Inhibitors of switch kinase ‘spleen tyrosine kinase’ in inflammation and immune-mediated disorders: A review

Accepted Manuscript Inhibitors of switch kinase ‘Spleen Tyrosine Kinase’ in Inflammation and Immunemediated Disorders: A Review Maninder Kaur, Om Silakari PII:

S0223-5234(13)00426-1

DOI:

10.1016/j.ejmech.2013.04.070

Reference:

EJMECH 6286

To appear in:

European Journal of Medicinal Chemistry

Received Date: 21 February 2013 Revised Date:

17 April 2013

Accepted Date: 18 April 2013

Please cite this article as: M. Kaur, O. Silakari, Inhibitors of switch kinase ‘Spleen Tyrosine Kinase’ in Inflammation and Immune-mediated Disorders: A Review, European Journal of Medicinal Chemistry (2013), doi: 10.1016/j.ejmech.2013.04.070. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Graphical abstract

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Molecular Modeling Lab (MML), Department of Pharmaceutical Sciences and Drug Research, Punjabi University, Patiala, Punjab, 147002, India. Abstract: Spleen tyrosine kinase (Syk),non-receptor protein tyrosine kinase plays a significant role in the immune cell signaling, that can be sited as a therapeutically relevant target for various allergic and autoimmune disorders.

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Inhibitors of switch kinase ‘Spleen Tyrosine Kinase’ in Inflammation and Immune-mediated Disorders: A Review Maninder Kaur, Om Silakari*

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Highlights

2. Role of Syk in immune cell signaling has been discussed.

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1. The structure and biology of Syk has been described.

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3. This review also includes data regarding small molecule Syk inhibitors.

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TITLE PAGE

mediated Disorders: A Review

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Maninder Kaur, Om Silakari*

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Inhibitors of switch kinase ‘Spleen Tyrosine Kinase’ in Inflammation and Immune-

Molecular Modeling Lab (MML), Department of Pharmaceutical Sciences and Drug

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Research, Punjabi University, Patiala, Punjab, 147002, India.

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*Corresponding authors E-mail: [email protected]; Tel.: +919501542696

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KEYWORDS

Autoimmune diseases; Inflammation; Spleen tyrosine kinase; Asthma; Psoriasis; B-cell

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lymphoma

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ABSTRACT

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Spleen tyrosine kinase (Syk), a member of Syk family of non-receptor protein tyrosine kinases plays a significant role in the immune cell signaling in B cells, mast cells, macrophages and neutrophils. Anomalous regulation of this kinase can lead to different allergic disorders and

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antibody- mediated autoimmune diseases such as rheumatoid arthritis, asthma, psoriasis and allergic rhinitis. Being involved in the growth and survive mechanism of B cells, its inhibition

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can be beneficial in B-cell lymphoma. Thus, Syk can be sited as a therapeutically relevant target for various allergic and autoimmune disorders. This review article describes the structure of Syk and its role in B-cell signaling. In addition to this, data regarding small molecule inhibitors of

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Syk has also been reviewed from different papers and patents published.

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1. Introduction Immune system serves as body’s defense against bacteria, viruses, and other potential

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illness causing agents. It is composed of a complex system of cells, organs, and tissues arranged in an elaborate network designed to optimize body’s response against invasion by the many pathogens we come in contact with every day. The immune system is able to detect invaders

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(such as bacteria) and then figure out how to go about destroying or disabling the threat.

Inflammation is generally the body’s first immune response to infection and irritation,

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and is often referred to as the innate cascade [1]. It is characterized by an influx of white blood cells, redness, heat, swelling, pain, and the dysfunction of those organs involved. The inflammation can be classified as either acute or chronic. Acute inflammation is the initial response of the body to harmful stimuli and is achieved by the increased movement of plasma

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and leukocytes (especially granulocytes) from the blood into the injured tissues [2]. A cascade of biochemical events propagates and matures the inflammatory response, involving the local vascular system, the immune system and various cells within the injured tissue. The acute

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inflammatory response requires constant stimulation to be sustained. Inflammatory mediators have short half lives and are quickly degraded in the tissue. Hence, acute inflammation ceases

