse serum and Trypsin/ EDTA were purchased from Thermo Scientific HyClone. Pen/Strep, L-Glutamine and DMSO were purchased from Sigma, UK. Tritiated deoxy-2-glucose and Ultima Gold scintillation fluid were both purchased from Perkin Elmer. Culture flasks and plates were purchased from Sarstedt.

C57BI/6 background mice were purchased from Charles River Laboratories UK. Chow diet from Special Diet Services and high fat diet from Bio- Serv. The ACCU-CHEK® Aviva meter system and insulin strips were from Roche. Gavage needles and syringes from Vet-Tech Solutions Ltd. DMSO and glucose (Sigma). Insulin (Actrapid human rDNA, from NovoNordisk, Bagsvaerd, Denmark. 1 x 10 ml) Expression and purification of recombinant human sRBP in P. pastoris

The sRBP was expressed with a N-terminal His-tag using the P. pastoris expression system as described by Wysocka-Kapcinska et al. (Wysocka-Kapcinska, M.; Campos-Sandoval, J.; Pal, A.; Findlay, J. Expression and characterization of recombinant human retinol-binding protein in Pichia pastoris. Protein Expr Purif 2010, 71 , 28-32). The sRBP coding sequence was amplified by PCR using the oligonucleotide primers EcoRI (forward; SEQ ID NO 7) Forward (EcoRI site underlined): 5^'- GAA TTC CAT CAT CAT CAT CAT CAT GAG CGC GAC TGC CGA GTG-3^'. The underlined codon is the first codon in mature sRBP and Reverse (Xbal site underlined, stop codon in bold, next to Xbal site): 5^'-TCT AGA CTA CAA AAG GTT TCT TTC TGA TCT GCC-3^'. The forward primer included the sequence for a 6xHis-tag. The PCR product was ligated into pCR-Blunt II pTOPO vector, and then digested with EcoRI and Xbal. The EcoRI-Xbal product was ligated then into pPICZalpha-A previously digested with the same enzymes. The PCR product was ligated in the pCR-Blunt II TOPO vector and subsequently introduced into the EcoRI/Xbal sites of pPICZo A vector under the control of the AOX1 promoter.

SEQ ID NO 9 defines sRBP PCR product with primers EcoRI forward and Xbal reverse (for subcloning into pPICZoA). The two restrictions sites are underlined. Struck through, the stop codon.

SEQ ID NO 9:

GAATTCCATC ATCATCATCA TCATGAGCGC GACTGCCGAG TGAGCAGCTT CCGAGTCAAG GAGAACTTCG ACAAGGCTCG CTTCTCTGGG ACCTGGTACG CCATGGCCAA GAAGGACCCC GAGGGCCTCT TTCTGCAGGA CAACATCGTC GCGGAGTTCT CCGTGGACGA GACCGGCCAG ATGAGCGCCA CAGCCAAGGG CCGAGTCCGT CTTTTGAATA ACTGGGACGT GTGCGCAGAC ATGGTGGGCA CCTTCACAGA CACCGAGGAC CCTGCCAAGT TCAAGATGAA GTACTGGGGC GTAGCCTCCT TTCTCCAGAA AGGAAATGAT GACCACTGGA TCGTCGACAC AGACTACGAC ACGTATGCCG TGCAGTACTC CTGCCGCCTC CTGAACCTCG ATGGCACCTG TGCTGACAGC TACTCCTTCG TGTTTTCCCG GGACCCCAAC GGCCTGCCCC CAGAAGCGCA GAAGATTGTA AGGCAGCGGC AGGAGGAGCT GTGCCTGGCC AGGCAGTACA GGCTGATCGT CCACAACGGT TACTGCGATG GCAGATCAGA AAGAAACCTT TTGT-AGTCTA GA SEQ ID NO 10:

His- sRBP

EFHHHHHHERDCRVSSFRVKENFDKARFSGTWYAMAKKDPEGLFLQDNIVAEFSVD ETGQMSATAKGRVRLLNNWDVCADMVGTFTDTEDPAKFKMKYWGVASFLQKGNDDHWIVD TDYDTYAVQYSCRLLNLDGTCADSYSFVFSRDPNGLPPEAQKIVRQRQEELCLARQYRLI VHNGYCDGRSERNLL

P. pastoris cells were transformed with the expression vector pPICZa A-His-sRBP and grown at 30°C in phosphate-buffered YP medium (1 % yeast extract, 2% peptone, pH 7.5) in the presence of 2% glycerol (w/v) until the OD₆₀o was 6.0. The cells were harvested by centrifugation at 7000 g for 10 min and used to inoculate YP medium to an OD₆₀o of 1.0 supplemented with 1 % methanol (v/v) to induce expression. In each of 2 days, methanol was added to a final concentration of 1 % (v/v). After 48 h of induction, the culture supernatant containing the His-sRBP was incubated with Ni-NTA resin and the bound proteins eluted with elution buffer (20 mM Na₂HP0₄, 500 mM NaCI, 250 mM imidazole, pH 7.4). The purified His-sRBP was dialysed against PBS buffer and then concentrated by ultrafiltration.

Expression and purification of recombinant human His-TTR

The coding sequence of human TTR in the vector pMMHa was a gift of Dr. J.W. Kelly. (Campos- Sandoval JA et al., Fenretinide Derivatives Act as Disrupters of Interactions of Serum Retinol Binding Protein (sRBP) with Transthyretin and the sRBP Receptor, J Med Chem. 201 1 Jul 14;54(13):4378- 4387). In order to facilitate the subsequent purification of the holo-sRBP-TTR complex, TTR was expressed with a N-terminal His-tag. The coding sequence of mature TTR (corresponding to residues 21-147) was inserted into the BamHI and Hindlll restriction sites in the pQE-30 vector. The oligonucleotide primers used in the PCR were BamHI (forward; SEQ ID NO 1 1 ) 5^'- GGATTCGGCCCTACGGGCACCG-3^'and Hindlll (reverse; SEQ ID NO 12) 5^'- AAGCTTTCATTCCTTGGGATTGGTGACG-3^'. The PCR product was ligated in the pCR-Blunt II TOPO vector, digested with BamHI and Hindlll and inserted into the pQE-30 vector.

SEQ ID NO 13 defines TTR PCR product with primers BamHI forward and Hindlll reverse (for subcloning into pQE30). The two restrictions sites are underlined. Struck-through, the stop codon: SEQ ID NO 13:

GGATCCG GCCCTACGGG CACCGGTGAA TCCAAGTGTC CTCTGATGGT CAAAGTTCTA GATGCTGTCC GAGGCAGTCC TGCCATCAAT GTGGCCGTGC ATGTGTTCAG AAAGGCTGCT GATGACACCT GGGAGCCATT TGCCTCTGGG AAAACCAGTG AGTCTGGAGA GCTGCATGGG CTCACAACTG AGGAGGAATT TGTAGAAGGG ATATACAAAG TGGAAATAGA CACCAAATCT TACTGGAAGG CACTTGGCAT CTCCCCATTC CATGAGCATG CAGAGGTGGT ATTCACAGCC AACGACTCCG GCCCCCGCCG CTACACCATT GCCGCCCTGC TGAGCCCCTA CTCCTATTCC ACCACGGCTG TCGTCACCAA TCCCAAGGAA TGAAAGCTT

SEQ ID NO 14:

His-TTR

MRGSHHHHHHGSGPTGTGESKCPLMVKVLDAVRGSPAINVAVHVFRKAADDTWEPFASG KTSESGELHGLTTEEEFVEGIYKVEIDTKSYWKALGISPFHEHAEVVFTANDSGPRRYTI AALLS P YS YSTTAVVTN P KE

After overnight induction with IPTG, the cells were collected and sonicated. The supernatant containing the soluble TTR was incubated with Ni-NTA resin and the recombinant protein eluted with elution buffer. The purified His-TTR was dialysed against PBS buffer. Purification of ROH-sRBP-TTR complex

Purified His-TTR was added to the refolded, untagged holo-sRBP solution and incubated overnight at 4°C in the presence of 100 μΜ ROH. Then, the protein solution was applied to a cobalt affinity column equilibrated with buffer I (20 mM Na₂HP0₄, 500 mM NaCI, 10 mM imidazole, pH 7.4). The column was washed with the same buffer and then eluted with elution buffer.

Surface Plasmon Resonance (SPR)

Interaction analyses were performed in SPR running buffer (HBS with 50μΜ EDTA) at a flow rate of Ι ΟμΙ/min, at constant temperature (25°C), using a Biacore 3000 system (GE Healthcare). Ni-NTA sensor chips were used for all experiments and general procedures were performed according to the manufacturer's instructions for this sensor-chip. Briefly, each cycle consisted of: surface activation with 20μΙ of nickel (2 min), followed by a 5 min injection of recombinant sRBP (His-tagged, 200 nM solution, prepared in SPR running buffer), and followed by a 1 min pulse of a specific concentration of compound (diluted in SPR running buffer), culminating with (i) a 5 min injection of untagged, native TTR (1 μΜ in running buffer incubated with 10 μΜ each compound), or (ii) a 5 min injection of solubilised HEK cells membranes (50μg ml in running buffer incubated with 10 μΜ of each compound); finally sensorchip regeneration was achieved by stripping all proteins from the surface, performing a 2 min injection of 0.3 mM EDTA in HBS, at the end of each cycle (max. number of regenerations/chip: n=100). Sensorchips were used for 2 weeks, or until nonspecific binding increased (>= 5%). All experiments were performed in parallel with an inactivated flow cell not coated with protein. Each independent experiment was repeated 3-5 times. sRBP-TTR interaction by pull-down assay The capacity of compounds capable of binding sRBP to disrupt the interaction between this protein and its partner TTR was examined by a pull-down assay. In this assay, the holo-sRBP-TTR complex (0.2 mg/mL in PBS buffer pH 7.4) was incubated in the presence of 20μΙ_ of NiNTA resin. Then, 0.5 μΜ Retinol and 100 μΜ of each compound was added and incubated for 1 hour at 37°C. The resin was centrifuged at 380 x g for 2 min to separate the fraction of free TTR from the fraction bound to RBP. The flow-throughs, containing the free TTR, were examined by SDS-PAGE and silver staining. A control with fenretininde (FEN), which is known to disrupt the interaction between sRBP and TTR, was included in the assay for comparison (Campos-Sandoval JA et al., Fenretinide Derivatives Act as Disrupters of Interactions of Serum Retinol Binding Protein (sRBP) with Transthyretin and the sRBP Receptor, J Med Chem. 201 1 Jul 14;54(13):4378-4387).

Data Processing

Prism 5.00 (GraphPad Software Inc.) was used to determine EC₅₀ values. All SPR sensorgrams were corrected for buffer induced refractive index changes at an uncoated reference surface, analysed using BIAEVALUATION software (BIAEVAL 3.2, GE Healthcare).

Glucose uptake Assay

Mouse muscle cells (C2C12) were seeded in DMEM (plus 10% FCS, Pen/Strep (100U) and L-Glut (2mM)) at 1 x10⁴cells/ml in 6-well plates. When 70-80% confluent, cells were differentiated in DMEM plus 2% horse serum for 3 days. Compounds of the present invention (50mM) were dissolved in 100% DMSO and diluted to 1 mM in Krebs Ringers Buffer (KRB; NaCI, 136mM; Hepes, 20mM; KCI, 4.7mM; MgS0₄, 1 mM; CaCI₂, 1 mM; Na₂HP0₄, 4.05mM; NaH₂P0₄, 0.95mM; pH7.4) including glucose (5mM). The cells were treated with 10μΜ compounds in DMEM plus 2% horse serum overnight at 37°C. Glucose uptake was measured, with modifications, as per Yun and colleagues. (Yun J, Rago C, Cheong I, Pagliarini R, Angenendt P, Rajagopalan H, Schmidt K, Willson JK, Markowitz S, Zhou S, Diaz LA Jr, Velculescu VE, Lengauer C, Kinzler KW, Vogelstein B, Papadopoulos N; Science, 2009, 325(5947): 1555-9). The cells were washed in KRB and ³H deoxy- 2-glucose (1 Ci/ml; specific activity 8mCi/mmol) added for 10min at 37°C. The cells were washed 3 times in ice-cold KRB and solubilized in 0.1 % SDS for 30min. The cells were harvested and 500μΙ (~250ug protein) added to 2ml Ultima Gold scintillation fluid. Counts were read, per minute, using a Wallac MicroBeta scintillation counter (Perkin Elmer) and results are expressed as counts per minute per mg protein (cpm/ mg protein) as assayed by the BCA method (Smith, P.K., ef al. (1985). "Measurement of protein using bicinchoninic acid". Anal. Biochem. 150 (1 ): 76-85).

