UNIVERSITÀ DI PISA
Dipartimento Di Farmacia
Corso di Laurea Magistrale in Farmacia
Tesi di laurea:
DESIGN AND SYNTHESIS OF NOVEL INDAZOLE-BASED GRK2
INHIBITORS
Relatori Candidato
Prof. Taliani Sabrina Francesco Giampietro
Dr. Barresi Elisabetta
Index
1
INTRODUCTION ... .1
GPCR AND PROTEIN KINASES ... 1
GRK: isoforms, tissue expression and role ... 2
GRK2 ... 5
GRK2 regulates GPCR internalization ... 5
GRK2 INVOLVMENT IN HUMAN DISEASES ... 8
1.5.1 GRK2 involvment in insuline-resistance and obesity ... 8
1.5.2 GRK2 involvement in immune system ... 8
1.5.3 GRK2 involvement in cell proliferation ... 9
1.5.4 GRK2 Involvement in Heart Failure ... 10
1.5.5 GRK2 involvement in depression ... 12
1.5.6 GRK2 involvement in Cancer ... 12
GRK2 STRUCTURAL FEATURES ... 15
GRK2 INHIBITORS ... 17
1.7.1 Polyanions and Polycations ... 17
1.7.2 Balanol ... 17
1.7.3 Takeda Inhibitors ... 18
1.7.4 RNA Aptamers ... 20
1.7.5 Paroxetine and Derivates... 23
1.7.6 Peptides ... 26
2
INTRODUCTION OF EXPERIMENTAL SECTION ... 30
4
ABBREVIATIONS USED ... 49
5
FIGURES AND TABLES ... 50
6
REFERENCES ... 52
1
GPCR AND PROTEIN KINASES
G Protein-Coupled Receptors (GPCRs) are a group of seven-transmembrane receptors, inclused in the largest family of cell-surface receptors.[1] Upon agonist stimulation, GPCRs activate heterotrimeric G proteins, which exchange bound Guanosine diphosphate (GDP) for Guanosine triphosphate (GTP), creating the dissociation of the G protein into Gα subunit and Gβγ subunit (figure 1). This dissociation causes intracellular signals through specific effector proteins and second messengers, including Adenylyl cyclase, Protein Kinase A (PKA) and other protein kinases.[2]
Figure 1.GPCR signal transduction. GPCR forms a complex with a unique Gα subunit. When the receptors are
inactive the Gα subunit is inactive, bound to GDP and in a heterotrimeric conformation with βγ-subunits. The α and γ subunits are attached to the plasma membrane by lipid anchors. Once bound to a ligand, the receptor is activated and undergoes a conformational change, and the Gα subunit releases GDP, binds to GTP and is activated. The Gα subunit then releases the βγ complex leading to the activation of a variety of downstream effector molecules by the Gα subunit and βγ complex separately.
2
Following continuos agonist-GPCR stimulation physical intracellular process can be initiated to maintain homeostasis and prevent various diseases.[3,4] Protein kinases are involved in GPCR homologous desensitization and are a ubiquitous group of enzymes that catalyze the phosporylation of specific substrates by transferring the γ-phosphate group from ATP (or GTP) to serine, threonine, or tyrosine residues in the cytoplasmic tails and loops of the target. This phosphorylation process causes conformational changes that affect protein functions.[5] Furthermore, protein kinases play a key role in a variety of cellular processes, including apoptosis, cell proliferation, gene expression, glycogen metabolism, immune response, neurotrasmission and oncogenesis.[6,7] AGC kinases, called AGC because include the Protein Kinase A, G and C, are one of the seven major groups of human protein kinase superfamilies and consists of 60 members, including G-Protein-Coupled-Receptor Kinases (GRKs), which are a family of seven serine/threonine kinases that regulate the activity of GPCRs.[8,9]
GRK: isoforms, tissue expression and role
The GRKs are cytolitic kinases that consist of seven isoforms (GRK1-GRK7); they are subdivided into three main groups based on their sequence homology [10,11]:
a. GRK subfamily (GRK1 and GRK7);
b. Beta-adrenergic receptor kinase (β-ARK) subfamily (GRK2 and GRK3); c. and GRK4 subfamily (GRK4,GRK5 and GRK6).
All GRK isoforms present multidomain proteins that consist of an amino-terminal region, an homology (RH) domain necessary to regulate G protein signaling by phosphorylation-indipendent mechanisms, a serine/threonine protein kinase domain (KD), and a carboxyl-terminal domain.[12](figure 2)
GRKs were initially identified as serine/threonine kinases that partecipate together with arrestins in the regulation of multiple GPCR.[13]
3
GRKs have many differences in tissue expression, molecular structure and function, playing a critical role in different signaling pathways of GPCR or non-GPCR substrates. GRK1, GRK4 and GRK7 are expressed in different tissues: GRK1 and GRK7 are expressed in the retina[14]; GRK4 is expressed in the testis, and at lower levels in the brain, kidney, and uterus myometrium. GRK2, GRK3, GRK5 and GRK6 are expressed in mammalian tissues.[4]
Normally, GPCR-agonist stimulation produces a conformational change in the GPCR, which promotes the binding of G protein to the intracellular binding site on the receptor. The actived Gα and Gβγ subunits are responsible for the activation of specific effectors, which produce different intracellular second messengers, that can generate cellular responses. After continuos agonist stimulation, GPCRs can undergo a desensibilizzation process mediated by specific proteins.(figure 3)
Figure 2. The structure of GRK. (a) Schematic representation of the GRK structure. GRKs
have a conserved regulator of G-protein signaling homology (RH) domain (shown in blue). The conserved catalytic domain is shown in red. In yellow is the C-terminal domain: GRK2 and GRK3 possess pleckstrin homology (PH) domain whereas GRK5 contains a polybasic region involved in phospholipid binding. Other GRKs are posttranslationally modified: GRK1 is farnesylated, GRK4 and 6 are palmitoylated, and GRK7 is geranylgeranylated. (b) Crystal structure of GRK2. The structure was solved as part of the Gαq-GRK2-Gβγ complex. The RH domain is shown in blue, the catalytic domain in red, and the PH domain in yellow.
4
The GRKs recognize and phosporylate agonist-activated GPCRs on serine and threonine residues and this process is crucial for desensitization, which happens through two-pro mechanism:
a. a phosphorylation reaction by GRK kinases to specific domain of GPCR-agonist; b. recruitment of β-arrestin to the phosphorylated receptor and receptor-G protein
uncoupling.
Following β-arrestin binding, GPCRs are targeted for clathrin-mediated endocytosis and internalized through coated pits. In the endosome, receptors can undergo various processes:
a. receptor-dephosphorylation and arrestin dissociation and recycling of GPCR to
the cell surface;
b. lysosomal proteolysis;
c. activation of further intracellular signaling pathways.[15,16]
As a result of arrestin binding and clathrin-mediated endocytosis, GPCR become not rexponsive to agonist stimulation and subsequently we have the arrest of signal propagation.
Figure 3. GRK-mediated desensitization and trafficking of GPCRs. Ligand binding to GPCRs not only
promotes G protein activation but makes the receptor a substrate for phosphorylation by GRKs (G protein- coupled receptor kinases). GRK phosphorylation promotes arrestin binding, which causes G protein uncoupling and promotes receptor internalization via clathrin-mediated endocytosis.
5
Beta-Arrestin also interacts with many other signaling molecules and promotes different arrestin-dependent and arrestin-independent signaling pathways. For example, β-arrestin can interact with Mitogen Activated Protein Kinase (MAPK) cascade components and non-receptor tyrosine kinases, other components of Raf/MEK/ERK cascade, components of the NfkB signaling pathway, and so on.[17]
Therefore, GRKs control a great number of pathways that regulate various steps of cell cycle, such as cellular growth, death, development and immune response.
GRK2
GRK2 is a serine/threonine intracellular kinase, member of β-adrenergic receptor kinase (β-ARK) subfamily, that can phosphorylate agonist-activated GPCRs on serine and threonine residues. It is expressed in many tissues; specifically, it plays a key role in embryonic development and heart function. In recent studies new physiological processes are also described that involve GRK2, such as the phosphorylation of non-receptor substrates and their crucial role in specific pathways that involve cellular tumor growth. This kinase especially promotes modulation of cellular functions such cell proliferation, survival and is involved in inflammation or angiogenic processes.