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once the stimulus has been removed. An acute inflammation will become chronic if the immune system is unable to rid the body of the offending foreign agent or if the agent is constantly able to re-enter the body. The chronic inflammation is a result of many autoimmune and inflammatory diseases like Alzheimer's disease, Rheumatoid arthritis, Cancer, Lupus, Severe combined immune deficient (SCID), Asthma etc [3]. The root of chronic inflammation is imbalance in immune system. Two branches of the immune system i.e. innate and adaptive are constantly communicating with each other to maintain balance in the body [4]. Their 4

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communication system involves specialized sensors and signals that unleash a cascade of biochemical reactions, producing metabolites that activate genes to relay protein messages that communicate an inflammatory call-to-action. Most critically, they are designed to turn that

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action off when they aren’t needed anymore. But patients with chronic inflammation may show increased levels of certain pro-inflammatory markers, even when there is no obvious reason for inflammation. Some of these markers include C-reactive protein, IFN-gamma, IL-1, IL-6, and

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TNF-alpha. Once the balance is disrupted, the immune system’s hyperactivity can self-

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perpetuate and quickly spiral into disease.

The inflammation is a major component of damage caused by autoimmune diseases, and is also a fundamental contributor to diseases such as cancer, diabetes, CNS disorders (stroke, traumatic brain injury, and epilepsy, multiple sclerosis, Alzheimer’s disease, Parkinson’s disease,

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depression, schizophrenia) and cardiovascular diseases.

Nonsteroidal anti-inflammatory drugs (NSAIDs) form a class of therapeutic substances that are most widely used because of their analgesic, antipyretic and antithrombogenic effects

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besides anti-inflammatory activity. While NSAIDs are effective in the management of pain and inflammation in a large number of conditions, it is well established that prolong use of these

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drugs cause some common side effects especially upper gastrointestinal (GI) damage including lesions, ‘silent’ ulcers, and life threatening perforations and hemorrhage [5-7], renal disorder [810], inhibition of the platelet aggregation and broncho constriction with resultant asthmatic problem etc [11]. Because of the substantial risks involved with the long-term use of NSAIDs there is an increasing demand for the development of newer agents with better pharmacological profile.

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In the past decade, important progress has been made in understanding the pathogenic mechanisms and defining the roles of relevant cells involved in allergic and autoimmune disorders. These breakthroughs facilitated the development of novel and effective drugs, such as

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the anti-IgE monoclonal antibody omalizumab (Xolair), tumor necrosis factor-alpha (TNF-a) inhibitor biologics [12,13]. Although these protein therapies are highly effective, they are difficult and expensive to develop, manufacture and administer. Moreover, some of these

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targeted biological treatments are associated with relevant side-effects, such as the reactivation of tuberculosis and other latent infections. For these reasons, the current research goal is to

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explore new therapeutic approaches and generate safer, more efficacious and more cost-effective therapies with improved dosing schedules. Several targets are candidates, and these include cytokines, chemokines and factors that participate in the signal transduction pathways, such as proteins of complement, adhesion molecules and kinases [14]. Most protein kinases play roles in

inflammation, and ageing.

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2. Syk

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a wide range of intracellular signaling that regulate cell proliferation, differentiation, apoptosis,

Spleen Tyrosne kinase (Syk), a cytoplasmic tyrosine kinase, is a member of non-receptor-type

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protein tyrosine kinase Syk family. It was first obtained as a 40 kDa proteolytic fragment derived from a p72 tyrosine kinase present in spleen, thymus and lung [15]. But later in 1991 Syk was cloned from porcine spleen as 72 kDa kinase [16]. Syk is entirely expressed in different cells of immune system including B-cells, mast cells, macrophages, platelets and neutrophils and non immune system including osteoclasts and breast

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cancer cells [17, 18]. On activation of Syk in these receptors, various processes including cytokine production, cell proliferation, differentiation, survival and phagocytosis orchestrates.

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2.1 Structure Syk along with Zeta associated 70 kDa protein tyrosine kinase (ZAP-70) belongs to the Syk family of cytosolic protein kinases thus sharing some common structural features. Both kinases

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are characterised by the presence of a C-terminal kinase domain and two Src homology 2 (SH2) domains separated by a linker domain. Additionally, two intervening domains named A and B

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are also present. First is located as helical coil between two SH2 domains and the second is located in the linker region that connects SH2 domain to the kinase domain (Fig. 1)[19]. The SH2 domains of Syk and ZAP-70 specifically binds to diphosphorylated immunoreceptor tyrosine-based activating motifs (ITAMS) located in cytoplasmic region of the various

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immunoreceptors such as Fc receptors, NK cell receptors, B cell and T cell receptors. ITAM is composed of a consensus motif carrying specific binding site of SH2 domains surrounded by two precisely spaced tyrosine residues.