Compound Synthesis

Ή NMR H NMR spectra were acquired at 298 K Bruker Avance (300 MHz) as solutions in CDCI₃ (1.75 ml), where residual CHCI₃ (δ 7.26 ppm) were used as respective internal references. Data are reported as chemical shifts (δ ppm), multiplicity (s = singlet, d = doublet, dd = doublet of doublets, t = triplet and m = multiplet), coupling constants (J, Hz) and relative integral.

Mass. Spec.

LC-MS grade Acetonitrile and Water were obtained from Sigma Aldrich. Formic acid was obtained from Agilent.

LC TOF-MS

The LC was a model 1200 Series (Agilent Corp, Santa Clara, CA), injection volume was 1 μΙ_, with a mobile phase of A = ACN with 0.1 % formic acid and B = 0.1 % formic acid in water. The LC/TOF- MS was a model 6210 Time-Of-Flight LC/MS (Agilent Corp, Santa Clara, CA) with an electrospray source both positive and negative (ESI+/-), capillary 3,500 V, nebuliser spray 30 psig, drying gas 5 L/min, source temperature 325 °C. The fragmentor voltage was used at 175 V. Reference masses (Agilent Solution) were 121.050873, 149.02332, 322.048121 , 922.009798, 1221.990633, 1521 .971475 and 2421.91399 m/z.

Materials

1- ((4-Trifluoromethyl)phenyl)piperazine was obtained from Fluorochem. 4-(thiophen-2-yl)butanoic acid was obtained from Aldrich. 4-Oxo-4-(2-thienyl)butyric acid was obtained from Aldrich. 1-(5- (Trifluoromethyl)pyridin-2-yl)piperazine was obtained from Maybridge. 1-(3-Chloro-5-trifluoromethyl- pyridin-2-yl)-piperazine was obtained from Maybridge. 5-(Thiophen-2-yl)pentanoic acid was obtained from Alfa Aesar. 4-[4-(Trifluoromethyl)phenyl]piperidine. HCI was obtained from Fluorochem. 4-(1 H- Pyrazol-4-yl)butanoic acid was obtained from ChemBridge. 6-(Thiophen-2-yl)hexanoic acid was obtained from Ukraine Organic Synthesis. Octanoic acid was obtained from Alfa Aesar. 3-(Thiophen-

2- yl)propanoic acid was obtained from Astatech, Inc. 3-(Furan-2-yl)propanoic acid was obtained from Alfa. 2-(Thiophen-2-yl)acetic acid was obtained from Apollo Scientific. 2-Thiophenethiol was obtained from Aldrich. 3-Bromopropionic acid was obtained from Aldrich. Valeric acid and 2- methoxyethylamine from Aldrich. Azelaic acid from Lancaster Synthesis. 3-Chloro-4- fluorobenzotrifluoride was obtained from Fluorochem. Pipeazine was obtained from Alfa Aesar. Acetic Acid was obtained from Aldrich. 1-(6-chloro-5-trifluoromethyl)pyridine-2-yl)piperazine was obtained from Key Organics. 3-Bromo-4-chloro-benzotrifluoride was obtained from Acros Organics.

4-(Thiophen-2-yl)-1-{4-[4-(trifluoromethyl)phenyl]piperazin-1-yl}butan-1-one (RTC1)

4-(Thiophen-2-yl)butanoic acid (0.05 ml, 0.34 mmol), HOBt (58 mg, 0.43 mmol), TBTU (140 mg, 0.43 mmol), anhydrous triethylamine (0.1 ml, 0.69 mmol) and dry DMF (2 ml) were placed in an oven-dried Schlenk tube under a nitrogen atmosphere. The resulting solution was stirred at room temperature for 15 minutes. A second Schlenk tube was prepared containing 1-(4-trifluoromethyl phenyl) piperazine (100 mg, 0.43 mmol) and dry DMF (1 ml) under a nitrogen atmosphere. The resulting solution was stirred until complete dissolution of the piperazine had occurred. The piperazine solution was then transferred, via a cannula, to the first Schlenk tube containing the carboxylic acid. The resulting solution was stirred for 24 hrs, under nitrogen, and monitored by TLC. After 24 hrs, the DMF was removed under reduced pressure and the resulting oil was acidified using a 0.1 M hydrochloric acid solution. The aqueous mixture was extracted with dichloromethane (20 ml, followed by 4 x 10 ml) and the organic layer washed with a saturated sodium bicarbonate solution (3 x 20 ml) and brine (3 x 20 ml). The organic layer was dried over magnesium sulphate and evaporated under reduced pressure. The residue was purified using flash chromatography (3:2, EtOAc:n-hexane) to obtain the desired product in an 80% yield. H NMR (300 MHz, CDCI₃) δ 7.49 (d, 2H), 7.12 (dd, 1 H), 6.90-6.94 (m, 3H), 6.80-6.82 (m, 1 H), 3.76 (t, 2H), 3.56 (t, 2H), 3.24 (t, 4H), 2.91 (t, 2H), 2.38 (t, 2H), 2.0-2.1 1 (m, 2H). MS (+ESI) calcd for C₁₈ H₂₁ F₃ N₂ O S m/z: [M + H]⁺, 382.1329; found 383.1399 [Diff(ppm) = 0.61].

1-(Thiophen-2-yl)-4-{4-[4-(trifluoromethyl)phenyl]piperazin-1-yl}butane-1,4-dione (RTC2)

4-Oxo-4-(2-thienyl) butyric acid (63 mg, 0.34 mmol), HOBt (58 mg, 0.43 mmol), TBTU (140 mg, 0.43 mmol), anhydrous triethylamine (0.1 ml, 0.69 mmol) and dry DMF (2 ml) were placed in an oven- dried Schlenk tube under a nitrogen atmosphere. The resulting solution was stirred at room temperature for 15 minutes. A second Schlenk tube was prepared containing 1-(4-trifluoromethyl phenyl) piperazine (100 mg, 0.43 mmol) and dry DMF (1 ml) under a nitrogen atmosphere. The resulting solution was stirred until complete dissolution of the piperazine had occurred. The piperazine solution was then transferred, via a cannula, to the first Schlenk tube containing the carboxylic acid. The resulting solution was stirred for 24 hrs, under nitrogen, and monitored by TLC. After 24 hrs, the DMF was removed under reduced pressure and the resulting oil was acidified using a 0.1 M hydrochloric acid solution. The aqueous mixture was extracted with dichloromethane (20 ml, followed by 4 x 10 ml) and the organic layer washed with a saturated sodium bicarbonate solution (3 x 20 ml) and brine (3 x 20 ml). The organic layer was dried over magnesium sulphate and evaporated under reduced pressure. The residue was purified using flash chromatography (3:2, EtOAc:n-hexane) to obtain the desired product in an 81 % yield. H NMR (300 MHz, CDCI₃) δ 7.81 (d, 1 H), 7.63 (d, 1 H), 7.49 (d, 2H), 7.13-7.16 (m, 1 H), 6.92 (d, 2H), 3.72-3.81 (m, 4H), 3.28 (t, 4H), 3.25 (t, 2H), 2.81 (t, 2H). MS (+ESI) calcd for C₁₉ H₁₉ F₃ N₂ 0₂ S m/z: [M + H]⁺ , 396.1 1 15; found 396.1 1 19 [Diff(ppm) = -1.1 1].

1-(Thiophen-2-yl)-4-{4-[5-(trifluoromethyl)pyridin-2-yl]piperazin-1-yl}butane-1,4-dion (RTC3). 4-Oxo-4-(2-thienyl) butyric acid (63 mg, 0.34 mmol), HOBt (58 mg, 0.43 mmol), TBTU (140 mg, 0.43 mmol), anhydrous triethylamine (0.1 ml, 0.69 mmol) and dry DMF (2 ml) were placed in an oven- dried Schlenk tube under a nitrogen atmosphere. The resulting solution was stirred at room temperature for 15 minutes. A second Schlenk tube was prepared containing 1-(5-trifluoromethyl-2- pyridinyl) piperazine (99 mg, 0.43 mmol) and dry DMF (1 ml) under a nitrogen atmosphere. The resulting solution was stirred until complete dissolution of the piperazine had occurred. The piperazine solution was then transferred, via a cannula, to the first Schlenk tube containing the carboxylic acid. The resulting solution was stirred for 24 hrs, under nitrogen, and monitored by TLC. After 24 hrs, the DMF was removed under reduced pressure and the resulting oil was acidified using a 0.1 M hydrochloric acid solution. The aqueous mixture was extracted with dichloromethane (20 ml, followed by 4x10 ml) and the organic layer washed with a saturated sodium bicarbonate solution (3 x 20 ml) and brine (3 x 20 ml). The organic layer was dried over magnesium sulphate and evaporated under reduced pressure. The residue was purified using flash chromatography (3:2, EtOAc:n- hexane) to obtain the desired product in a 63% yield. H NMR (300 MHz, CDCI₃) δ 8.42 (s, 1 H), 7.8 (d, 1 H), 7.63-7.67 (m, 2H), 7.13 (t, 1 H), 6.63 (d, 1 H), 3.61-3.79 (m, 8H), 3.32 (t, 2H), 2.81 (t, 2H). MS (+ESI) calcd for C₁₈ H₁₈ F₃ N₃ 0₂ S m/z: [M + H]⁺ , 397.1079; found 397.1072 [Diff(ppm) = 1.79]

RTC3 1 -{4-[3-Chloro-5-(trifluoromethyl)pyridi^

(RTC4). 4-Oxo-4-(2-thienyl) butyric acid (63 mg, 0.34 mmol), HOBt (58 mg, 0.43 mmol), TBTU (140 mg, 0.43 mmol), anhydrous triethylamine (0.1 ml, 0.69 mmol) and dry DMF (2 ml) were placed in an oven- dried Schlenk tube under a nitrogen atmosphere. The resulting solution was stirred at room temperature for 15 minutes. A second Schlenk tube was prepared containing 1-[3-chloro-5- (trifluoromethyl)-2-pyridinyl] piperazine (1 14 mg, 0.43 mmol) and dry DMF (1 ml) under a nitrogen atmosphere. The resulting solution was stirred until complete dissolution of the piperazine had occurred. The piperazine solution was then transferred, via a cannula, to the first Schlenk tube containing the carboxylic acid. The resulting solution was stirred for 24 hrs, under nitrogen, and monitored by TLC. After 24 hrs, the DMF was removed under reduced pressure and the resulting oil was acidified using a 0.1 M hydrochloric acid solution. The aqueous mixture was extracted with dichloromethane (20 ml, followed by 4 x 10 ml) and the organic layer washed with a saturated sodium bicarbonate solution (3 x 20 ml) and brine (3 x 20 ml). The organic layer was dried over magnesium sulphate and evaporated under reduced pressure. The residue was purified using flash chromatography (3:2, EtOAc:n-hexane) to obtain the desired product in a 70% yield. H NMR (300 MHz, CDCI₃) δ 8.40 (s, 1 H), 7.79-7.83 (m, 2H), 7.63 (dd, 1 H), 7.13-7.16 (m, 1 H), 3.72-3.79 (m, 4H), 3.48-3.58 (m, 4H), 3.32 (t, 2H), 2.81 (t, 2H). MS (+ESI) calcd for C₁₈ H₁₇ CI F₃ N₃ 0₂ S m/z: [M + H]⁺ , 432.0755; found 432.0765 [Diff(ppm) = 2.34].