GRK2 regulates GPCR internalization
GRK2 plays a key role in GPCRs-desensibilization mechanism and are responsible of GPCRs internalization. As previously described, this kinase can phosphorylate agonist-GPCRs on serine or threonine residues, thus allowing the recruitment of β-arrestins. This process leads to uncoupling from G proteins and GPCR and leads to the attenuation of GPCR signaling.
GRK-dependent recruitment of β-arrestins is necessary for endocytosis, which is mediated by clathrin, a protein involved in the formation of coated vesicles.
6
Also, GRK2 utilizes other mechanisms to mediate receptors internalization. For example, GRK2 can bind phosphoinositide 3-kinase (PI3K), leading to the recruitment of PI3K itself on cellular surface upon agonist stimulation of the β-adrenergic receptor. This is an important way for -Adrenergic receptor endocytosis, through Adaptor Protein 2 (AP2) recruitment on receptor that works on cell membrane to internalize cargo and it is involved with clathrin in the formation of coated pils. In addition, the Carboxy-terminal domain of GRK2 binds clathrin, improving receptors internalization, and resulting in the co-localization of the receptor and GRK2 in endosomes. Thus, this interaction between GRK2 and clathrin plays another role in -arrestin-independent and Dynamin-dependent internalization, which is a GTPase involved in endocytosis of eucaryotic cells.
GRK2 promotes GIT-1 and GIT-2 (GRK interactor-1 and -2) by binding to the membrane, leading to inhibition of the agonist-promoted clathrin-mediated endocytosis of GPCRs and the accumulation of phosphorylated inactive receptors at the plasma membrane.[18] Recent studies indicate that GRKs, and other proteins such as arrestins, can regulate a number of intracellular processes and also play physiological role in regulation of signalling mediated by other receptors, such as tyrosine kinase receptors, Insulin and Platelet-derived growth factor (PDGF). Literature reports demonstrated that GRK2 was recruited upon activation of EGF receptors and then was phosphorylated at tyrosine residues of GRK2 itself, leading to opiod receptor transregulation. These results showed how GRK2 can regulate downstream activity of certain receptors at different levels or promote signal trasduction cascades through interactions with some signaling molecules.
7
Figure 4. The complex G protein-coupled receptor kinase 2 (GRK2) interactome.
In addition, GRK2 interacts with a growing number of non-GPCR substrates that include tubulin, phosducin, ribosomal protein P2, ezrin protein, the calcium binding protein DREAM and p38 mitogen-activated protein kinases.(figure 4)
Furthermore, GRK2 inhibits cell growth arrest and apoptosis mediated by Transforming growth factor beta 1 (TGF-β1) via phosphorylation of SMADs, that are a group of proteins that transduce extracellular signals from TGF-β1 ligands to the nucleus, leading to activate gene transcription.
These findings suggest that GRK2 also partecipates in many processes as an effector, through the phosphorylation of subtrates.
Other recent data evidenced that GRK2 enstablishes interactions with Adenomatous polyposis coli protein (APC) in osteoblasts, clathrin, calmodulin, caveolin or Raf kinase inhibitor protein (RKIP) that seems to partecipate in controlling GRK2 activity.
It was shown the possibility for GRK2 to be rapidly degraded by the proteasome pathway and that GRK2 turnover is enhanced by activation of β2-adrenergic receptor upon
phosphorylation of GRK2 mediated by Proto-oncogene tyrosine-protein kinase (c-Src) and MAPK in a β-arrestin-dependent manner. Another protein involved in GRK2 degradation is Mouse double minute 2 homolog (Mdm2), a negative regulator of the tumor suppressor p53.
8
In opposite manner, the activation of the PI3K/Akt pathway promoted by agonists leads to the preclusion of GRK2 degradation mediated by Mdm2 and to enhanced GRK2 stability.[19]
GRK2 INVOLVMENT IN HUMAN DISEASES
1.5.1 GRK2 involvment in insuline-resistance and obesity
GRK2 is involved in the regulation of insulin signaling. It was shown that increased kinase levels are responsible of decreased insulin-stimulated GLUT-4 translocation.
GRK2 interacts with Gαq in 3T3L1 adipocytes and can act as an inhibitor of insulin-mediated glucose transport GLUT-4 insulin-mediated, independently of its kinase activity. GRK2 plays several physiological roles in the modulation of insulin responses in vivo, because GRK2 RGS domain-Gαq subunit binding inhibits directly GLUT-4 translocation and lead to hypothesize that GRK2 may also play a role in insulin's metabolic signals. It was demonstrated that GRK2 increased expression in key tissues in different experimental models of insulin resistance; specifically, a 50% down-regulation of GRK2 levels in hemizygous mice is sufficient to protect against TNFα, aging or high fat diet-induced alterations insulin signaling. Particularly, reduced levels of GRK2 lead to a decrease of age-related adiposity and to a lean phenotype.[20]
1.5.2 GRK2 involvement in immune system
GRK2 has an important role in development of inflammatory processes and regulates epithelial and immune cell migration. It was showed that GRK2 high levels in epithelial cells and fibroblasts promotes their migration in response to fibronectin, by activation of Sphingosine-1-phosphate receptor (S1PR). GRK2 is also involved in lymphocyte migration into infected tissue during an inflammatory process, as well as high levels in endothelial cells of liver cells reveal the ability to inhibit the activity of AKT signaling, reducing nitric oxide synthase (eNOS) phosphorylation and nitric oxide (NO) production,
9
playing a role in portal hypertension. The inibition of AKT, a serine/threonine-specific protein kinase, leads to GRK2 desensitization of endothelyal receptor type B (ETb) and reduction of the downstream PI-3 kinase-Akt-eNOS pathway.[21]
Recent studies evidenced that GRK2 is highly expressed in different cell types of immune system, because it can phosphorylates chemokine and chemotactic receptors, leading to leukocyte accumulation in the inflammed area and a recall of T cells from lymphoid organs. In patients with Rheumatoid arthritis, peripheral blood mononuclear cells showed an attenuation of GRK2 expression and activity.[22]
GRK2 is also expressed in brain, where regulates the duration of microglial activation in the spinal cord, limiting microglial cytokine release and blocking the extent of nociceptive sensitization, suggesting that GRK2 has a critical role in chronic inflammatory pain and thus the regulation of pain sensitization.[8]
1.5.3 GRK2 involvement in cell proliferation
GRK2 regulates cell proliferation because it is involved in some phases of cell cycle progression, playing an importan role in the transition from the G2 to M phase.
In normal conditions GRK2 controls cell cycle progression and arrest it in a receptor-independent manner. However, cyclin-dependent kinase 2 (CDK2)-mediated phosphorylation of carboxy-terminal residues of GRK2 induces down-regulation of this kinase during the G2/M phase, because it leads to the binding of GRK2 to prolyl isomerase and subsequent degradation. Therefore, prevention of GRK2 phosphorylation at Ser670 of its C-terminus residue is an important way to prevent GRK2 down-regulation and delays cell cycle progression. GRK2 is a protein kinase that can interact with a growing number of non-GPCR substrates, in particular with PDGF and EGF receptors, and a great number of proteins involved in pathways controlling cell migration and proliferation. This kinase has a critical role in human hepatocarcinoma cells, where is able to inhibit cell growth arrest and apoptosis mediated by TGF, that is accompanied by increased levels of p53 phosphorylation and cyclin B, an effect dependent by GRK2 activity.