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The relative distance between two tyrosine residues of ITAM belonging to different receptors

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can be assessed by high conformational flexibility possessed by SH2 domains of Syk [20]. Also SH2 domains can adjust their relative orientations to get fit into the space between the tyrosine residues. This distinct property of Syk enables it to interact with different immunoreceptors (Bcell and T- cell antigen receptors, NK- cell receptors) and with some G-protein-coupled receptors and modulate their activity. Thus Syk can be considered not only a protein kinase but also a real protein adaptor.

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Domain B that links SH2 domain with kinase domain seems to be crucial for Syk immunoregulatory activity [21]. Syk lacking a sequence of 23 amino acids (Syk B) is capable of comparable intrinsic enzymatic activity whereas it exhibits reduced ability to bind

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phosphorylated ITAM motif [22]. ZAP-70 binds less avidly to phosphorylate ITAM than Syk corresponding to its structural similarity to Syk B.

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2.2 Mechanism of action

Syk kinase regulates signaling in B-cells, mast cells, macrophages, neutrophiles, platelets and

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erythrocytes. Specific immunoreceptor interactions present on the membrane for example B-cell receptor antigen interaction in the B-cell, FcεRI receptor IgE interaction in mast cells and FcγR receptor interaction in macrophages initiates Syk activation [23, 24, 25]. In B-cells, after BCR ligation by the antigen, the Lyn protein tyrosine kinase (PTK),

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member of Src family, is activated and phosphorylates the ITAM motif in the cytoplasmic tail of receptor (Fig. 2). Subsequent association of phosphorylated ITAM with SH2 domain of Syk leads to the activation of Syk by autophosphorylation. The major autophosphorylation sites of

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Syk have been identified in the A and B interdomains. Activated Syk then phosphorylates several other substrates, many of which are adapter type proteins, such as SLP-65, which in turn

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recruit additional molecules, such as Vav, Grb-2, Gads and others like phospholipase Cγ (PLCγ) and ADAP (adhesion and degranulation promoting adaptor protein)], leading to stimulation of downstream pathways such as actin polymerization and mitogen-activated protein kinase (MAPK) activation. The interdomain B acts as a docking region that provides phosphotyrosyl residues for the binding to SH2 domains of other signaling molecules, such as Vav1 (Tyr342 of

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murine Syk), cCbl (Tyr317) and PLC-γ (Tyr342 or Tyr346), even if it is mediated by adapter proteins such as LAT, Gads, SLP-76 or BLNK [26-28].

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Downstream signaling: Activated lipase PLCγ1 hydrolyzes the membrane bound PIP2 into two secondary messengers inositol triphosphate (IP3) and diacyl glycerol (DAG). These secondary messengers further initiate their two different pathways such as IP3 and DAG pathway.

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IP3 mediated pathway: IP3, a product of phosphoinositol diphosphate (PIP2) hydrolysis, translocates into cytoplasm and binds to the IP3 receptors present on endoplasmic reticulum(ER)

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which in turn promotes the efflux of calcium ions from ER to the cytoplasm [29]. Cytoplamic calcium binds to a multifunctional intermediate messenger calmodulin (Calcium modulated protein) and this complex of calcium and calmodulin activates a calcium dependent serine threonine phosphatase i.e. calcineurin. Activated calcineurin dephosphorylates cytoplasmic

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Nuclear factor of activated T-cells (NFAT, a transcription factor) and activates it, thereafter the activated cNFAT translocates into nucleus and regulates the expression of pro-inflammatory cytokines such as interleukin 2 (IL-2) that in turn promotes the growth and differentiation of T-

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cells [30].

DAG mediated pathway: DAG, the second product of PIP2 hydrolysis, regulates the activity of

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protein kinase C theta and mitogen activated protein kinases (MAPKs). PKC-θ activates inhibitor of kappa B kinase (IKK) which degrades an inhibitory protein inhibitor of kappa B (IκBα) bound to NFκB and release free (activated) NFκB that translocates into the nucleus to perform transcriptional role for genetic modulation [31, 32]. DAG also phosphorylates Ras guanyl nucleotide releasing protein (RasGRP) and release Ras that activates Jun N-terminal kinase (JNK) and p38 kinase which phosphorylates c-Jun and c-Fos respectively [33]. Activated c-Jun

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and c-Fos translocates into the nucleus and in combination form a transcription factor Activating Protein-1 (AP-1) that is important for the genetic modulation [34].