RTC4 4-(Thiophen-2-yl)-1 -{4-[5-(trifluoromethyl)pyridin-2-yl]piperazin-1 -yl}butan-1 -one (RTC5)

4-(Thiophen-2-yl)butanoic acid (63 mg, 0.34 mmol), HOBt (58 mg, 0.43 mmol), TBTU (140 mg, 0.43 mmol), anhydrous triethylamine (0.1 ml, 0.69 mmol) and dry DMF (2 ml) were placed in an oven- dried Schlenk tube under a nitrogen atmosphere. The resulting solution was stirred at room temperature for 15 minutes. A second Schlenk tube was prepared containing 1-(5-trifluoromethyl-2- pyridinyl) piperazine (99 mg, 0.43 mmol) and dry DMF (1 ml) under a nitrogen atmosphere. The resulting solution was stirred until complete dissolution of the piperazine had occurred. The piperazine solution was then transferred, via a cannula, to the first Schlenk tube containing the carboxylic acid. The resulting solution was stirred for 24 hrs, under nitrogen, and monitored by TLC. After 24 hrs, the DMF was removed under reduced pressure and the resulting oil was acidified using a 0.1 M hydrochloric acid solution. The aqueous mixture was extracted with dichloromethane (20 ml, followed by 4 x 10 ml) and the organic layer washed with a saturated sodium bicarbonate solution (3 x 20 ml) and brine (3 x 20 ml). The organic layer was dried over magnesium sulphate and evaporated under reduced pressure. The residue was purified using flash chromatography (3:2, EtOAc:n-hexane) to obtain the desired product in an 80% yield. H NMR (300 MHz, CDCI₃) δ 8.40 (s, 1 H), 7.64 (dd, 1 H), 7.12 (dd, 1 H), 6.91-6.94 (m, 1 H), 6.81 (dd, 1 H), 6.63 (d, 1 H), 3.51-3.76 (m, 8H), 2.9 (t, 2H), 2.39 (t, 2H), 2.01-2.1 1 (m, 2H). MS (+ESI) calcd for C₁₈ H₂₀ F₃ N₃ O S m/z: [M + H]⁺ , 384.1352; found 384.1365 [Diff(ppm) = 3.38].

1 -{4-[3-Chloro-5-(trifluoromethyl)pyridin-2-yl]piperazin-1 -yl}-4-(thiophen-2-yl)butan-1 -one (RTC6)

4-(Thiophen-2-yl)butanoic acid (63 mg, 0.34 mmol), HOBt (58 mg, 0.43 mmol), TBTU (140 mg, 0.43 mmol), anhydrous triethylamine (0.1 ml, 0.69 mmol) and dry DMF (2 ml) were placed in an oven- dried Schlenk tube under a nitrogen atmosphere. The resulting solution was stirred at room temperature for 15 minutes. A second Schlenk tube was prepared containing 1-[3-chloro-5- (trifluoromethyl)-2-pyridinyl] piperazine (1 14 mg, 0.43 mmol) and dry DMF (1 ml) under a nitrogen atmosphere. The resulting solution was stirred until complete dissolution of the piperazine had occurred. The piperazine solution was then transferred, via a cannula, to the first Schlenk tube containing the carboxylic acid. The resulting solution was stirred for 24 hrs, under nitrogen, and monitored by TLC. After 24 hrs, the DMF was removed under reduced pressure and the resulting oil was acidified using a 0.1 M hydrochloric acid solution. The aqueous mixture was extracted with dichloromethane (20 ml, followed by 4 x 10 ml) and the organic layer washed with a saturated sodium bicarbonate solution (3 x 20 ml) and brine (3 x 20 ml). The organic layer was dried over magnesium sulphate and evaporated under reduced pressure. The residue was purified using flash chromatography (3:2, EtOAc:n-hexane) to obtain the desired product in a 64 % yield. H NMR (300 MHz, CDCI₃) δ 8.40 (s, 1 H), 7.8 (d, 1 H), 7.12 (dd, 1 H), 6.91-6.94 (m, 1 H), 6.81-6.82 (m, 1 H), 3.76- 3.79 (m, 2H), 3.56-3.59 (m, 2H), 3.46-3.49 (m, 4H), 2.91 (t, 2H), 2.39 (t, 2H), 2.04-2.09 (m, 2H). MS (+ESI) calcd for C₁₈ H₁₉ CI F₃ N₃ O S m/z: [M + H]⁺ , 417.0898; found 417.0889 [Diff(ppm) = 2.06].

3-(Thiophen-2-ylthio)-1 -(4-(4-(trifluoromethyl)phenyl)piperazin-1 -yl)propan-1 -one (RTC7) 3-(Thiophen-3-ylthio) propanoic acid (37 mg, 0.19 mmol), HOBt (30 mg, 0.219 mmol), TBTU (70 mg, 0.219 mmol) and anhydrous triethylamine (44 μΙ, 0.316 mmol) and dry DMF (1 ml) were placed in an oven-dried Schlenk tube under a nitrogen atmosphere. The resulting solution was stirred at room temperature for 15 minutes. A second Schlenk tube was prepared containing 1-(4-trifluoromethyl phenyl) piperazine (50 mg, 0.219 mmol) and dry DMF (1 ml) under a nitrogen atmosphere. The resulting solution was stirred until complete dissolution of the piperazine had occurred. The piperazine solution was then transferred, via a cannula, to the first Schlenk tube containing the carboxylic acid. The resulting solution was stirred for 24 hrs, under nitrogen, and monitored by TLC. After 24 hrs, the DMF was removed under reduced pressure and the resulting oil was acidified using a 0.1 M hydrochloric acid solution. The aqueous mixture was extracted with dichloromethane (20 ml, followed by 4 x 10 ml) and the organic layer washed with a saturated sodium bicarbonate solution (3 x 20 ml) and brine (3 x 20 ml). The organic layer was dried over magnesium sulphate and evaporated under reduced pressure. The residue was purified using flash chromatography (3:2, EtOAc:n-hexane) to obtain the desired product in a 63 % yield. H NMR (300 MHz, CDCI₃) δ 7.47 (d, 2H), 7.34 (dd, 1 H), 7.13 (dd, 1 H), 6.96-6.99 (m, 1 H), 6.88 (d, 2H), 3.75 (t, 2H), 3.54 (t, 2H), 3.22-3.27 (m, 4H), 3.06-3.14 (m, 2H). MS (+ESI) calcd for C₁₈ H₂₉ F₃ N₂ O S₂ m/z: [M + H]⁺ , 400.0952; found 401 .1665 [Diff(ppm) = 3.48].

9-Oxo-9-(4-(4-(trifluoromethyl)phenyl)piperazin-1 -yl)nonanoic acid (RTC15) Azelaic acid (326 mg, 1.74 mmol), HOBt (1 17 mg, 0.87 mmol), TBTU (279 mg, 0.87 mmol), anhydrous triethylamine (121 μΙ, 1 .39 mmol) and dry DMF (2 ml) were placed in an oven-dried Schlenk tube under a nitrogen atmosphere. The resulting solution was stirred at room temperature for 15 minutes. A second Schlenk tube was prepared containing 1-(4-trifluoromethyl phenyl) piperazine (200 mg, 0.87 mmol) and dry DMF (1 ml) under a nitrogen atmosphere. The resulting solution was stirred until complete dissolution of the piperazine had occurred. The piperazine solution was then transferred, via a cannula, to the first Schlenk tube containing the carboxylic acid. The resulting solution was stirred for 24 hrs, under nitrogen, and monitored by TLC. After 24 hrs, the DMF was removed under reduced pressure and the resulting oil was acidified using a 0.1 M hydrochloric acid solution. The aqueous mixture was extracted with dichloromethane (20 ml, followed by 4 x 10 ml) and the organic layer washed with a saturated sodium bicarbonate solution (3 x 20 ml) and brine (3 x 20 ml). The organic layer was dried over magnesium sulphate and evaporated under reduced pressure. The residue was purified using flash chromatography (DCM followed by 9.5: 0.5, DCM:MeOH) to obtain the desired product as a white solid in a 60% yield. H NMR (300 MHz, CDCI₃) δ 7.49 (d, 2H), 6.91 (d, 2H), 3.77-3.78 (m, 2H), 3.62-3.64 (m, 2H), 3.24-3.31 (m, 2H), 2.33- 2.39 (m, 2H), 1.60-1.68 (m, 4H), 1.33-1.38 (m, 6H). MS (+ESI) calcd for C₂₀ H₂₇ F₃ N₂ 0₃ m/z: [M + H]⁺, 401 .2047; found 401.2047 [Diff(ppm) = 0].

N-(2-Methoxyethyl)-9-oxo-9-(4-(4-(trifluoromethyl)phenyl)piperazin-1 -yl)nonanamide (RTC18) RTC15 (137 mg, 0.34 mmol), HOBt (58 mg, 0.43 mmol), TBTU (140 mg, 0.43 mmol), anhydrous triethylamine (0.1 ml, 0.69 mmol) and dry DMF (1 ml) were placed in an oven-dried Schlenk tube under a nitrogen atmosphere. The resulting solution was stirred at room temperature for 15 minutes. A second Schlenk tube was prepared containing 2-methoxyethylamine (29 μΙ, 0.43 mmol) and dry DMF (1 ml) under a nitrogen atmosphere. The resulting solution was stirred until complete dissolution of the piperazine had occurred. The piperazine solution was then transferred, via a cannula, to the first Schlenk tube containing the carboxylic acid. The resulting solution was stirred for 24 hrs, under nitrogen, and monitored by TLC. After 24 hrs, the DMF was removed under reduced pressure and the resulting oil was acidified using a 0.1 M hydrochloric acid solution. The aqueous mixture was extracted with dichloromethane (20 ml, followed by 4 x 10 ml) and the organic layer washed with a saturated sodium bicarbonate solution (3 x 20 ml) and brine (3 x 20 ml). The organic layer was dried over magnesium sulphate and evaporated under reduced pressure. The residue was purified using flash chromatography (1 : 1 , EtOAc:n-hexane followed by 4: 1 EtOAc:MeOH) to obtain the desired product as a white solid in a 34% yield. H NMR (300 MHz, CDCI₃) δ 7.48 (d, 2H), 6.91

(d, 2H), 5.86 (bs, 1 H), 3.76-3.79 (m, 2H), 3.62-3.65 (m, 2H), 3.43-3.45 (m, 4H), 3.36 (s, 3H), 3.24- 3.30 (m, 4H), 2.33 (t, J = 7.9 Hz, 2H), 2.15 (t, J = 7.9 Hz, 2H), 1.63-1.65 (m, 4H), 1.31-1.38 (m, 6H). MS (+ESI) calcd for C₂₃ H₃₄ F₃ N₃ 0₃ m/z: [M + H]⁺, 458.2625; found 458.2631 [Diff(ppm) = 1 .31].