10
On the other hand, GRK2 leads to the attenuation of proliferation of hormone-dependent thyroid cancer cells by stimulating PDGF. Recent studies showed that in presence of DNA damage induced by agents such as Doxorubicin, GRK2 reaches an higher steady-state level that, in such conditions, is inversely correlate with the p53 response and the induction of the apoptosis. These results suggest that GRK2 plays a critical role into cell cycle and contributes to control G2/M phase, leading to a less apoptotic process of arrested cells. Another recent study marks the importance of GRK2 in cell cycle, highlighting the involvment of GRK2 in Smoothened (Smo) signaling in zebrafish embryos that regulates the formation of neural tube and slow muscles. GRK2 also interacts directly withProtein patched homolog 1 (PTCH1), in a phosphorylation-independent manner, leading to cyclin B1 accumulation, that is a nuclear protein required for successful entry in mitosis during early development. It was instead observed a retarded growth and arrest of development in GRK2-knockdown zebrafish embryos, thus suggesting the importance of its role in the Smo pathway.[23]
1.5.4 GRK2 Involvement in Heart Failure
Heart Failure is characterized by a marked decrease of in myocardial contractility and loss of pump function. GRK2 is one of the most important biomarker that is up-regulated in this human disease and in animal models. It has been shown that reducted GRK2 levels can improve pump function in several animal models and play a critical role into myocite, because influence cardiac contractile function and cell metabolism. It works in desensitization mechanisms of Beta-adrenergic receptors (-AR) and its up-regulation occurs initially after cardiac injury or stress. It is necessary to shut-down over-activated -ARs, that appear as a result of compensated increases in chatecolamines. A prolonged up-regulation leads to a disregulation of -AR system and to a loss of inotropic reserve and, after a long time, to Heart Failure.
Recent studies has been shown that heterozygous GRK2 knockout mice with 50% less GRK2 in all tissues have increased myocardial function, while homozygous embryos mice where GRK2 is ablated die during gestation, suggesting a possible prevention of heart failure development after a myocardial infarction.
11
Moreover, homozygous GRK2 knockout mice showed a pronunced hypoplasia of ventricular myocardium and dysplasia of the interventricular septum, suggesting that ablation of GRK2 early in development leads to abnormalities in adulthood, as a result of chronic chatecolamine activity.
Overexpression of this kinase is related to reduced myocardial contractility and leads to a lesser responsivity of -ARs. Thus, GRK2 is considered as an important modulator of myocardial contractility and plays an important role in heart development.[24]
In Heart failure, incresased expression/activity of GRK2 leads to uncoupling β-AR and to the decrease of diastolic and sistolic ventricular function.(figure 5)
It is known that GRK2 modulation by the use of inhibitor peptide βARKct, it is able to enhance cardiac response in ischemic injury. In addition, βARKct inhibits GRK2 migration to cellular membrane leading to an increased receptor density of β-AR and promoting myocardial contractility mediated by Gs/PKA.[25]
Figure 5.Chronic heart failure leads to upregulation of GRK2, both in cardiac myocytes and in adrenal chromaffin cells.
It has been demonstrated that patients with heart failure have reduced β1-adrenergic receptors density, and a consequent increase in GRK2 expression and activity.
12
Enhanced GRK2 levels in myocite and increased mRNA levels, which codify for this protein may be related to the diminished response of failing hearts to β-receptor agonists. This result should be encouraging to resource selective GRK2 inhibitors to treat Heart failure.[26]
1.5.5 GRK2 involvement in depression
Recent studies indicated that depressive disorders are characterized by altered levels of GRK2, expecially these disorders associated to altered expression of GPCR in the brain and blood cells.
Depression disorder is accompanied by a variety of symptoms such as fatigue, anorexia, lack of motivation, apathy and low sociability that are associated with GRK2 abnormal expression. An after death analysis of suicidal patients and non suicidal patients with depression evidenced that GRK2 levels were increased in the prefrontal cortex; whereas, a long treatment of these patients with antidepressants such as Paroxetine and Fluoxetine restored the normal GRK2 values, suggesting that high GRK2 levels may contribute to the pathogenesis of depression. On the contrary, other studies showed that GRK2 low levels may contribute to the onset of this pathological disease. Based on the knowledge of this protein it is not clear which role GRK2 has in depression development, but can be a starting point for its identification as a new clinical target in this disease.[27]
1.5.6 GRK2 involvement in Cancer
Tumor has important features such as substained proliferation, refractoriness to growth suppressors, resistance to cell death or aberrant motility, and metastasis and it can be triggered by mutations and signaling adaptions. GRK2 has an important role in specific pathways that regulate cancer growth and partecipate in many processes that governate oncogenic envelopment. Altered GRK2 levels show an implication in different tumoral contexts and demonstrated to promote breast tumorigenesis or to trigger the tumoral angiogenic switch. There are many features that characterize tumor cells such oncogen-driven proliferation, which is assisted by secreting of their own growth factors (GF) or
13
promoting their relais by cells within the stroma. Moreover, GF-receptors can also be mutated or over-expressed, leading to hyperesponsive cells.
In this context, GRK2 appears as a potentially onco-modulator, that partecipates in functional connections with signaling network involved in homeostasis and cell proliferation. It was demonstrated that altered levels of GRK2 are responsible to activate MAPK/ERK pathway, which has an important role in cell proliferation. It is triggered by desensitization mechanisms of GPCR and modulation of GPCR-β-arrestin-MAPK cascades or EGFR pathways. Upon GRK2-downregulation, the response of endhotelial cells to angiogenic stimuli (VEGF, S1P, Serum) is increased, modifying endothelial cells capacity to organize into tubular structure, as well as interrumpting the balance in the secretion of inflammatory and angiogenic factors. Furthermore, reducted levels of GRK2 can alterate TGF-β signaling, a tumoral growth factor which controls activation and evolution of angiogenic phases through the modulation of ALK1 and ALK5 receptor effects. Depressed levels of GRK2 hinder endothelial cells differentiation and their fusion in tubular structure, impeding the recruitment of pericytes that are around them and leading to immature and leaky vessels. Recent studies have shown that the vessels of tumors growth in mice increased size and reduced pericyte recruitment, important features of the tumor microvasculature. A down-modulation of GRK2 in endothelium seems to promote the recruitment of macrophages to the cancer area through directly alteration of chemotactic secretome of endhotelial cells and formation of leaky vessels, leading to a gradient chemoattractans for myeloid cells.
GRK2 in breast tumorigenesis
It has been shown recently that GRK2 plays a critical role as an oncomodulator in Breast cancer and its tumorigenesis. In luminal tumors and in certain non-luminal tumors, there are an hyper-activation of estrogen or EGFR receptors, the Ras-HER2 and the PI3K-AKT cascades, that lead to enhanced GRK2 expression in transformed breast epithelial cells through increased stimulation of the AKT pathway.
Cancer cells present genetic alterations in PI3K/AKT/mTOR pathway or hyperstimulation of Estrogen receptors that can trigger AKT stimulation. Increased levels of GRK2 play a key role in acquisition of oncogenic features by luminal and basal breast cancer cells and
14
GRK2 up-regulation promotes a reinforcement of EGF or ERK1/2 and AKT pathways, regulating cellular growth in positive manner and leading to resistance to the induction of cell death by agent therapeutics. Conversely, decreased levels of GRK2 have opposite effects in tumoral breast cells and sensibilize them to chemotherapeutic agents. GRK2 up-regulation promotes anchorage-independent growth of luminal MCF7 or MDA-MB-231 basal cancer cells and enhances their possibility to grow in vivo.
Figure 6. Physiological integration of cell-type specific GRK2 effects in breast cancer tumorigenesis. Concurrent and
opposite changes in GRK2 expression taking place in epithelial and endothelial components of breast tumors might act synergistically to promote tumor growth.
Furthermore, increased levels of GRK2 observed in breast cancer patients favor phosphorylation and activation of Histone-Deacetilase-6 (HDAC6), an enzime that leads to de-acetylation of the Prolyl Isomerase (Pin-1), which in turn is a central modulator of tumor progression and triggers a positive feedback loop of growth factor transduction cascades leading to continuos cell survival and proliferation. Therefore, GRK2 up-regulation appears as the concequence of stimulation of different pathways altered in luminal breast cancer and the activation of GRK2-HDAC6-Pin1 signaling suggests that it would be a possible target for therapies.[28](figure 6)
15
GRK2 in Pancreatic cancer
Inside pancreatic cancer GRK2/ADRBK gene was identified as a critical target employing a screening approach. The ablation of GRK2 through RNA interfence mechanism leads to growth inibition and arrest of cell cycle on G1/S phase.