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2.3 Synthetic derivatives of Syk inhibitors 2.3.1 Naphthyridines- [1,6]naphthyridine is the basic nucleus that is essential to exhibit Syk inhibitory activity (Fig. 3A)[35]. Substitutions can be done at 5th and 7th position in order to get

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potent Syk inhibitors. At 7th position an aryl substituent is essentially required for the activity. Phenyl group at position 7 (1, Table 1) seems to be important for the activity which can further

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be substituted at ortho, meta and para position. Substitutions at ortho (2) and meta (3) position of phenyl ring leads to decrease in activity whereas substitution at para position gives molecules with greater activity (4). Substituent at the para position should be of small size because the activity decreases as the size increases (5). Larger group can only be tolerated if it is additionally

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supported with polar functionality (6).

At 5th position alkylene diamine chain should be present. Chain with three to four carbons is optimal for the activity whereas compound with 5 carbons exhibit decreased activity (7). Mono

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methylation at either amine can be tolerated (8, 9) whereas di or trimethylation results in decreased activity (10, 11, 12). Replacement of either nitrogen of amine group by oxygen results

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in decreased activity by one log order (13, 14, and 15). At position 3 small groups like bromine can be tolerated (16) whereas large groups like phenyl cannot be tolerated (17). The reported docking studies of naphthyridines showed four essential hydrogen bond interactions such as terminal amino (-NH2 ) group of ligand with Asp512 (N…..O; 2.52Å) and Asn499 (N…..O; 2.63Å) amino acid residues of protein. Other H-bonds are between N atom

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of napthyridine ring (1st

position) with Ala451 (N…..N; 3.02Å) and oxygen of

morpholine ring with Gln462 (O…..N; 2.90) [35].

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2.3.2 Pyrimidine-5-carboxamide- Substitutions can be made at three positions i.e. 2nd, 4th and 5th position of pyrimidine nucleus(Fig. 3B)[36]. At 2nd position an alkylene diamine chain of two to four carbons is essential for Syk inhibitory activity for example compounds with ethylene diamine (18, Table 1) and butylene diamine (19) chain shows IC50 of 0.041µM and 0.047 µM

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whereas compound with propylene diamine chain exhibits activity of 0.23 µM. Methylation at

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either N of diamino group results in greater loss in potency (20 and 21).

Anilino group substituted or unsubstituted at position 4 is must for the activity. Any other group at this position results in complete loss in the activity (22). Meta substitution of anilino group (23) gives compounds with good inhibitory activity as compared to ortho or para substituted

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compounds (24, 25). Ortho and para substituted compounds show similar activity to that of unsubstituted anilino group. Bromo substituted anilino group at position 4 shows the highest potency i.e. 0.023 µM (26).

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Carboxamide group at position 5 is necessary for making the interactions with the receptor via

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NH2 group. N methylated carboxamide derivatives resulted in complete loss of activity (27, 28). SAR is complementary to the docking study reported by Hisamichi et al. Three docking interactions were exhibited by the molecule i. e. NH of the carboxamide group forms bond with carbonyl O of Glu449, O of carbonyl group of carboxamide group forms a bond with NH of Ala 451 and NH of aniline group forms a bond with carbonyl group of Ala 451. These interactions results in potent Syk inhibitors.

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2.3.3 Imidazo[1,2-c]pyrimidine- 5th and 7th position of the imidazo[1,2-c]pyrimidine can be substituted with different groups to get potent Syk inhibitors (Fig. 3C)[37]. Ethylene diamino chain at 5th position gives compound with good inhibitory activity (29, Table 1). The activity

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decreases if the length of carbon chain increases (30). Simple amino derivatives exhibit very weak Syk inhibition (31). Cylcohexyldiamino group of specific stereochemistry can also be tolerated at this position. For instance cis- cyclohexyldiamino derivative shows excellent

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inhibitory activity whereas trans- cyclohexyldiamino derivatives showed very weak Syk inhibition. Anilino group at position 7 is essential for good inhibitory activity. Phenyl ring of