4-(1 H-Pyrazol-4-yl)-1 -(4-(4-(trifluoromethyl)phenyl)piperazin-1 -yl)butan-1 -one (RTC193)

4-(1 H-Pyrazol-4-yl)butanoic acid (100 mg, 0.65 mmol), HOBt (96 mg, 0.71 mmol), TBTU (228 mg, 0.71 mmol), anhydrous triethylamine (144 μΙ, 1.04 mmol) and dry DMF (2 ml) were placed in an oven-dried Schlenk tube under a nitrogen atmosphere. The resulting solution was stirred at room temperature for 15 minutes. A second Schlenk tube was prepared containing 1-(4-trifluoromethyl phenyl) piperazine (100 mg, 0.71 mmol) and dry DMF (1 ml) under a nitrogen atmosphere. The resulting solution was stirred until complete dissolution of the piperazine had occurred. The piperazine solution was then transferred, via a cannula, to the first Schlenk tube containing the carboxylic acid. The resulting solution was stirred for 24 hrs, under nitrogen, and monitored by TLC. After 24 hrs, the DMF was removed under reduced pressure and the resulting oil was acidified using a 0.1 M hydrochloric acid solution. The aqueous mixture was extracted with dichloromethane (20 ml, followed by 4 x 10 ml) and the organic layer washed with a saturated sodium bicarbonate solution (3 x 20 ml) and brine (3 x 20 ml). The organic layer was dried over magnesium sulphate and evaporated under reduced pressure. The residue was purified using flash chromatography (3:2, EtOAc:n-hexane) to obtain the desired product in an 10% yield. H NMR (300 MHz, CDCI₃) δ 7.49 (d, 4H), 6.91 (d, 2H), 3.77 (t, 2H), 3.58 (t, 2H), 3.24 (t, 4H), 2.57 (t, 2H), 2.37 (t, 2H), 1.91-2.01 (m, 2H). MS (+ESI) calcd for C₁₈ H₂₁ F₃ N₄ O m/z: [M + H]⁺ , 366.1677; found 368.177 [Diff(ppm) = 1 .55].

1 -(4-(4-(Trifluoromethyl)phenyl)piperazin-1 -yl)pentan-1 -one (RTC194)

Valeric acid (106 μΙ, 0.979 mmol), HOBt (144 mg, 0.979 mmol), TBTU (343 mg, 0.979 mmol), anhydrous triethylamine (216 μΙ, 1 .56 mmol) and dry DMF (2 ml) were placed in an oven-dried Schlenk tube under a nitrogen atmosphere. The resulting solution was stirred at room temperature for 15 minutes. A second Schlenk tube was prepared containing 1-(4-trifluoromethyl phenyl) piperazine (246 mg, 1.07 mmol) and dry DMF (1 ml) under a nitrogen atmosphere. The resulting solution was stirred until complete dissolution of the piperazine had occurred. The piperazine solution was then transferred, via a cannula, to the first Schlenk tube containing the carboxylic acid. The resulting solution was stirred for 24 hrs, under nitrogen, and monitored by TLC. After 24 hrs, the DMF was removed under reduced pressure and the resulting oil was acidified using a 0.1 M hydrochloric acid solution. The aqueous mixture was extracted with dichloromethane (20 ml, followed by 4 x 10 ml) and the organic layer washed with a saturated sodium bicarbonate solution (3 x 20 ml) and brine (3 x 20 ml). The organic layer was dried over magnesium sulphate and evaporated under reduced pressure. The residue was purified using flash chromatography (3:2, EtOAc:n-hexane) to obtain the desired product in an 57 % yield. H NMR (300 MHz, CDCI₃) δ 7.48 (d, 2H), 6.91 (d, 2H), 3.76 (t, 2H), 3.63 (t, 2H), 3.24-3.30 (m, 4H), 2.35 (t, 2H), 1.59-1.69 (m, 2H), 1 .33-1.45 (m, 2H), 0.92 (t, 3H). MS (+ESI) calcd for C₁₆ H₂₁ F₃ N₂ O m/z: [M + H]⁺ , 314.1596; found 315.1679 [Diff(ppm) = - 3.06].

5-(Thiophen-2-yl)-1-(4-(4-(trifluoromethyl)phenyl)piperazin-1-yl)pentan-1-one (RTC195) 5-(Thiophen-2-yl)pentanoic acid (72 mg, 0.39 mmol), HOBt (58 mg, 0.43 mmol), TBTU (138 mg, 0.43 mmol), anhydrous triethylamine (86 μΙ, 0.62 mmol) and dry DMF (2 ml) were placed in an oven-dried Schlenk tube under a nitrogen atmosphere. The resulting solution was stirred at room temperature for 15 minutes. A second Schlenk tube was prepared containing 1-(4-trifluoromethyl phenyl) piperazine (100 mg, 0.43 mmol) and dry DMF (1 ml) under a nitrogen atmosphere. The resulting solution was stirred until complete dissolution of the piperazine had occurred. The piperazine solution was then transferred, via a cannula, to the first Schlenk tube containing the carboxylic acid. The resulting solution was stirred for 24 hrs, under nitrogen, and monitored by TLC. After 24 hrs, the DMF was removed under reduced pressure and the resulting oil was acidified using a 0.1 M hydrochloric acid solution. The aqueous mixture was extracted with dichloromethane (20 ml, followed by 4 x 10 ml) and the organic layer washed with a saturated sodium bicarbonate solution (3 x 20 ml) and brine (3 x 20 ml). The organic layer was dried over magnesium sulphate and evaporated under reduced pressure. The residue was purified using flash chromatography (3:2, EtOAc:n-hexane) to obtain the desired product in an 25 % yield. H NMR (300 MHz, CDCI₃) δ 7.49 (d, 2H), 7.09 (dd, 1 H), 6.89-7.01 (m, 3H), 6.79-6.84 (m, 1 H), 3.76 (t, 2H), 3.59 (t, 2H), 3.23 (t, 4H), 2.85 (t, 2H), 2.37, (t, 2H), 1.74-1.82 (m, 4H). MS (+ESI) calcd for C₂₀ H₂₃ F₃ N₂ O m/z: [M + H]⁺ , 396.1468; found 397.1556 [Diff(ppm) = -3.75].

4-(Thiophen-2-yl)-1 -(4-(4-(trifluoromethyl)phenyl)piperidin-1 -yl)butan-1 -one (RTC196)

4-(Thiophen-2-yl)butanoic acid (50 μΙ, 0.34 mmol), HOBt (51 mg, 0.38 mmol), TBTU (122 mg, 0.38 mmol), anhydrous triethylamine (55 μΙ, 0.544 mmol) and dry DMF (2 ml) were placed in an oven- dried Schlenk tube under a nitrogen atmosphere. The resulting solution was stirred at room temperature for 15 minutes. A second Schlenk tube was prepared containing 4[4-(trifluoromethyl) phenyl] piperidine Hydrochloride (100 mg, 0.38 mmol), anhydrous triethylamine (55 μΙ, 0.544 mmol) and dry DMF (1 ml) under a nitrogen atmosphere. The resulting solution was stirred until complete dissolution of the piperidine had occurred. The piperidine solution was then transferred, via a cannula, to the first Schlenk tube containing the carboxylic acid. The resulting solution was stirred for 24 hrs, under nitrogen, and monitored by TLC. After 24 hrs, the DMF was removed under reduced pressure and the resulting oil was acidified using a 0.1 M hydrochloric acid solution. The aqueous mixture was extracted with dichloromethane (20 ml, followed by 4 x 10 ml) and the organic layer washed with a saturated sodium bicarbonate solution (3 x 20 ml) and brine (3 x 20 ml). The organic layer was dried over magnesium sulphate and evaporated under reduced pressure. The residue was purified using flash chromatography (3:2, EtOAc:n-hexane) to obtain the desired product in an 48 % yield. H NMR (300 MHz, CDCI₃) δ 7.55 (d, 2H), 7.29 (d, 2H), 7.1 1 (dd, 1 H), 6.91-6.94 (m, 1 H), 6.81- 6.82 (m, 1 H), 4.8 (d, 1 H), 3.90 (d, 1 H), 3.06-3.14 (m, 1 H), 2.91 (t, 2H), 2.74-2.85 (m, 1 H), 2.59-2.67 (m, 1 H), 2.39 (t, 2H), 2.01-2.1 1 (m, 2H), 1.87 (d, 2H), 1 .52-1.69 (m, 2H). MS (+ESI) calcd for C₂₀ H₂₂ F₃ N O S m/z: [M + H]⁺ , 381 .1361 ; found 382.1447 [Diff(ppm) = -3.39].

3-(Thiophen-2-yl)-1 -(4-(4-(trifluoromethyl)phenyl)piperazin-1 -yl)propan-1 -one (RTC532)

3-(2-Thienyl) propanoic acid (50 mg, 0.32 mmol), HOBt (47 mg, 0.35 mmol), TBTU (1 13 mg, 0.35 mmol), anhydrous triethylamine (71 μΙ, 0.51 mmol) and dry DMF (2 ml) were placed in an oven-dried Schlenk tube under a nitrogen atmosphere. The resulting solution was stirred at room temperature for 15 minutes. A second Schlenk tube was prepared containing 1-(4-trifluoromethyl phenyl) piperazine (81 mg, 0.35 mmol and dry DMF (1 ml) under a nitrogen atmosphere. The resulting solution was stirred until complete dissolution of the piperazine had occurred. The piperazine solution was then transferred, via a cannula, to the first Schlenk tube containing the carboxylic acid. The resulting solution was stirred for 24 hrs, under nitrogen, and monitored by TLC. After 24 hrs, the DMF was removed under reduced pressure and the resulting oil was acidified using a 0.1 M hydrochloric acid solution. The aqueous mixture was extracted with dichloromethane (20 ml, followed by 4 x 10 ml) and the organic layer washed with a saturated sodium bicarbonate solution (3 x 20 ml) and brine (3 x 20 ml). The organic layer was dried over magnesium sulphate and evaporated under reduced pressure. The residue was purified using flash chromatography (3:2, EtOAc:n-hexane) to obtain the desired product in an 44 % yield. H NMR (300 MHz, CDCI₃) δ 7.48 , (d, 2H), 7.1 1 (dd, 1 H), 6.88-6.93 (m, 2H), 6.84-6.85 (m, 2H), 3.76 (t, 2H), 3.57 (t, 2H), 3.17-3.26 (m, 6H), 2.69 (t, 2H). MS (+ESI) calcd for C₁₈ H₁₉ F₃ N₂ O m/z: [M + H]⁺ , 368.1 164; found 369.1243 [Diff(ppm) = -1.25].

1 -(4-(4-(Trifluoromethyl)phenyl)piperazin-1 -yl)octan-1 -one (RTC533)

Octanoic acid (62 μΙ, 0.39 mmol), HOBt (58 mg, 0.43 mmol), TBTU (138 mg, 0.43 mmol), anhydrous triethylamine (87 μΙ, 0.63 mmol) and dry DMF (2 ml) were placed in an oven-dried Schlenk tube under a nitrogen atmosphere. The resulting solution was stirred at room temperature for 15 minutes. A second Schlenk tube was prepared containing 1-(4-trifluoromethyl phenyl) piperazine (100 mg, 0.43 mmol and dry DMF (1 ml) under a nitrogen atmosphere. The resulting solution was stirred until complete dissolution of the piperazine had occurred. The piperazine solution was then transferred, via a cannula, to the first Schlenk tube containing the carboxylic acid. The resulting solution was stirred for 24 hrs, under nitrogen, and monitored by TLC. After 24 hrs, the DMF was removed under reduced pressure and the resulting oil was acidified using a 0.1 M hydrochloric acid solution. The aqueous mixture was extracted with dichloromethane (20 ml, followed by 4 x 10 ml) and the organic layer washed with a saturated sodium bicarbonate solution (3 x 20 ml) and brine (3 x 20 ml). The organic layer was dried over magnesium sulphate and evaporated under reduced pressure. The residue was purified using flash chromatography (3:2, EtOAc:n-hexane) to obtain the desired product in an 63 % yield. H NMR (300 MHz, CDCI₃) δ 7.48 (d, 2H), 6.91 (d, 2H), 3.77 (t, 2H), 3.62 (t, 2H), 3.24-3.30 (m, 4H), 2.34 (t, 2H), 1.61-1.71 (m, 2H), 1 .29-1.34 (m, 8H), 0.86-0.91 (m, 3H). MS (+ESI) calcd for C₁₉ H₂₇ F₃ N₂ O m/z: [M + H]⁺ , 357.2148; found 357.2136 [Diff(ppm) = -3.36].