With the use of microarray analysis it was possible to show that GRK2 levels were not detectable in non-tumoral tissues, whereas in pancreatic ductal adenocarcinoma they were easily detectable in 51% in epithelial cancer cells and in some groups of infiltrating immune cells.[28]
GRK2 in hepatocarcinoma
Contrary to breast cancer cells, GRK2 over-expression leads to less response of EGR-1, while GRK-2 downregulation show increased EGR-1 expression, where this nuclear protein plays several role into differentation and mitogenesis as a tumor suppressor gene. Overexpression of GRK2 causes a decrease in early growth response-1 (EGR1) expression and this kinase can also interact with insuline-like growth factor 1 receptor (IGF-1R). GRK2 may inhibit epatocellular carcinoma induced by IGF-1 via down-regulation of EGR-1, suggesting a potential therapeutic approach against hepatocellular carcinoma.[29]
GRK2 STRUCTURAL FEATURES
All GRKs maintain a central catalytic domain of 270 residues, flanked by a N-terminal domain of 185 aminoacids and a C-terminal domain characterized by 100-230 aminoacids, which is different in structure and lenght.
The N-terminal domain is important for receptor recognition, for intracellular membrane anchoring and includes an RH domain of 120 aa. In GRK2 the RH domain interacts with Gαq, thus blocking the activation of their effectors, Phospholipase C. Furthermore, it may function as an antagonist effector of GPCRs-G protein interaction in the absence of phosphorylation.
16
The C-terminal domain of GRK2 contains a pleckstrin homology domain (PH), which has the property to interact with phosphatidylinositol-4,5-biphosphate (PIP2) and free Gβγ subunits through binding sites into a PH domain, unique for GRK2 and GRK3. This interaction regulates the agonist-dependent translocation of GRK2 to the plasma membrane.[19]
The three GRK2 regions, located on the vertices of a triangle, show how this kinase blocks signal transmission through the binding to GPCR and G protein subunits, using different molecular sites.
Figure 7. Structural features of GRK2. GRK2 is oriented to show the ATP-binding site with the kinase domain colored
green and the regulator of G protein signaling homology (RH) and pleckstrin homology (PH) domains colored slate. ATP binds between the small and large lobes (connected via the hinge region) and is modeled on the basis of the GRK1-ATP structure.
The KD (Kinase Domain) of GRK2 is composed of a small lobe (residues 186-272 and 496-513) and a large lobe (residues 273-475). The small lobe is formed by six-stranded antiparallel Beta-sheets and three helices; the large lobe is characterized by alpha-helices and four antiparallel Beta-strands (figure 7).
GRK2 contains an ATP-binding site, which is located at the interface of small lobe and large lobe, and is rightly conserved in the majority of kinases. It includes the phosphate binding loop (P-loop), the alpha-C-helix, the hinge connecting the small and the large lobes, and the activation loop or phosphorylation site.
17
It represents a target for small molecule kinase inhibitors, which bind in similar mode of ATP-itself, and a point of start to research new inhibitors.[30]
GRK2 INHIBITORS
Recent studies have shown the complex of signaling pathways that involve GRK2 in human diseases and have led to the development of new selective GRK2 inhibitors, because this kinase represents a potential diagnostic marker and therapeutic target, especially in human diseases like heart failure, inflammation and cancer. A particular attention should be paid to the description of GRK2 inhibitors studied in recent years, their structure activity relationships and their mechanism of action.
1.7.1 Polyanions and Polycations
The first compounds that have shown a great ability to inhibit GRK2 phosphorylation on Rodopsin were Polyanionic and Polycationic compounds. The most potent inhibitors of this group were Heparin and Dextran sulfate with IC50 values of 0.15 μM, but they have
shown their inability to crossing the plasma membrane due to their high charge. It was examinated also the activity of Polyaspartic acid (IC50 = 1.3 μM), Polyglutamic acid (IC50
= 2.0 μM) and Inositol hexasulfate (IC50 = 13.5 μM).[31]
However, these compounds have proved to be not selective, as they also inhibit Casein kinase II and Low density lipoprotein receptor kinase II.[32]
1.7.2 Balanol
Balanol (1) is a fungal metabolite produced by Verticillium balanoides that act as a potent inhibitor of GRK2, competing with ATP site in GRK2 KD.[33](figure 8)
18
Figure 8. Structure of Balanol
It has been demonstrated that this compound can permeate cell membrane and it is characterized by four rings system, named A-D, linked by a linear fashion.
The 4-hydroxybenzoyl moiety (A ring) is connected to hexahydroazepine group (B ring) by an amide linkage, that in turn is connected to the benzophenone moiety (C and D) by an ester linkage. Balanol 1 acts as an ATP mimetic, establishing interactions with ATP binding subsites. The 4-hydroxybenzoil ring occupies the hydrophobic adenin subsite; the B ring occupies the ribose subsite and ring C interacts with polyphospate subsite. The D ring occupies a hydrophobic subsite.[34]
Recent studies which comparates apo-GRK2 and GRK2-Balanol complex revealed that Balanol 1 stabilized a "slighty more closed" conformation of the KD. Thus, this inhibitor seems to recognize and stabilize an unique inactive conformation of GRK2. This compound inhibits GRK2 at the nanomolar level (IC50 = 42 nM), but it is able to inhibits
other isoforms of kinase at high concentrations.
1.7.3 Takeda Inhibitors
They are compounds discovered by Takeda Pharmaceutical Company in 2007 and their name are CMPD103A (2a) and CMPD101 (2b).(figure 9)
They are characterized by four rings connected in a linear manner, that have been shown major selectivity for GRK2 and they are able to interacts with catalitic site of GRK2 and GRK3, demonstrating a certain selectivity compared to other compounds.
19
To determine their mechanism of action, two compounds were co-crystallized with the GRK2-Gβγ complex.
Figure 9. Structure of Takeda inhibitors (CMPD103A and CMPD101)
The A ring of both inhibitors is able to interact with adenine subsite through an H-bond between the pyridine/pyrimidine N4 atom and the amide nitrogen of Met274 in the
catalytic region. The substituted 1,2,4-triazole group binds GRK2 in the ribose subsite forming H-bond between the N9 atom and the side chain of Lys220.
The triphosphate subsite is occupied by C ring or aminobenzamide moiety and interacts through H-bonds between the NH and the side chains of Asp335 and Lys220. There are established also H-bonds between carbonyl oxigen of the amide linkage on C and D rings and the amide nitrogens of Gly201, Phe202 and Gly203 similar to Balanol.
The D ring differentiates Takeda inhibitors from other compounds and binds to the hydrophobic subsite through nonpolar interactions with Gly201, Phe202, Leu235, Glu239, Gly337 and Leu338. Balanol and Takeda inhibitors interact with GRK2 in an open, noncatalytic conformation, thus promoting a slight closure of the kinase KD. CMPD103A(2a) and CMPD101(2b) have been shown to inhibit GRK2 with IC50 =
290nM and IC50 = 54 nM, respectively.
Balanol (1) is considered more potent (IC50 value of 35 nM) than CMPD103A and
CMPD101, but these compounds were inactive against GRK1 and GRK5 isoforms, thus demonstrating a better selectivity towards GRK2.[30](Table 1)
20
Table 1. IC50 Values of Balanol 1 and Takeda Inhibitors towards GRK1, GRK2 and
GRK5 IC50 nM Compd GRK2 GRK1 GRK5 2a 290 ± 98 n.i. n.i. 2b 54 ± 14 n.i. n.i. 1 35 ± 8 4100 ± 600 440 ± 150
Balanol 1 structure present oxygen-rich substituens that produce conformational changes in the active site, whereas Takeda inhibitors are characterized by five donor/acceptor groups less than Balanol 1 that are able to establishing nonpolar interactions and that justify their selectivity towards GRK2.
CMPD103A was tested in recent studies on μ-opiod receptor (μOPr), evaluating its effects when these receptors in locus coeruleus neurons were desensitize by agonists.