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anilino group can be substituted at meta and para positions and meta position was found to be more favourable as compared to para position (32, 33). Further it was observed that di substitution is superior to mono substitution at meta position (34, 35). 2.3.4 1,2,4-triazolo[4,3-c]pyrimidine- Substitutions can be done at 5th and 3rd position of the

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skeleton (Fig. 3D)[38]. Ethylene diamine chain at position 5 gives the most potent compound (36, Table 1) i.e. IC50 of 0.009 µM. Activity decreases when the carbon chain length is varied (37) or when terminal amino group is replaced by hydroxy group or tertiary butyl group whereas

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the activity is retained if ethylene diamino group is replaced by cis-cyclohexyl diamino group

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(38). This suggests that proper position of amino group is necessary for Syk inhibitory activity. Dimroth rearrangement results in 1,2,4-triazolo[1,5-c]pyrimidine derivatives that exhibit similar inhibitory activity (39). At position 3 in 1,2,4-triazolo[4,3-c]pyrimidine nucleus and position 2 in 1,2,4-triazolo[1,5-c]pyrimidine nucleus incorporation of substituted (40) or unsubstituted phenyl group (41) results in decrease of inhibitory activity. 2.3.5 4- thiazolyl-2-phenylaminopyrimidines- Substitution can be done at aniline ring and thiazole ring (Fig. 3E) [39]. 3,5-disubstituted aniline ring derivatives are highly active 12

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compounds of this series. The most potent compound is with 3,5-dimethyl aniline ring (42, Table 1) i.e. IC50 0.550 µM whereas 3,6-dimethyl aniline ring analog shows very weak Syk inhibitory activity. Removal of one methyl group (43) or changing the position of methyl group (44) results

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in weak Syk inhibitors. Replacement of one methyl group with triflouromethyl and other one either with methoxy or with hydroxy results in decrease of Syk inhibitory activity value (45, 46). In order to increase cellular potency substitutions can be done at position 4 and 5 of thiazole ring

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like hydroxy methyl (47). Replacement of hydroxy methyl with alkanol side chain enhances the

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stability (48).

It has been revealed from the docking studies reported by Farmer et al. that aniline ring should be present in the same plane as that of pyrimidine core. The substituent that causes the aniline ring to lie out of the plane leads to loss of the activity.

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2.3.6 Oxindoles- The highest active oxindole derivative exhibit IC50 of 5 nM (49, Table 1) [40]. Saturation of the double bond leads to decrease in the potency i.e. IC50 of 8100 nM (50). Also replacement of both hydrogens of sulphonamide results in lower activity showing the importance

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of hydrogen at that position (51, 52) (Fig. 3F).

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2.4 Natural inhibitors of Syk

Curcumin is reported as Syk inhibitor by Gururajan et al. in 2007. It was observed that curcumin modifies Syk signaling pathway hence can be used in the treatment of B-cell lymphoma [41]. Inhibitory activity of piceatannol against Syk is not clear yet [42]. (Table 1) 2.5 Functions of Syk

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A number of in vivo animal models and Syk knockout experiments have been studied for inflammation, rheumatoid arthritis, asthma, allergic rhinitis, B-cell lymphoma, Intestinal Ischemia reperfusion injury, Systemic lupus erythematosus, Idiopathic thrombocytopenic

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purpura and these experimental reports have clearly demonstrated the benefits of Syk inhibition in these mentioned pathological conditions [43-49].

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2.5.1 Rheumatoid Arthritis: Despite extensive efforts, the etiology and pathogenesis of RA remain poorly understood, and numerous cell populations have been implicated including T

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cells, B cells, monocytes/macrophages, mast cells, dendritic cells and fibroblasts. Chemokines, matrix metalloproteinases, adhesion molecules, angiogenic growth factors, and dysregulated intra-articular expression of proinflammatory cytokines (in particular IL-1β and TNFα) play key role in pathogenesis of RA [50]. Immune complexes are also deposited at the cartilage level in RA [51]. FcR polymorphism was found to be an important disease susceptibility factor by

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genetic linkage studies. Additional factors that contribute to pathogenesis and susceptibility of RA are functional polymorphic variants within FcRG2A or FcRG2B, or within FcRG3A or FcGR3B. In the past decade, treatment of RA included non steroidal anti-inflammatory drugs