3-(Furan-2-yl)-1-(4-(4-(trifluoromethyl) phenyl) piperazin-1-yl)propan-1-one (RTC535)

3-(2-Furyl)propanoic acid (54 mg, 0.39 mmol), HOBt (58 mg, 0.43 mmol), TBTU (138 mg, 0.43 mmol), anhydrous triethylamine (87 μΙ, 0.63 mmol) and dry DMF (2 ml) were placed in an oven-dried Schlenk tube under a nitrogen atmosphere. The resulting solution was stirred at room temperature for 15 minutes. A second Schlenk tube was prepared containing 1-(4-trifluoromethyl phenyl) piperazine (100 mg, 0.43 mmol and dry DMF (1 ml) under a nitrogen atmosphere. The resulting solution was stirred until complete dissolution of the piperazine had occurred. The piperazine solution was then transferred, via a cannula, to the first Schlenk tube containing the carboxylic acid. The resulting solution was stirred for 24 hrs, under nitrogen, and monitored by TLC. After 24 hrs, the DMF was removed under reduced pressure and the resulting oil was acidified using a 0.1 M hydrochloric acid solution. The aqueous mixture was extracted with dichloromethane (20 ml, followed by 4 x 10 ml) and the organic layer washed with a saturated sodium bicarbonate solution (3 x 20 ml) and brine (3 x 20 ml). The organic layer was dried over magnesium sulphate and evaporated under reduced pressure. The residue was purified using flash chromatography (3:2, EtOAc:n-hexane) to obtain the desired product in an 31 % yield. H NMR (300 MHz, CDCI₃) δ 7.48 (d, 2H), 7.31 (s, 1 H), 6.89 (d, 2H), 6.28 (t, 1 H), 6.04 (d, 1 H), 3.77 (t, 2H), 3.58 (t, 2H), 3.21-3.26 (m, 4H), 3.00 (t, 2H), 2.69 (t, 2H). MS (+ESI) calcd for C₁₈ H₁₉ F₃ N₂ 0₂ m/z: [M + H]⁺ , 353.1471 ; found 353.1461 [Diff(ppm) = - 2.83].

2-(Thiophen-2-yl)-1 -{4-[4-(trifluoromethyl)phenyl]piperazin-1 -yl}ethan-1 -one (RTC536) 2-Thienylacetic acid (54 mg, 0.39 mmol), HOBt (58 mg, 0.43 mmol), TBTU (138 mg, 0.43 mmol), anhydrous triethylamine (87 μΙ, 0.63 mmol) and dry DMF (2 ml) were placed in an oven-dried Schlenk tube under a nitrogen atmosphere. The resulting solution was stirred at room temperature for 15 minutes. A second Schlenk tube was prepared containing 1-(4-trifluoromethyl phenyl) piperazine (100 mg, 0.43 mmol and dry DMF (1 ml) under a nitrogen atmosphere. The resulting solution was stirred until complete dissolution of the piperazine had occurred. The piperazine solution was then transferred, via a cannula, to the first Schlenk tube containing the carboxylic acid. The resulting solution was stirred for 24 hrs, under nitrogen, and monitored by TLC. After 24 hrs, the DMF was removed under reduced pressure and the resulting oil was acidified using a 0.1 M hydrochloric acid solution. The aqueous mixture was extracted with dichloromethane (20 ml, followed by 4 x 10 ml) and the organic layer washed with a saturated sodium bicarbonate solution (3 x 20 ml) and brine (3 x 20 ml). The organic layer was dried over magnesium sulphate and evaporated under reduced pressure. The residue was purified using flash chromatography (3:2, EtOAc:n-hexane) to obtain the desired product in an 20 % yield. H NMR (300 MHz, CDCI₃) δ 7.47 (d, 2H), 7.19 (dd, 1 H), 6.88-6.97 (m, 4H), 3.96 (s, 2H), 3.79 (t, 2H), 3.66 (t, 2H), 3.24 (t, 2H), 3.15 (t, 2H). MS (+ESI) calcd for C₁₇ H₁₇ F₃ N₂ 0₂ S m/z: [M + H]⁺ , 355.1086; found 355.1084 [Diff(ppm) = -0.56].

6-(Thiophen-2-yl)-1 -(4-(4-(trifluoromethyl)phenyl)piperazin-1 -yl)hexan-1 -one (RTC537) 6-(Thiophen-2-yl) hexanoic acid (77 mg, 0.39 mmol), HOBt (58 mg, 0.43 mmol), TBTU (138 mg, 0.43 mmol), anhydrous triethylamine (87 μΙ, 0.63 mmol) and dry DMF (2 ml) were placed in an oven-dried Schlenk tube under a nitrogen atmosphere. The resulting solution was stirred at room temperature for 15 minutes. A second Schlenk tube was prepared containing 1-(4-trifluoromethyl phenyl) piperazine (100 mg, 0.43 mmol and dry DMF (1 ml) under a nitrogen atmosphere. The resulting solution was stirred until complete dissolution of the piperazine had occurred. The piperazine solution was then transferred, via a cannula, to the first Schlenk tube containing the carboxylic acid. The resulting solution was stirred for 24 hrs, under nitrogen, and monitored by TLC. After 24 hrs, the DMF was removed under reduced pressure and the resulting oil was acidified using a 0.1 M hydrochloric acid solution. The aqueous mixture was extracted with dichloromethane (20 ml, followed by 4 x 10 ml) and the organic layer washed with a saturated sodium bicarbonate solution (3 x 20 ml) and brine (3 x 20 ml). The organic layer was dried over magnesium sulphate and evaporated under reduced pressure. The residue was purified using flash chromatography (3:2, EtOAc:n-hexane) to obtain the desired product in an 75 % yield. H NMR (300 MHz, CDCI₃) δ 7.48 (d, 2H), 7.08 (dd, 1 H), 6.88-6.93 (m, 3H), 6.76-6.77 (m, 1 H), 3.75 (t, 2H), 3.59 (t, 2H), 3.22-3.28 (m, 4H), 2.81 (t, 2H), 2.34 (t, 2H), 1.65-1.77 (m, 4H), 1.44-1.48 (m, 4H). MS (+ESI) calcd for C₁₈ H₂₁ F₃ N₄ O m/z: [M + Na]⁺ , 410.1621 ; found 433.1532 [Diff(ppm) = -1.15].

3-(Thiophen-3-ylthio)propanoic acid (RD 23)

Thienyl-2-thiol (48 μΙ, 0.52 mmol), 3-bromopropanoic acid (80 mg, 0.52 mmol) and sodium bicarbonate (130 mg, 1.56 mmol) were refluxed in ethanol (2 ml) for 5 hrs. The resulting reaction mixture was dissolved in water and washed with diethyl ether (20 ml, followed by 4 x 10 ml). The aqueous layer was subsequently acidified with a 0.1 M HCI solution (~ 30 ml) and extracted with diethyl ether (20 ml, followed by 4 x 10 ml) to obtain the desired product in an 85 % yield. H NMR (300 MHz, CDCI₃) δ 10.69 (br s, 1 H), 7.36 (dd, 1 H), 7.15 (dd, 1 H), 6.97-6.99 (m, 1 H), 2.97 (t, 2H), 2.64 (t, 2H). MS (+ESI) calcd for C₁₈ H₂₁ F₃ N₄ O m/z: [M + H]⁺ , 189.0038; found 189.0040 [Diff(ppm) = 1 .06].

1 -(4-(3-Chloro-5-(trifluoromethyl)pyridin-2-yl)piperazin-1 -yl)-3-(thiophen-2-ylthio)propan-1 -one (RTB 70)

3-(Thiophen-3-ylthio)propanoic acid (450 mg, 2.39 mmol), HOBt (698 mg, 2.63 mmol), TBTU (767 mg, 2.63 mmol), anhydrous triethylamine (532 μΙ, 3.82 mmol) and dry DMF (2 ml) were placed in an oven-dried Schlenk tube under a nitrogen atmosphere. The resulting solution was stirred at room temperature for 15 minutes. A second Schlenk tube was prepared containing 1-[3-chloro-5- (trifluoromethyl)-2-pyridinyl] piperazine (698 mg, 2.63 mmol) and dry DMF (1 ml) under a nitrogen atmosphere. The resulting solution was stirred until complete dissolution of the piperazine had occurred. The piperazine solution was then transferred, via a cannula, to the first Schlenk tube containing the carboxylic acid. The resulting solution was stirred for 24 hrs, under nitrogen, and monitored by TLC. After 24 hrs, the DMF was removed under reduced pressure and the resulting oil was acidified using a 0.1 M hydrochloric acid solution. The aqueous mixture was extracted with dichloromethane (20 ml, followed by 4 x 10 ml) and the organic layer washed with a saturated sodium bicarbonate solution (3 x 20 ml) and brine (3 x 20 ml). The organic layer was dried over magnesium sulphate and evaporated under reduced pressure. The residue was purified using flash chromatography (3:2, EtOAc:n-hexane) to obtain the desired product in a 71 % yield. H NMR (300 MHz, CDCI₃) δ 8.40-8.41 (m, 1 H), 7.79 (d, 1 H), 7.35 (dd, 1 H), 7.14 (dd, 1 H), 6.97-7.00 (m, 1 H), 3.75- 3.79 (m, 2H), 3.46-3.59 (m, 6H), 3.09 (t, 2H), 2.66 (t, 2H). MS (+ESI) calcd for C₁₇ H₁₇ CI F₃ N₃ O S m/z: [M + H]⁺ , 436.0526; found 436.0531 [Diff(ppm) = 1 .15].

4-(Thiophen-2-yl)butan-1 -ol (RD28) UAIH₄ (26 mg, 0.68 mmol) was added to THF (500 μΙ_) and a solution of 4-(thiophen-2-yl)butanoic acid (100 μΙ_, 0.68 mmol) in THF (500 μΙ_) was subsequently added. The resulting solution was stirred at room temperature and monitored by TLC. After 6 hours, the THF was removed under reduced pressure and the residue purified using flash chromatography (DCM, R_f = 0.25) to give the desired product in a 90% yield. H NMR (300 MHz, CDCI₃) δ 7.09 (dd, J = 5.1 Hz, J = 1 .2 Hz, 1 H), 6.92-6.89 (m, 1 H) 6.79-6.77 (m, 1 H), 3.68 (t, J = 6.6 Hz, 2H), 2.88 (t, J = 7.8 Hz, 2H), 1.78-1.70 (m, 2H), 1.65-1.62 (m, 2H), 1.60 (br s, 1 H). 4-(Thiophen-2-yl)butyl methanesulfonate (RD31)

To a solution of 4-(thiophen-2-yl)butan-1-ol (RD28; 55 mg, 0.35 mmol) and triethylamine (58 μΙ_, 0.42 mmol) in anhydrous DCM kept at 0°C, was added mesyl chloride (29 μΙ_, 0.38 mmol). The reaction mixture was maintained at 0°C for 1 hour, followed by warming to room temperature. The mixture was stirred at room temperature under a nitrogen atmosphere and monitored by TLC. After 3 hours, the solvent was removed and the residue purified using flash chromatography (3:2 EtOAc:n-hexane) to give the desired compound in an 89% yield. H NMR (300 MHz, CDCI₃) δ 7.13 (dd, J = 5.1 Hz, J = 1.2 Hz, 1 H), 6.93-6.90 (m, 1 H) 6.80-6.78 (m, 1 H) 4.25-4.21 (m, 2H), 2.98 (s, 3H), 2.88-2.86 (m, 2H), 1.83-1.78 (m, 4H). ³C NMR (75 MHz, CDCI₃) 143.2, 125.8, 123.4, 122.2, 68.7, 36.3, 28.1 , 27.4, 26.5. MS (+ESI) calcd for C₉ H₁₄ 0₃ S₂ m/z: [M + Na]⁺, 257.0277; found 257.0273 [Diff(ppm) = -1.6].