Another activity was studied in HEK293 cells, where CMPD103A 2a inhibited receptor phosphorylation at Ser375. Furthermore, CPMD101 2b was tested on G-protein coupled desensitization activated by opiods that involves rectifying potassium currents, justifying the involvment of GRK2 in this process.[35]
1.7.4 RNA Aptamers
Aptamers are short single-stranded oligonucleotides that are able to bind various molecules with high affinity and specificity and are usually selected from the oligonucleotide collection known as the initial oligonucleotide pool (IOP) that is often called a “combinatorial library".[36]
They rapresent a start point for the development of new inhibitors through the SELEX process, that allowed to identify a specific RNA aptamer capable to bound and inhibit GRK2. Usually, the identification of RNA aptamers was made by designing an RNA libray formed by a region of 20 random nucleotides in a loop structure, that is flanked by
21
13 nucleotides which pair the complementary bases of antiparallel strand.
The central stem is characterized by two consecutive G-U base pairs that contain in the center the couple A-A to reduce its stability. On the side of the stem there are also two restriction sites, that correspond to restriction endonucleases obtained from Providencia
startui and Haemophilus influenza.(figure 10)
a
b
Figure 10. a) General structure of RNA aptamers. In red: 20-nucleotides (N20) central random region, arranged in a
loop structure. In green: 13-nucleotides stem structure with a central A-A mismatch. In orange and brown respectively: PstI and HindII restriction sites; b) Sequences of selected aptamers.
The stem is also flanked by single-stranded regions (C20 and C47) through primer hybridization. Four RNA aptamers were examinated to evaluate their activity against GRK2 and their selectivity was demonstrated by the poor affinities of 3a against PKA and Erk-2. Compound 3a (figure 11) called C13 strongly inhibits GRK2, with IC50 =
22
4.1±1.2 nM, and confirmed that is a more potent inhibitor than Balanol 1 and Takeda inhibitors 2a and 2b. It was able to act against GRK2-catalyzed rodopsin phosphorylation. Starting from compound 3a, with a series of truncations and modifications, important results were obtained with compound 3c, a truncated form that inhibits GRK2 with IC50 = 11±4 nM.[37]
Figure 11. Structure of C13 (3a).
It was demonstrated that RNA aptamer instaured hydrophobic and electrostatic interactions with the catalytic core of the KD through a small portion of its nucleotidic sequence (49-AUAC-52) and the truncation form it was able to interact with GRK2 mimicking the binding of the ATP in the active site of the kinase. In particular, the truncated aptamers established binding with GRK2 through the position A51, that ensure affinity and selectivity. The aptamer 3a stabilizes a unique inactive conformation of GRK2 and establishes multiple interactions within and outside of the catalytic core.
23
It represents a new tool to understand GRK2 function and activity, but it has many problem about the use due to its high molecular weight.[38]
1.7.5 Paroxetine and Derivates
Paroxetine (4) is an antidepressant belonging to the selective serotonine re-uptake inhibitor (SSRI) class and is approved by FDA to treat depression, obsessive-compulsive, anxiety, panic and post-traumatic stress disorders.
Figure 12. Structure of Paroxetine (4)
Paroxetine is a selective GRK2 inhibitor discovered during an aptamer displacement assay intented to identify small molecules able to inhibit GRK2.
The molecular structure (figure 12) has a dioxole moiety that binds in the adenine subsite forming an H-bond between one of the oxygens and the amide backbone nitrogen of Met274.
The oxygen of dioxole moiety mimes the N1 atom of ATP; whereas the carbon of the
benzodioxole group and the backbone carbonyl oxygen of Asp272 interact with a low free energy carbon-oxygen H-bond. The methylene moiety establishes van der Waals interactions in the adenine subsite and the piperidine moiety is able to bind in the ribose subsite creating a network of H-bonds. The endocyclic NH interacts with the carboxylic acid of Asp278, the carbonyl oxygen of Ala321 and Asn322. The fluorophenyl ring binds a portion characterized by P-loop residues and the side chains of Lys220 and Leu222, establishing non polar interactions which stabilize GRK2-paroxetine binding.
24
In Paroxetine derivates the loss of fluorine is unfavorable for kinase inhibition.
Paroxetine was tested for its ability to inhibit phosphorylation of rhodopsin in rod outer segment (ROS) membranes mediated by GRK2. It resulted able to inhibit GRK2 with an IC50 = 19.9 μM in presence of 5 μM ATP and it was observed a 16- and 13-fold lower
potency toward GRK1 and GRK5. Paroxetine also showed to inhibit GRK2 phosphorylation into the tubulin phosphorylation assay with an IC50 = 2.5 μM.[39]
This compound was also tested in vivo by treating wild-type mice 2 weeks after a myocardial infarction, in comparison with fluoxetine-treated mice which do not inhibit GRK2. All mice showed initially similar left ventricular dysfunction, and after treatment with Paroxetine had a great envelopment of myocardial function and structure. It was demonstrated that Paroxetine was able to inhibit GRK2 improving cardiac function after myocardial infarction and this thing rapresented a new starting point for the development of novel GRK2 inhibitors.[40]
Recent in vivo studies performed by Tang et al. validated the ability of Paroxetine to modulate GRK2 expression in superior cervical ganglion (SCG) neurons in rat models characterized by limb ischemia-reperfusion injury that is associated with a complex regional pain syndrome type. It was shown how, in these mice, inaltered levels of GRK2 in the ipsilateral and controlateral SGRs were regulated and normalized upon Paroxetine administration.
These results also show Paroxetine ability to normalize altered mRNA levels and protein expression in ischemia-reperfusion injury. It was also demonstrated the involvment of GRK2 in neuropathic pain syndromes and Paroxetine capacity to reduce sensory abnormalities, such as allodynia, by inducing GRK2 up-regulation in SCGs.[41]
25 F R 5a 5b HCl N H OR OH OH NH O
Figure 13. Structure of Paroxetine derivates with different R chain (5a and 5b)
Another derivate of Paroxetine that was synthesized to study structure-activity relationships (figure 13) is characterized by a benzodioxole nucleus and a benzolactam ring. The substitution leads to a major inhibitor potency due to the formation of a stronger H-bond with the hinge region compared with uncoventional carbon-oxygen H-bonds. The interactions between Paroxetine and GRK2 are conserved in the complex GRK2-5a, with the exception of Met274, which rotates to avoid a steric clash with the benzolactam carbonyl. The benzolactam inhibitor is less selective than Paroxetine because it strongly inhibits PKA and PKC.
These results show that the presence of group able to engage conventional H-bonds (NH) increases the activity toward other kinases and decreases the selectivity toward GRK isoforms.
However, indazole/dihydropyrimidine compounds were preserved to select new scaffolds that maintain a similar pharmacokinetic and selectivity with respect to Paroxetine. [42] Other studies identified five compounds that maintained three rings: an indazole ring, a dihydropyrimidine/dihydropyridone ring, and a substituted phenyl ring; these compounds had the property to inhibit GRK2 in similar manner with respect to Paroxetine. One of this compounds was used to study its binding and structure-activity relationships (figure 14).