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(NSAIDS), disease- modifying antirheumatic drugs (DMARDS) [52]. These drugs were

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associated with adverse side effects such as gastrointestinal and renal side-effects and liver toxicity. Due to unmet desired needs these drugs were replaced by biological agents such as TNFα blockers (etanercept, infliximab and adalimumab), IL-1 receptor antagonists (anakinra and AMG 108), monoclonal antibodies against B cells (rituximab, ocrelizumab and HuMax), T-cell co-stimulation blockers (abatacept) and IL-6 receptor antagonists (tocilizumab) [53]. Concerns still prevailed regarding their immunosuppressive effects and increased risk of infection. Thus, there was a need to develop small kinase inhibitors that participate in signal transduction. Based 14

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on the finding that Syk-deficient bone marrow murine chimeras do not allow the development of arthritis following the injection of arthritogenic K/BxN serum, Syk-dependent signaling was

Fostamatinib

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prodrug of R406

Syk

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considered important in the development of arthritis [54]. inhibitor,

developed

by Rigel

Pharmaceuticals Inc. has undergone preclinical studies, Phase I and Phase II clinical trials (Fig.

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4). Both R788 and R406 inhibited local inflammatory injury mediated by immune complex in reverse-passive arthus reaction and two-antibody-induced arthritis models by inhibiting the Fc

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receptor signaling [56]. Also, progression and severity of clinically induced arthritis was inhibited in R788/R406 treated mice. Administration of R788/R406, after disease onset, completely suppressed all manifestations including tissue swelling, joint space narrowing, inflammatory cell infiltration into the synovial fluid and tissue, inflammatory cytokine secretion, IL-8 accumulation in synovium, bone erosion and accumulation of cartilage degradation

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products in the serum. A small Phase I study demonstrated clinical efficacy of R788 without any serious side effects. In Phase II studies, twice-daily oral doses of 100mg and 150mg of R788 showed superior efficacy over the placebo at ACR20 (American College of Rheumatology 20%

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improvement criteria [56]) (65, 72%), ACR50 (49, 57%) and ACR70 (33, 40%) [55]. Diarrhea,

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nausea, gastritis, neutropenia and elevated liver alanine aminotransferase levels were reported as major side effects. AstraZeneca designed a global phase III program with the goal of filing New Drug Applications with the US Food and Drug Administration and European Medicines Agency in 2013(AstraZeneca and Rigel Pharmaceuticals sign worldwide license agreement for late stage development product – fostamatinib disodium (r788) – for the treatment of rheumatoid arthritis (RA). Rigel Pharmaceuticals Inc. Press release, February 16, 2010.). Thus, R788 can be used as potential treatment of RA in future. 15

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2.5.2 Allergic conditions: Pathophysiology of allergic conditions including asthma and allergic rhinitis is associated with production of allergen-specific IgE. IgE then binds to FcεRI receptor, present on the surface of mast cells and basophils in the mucosal linings of the airways. This

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binding sensitizes the cells to specific allergen, stabilizes the receptor complex and causes the substantial increase in the expression of FcεRI on the cell surface. Exposure to various allergens causes the cross linking of IgE- FcεRI complex resulting in the release and production of various

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mediators that play major role in allergic responses [57]. Current therapies in allergic conditions treatment include single mediator antagonists example antihistamine H1 receptor antagonists,

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leukotriene modifiers. Another therapy is to aim at inhibiting the production and release of all mediators by antagonizing IgE action. This includes human recombinant monoclonal antibody Omalizumab [58]. Alternatively, targeting the intracellular signaling cascade may represent an attractive approach. Syk represents the most attractive target because studies with mast cells

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derived from Syk are important in the activation of mediators of degranulation, eicosanoid, and cytokine production [59]. BAY 61-3606 (Fig. 4), potent and selective Syk inhibitor, developed by Bayer researchers, blocked both degranulation and cytokine synthesis in mast cells and

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suppressed antigen-induced passive coetaneous reaction, bronchoconstriction, bronchial edema and airway inflammation [60]. Another Syk kinase inhibitor NVP-QAB205-AA (Fig. 4) showed

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efficacy in vivo asthma models [61]. R406 inhibited globet cell metaplasia and airway hyperresponsiveness, developed in mice exposed to aerosolized 1% ovalbumin for 10 days [62]. This preclinical study justifies clinical significance of Syk inhibition. Rigel developed R112 (Fig. 4), which was tested clinically for the safety and efficacy It significantly reduces the levels of prostaglandin D2, key mediator in nasal congestion [63]. In Phase II studies, double blind, randomized, placebo controlled testing was carried out in patients with symptomatic seasonal

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allergic rhinitis [64]. Improvement in the symptoms as compared to placebo was observed after intranasal application [65]. The most important feature of R112 was noted to be the rapid onset of action. Rigel announced R343 (Fig. 4) for advanced preclinical development in allergic

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asthma.