1-(2-Chloro-4-(trifluoromethyl)phenyl) piperazine (RD20)

3- Chloro-4-fluorobenzotrifluoride (100 μΙ_, 0.74 mmol) and piperazine (129 mg, 1 .49 mmol) were dissolved in NMP (2 mL) and heated at 200°C for 30 mins in a microwave reactor. The reaction mixture was purified using flash chromatography (1 : 10 MeOH:DCM) to obtain the desired product in a 61 % yield. H NMR (300 MHz, CDCI₃) δ 7.61 (d, J = 1 .5 Hz, 1 H), 7.45 (dd, J = 8.4, J = 1.5 Hz, 1 H), 7.07 (d, J = 8.4 Hz, 1 H), 3.09 (s, 8H), 2.68 (br s, 1 H). ³C NMR (75 MHz, CDCI₃) 151.6, 128.8, 127.7 (q, J = 3.75 Hz), 125.3 (q, J = 33 Hz), 124.6 (q, J = 3.75 Hz), 120.1 , 1 18.1 (q, J = 270 Hz), 52.0, 45.9. MS (+ESI) calcd for Cn H₁₂ CI F₃ N₂ m/z: [M + H] ⁺, 264.06; found 265.071 1 [Diff(ppm) = -1.13].

1-(4-(2-Chloro-4-(trifluoromethyl)phenyl)piperazin-1-yl)-4-(thiophen-2-yl)butan-1-one (RTC8)

4- (Thiophen-2-yl)butanoic acid (46 μΙ_, 0.31 mmol), HOBt (47 mg, 0.34 mmol), TBTU (1 1 1 mg, 0.34 mmol), anhydrous triethylamine (0.131 ml, 0.5 mmol), and anhydrous DMF (5 mL) were placed in an oven-dried Schlenk tube under a nitrogen atmosphere. The resulting solution was stirred at room temperature for 15 minutes. A second Schlenk tube was prepared containing 1-(2-chloro-4- (trifluoromethyl)phenyl) piperazine (RD20; 90 mg, 0.34 mmol) and anhydrous DMF (1 mL) under a nitrogen atmosphere. The resulting solution was stirred until complete dissolution of the piperazine had occurred. The piperazine solution was then transferred, via a cannula, to the first Schlenk tube containing the carboxylic acid. The solution was stirred under nitrogen and monitored by TLC. After 24 hours, the DMF was removed under reduced pressure and the resulting oil was acidified using a 0.1 M HCI solution. The aqueous mixture was extracted with DCM (20 mL, followed by 4 x 10 mL) and the organic layer washed with a saturated sodium bicarbonate solution (3 x 20 mL) and brine (3 x 20 mL). The organic layer was dried over magnesium sulphate and the solvent removed in vacuo. The residue was purified using flash chromatography (1 : 1 , EtOAc:Pet. Ether) to obtain the desired product in an 80% yield. H NMR (300 MHz, CDCI₃) δ 7.62 (d, J = 1 .5 Hz, 1 H), 7.46 (dd, J = 8.4 Hz, J = 1.2 Hz, 1 H), 7.1 1 (dd, J = 5.1 Hz, J = 1.2 Hz, 1 H), 7.03 (d, J = 8.4 Hz, 1 H), 6.93-6.90 (m, 1 H), 6.81-6.80 (m, 1 H), 3.82-3.79 (m, 2H), 3.61-3.57 (m, 2H), 3.07-3.04 (m, 4H), 2.90 (t, J = 7.2 Hz, 2H), 2.38 (t, J = 7.2 Hz, 2H), 2.15-2.01 (m, J = 7.2 Hz, 2H). ^1JC NMR (75 MHz, CDCI₃) 171 .1 , 151 .6, 144.3, 128.8, 127.8 (q, J = 3.75 Hz), 126.8, 125.3 (q, J = 33 Hz), 124.7 (q, J = 3.75 Hz), 124.5, 123.2, 120.3, 1 18.1 (q, J = 270 Hz), 51 .1 , 50.7, 45.5, 41.6, 32.0, 29.2, 27.0. MS (+ESI) calcd for C₁₉ H₂₀ CI F₃ N₂ O S m/z: [M + H] ⁺, 416.0941 ; found 417.1017 [Diff(ppm) = 1.74].

1-(4-(4-(Trifluoromethyl)phenyl)piperazin-1-yl)ethanone (RTC12) Acetic acid (33 μΙ_, 0.59 mmol), HOBt (87 mg, 0.65 mmol), TBTU (208 mg, 0.65 mmol), anhydrous triethylamine (131 μΙ_, 0.94 mmol) and anhydrous DMF (2 mL) were placed in an oven-dried Schlenk tube under a nitrogen atmosphere. The resulting solution was stirred at room temperature for 15 minutes. A second Schlenk tube was prepared containing 1-(4-(trifluoromethyl)phenyl)piperazine (150 mg, 0.65 mmol) and anhydrous DMF (1 mL) under a nitrogen atmosphere. The resulting solution was stirred until complete dissolution of the piperazine had occurred. The piperazine solution was then transferred, via a cannula, to the first Schlenk tube containing the carboxylic acid. The resulting solution was stirred under nitrogen and monitored by TLC. After 24 hours, the DMF was removed under reduced pressure and the resulting oil was acidified using a 0.1 M HCI solution. The aqueous mixture was extracted with DCM (20 mL, followed by 4 x 10 mL) and the organic layer washed with a saturated sodium bicarbonate solution (3 x 20 mL) and brine (3 x 20 mL). The organic layer was dried over magnesium sulphate and the solvent removed in vacuo. The residue was purified using flash chromatography (4:1 , EtOAc:n-hexane) to obtain the desired product in a 57% yield. H NMR (300 MHz, CDCI₃) δ 7.49 (d, J = 8.7 Hz, 2H), 6.91 (d, J = 8.7 Hz, 2H), 3.79-3.76 (m, 2H), 3.65-3.61 (m, 2H), 3.31-3.24 (m, 4H), 2.14 (s, 3H). ³C NMR (75 MHz, CDCI₃) 168.0, 151.9, 125.4 (q, J = 3.75 Hz), 1 19.6 (q, J = 33 Hz), 1 18.2 (q, J = 268.5 Hz), 1 15.0, 48.3, 48.0, 45.8, 41.0, 21.3. MS (+ESI) calcd for C₁₃ H₁₅ F₃ N₂ O m/z: [M + H]⁺, 273.1209; found 273.1222 [Diff(ppm) = 4.8].

1-(Thiophen-2-yl)-4-(4-(4-(trifluoromethyl)phenyl)piperidin-1-yl)butane-1,4-dione (RTC39)

4-Oxo-4-(thiophen-2-yl)butanoic acid (63 [it, 0.34 mmol), HOBt (50 mg, 0.37 mmol), TBTU (120 mg, 0.37 mmol), anhydrous triethylamine (76 μΙ_, 0.55 mmol) and anhydrous DMF (2 mL) were placed in an oven-dried Schlenk tube under a nitrogen atmosphere. The resulting solution was stirred at room temperature for 15 minutes. A second Schlenk tube was prepared containing 4-(4- (trifluoromethyl)phenyl)piperidine.HCI (218 mg, 0.816 mmol), anhydrous triethylamine (95 μΙ_, 0.68 mmol) and anhydrous DMF (1 mL) under a nitrogen atmosphere. The resulting solution was stirred until complete dissolution of the piperidine had occurred. The piperidine solution was then transferred, via a cannula, to the first Schlenk tube containing the carboxylic acid. The resulting solution was stirred under nitrogen and monitored by TLC. After 24 hours, the DMF was removed under reduced pressure and the resulting oil was acidified using a 0.1 M HCI. The aqueous mixture was extracted with DCM (20 mL, followed by 4 x 10 mL) and the organic layer washed with a saturated sodium bicarbonate solution (3 x 20 mL) and brine (3 x 20 mL). The organic layer was dried over magnesium sulphate and the solvent removed in vacuo. The residue was purified using flash chromatography (3:2, EtOAc:n-hexane) to obtain the desired product in a 70% yield. H NMR (300 MHz, CDCI₃) δ 7.81 (dd, J = 3.9 Hz, J = 0.9 Hz, 1 H), 7.64 (dd, J = 4.8 Hz, J = 0.9, 1 H), 7.55 (d, J = 8.4 Hz, 2H), 7.30 (d, J = 8.4 Hz, 2H), 7.12 (m, 1 H), 4.81-4.76 (m, 1 H), 4.15-4.10 (m, 1 H), 3.34- 3.26 (m, 2H), 3.23-3.13 (m, 1 H), 2.86-2.75 (m, 3H), 2.75-2.62 (m, 1 H), 1.96-1.85 (m, 2H), 1.76-1.55 (m, 2H). ³C NMR (75 MHz, CDCI₃) 192.1 , 169.8, 149.1 , 144.0, 132.4, 132.0, 128.2 (q, J = 32 Hz), 128.1 , 127.1 , 125.4 (q, J = 3.75), 1 18.8 (q, J = 268.5 Hz), 45.9, 42.7, 42.4, 34.3, 33.4, 32.6, 27.2. MS (+ESI) calcd for C₂₀ H₂₀ F₃ N 0₂ S m/z: [M + H]⁺, 396.1229; found 396.124 [Diff(ppm) = -2.67].

1 -(4-(6-Chloro-5-(trifluoromethyl)pyridin-2-yl)piperazin-1 -yl)-4-(thiophen-2-yl)butan-1 -one (RTC44)

4-(Thiophen-2-yl)butanoic acid (100 μί, 0.68 mmol), HOBt (1 10 mg, 0.816 mmol), TBTU (262 mg, 0.816 mmol), anhydrous triethylamine (152 μί, 1.08 mmol) and anhydrous DMF (2 mL) were placed in an oven-dried Schlenk tube under a nitrogen atmosphere. The resulting solution was stirred at room temperature for 15 minutes. A second Schlenk tube was prepared containing 1-(6-chloro-5- (trifluoromethyl) pyridin-2-yl)piperazine (218 mg, 0.816 mmol) and anhydrous DMF (1 mL) under a nitrogen atmosphere. The resulting solution was stirred until complete dissolution of the piperazine had occurred. The piperazine solution was then transferred, via a cannula, to the first Schlenk tube containing the carboxylic acid. The resulting solution was stirred under nitrogen and monitored by TLC. After 24 hours, the DMF was removed under reduced pressure and the resulting oil was acidified using a 0.1 M HCI solution. The aqueous mixture was extracted with DCM (20 mL, followed by 4 x 10 mL) and the organic layer washed with a saturated sodium bicarbonate solution (3 x 20 mL) and brine (3 x 20 mL). The organic layer was dried over magnesium sulphate and the solvent removed in vacuo. The residue was purified using flash chromatography (3:2, EtOAc:n-hexane) to obtain the desired product in a 68% yield. H NMR (300 MHz, CDCI₃) δ 7.66 (d, J = 8.7 Hz, 1 H), 7.10 (dd, J = 5.1 Hz, J = 1 .2 Hz, 1 H), 6.92-6.89 (m, 1 H), 6.80-3.79 (m, 1 H), 6.46 (d, J = 8.7 Hz, 1 H), 3.72- 3.67 (m, 4H), 3.97-3.57 (m, 2H), 3.52-3.49 (m, 2H), 2.88 (t, J = 7.5 Hz, 2H), 2.31 (t, J = 7.5 Hz, 2H), 2.09-1.99 (m, J = 7.2 Hz, 2H). ³C NMR (75 MHz, CDCI₃) 171.2, 158.9, 147.5, 144.2, 137.6 (q, J = 3.75 Hz), 126.8, 124.5, 123.2, 1 17.8 (q, J = 268.5 Hz), 1 12.0 (q, J = 33 Hz), 103.3, 44.7, 44.5, 44.1 , 40.7, 31 .9, 29.2, 26.9. MS (+ESI) calcd for C₁₈ H₁₉ CI F₃ N₃ O S m/z: [M + H]⁺, 418.0962; found 418.0953 [Diff(ppm) = -2.23].