26
Figure 14. Structure of compound GSK180736A with R1 and R2 chain. (6)
GSK180736A (6) is capable to bind the active site of the KD in similar manner to Paroxetine. The indazole ring binds to the adenine subsite through H-bonds with the hinge residues backbone atoms, in similar manner to Paroxetine benzodioxole ring. The amide linker instaured a bond with Ser334, whereas the dihydropyrimidine ring formed an H-bond with the carbonyl of Arg199 in the P-loop and low interactions with the large lobe. The fluorophenyl moiety interacts with a region formed by the P-loop and the side chain of the active site Lys220. This compound showed an inhibition potency toward GRK2 with IC50 = 0.25 μM and a good selectivity for this kinase.[43]
1.7.6 Peptides
Peptides represent an another class of GRK2 inhibitors, which were delevoped based on the knowledge that the main function of GRKct peptide, the C-terminal region of GRK2 (GRK2ct), is to inhibit GRK2.[44]
Recent studies on transgenic mice with cardiac-restricted overexpression of GRKct or beta2 Adrenergic Receptor, that were mated to create different genetic models of murine
27
HF, have shown that the cardiac overexpression of β2-AR did non influence the
development of dilated cardiomyopathy phenotype. However, the overexpression of GRKct in transgenic mice leaded to cardiac-targeted GRK2 inhibition and reduction of
β1-AR desensitization, which preserved a worsening of heart function.[45]
The development of new short peptides such as GRK2 inhibitors are needed and these molecules were identified in synthetic peptides containing intracellular and extracellular domains of hamster β2-AR. Four inhibitors were identified, where each molecule showed
a decreasing sequence of peptides and decreased activity according to its structure. These inhibitors derived from the first intracellular loop of β2-AR: β2-AR 56-74(a); β2-AR
57-71(b); β2-AR 59-69(c); and β2-AR 60-66(d).(Table 2)
β2-AR 56-74 was considered the most potent inhibitor and was used as a start point for the development of new inhibitors. The truncated form was used to evaluate the peptide/GRK2 interactions and to clarify which peptide end was responsible for activity. β2-AR 56-74 was able to inhibit GRK2 with IC50 = 40 μM.[46]
Table 2. GRK2 peptide sequences and their activity
Peptide Sequence GRK2 IC50μM (a)β2-AR (56-74) (b)β2-AR (57-71) (c)β2-AR (59-69) (d)β2-AR (60-66) TAIAKFERLQTVTNYFITS AIAKFERLQTVTNYF AKFERLQTVTN KFERLQT 40 62 1600 2600
After these compounds, new peptides were developed that present the addition of polar amino acids at both ends of the prototype sequence and they showed an increase of potency of 9-fold toward GRK2. The peptides were tested for their activity toward GRK3 and GRK5 isoforms, highlighting their lowest selectivity against these two isoforms. A truncated peptide inhibitor (59-74E) was used to study structure-activity relationships and some studies have shown that this peptide established one contact point with GRK2 by mimicking the first intracellular loop of the receptor. This peptide presented a sequence
28
AKFERLQTVTNYFITSE; studies in vivo on human cell lines demonstrated that it inhibited desensitization of β2-AR induced by agonists and it was able to enhance their receptorial responce. Thus, these results support the use of these inhibitors for the treatment of specific cardiac diseases, by a modulation of β-adrenergic signaling pathway in cardiomyocytes.[47]
Another compound that revealed to be a selective inhibitor was a small peptide derived from the first intracellular loop of β2-AR and consisted of the sequence MAKFERLQTVTNYFITSE.
Recent studies on this peptide demonstrated that its GRK2 inhibition leaded to a cardioprotective effect due to an increased activation of the MAPK signaling pathway. This compound administered to mice overexpressing RAF kinase inhibitor protein (RKIP), which is responsible for the inhibition of GRK2 and MAPK pathways, produced cardiomyocite apoptosis, heart dilatation and cardiac lipid overload. However, in mice overexpressing this peptide inhibitor, less cardyomiocite apoptosis and reducted cardiac dilatation was observed, thus demonstrating the cardioprotective effect due to GRK2 inhibition and MAPK pathway.[48]
Another result obtained from peptide inhibitors that act toward GRK2 is the prevention of cardiac lipid accumulation and reduced FASN (fatty-acid synthase) levels, leading to delay Heart failure symptoms.[49]
Another group of inhibitors is represented by acylated glycine derivates of short peptides, such as myristyl GLLRrHS and lauryl GLLRrHSI that have a sequence similar to that of the catalytic fragment 383-390 KLLRGHSP of GRK2, utilized to realyze novel sequence of peptides able to inhibit effectively GRK2.
Thus, these results leaded to peptide GLLRrHS and GLLRrHSI, that are identical to previously compounds with the exception of the N-terminal acylation at Gly1; they selectively inhibited GRK2 in vitro with an inhibition of 47.6% and 49.6%, respectively.[10,11]
The development of all these compounds suggests that inhibitors of GRK2 could act selectively with the active site if they present an indazole-based group, that is fondamental for binding of adenine subsite, establishing H-bond with aminoacidic residues.
29
The conservation of this scaffold was suggested by molecular-modelling studies of structure-activity about Balanol and Indazole derivates, that contain groups similar to imidazole moiety mimicking adenine and ribose residues. However, the introduction of aminobenzamide and substituted benzene rings gives the structure some flexibility; this evidence derives from docking studies about CMPD103A and CMPD101 inhibitors, that contain similar chain able to establish chemical bond with polyphosphate subsite and aminoacidic residues of hydrophobic subsite.
30
2
INTRODUCTION OF
31
The G protein-coupled receptor kinase GRK2 is ubiquitously expressed in many tissues and plays a key role in several signal transduction pathways and in the regulation of many important cellular processes.
A number of studies have evidenced that GRK2 levels and functions are altered in several pathological disorders. GRK2 plays an important role in embryonic cardiac development and function, and it is required for the proper development and maintenance of heart structure. As a matter of fact, an up-regulation of GRK2 mRNA was observed in patients with cardiac ischemia and left ventricular hypertrophy.
GRK2 is considered a regulator of cell proliferation and, in this regard, alterations of its levels and activity has been noticed in different types of tumors, such as ovarian, prostate and thyroid cancers, mainly based on hormonal mechanisms.
GRK2 was proven to play a key role also in autoimmune disorders: a reduced GRK2 expression was observed in an experimental autoimmune encephalomyelitis and in an animal model of Multiple Sclerosis (MS) and of arthritis.
A critical role to this kinase was also attributed in the pathogenesis of Alzheimer's disease (AD), because of its high expression in the cardiac, vascular and cerebral tissues. In this context, increased levels of GRK2 were correlated with the degree of cognitive impairment of AD patients.
In view of all the described pathways in which the kinase is involved, the selective targeting of GRK2 may represent a valid and innovative approach to treat a panel of human disorders.
The main problem with the majority of GRK2 inhibitors developed to date is that they potently inhibit not only the target kinase, but also other GRK isoforms, as well as other AGC family kinases.
In this context, my thesis work was focused on the design and synthesis of new potent and selective GRK2 inhibitors, exploiting a heterocyclic derivative characterized by a
32
Figure 15. Structure of lead compound LR-151-3
The pyrazolo[3,4-d]pyrimidine moiety is mimetic of adenine residue of ATP, whereas the substituent at position 6 of the scaffold is mimetic of the ribose ring. This compound showed to inhibit GRK2 subfamily with an IC50 value of 0.12 µM; although it lacks of
selectivity versus GRK3, the inhibition of GRK5 at 0.1 mM concentration was 45%. In order to rationally design suitable substituents to be introduced on this lead compound, three potent reported GRK2 inhibitors were initially examined: the natural product balanol, potent but non-selective, and two Takeda heterocyclic compounds, higher selective for GRK2, CMPD101 and CMPD103A.(figure 16)
Figure 16. Structure of Balanol, CMPD101 and CMPD103A
Balanol acts as ATP competitive GRK2 inhibitor, as a result of specific interactions between its four rings and the ATP binding subsites: the A ring occupies the hydrophobic
33
adenine subsite; the B ring occupies the ribose subsite; the aromatic C ring binds to the polyphosphate subsite; lastly, the D ring occupies a hydrophobic subsite.[34]
The potent inhibitors CMPD101 and CMPD103A also feature four ring systems (A, B, C and D), able to generate the same interactions of balanol in the ATP binding site. The difference in the selectivity profile of these three inhibitorscan be explained by evaluating the structural differences of C and D rings
.
In fact, the benzophenone moiety of balanol is characterized by oxygen-rich substituents, which are able to produce conformational changes in the active sites of different kinases. On the contrary, interactions generated by the C and D rings of Takeda inhibitors are mostly non-polar, thus being less able to interact with the residues of the KD of other GRK isoforms and different types of kinases.[30] Actually, molecular modeling studies were subsequently performed and clarified the importance of the positions 1 and 2 of the pyrazolo[3,4-d]pyrimidine system and the amino group of the substituent at position 6, which are involved in H-bond interactions that are critical for the proper orientation of the molecule in the GRK2 KD. On the contrary, the introduction of substituents on the amino group at position 4 of the lead compound seemed to be advantageous.In this view, starting from LR-151-3, in order to improve the selectivity profile, it was thought to synthetize new indazole derivatives, as indazole nucleus is synthetically more accessible with respect to the pyrazolo[3,4-d]pyrimidine one. Position 4 of this central scaffold was then modified by inserting side chain (A or B) featuring: (i) a two-atom linker to allow flexibility to the structure; (ii) a piperidine ring able to realize H-bond interactions; (iii) an aromatic system able to generate interactions with the allosteric site linked to the 3-position of piperidine by an ester or amido linker (figure 17).