Methyl 9-oxo-9-(4-(4-(trifluoromethyl)phenyl)piperazin-1 -yl)nonanoate (RTC56)

Methyl hydrogen azelate (479 mg, 2.3 mmol), BOP (1 .14 g, 2.6 mmol), anhydrous triethylamine (512 [it, 3.6 mmol) and anhydrous DCM (40 mL) were placed in an oven-dried three neck flask under a nitrogen atmosphere. The resulting solution was stirred at room temperature for 15 minutes. 1-(4- (Trifluoromethyl)phenyl)piperazine (600 mg, 2.6 mmol) was added and the reaction mixture stirred under nitrogen and monitored by TLC. After 16 hours, the DCM was removed under reduced pressure and the resulting oil was acidified using a 0.1 M HCI. The aqueous mixture was extracted with DCM (20 mL, followed by 4 x 10 mL) and the organic layer washed with a saturated sodium bicarbonate solution (3 x 20 mL) and brine (3 x 20 mL). The organic layer was dried over magnesium sulphate and the solvent removed in vacuo. The residue was purified using flash chromatography (3:2, EtOAc et. Ether) to obtain the desired product as an off white solid in a 98% yield. H NMR (300 MHz, CDCI₃) δ 7.41 (d, J = 8.7 Hz, 2H), 6.85 (d, J = 8.7 Hz, 2H), 3.73-3.69 (m, 2H), 3.58 (overlapping -OCH3 and piperazine -CH2 signal, m, 5H), 3.23-3.19 (m, 4H), 2.28 (t, J = 7.8 Hz, 2H), 2.21 (t, J = 7.8 Hz, 2H), 1.58-1.56 (m, 4H), 1.30-1.26 (m, 6H). ³C NMR (75 MHz, CDCI₃) 174.2, 171 .7, 152.9, 126.4 (q, J = 3.75 Hz), 120.6 (q, J = 33 Hz), 1 19.2 (q, J = 271.5 Hz), 1 14.9, 51.4, 48.4, 48.1 , 45.1 , 41.1 , 34.0, 33.2, 29.2, 29.0, 28.9, 25.1 , 24.8. MS (+ESI) calcd for C₂i H₂₉ F₃ N₂ 0₃ m/z: [M + H]⁺, 415.2203; found 415.219 [Diff(ppm) = -3.14].

1-(4-(2-Bromo-4-(trifluoromethyl)phenyl)piperazin-1-yl)-4-(thiophen-2-yl)butan-1-one (RTC62) 3-Bromo-4-chlorobenzotrifluoride and piperazine were dissolved in NMP (2 mL) and heated to 200°C for 30 mins in a microwave reactor. NMP was removed using flash chromatography (1 :10 MeOH:DCM) to give 1-(2-bromo-4-(trifluoromethyl)phenyl) piperazine (RD71 ) which was used without further purification. 4-(thiophen-2-yl)butanoic acid (41 μί, 0.28 mmol), HOBt (41 mg, 0.3 mmol), TBTU (98 mg, 0.3 mmol), anhydrous triethylamine (62 μί, 0.44 mmol) and anhydrous DMF (2 mL) were placed in an oven-dried Schlenk tube under a nitrogen atmosphere. The resulting solution was stirred at room temperature for 15 minutes. A second Schlenk tube was prepared containing RD71 (95 mg, 0.3 mmol) and anhydrous DMF (1 mL) under a nitrogen atmosphere. The resulting solution was stirred until complete dissolution of the piperazine had occurred. The piperazine solution was then transferred, via a cannula, to the first Schlenk tube containing the carboxylic acid. The resulting solution was stirred under nitrogen, and monitored by TLC. After 24 hours, the DMF was removed under reduced pressure and the resulting oil was acidified using a 0.1 M HCI solution. The aqueous mixture was extracted with DCM (20 mL, followed by 4 x 10 mL) and the organic layer washed with a saturated sodium bicarbonate solution (3 x 20 mL) and brine (3 x 20 mL). The organic layer was dried over magnesium sulphate and the solvent removed in vacuo. The residue was purified using flash chromatography (1 :1 EtOAc:n-hexane) to obtain the desired product in a 50% yield. H NMR (300 MHz, CDCI₃) δ 7.82 (d, J = 1 .5 Hz, 1 H), 7.51 (dd, J = 8.4 Hz, J = 1.5 Hz, 1 H), 7.1 1 (dd, J = 5.1 Hz, J = 1 .2 Hz, 1 H), 7.03 (d, J = 8.4 Hz, 1 H), 6.94-6.91 (m, 1 H), 6.82-6.80 (m, 1 H), 3.83-3.80 (m, 2H), 3.61-3.58 (m, 2H), 3.06-303 (m, 4H), 2.90 (t, J = 7.2 Hz, 2H), 2.34 (t, J = 7.2 Hz, 2H), 2.01 (m, J = 7.5 Hz, 2H). ³C NMR (75 MHz, CDCI₃) 171.1 , 151.6, 144.3, 131.0 (q, J = 3.5 Hz), 126.8, 125.9 (q, J = 33 Hz), 125.4 (q, J = 3.5 Hz), 124.5, 123.2, 120.8, 1 19.4, 1 17.9 (q, J = 270 Hz), 51.6, 51 .1 , 45.6, 41.6, 32.0, 29.3, 27.0. MS (+ESI) calcd for C₁₉ H₂₀ Br F₃ N₂ O S m/z: [M + K] ⁺, 499.0079; found 499.0063 [Diff(ppm) = 3.1].

5-Phenyl-1-(4-(4-(trifluoromethyl)phenyl)piperazin-1-yl)pentan-1-one (RTC16)

5-Phenylpentanoic acid (105 mg, 0.59 mmol), HOBt (87 mg, 0.65 mmol), TBTU (208 mg, 0.65 mmol), anhydrous triethylamine (131 μΙ_, 0.94 mmol) and anhydrous DMF (2 mL) were placed in an oven-dried Schlenk tube under a nitrogen atmosphere. The resulting solution was stirred at room temperature for 15 minutes. A second Schlenk tube was prepared containing 1-(4- (trifluoromethyl)phenyl)piperazine (150 mg, 0.65 mmol) and anhydrous DMF (1 mL) under a nitrogen atmosphere. The resulting solution was stirred until complete dissolution of the piperazine had occurred. The piperazine solution was then transferred, via a cannula, to the first Schlenk tube containing the carboxylic acid. The resulting solution was stirred under nitrogen and monitored by TLC. After 24 hours, the DMF was removed under reduced pressure and the resulting oil was acidified using a 0.1 M HCI solution. The aqueous mixture was extracted with DCM (20 mL, followed by 4 x 10 mL) and the organic layer washed with a saturated sodium bicarbonate solution (3 x 20 mL) and brine (3 x 20 mL). The organic layer was dried over magnesium sulphate and the solvent removed in vacuo. The residue was purified using flash chromatography (3:2, EtOAc:n-hexane) to obtain the desired product in an 44 % yield. H NMR (300 MHz, CDCI₃) δ 7.50-7.46 (m, 2H), 7.29- 7.14 (m, 5H), 6.91-6.87 (m, 2H), 3.74 (d, J = 4.5 Hz, 2H), 3.55 (d, J = 4.5 Hz, 2H), 3.20 (d, J = 3.6 Hz, 4H), 2.63 (d, J = 4.8 Hz, 2H), 2.35 (d, J = 4.8 Hz, 2H), 1.68 (d, J = 4.5 Hz, 4H). ³C NMR (75 MHz, CDCI₃) 171.5, 152.9, 142.1 , 128.4, 128.3, 126.4 (q, J = 3 Hz), 125.8, 120.6 (q, J = 33 Hz), 1 19.2 (q, J = 271.5 Hz), 1 14.9, 48.3, 48.1 , 45.1 , 41.1 , 35.7, 33.1 , 31.1 , 24.8. MS (+ESI) calcd for C₂₂ H₂₅ F₃ N₂ O m/z: [M + Na]⁺, 413.181 1 ; found 413.1794 [Diff(ppm) = - 4.08].

4-Hydroxy-4-(thiophen-2-yl)-1 -(4-(4-(trifluoromethyl)phenyl)piperazin-1 -yl)butan-1 -one (RTC29) 1-(Thiophen-3-yl)-4-(4-(4-(trifluoromethyl)phenyl)piperazin- (RTC2; 350 mg,

0.88 mmol) was added to MeOH (3 mL) followed by NaBH₄ (66 mg, 1 .76 mmol) and the suspension was heated at 50°C overnight. MeOH was removed under reduced pressure and the resulting solid re-dissolved in EtOAc (15 mL). The solution was washed with H₂0 (10 mL) and extracted with EtOAc (3 x 10 mL) and the combined organic layers dried over magnesium sulphate the solvent removed in vacuo. The residue was purified using flash chromatography (19: 1 , DCM:MeOH) to obtain the desired product in an 81 % yield. H NMR (300 MHz, CDCI₃) δ 7.48 (d, J = 8.7 Hz, 2H), 7.23-7.21 (m, 1 H), 6.98-6.69 (m, 2H), 6.90 (d, J = 8.7 Hz, 2H), 5.08-5.04 (m, 1 H), 3.80-3.77 (m, 2H), 3.63-3.60 (m, 2H), 3.32-3.26 (m, 4H), 2.57-2.53 (m, 2H), 2.27-2.15 (m, 2H). ³C NMR (75 MHz, CDCI₃) 170.9, 151 .7, 147.8, 125.7, 125.4 (q, J = 3.5 Hz), 123.2, 122.2, 1 19.7 (q, J = 33 Hz), 1 18.1 (q, J = 270 Hz), 1 13.9, 68.6, 47.2, 46.9, 44.1 , 40.3, 32.9, 28.3. MS (+ESI) calcd for C₁₉ H₂₁ F₃ N₂ 0₂ S m/z: [2M + Na] ⁺, 819.2484; found 819.2444 [Diff(ppm) = 4.86]

methyl 9-oxo-9-(4-(4-(trifluoromethyl)phenyl)piperidin-1 -yl)nonanoate (RTC78)

Methyl hydrogen azelate (31 1 μί, 1.6 mmol), HOBt (238 mg, 1.7 mmol), TBTU (568 mg, 1 .76 mmol), anhydrous triethylamine (394 μί, 2.8 mmol) and anhydrous DMF (4 mL) were placed in an oven- dried Schlenk tube under a nitrogen atmosphere. The resulting solution was stirred at room temperature for 15 minutes. A second Schlenk tube was prepared containing 4[4-(trifluoromethyl) phenyl] piperidine.HCI (470 mg, 1.7 mmol), anhydrous triethylamine (394 μΙ_, 2.8 mmol) and anhydrous DMF (2 mL) under a nitrogen atmosphere. The resulting solution was stirred until complete dissolution of the piperidine had occurred. The piperidine solution was then transferred, via a cannula, to the first Schlenk tube containing the carboxylic acid. The resulting solution was stirred for 24 hrs, under nitrogen, and monitored by TLC. After 24 hrs, the DMF was removed under reduced pressure and the resulting oil was acidified using a 0.1 M HCI solution. The aqueous mixture was extracted with DCM (20 mL, followed by 4 x 10 mL) and the organic layer washed with a saturated sodium bicarbonate solution (3 x 20 mL) and brine (3 x 20 mL). The organic layer was dried over sodium sulphate and the solvent removed in vacuo. The residue was purified using flash chromatography (3:2, EtOAc:n-hexane) to obtain the desired product in an 66 % yield. H NMR (300 MHz, CDCI₃) δ 7.58 (d, J = 8.1 Hz, 2H), 7.32 (d, J = 8.1 Hz, 2H), 4.84 (d, J = 13 Hz, 1 H), 4.02 (d, J = 13 Hz, 1 H), 3.65 (s, 3H), 3.18 (t, J = 13 Hz, 1 H), 2.85-2.77 (m, 1 H), 2.66 (t, J = 13 Hz, 1 H), 2.39-2.81 (m, 4H), 1.94-1.86 (m, 2H), 1.67-1.60 (m, 6H), 1.35-1.34 (m, 6H). ³C NMR (75 MHz, CDCI₃) 174.2, 171 .4, 149.2, 129.4 (q, J = 32 Hz), 127.1 , 125.5 (q, J = 3.75 Hz), 1 18.7 (q, J = 270 Hz), 51.4, 46.0, 42.6, 42.0, 34.0, 33.7, 33.3, 32.6, 29.2, 29.0, 28.9, 25.3, 24.8. C₂₂ H₃₀ F₃ N 0₃ m/z: [M + H]⁺ , 414.2251 ; found 414.2259 [Diff(ppm) = 1.9]