34 HN N HN R N O O N N H O A B Ethylene
linker Piperidine ring
Aromatic system N N N N N N H H N O F A B C D F Hydrophobic adenine subsite Ribose subsite Polyphosphate binding site Hydrophobic subsite Critical H-bond
interactions H-bondCritical
interactions N N HN N NH2 R = Molecular modeling studies CMPD103A NH2 NH N NH2 LR-151-3
Figure 17. Structure of designed GRK2 inhibitors.
The convergent procedure applied for the synthesis of A and B chain 6 and 11 is outlined in Scheme 1. Derivatives 3 and 9 were obtained in two steps starting from the N-Boc-piperidine 3-methanol 1 and N-Boc-3 aminomethylN-Boc-piperidine 7, that were reacted with benzoyl chloride in the presence of triethylamine to obtain the ester derivatives 2 and 8, purified by flash chromatography (eluent mixture: petroleum ether/AcOEt 9/1), and then deprotected by treatment with trifluoroacetic acid in dichloromethane leading to amine derivatives 3 and 9. Treatment of benzyl alchool with chloroacetic acid in toluene and in presence of PTSA yielded derivative 4. Reaction of derivative 4 with compound 3 or 9 in
35
presence of triethylamine gave compound 5 and 10, that were purified by flash chromatography (eluent mixture:AcoEt/petroleum ether 7,5/2,5).
The ester derivatives 5 and 10 were catalytically hydrogenated over palladium to obtain the corresponding acids 6 and 11, sufficiently pure to be used in the next step without further purifications. Scheme 1 N X Boc N X O Boc N H X O N X O COOCH2Ph OH + Cl OH O O Cl O I II III N X O COOH IV
Reagents and Conditions: I: Benzoyl chloride, NEt3, CHCl3, 0°C, 4h; II: TFA, DCM, rt, 2h; III: DMF, NEt3, rt, 24h; IV: H2/Pd-C, EtOH ass, rt; V: PTSA, Toluene, , 2h.
V 2, 3, 5, 6: X=O 8, 9, 10, 11: X=NH 1 X=OH 7 X=NH2 4 2, 8 3, 9 5, 10 6, 11
36
The synthesis of the 6-(4-nitrophenyl)-1H-indazol-4-amine 12 scaffold was performed starting from commercially available 4-amino-6-bromo-1H-indazole, which was subjected to a Suzuki cross-coupling reaction with 4-nitrophenyl boronic acid and Pd(dppf)Cl2-DCM as catalyst (Scheme 2). Numerous different attempts were
accomplished to perform the coupling between derivative 12 and the lateral chain A or B in order to obtain derivatives 13a-b, by varying coupling reagents (TBTU, HATU, CDI and EDCI), solvent and temperature conditions, as illustrated in Table 3. In all these attemps a great quantity of starting product 12 remains unreacted.
Then we tried an alternative approach by activating the carboxylic acid of the A and B chains of compound 6 and 11 with thionyl chloride or oxalyl chloride and then adding derivative 12 in the presence of triethylamine. Also in this case, various reaction conditions were experimented but, unfortunately, the starting material remains always unreacted.
Table 3. Procedures for the synthesis of 13a, 13b and 14. Procedure Lateral Chain Reagents and Conditions a A HATU, DIPEA, DMF r.t., overnight b A TBTU, DIPEA, DMF 0 °C, overnight c A SOCl2, NEt3, 2h d B
Oxalyl chloride, NEt3
Toluene, r.t., overnight
e B
CDI, DMF, r.t., overnight
f B
EDCI, HOBt, DIPEA, DCM, r.t., overnight
37
The only positive result has been obtained using procedure e, in which derivative 11 was condensed with compound 12, in the presence of CDI in anhydrous DMF. After work-up of the reaction mixture a unique product was isolated. Structural characterization by 1 H-NMR led, unfortunately and surprisingly, to the identification of compound 14 and not the desired compound 13a.(Scheme 2).
Scheme 2 N H N NH2 Br N H N NH2 O2N N X O COOH 6,11 N H N HN O2N O N X O N N NH2 O2N O N NH O 12 13a 14 X X=O X=NH 13b I
Reagents and Conditions: I: 4-nitrophenyl boronic acid, Pd(dppf)Cl2-DCM, Na2CO3, dioxane, H2O at 100° overnight;
Based on these failed attemps, it was thought to change the synthetic procedure, as illustrated in Scheme 3. Compound 3 and 9 was treated with 2-bromoacetaldeyhde diethyl acetal and potassium carbonate to obtain the correspondent diethyl acetal derivatives 15 and 16, purified by flash chromatography (eluent mixture: dichloromethane/methanol
38
9.5/0.5). Derivative 15 and 16 were then reacted with compound 12 in the presence of trifluoroacetic acid and NaBH3CN, obtaining compounds 17 and 18 which were purified
by flash chromatography (eluent mixture:dichloromethane/methanol 9,5/0,5). Finally, the nitro group of compounds 17 and 18 was catalytically hydrogenated over palladium, thus obtaining the desired compounds 19 and 20.
Scheme 3 NH2 N H N O2N HN N H N O2N N X O HN N H N H2N N X O II III 12 17, 18 19, 20 N H X O N X O OEt OEt 15, 16 3, 9 3, 15, 17, 19: X = O 9, 16, 18, 20: X = NH I
Reagents and Conditions: I: 2-bromoacetaldeyhde diethyl acetal, K2CO3, 120 °C overnight; II: TFA, at 0° for 5';
39
40
Melting points were determined using a Reichert Köfler hot-stage apparatus and are uncorrected. Routine nuclear magnetic resonance spectra were recorded in DMSO-d6 and MeOD solution on a Bruker spectrometer operating at 400 MHz. Evaporation was performed in vacuo (rotary evaporator). Analytical TLC was carried out on Merck 0.2 mm precoated silica gel aluminum sheets (60 F-254). Silica gel 60 Merck (230-400 mesh ASTM) was used for column chromatography. Anhydrous reactions were performed in flame-dried glassware under N2. All compounds showed ≥ 95% purity. All reagents used
were obtained from commercial sources. All solvents were of an analytical grade.
General procedure for the synthesis of Tert-butyl
3-((benzoyloxy)methyl)piperidine-1-carboxylate (2) and tert-butyl 3-((benzamido)methyl)piperidine-1-3-((benzoyloxy)methyl)piperidine-1-carboxylate (8).
N-Boc-piperidine-3-methanol 1 or N-Boc-3-aminomethyl-piperidine 7 (0.002 mol) was
dissolved in CHCl3 coleedat 0°C. Benzoyl chloride (0.27 ml, 0.002 mol) and NEt3 (0.33
ml, 0.0024 mol) were added. The mixture was successively stirred at room temperature for 4 hours (TLC analysis: petroleum ether 30-60 °C/AcoEt 9/1).
The organic solvent was removed under reduced pressure and the residue dissolved in CHCl3 was washed with NaHCO3 5%, H20, HCl 10% and H20. The solution obtained was
dried over Na2SO4, then filtered and concentrated under reduced pressure.
The crude compound was purified by flash chromatography (petroleum ether 30-60 °C /AcOEt: 9/1).
tert-butyl 3-((benzoyloxy)methyl)piperidine-1-carboxylate 2: yield = 88%; m.p. = 66-68
°C; 1H-NMR (DMSO-d6, ppm): 8-7.97 (m, 2H); 7.70-7.66 (m, 1H); 7.55 (t, 2H, J = 7.6
Hz); 4.22-4.10 (m, 2H); 3.94-3.85 (m, 1H); 3.73-3.69 (m, 1H); 2.88-2.67 (m, 2H);
1.89-1.88 (m, 1H); 1.81-1.78 (m, 1H); 1.64-1.61 (m, 1H); 1.42-1.16 (m, 11H).
tert-butyl 3-((benzamido)methyl)piperidine-1-carboxylate 8. Yield = quantitative; m.p. =
98-100 °C; 1H-NMR (DMSO-d6, ppm): 8.53 (t, 1H, J = 5.4 Hz); 7.84 (d, 2H, J = 6.8 Hz); 7.55-7.45 (m, 3H); 3.94-3.75 (m, 2H); 3.16-3.13 (m, 2H); 2.80-2.74 (m, 1H); 1.76-1.60 (m, 3H); 1.35 (s, 11H).