9-oxo-9-(4-(4-(trifluoromethyl)phenyl)piperidin-1 -yl)nonanoic acid (RTC79)

Methyl 9-oxo-9-(4-(4-(trifluoromethyl)phenyl)piperidin-1-yl)nonanoate (RTC78) (315 mg, 0.76 mmol) was added to a solution of potassium hydroxide (213 mg, 3.81 mmol) in EtOH (15 mL). The solution was brought to reflux and monitored by TLC. After 5 hrs, the EtOH was removed under reduced pressure and the residue dissolved in H₂0. The aqueous layer was acidified to pH = 6 using 1 M HCI and extracted with DCM (20 mL, followed by 4 x 10 mL). The organic layer was dried over sodium sulphate and the solvent removed in vacuo. The residue was purified using flash chromatography (1 :9, MeOH:DCM) to obtain the desired product in an 86 % yield. H NMR (300 MHz, CDCI₃) δ 10.43 (s, 1 H), 7.58 (d, J = 8.1 Hz, 2H), 7.33 (d, J = 8.1 Hz, 2H), 4.84 (d, J = 13 Hz, 1 H), 4.03 (d, J = 13 Hz, 1 H), 3.20 (t, J = 13 Hz, 1 H), 2.86-2.79 (m, 1 H), 2.68 (t, J = 13 Hz, 1 H), 2.42-2.31 (m ,4H), 1.96-1.86 (m, 2H), 1.75-1.50 (m, 6H), 1.41-1.32 (m, 6H). ³C NMR (75 MHz, CDCI₃). 178.5, 172.1 , 149.1 , 128.1 (q, J = 32.25 Hz), 127.4, 125.5 (q, J = 3.75 Hz), 1 18.8 (q, J = 270 Hz), 46.2, 42.5, 42.2, 34.2, 33.6, 33.3, 32.9, 32.5, 29.2, 29.0, 28.90, 25.3, 24.7. MS (+ESI) calcd for C₂i H₂₈ F₃ N 0₃ m/z: [M + Na] ⁺, 422.19341 ; found 422.1913 [Diff(ppm) = 4.75].

Claims

1. A compound having the general formula (I):

X is an atom selected from carbon and nitrogen;

Y is selected from:

(i) a halide selected from fluoride, chloride, bromide, iodide, and astatide; and

(ii) a hydrogen atom;

Z is selected from:

(i) a short-chain alkylene, alkenylene, or alkynylene, which can be linear or branched, and which can be substituted or unsubstituted; and

(ii) a short-chain thio-alkylene, thio-alkenylene, or thio-alkynylene, which can linear or branched, and which can be substituted or unsubstituted;

R and R1 are each independently selected from:

(i) a hydrogen atom;

(ii) an oxygen atom; and

(iii) a hydroxyl group;

R2 is selected from:

(i) a five-membered heterocyclic moiety;

(iii) a carbonyl group, optionally which can be substituted or unsubstituted; and

(iv) a hydrogen atom; and R3 is an atom selected from carbon and nitrogen; or a pharmaceutically acceptable salt thereof.

2. A compound according to Claim 1 , wherein R2 is a five-membered heterocyclic moiety selected from thiophene, furan, imidazole, and pyrazole.

3. A compound according to Claim 1 , wherein R2 is a carboxyl group (-COOH), which can be substituted or unsubstituted.

4. A compound according to Claim 1 , wherein R2 is an alkoxyl substituted carbonyl group.

5. A compound according to Claim 1 , wherein R2 is an amide group, which can be substituted or unsubstituted.

6. A compound according to Claim 1 , wherein R2 is a carboxamide group, substituted with an ether group.

7. A compound according to Claim 1 , wherein R2 is a saturated or unsaturated six-membered cyclic hydrocarbon moiety.

8. A compound according to Claim 1 or 2, wherein the compound has the general formula (I); X is a nitrogen atom; Y is chloride attached to the carbon atom at position 3 of the pyridinyl moiety; Z is thioethylene; R and R1 are each a hydrogen atom; R2 is thiophene; and R3 is a nitrogen.

9. A compound according to Claim 1 or 2, wherein the compound has the general formula (I); X is a carbon atom; Y is a hydrogen atom; Z is propylene; R and R1 are each a hydrogen atom; R2 is thiophene; and R3 is nitrogen.

10. A compound according to Claim 1 or 2, wherein the compound has the general formula (I); X is a carbon atom; Y is a hydrogen atom; Z is propylene, wherein one R is an oxygen atom; R1 is a hydrogen atom; R2 is a thiophene; and R3 is nitrogen.

A compound according to Claim 1 or 2, wherein the compound has the general formula (I); X is a nitrogen atom; Y is a hydrogen atom; Z is propylene; R is an oxygen atom; R1 is a hydrogen atom; R2 is thiophene; and R3 is nitrogen.

12. A compound according to Claim 1 or 2, wherein the compound has the general formula (I); X is a nitrogen atom; Y is chloride attached to the carbon atom at position 3 of the pyridinyl moiety; Z is propylene; R is an oxygen atom; R1 is a hydrogen atom; R2 is thiophene; and R3 is nitrogen.

13. A compound according to Claim 1 or 2, wherein the compound has the general formula (I); X is a nitrogen atom; Y is a hydrogen atom; Z is propylene; R and R1 are each a hydrogen atom; R2 is thiophene; and R3 is nitrogen.

14. A compound according to Claim 1 or 2, wherein the compound has the general formula (I); X is a nitrogen atom; Y is chloride attached to the carbon atom at position 3 of the pyridinyl moiety; Z is a propylene; R and R1 are each a hydrogen atom; R2 is thiophene; and R3 is nitrogen.

15. A compound according to Claim 1 or 2, wherein the compound has the general formula (I); X is a carbon; Y is a hydrogen atom; Z is thioethylene; R and R1 are each a hydrogen atom; R2 is thiophene; and R3 is nitrogen.

16. A compound according to Claim 1 or 3, wherein the compound has the general formula (I); X is a carbon; Y is a hydrogen atom; Z is heptylene; R and R1 are each a hydrogen atom; R2 is an unsubstituted carboxyl group; and R3 is nitrogen.

17. A compound according to Claim 1 or 6, wherein the compound has the general formula (I); X is a carbon; Y is a hydrogen atom; Z is heptylene; R and R1 are each a hydrogen atom; R2 is a carboxamide group, substituted with a methoxyethanyl group; and R3 is nitrogen. 18. A compound according to Claim 1 or 2, wherein the compound has the general formula (I); X is a carbon; Y is a hydrogen atom; Z is propylene; R and R1 are each a hydrogen atom; R2 is pyrazole; and R3 is nitrogen.

19. A compound according to Claim 1 , wherein compound has the general formula (I); X is a carbon; Y is a hydrogen atom; Z is butylene; R and R1 are each a hydrogen atom; R2 is a hydrogen atom; and R3 is nitrogen.

20. A compound according to Claim 1 or 2, wherein the compound has the general formula (I); X is a carbon; Y is a hydrogen atom; Z is butylene; R and R1 are each a hydrogen atom; R2 is thiophene; and R3 is nitrogen.

21. A compound according to Claim 1 or 2, wherein the compound has the general formula (I); X is a carbon; Y is a hydrogen atom; Z is propylene; R and R1 are each a hydrogen atom; R2 is thiophene; and R3 is carbon.

22. A compound according to Claim 1 or 2, wherein the compound has the general formula (I); X is a carbon; Y is a hydrogen atom; Z is ethylene; R and R1 are each a hydrogen atom; R2 is thiophene; and R3 is nitrogen.

23. A compound according to Claim 1 , wherein the compound has the general formula (I); X is a carbon; Y is a hydrogen atom; Z is heptylene; R and R1 are each a hydrogen atom; R2 is a hydrogen atom; and R3 is nitrogen.

24. A compound according to Claim 1 or 2, wherein the compound has the general formula (I); X is a carbon; Y is a hydrogen atom; Z is ethylene; R and R1 are each a hydrogen atom; R2 is furan; and R3 is nitrogen.

25. A compound according to Claim 1 or 2, wherein the compound has the general formula (I); X is a carbon; Y is a hydrogen atom; Z is methylene; R and R1 are each a hydrogen atom; R2 is thiophene; and R3 is nitrogen.

26. A compound according to Claim 1 or 2, wherein the compound has the general formula (I); X is a carbon; Y is a hydrogen atom; Z is pentylene; R and R1 are each a hydrogen atom; R2 is thiophene; and R3 is nitrogen.

27. A compound according to Claim 1 or 2, wherein the compound has the general formula (I); X is a carbon; Y is a chloride attached to the carbon atom at position 2 of the benzene moiety; Z is propylene; R and R1 are each a hydrogen atom; R2 is thiophene; and R3 is nitrogen. 28. A compound according to Claim 1 , wherein the compound has the general formula (I); X is a carbon; Y is hydrogen; Z is methylene; R and R1 are each a hydrogen atom; R2 is hydrogen; and R3 is nitrogen.

A compound according to Claim 1 or 2, wherein the compound has the general formula (I); X is a carbon atom; Y is a hydrogen atom; Z is propylene, wherein one R is an oxygen atom; R1 is a hydrogen atom; R2 is a thiophene; and R3 is carbon.

A compound according to Claim 1 or 2, wherein the compound has the general formula (I); X is a nitrogen atom; Y is a chloride attached to the carbon atom at position 2 of the pyridinyl moiety; Z is propylene; R and R1 are each a hydrogen atom; R2 is a thiophene; and R3 is nitrogen.

31. A compound according to Claim 1 or 4, wherein the compound has the general formula (I); X is a carbon atom; Y is hydrogen; Z is heptylene; R and R1 are each a hydrogen atom; R2 is a methoxyl-substituted carbonyl group; and R3 is nitrogen.

32. A compound according to Claim 1 or 2, wherein the compound has the general formula (I); X is a carbon atom; Y is a bromide attached to the carbon atom at position 2 of the benzene moiety; Z is propylene; R and R1 are each a hydrogen atom; R2 is a thiophene; and R3 is nitrogen.

33. A compound according to Claim 1 , wherein the compound has the general formula (I); X is a carbon atom; Y is hydrogen; Z is butylene; R and R1 are each a hydrogen atom; R2 is an unsubstituted six-membered hydrocarbon moiety; and R3 is nitrogen.

34. A compound according to Claim 1 or 2, wherein the compound has the general formula (I); X is a carbon atom; Y is a hydrogen atom; Z is propylene, wherein one R is a hydroxyl group; R1 is a hydrogen atom; R2 is a thiophene; and R3 is nitrogen.

A compound according to Claim 1 or 4, wherein the compound has the general formula (I); X is a carbon atom; Y is hydrogen; Z is heptylene; R and R1 are each a hydrogen atom; R2 is a methoxyl-substituted carbonyl group; and R3 is carbon.

A compound according to Claim 1 or 3, wherein the compound has the general formula (I); X is a carbon atom; Y is hydrogen; Z is heptylene; R and R1 are each a hydrogen atom; R2 is an unsubstituted carboxyl group; and R3 is carbon.

37. A compound according to any preceding claim, wherein the pharmaceutically acceptable salt is selected from hydrochloride, hydrobromide, sulphate, methane-sulfonate, nitrate, maleate, acetate, citrate, fumarate, tartrate, succinate, benzoate, and salts with amino acids.

38. A pharmaceutical composition comprising a compound according to any one of Claims 1-37; and, optionally a pharmaceutically acceptable excipient.

39. A compound according to any one of Claims 1-37, or a pharmaceutical composition

according to Claim 38, for use in treating diabetes.

40. A compound according to any one of Claims 1-37, or a pharmaceutical composition according to Claim 38, for use in treating obesity.