41
General procedure for the synthesis of (piperidin-3-yl)methyl benzoate (3)
N-((piperidin-3-yl)methyl)benzamide (9)
Trifluoroacetic acid (2 ml, 0.002 mol) was added to a stirred solution of derivative 2 or 8 (0.002 mol) in 2 ml of DCM and the mixture was maintained at room temperature for 2 hours (TLC analysis DCM/MeOH: 9.5/0.5). Evaporation of the solvent under reduced pressure yielded a solid residue that was taken up with water. The solution obtained was cooled in an ice bath, made alkaline (pH = 10) by adding a 3M solution of NaOH, and extracted with DCM. After drying with Na2SO4, the organic solvent was eliminated under
vacuum and product 3 or 9 was isolated in the desired purity degree.
(piperidin-3-yl)methyl benzoate 3. Yield = quantitative; m.p. = 102-104 °C. 1H-NMR
(DMSO-d6, ppm): 8.49-8.43 (bs, 1H); 8.01-7.99 (m, 2H); 7.71-7.67 (m, 1H); 7.57-7.53 (m, 2H); 4.26-4.15 (m, 2H); 3.38-3.22 (m, 2H); 2.84-2.74 (m, 2H); 2.21-2.15 (m, 1H); 1.84-1.79 (m, 2H); 1.68-1.58 (m, 1H); 1.36-1.1 (m, 1H).
N-((piperidin-3-yl)methyl)benzamide 9. Yield = quantitative; oil; 1H-NMR (DMSO-d6, ppm): 8.47 (t, 1H, J = 5.6 Hz); 7.85-7.83 (m, 2H); 7.54-7.43 (m, 3H); 3.13-3.10 (m, 2H); 2.96-2.84 (m, 2H); 2.52-2.42 (m, 1H); 2.58-2.20 (m, 4H); 1.75-1.66 (m, 2H); 1.61-1.56 (m, 1H); 1.39-1.29 (m, 1H); 1.12-1.05 (m, 1H).
Procedure for the synthesis of benzyl 2-chloroacetate (4)
To a solution of chloroacetic acid (0.661 g, 0.007 mol) in 5.75 ml of toluene benzylic alchool (0.66 ml, 6.38 x 10-3 mol) and p-Toluenesulfonic acid (0.025 g, 1.31 x 10-4 mol) were added and the solution obtained was refluxed for 2 hours. (TLC analysis: petroleum ether 30-60 °C /AcOEt: 7/3)
The mixture was concentrated under reduced pressure, then extracted with dichloromethane and washed with a satured solution of NaHCO3. The organic phase was
dried over Na2SO4, filtered and the organic solvent was evaporated under reduced
42
Yield = quantitative; oil; 1H-NMR (DMSO-d6, ppm): 7.41-7.34 (m, 5H); 5.22 (s, 2H); 4.24 (s, 2H).
Procedure for the synthesis of (1-(((benzyloxy)carbonyl)methyl)piperidin-3-yl)methyl
benzoate (5) and benzyl 2-(3-((benzamido)methyl)piperidin-1-yl)acetate (10)
To a solution of benzyl 2-chloroacetate 4 (0.161 g, 0.00086 mol) in dimethylformamide, derivative 3 or 9 (0.0022 mol) and triethylamine (0.12 ml, 8.8 x 10-4 mol) were added. The mixture was then stirred overnight at room temperature (TLC analysis: AcoEt/petroleum ether 30-60 °C: 5/5).
The organic solvent was evaporated under reduced pressure. Ice was added to the solution and the resulting mixture was extracted with dichlorometane, dried over Na2SO4, filtered
and the organic solvent was evaporated under reduced pressure. The products were finally purified by flash chromatography (eluent mixture: AcoEt/petroleum ether 30-60 °C: 7,5/2,5).
(1-(((benzyloxy)carbonyl)methyl)piperidin-3-yl)methyl benzoate 5. Yield = 43 %; oil; 1H-NMR (DMSO-d6, ppm): 8.03-8.00 (m, 2H); 7.63-7.59 (m, 1H); 7.50-7.46 (m, 2H); 7.38-7.30 (m, 5H); 5.16 (s, 2H); 4.25-4.21 (m, 1H); 4.16-4.08 (m, 1H); 3.30 (s, 2H); 3.06-3.04 (m, 1H); 2.91-2.88 (m, 1H); 2.24-2.02 (m, 3H); 1.83-1.65 (m, 3H); 1.19-1.09 (m, 1H). 2-(3-((benzamido)methyl)piperidin-1-yl)acetate 10. Yield = 46%; m.p. = 97-99 °C; 1 H-NMR (DMSO-d6, ppm): 7.84-7.81 (m, 2H); 7.48-7.46 (m, 1H); 7.46-7.44 (m, 2H); 7.37-7.30 (m, 5H); 5.15 (s, 2H); 3.34-3.23 (m, 4H); 2.98-2.85 (m, 2H); 2.21-2.15 (m, 1H); 2.01-1.96 (m, 2H); 1.81-1.78 (m, 1H); 1.75-1.67 (m, 2H); 1.01-1.20 (m, 1H).
43
Procedure for the synthesis of 2-(3-((benzoyloxy)methyl)piperidin-1-yl)acetic acid (6) and 2-(3-((benzamido)methyl)piperidin-1-yl)acetic acid (11)
Pd/C 10 % was added to a solution of derivative 5 or 10 (0.001 mol) in absolute ethanol. The resulting mixture was hydrogenated under stirring for 6 hours at room temperature and reduced pressure (TLC analysis: AcOEt/petroleum ether 30-60 °C: 5/5).
Once hydrogen absorption ceased, the catalyst was filtered off and the ethanolic solution was evaporated to dryness at reduced pressure.
2-(3-((benzoyloxy)methyl)piperidin-1-yl)acetic acid 6. Yield = 92%; m.p. = 147-149 °C; 1H-NMR (DMSO-d6, ppm): 8.05-8.03 (m, 2H); 7.64-7.60 (m, 1H); 7.51-7.47 (m, 2H);
4.36-4.32 (m, 1H); 4.24-4.19 (m, 1H); 3.77-3.74 (m, 1H); 3.65-3.61 (m, 3H); 2.99-2.90 (m, 2H); 2.46-2.41 (m, 1H); 2.02-1.94 (m, 3H); 1.47-1.41 (m, 1H).
2-(3-((benzamido)methyl)piperidin-1-yl)acetic acid 11. Yield = 56%; m.p. = 196-198 °C; 1H-NMR (DMSO-d6, ppm): 7.86-7.84 (m, 2H); 7.58-7.54 (m, 1H); 7.50-7.46 (m, 2H);
3.68-3.56 (m, 4H); 3.45-3.40 (m, 1H); 3.34-3.29 (m, 1H); 2.94 (t, 1H, J = 11.4 Hz); 2.77 (t, 1H, J = 11.6 Hz); 1.97-1.86 (m, 3H); 1.36-1.31 (m, 1H).
Procedure for the synthesis of 6-(4-nitrophenyl)-1H-indazol-4-amine (12)
6-bromo-1H-indazol-4-amine (0.100 g, 4.7 x 10-4 mol), 4-nitro-phenylboronic acid (0.118 g, 7.05 x 10-4 mol), Na2CO3 (0.209 g, 1.97 x 10-3 mol) and Pd(dppf)Cl2-DCM (0.0038 g,
4 x 10-5 mol) were dissolved in a 2 ml of a mixture of dioxane : H2O (1:1).
The mixture obtained was stirred at 140 °C overnight (TLC analysis: petroleum ether 30-60 °C: AcoEt 3/7).
The mixture was concentrated under reduced pressure and then purified by flash chromatography (eluent mixture: petroleum ether 30-60 °C /AcoEt: 3/7).