2.5.3 Idiopathic thrombocytopenic purpura: ITP is characterized by increased clearance of

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circulating IgG-coated platelets through Fcγ receptor-bearing macrophages in the spleen and the liver [66]. It is an autoimmune disease which is mediated by production of IgG against specific

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antigen exposed on platelets. Syk plays a central role in FcγR mediated signal transduction, thus Syk can be blocked in order to decrease platelet destruction in patients with ITP. R406/R788, Syk inhibitor, was evaluated for amelioration of ITP by using mouse models to treat cytopenia [67]. After significant preclinical study results, single-center, open-label, dose-escalating phase II study was initiated in November 2007. R788 showed improved platelet counts in this

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autoimmune disorder with minor gastrointestinal related side effects and elevated blood pressure. Hence, targeting inhibition of Syk can replace already prevailing therapies including platelet

eltrombopag).

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infusion, splenectomy, anti-D IgG and agents that increase platelet production (romiplostim and

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2.5.4 Systemic lupus erythematosus (SLE): SLE is an autoimmune disease characterized by multisystem microvascular inflammation with generation of antibodies, affecting almost every organ system of body including joints, skin, blood vessels, liver, kidney and nervous system [68]. Pathogenesis of SLE has been associated with B-cell activation in which Syk may play an important role [69]. In SLE, the FcγR-Syk associated with TCR in lieu of ZAP-70 [70]. This rewiring of the TCR has been claimed to account, for the overactive T-cell phenotype observed in SLE [71]. Preclinical study was carried out for R788 using lupus prone NZB/NZW mice. 17

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Delayed onset of proteinuria and renal dysfunction, decreased kidney infiltrates and prolonged survival in these mice were observed [72]. Additionally, arthus responses and severity of established experimental glomerulonephritis was reduced with the administration of R788.

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Moreover, lupus prone MRL/lpr and BAX/BAK mice were treated with R788 that demonstrates prevention of development of skin and renal pathology [73]. Syk inhibition reduced splenomegaly and lymphadenopathy and other immune parameters. Thus, validation of SLE

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suppression by Syk inhibition in three animal models justifies its clinical value.

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2.5.5 Intestinal Ischemia reperfusion injury: Hematopoietic cells are involved in the expression of Ischemia reperfusion injury. Thus Syk inhibition can be of some value in IRI treatment [74]. R788 was tested in mice for the suppression in both local intestinal and remote lung injury. IgM and complement 3 depositions to the effected tissues and polymorphonuclear cell infiltration were reduced significantly. Thus, Syk inhibitors can have clinical value in IRI involving

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conditions such as organ transplant and coronary and carotid revascularization. 2.5.6 B cell Lymphoma: Lymphoma is characterized by uncontrollable growth of lymphocytes or

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white blood cells that build up in the lymphatic system and bone marrow, giving rise to malignant tumors. Lymphomas can be treated by combinations of various therapies including

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chemotherapy, monoclonal antibodies, immunotherapy, radiation and hematopoietic stem cell transplantation. But in all these therapies disease recurrence is common. The uncontrolled growth of tumor cells particularly B cells, is mediated by Syk, that is involved in the signal transduction cascade required for their proliferation. Upon Syk inhibition, survival mechanism mediated by Syk (B-cell antigen receptor signaling) might be important in various B-cell lymphomas, is halted [75]. Also, mammalian target of rapamycin (mTOR) is emerging as important target for antitumor therapy and Syk plays major role in mTOR activation. Hence, Syk 18

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inhibitors can be employed in the treatment of B-cell lymphoma [76]. Rigel's Syk inhibitor R788 was able to induce apoptosis of transformed B-cells in vitro and regression of tumor in vivo.

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2.6 Patent information 2, 4-pyrimidine-diamine was reported as Syk inhibitors first by research group of Rigel Pharmaceuticals (WO2003063794 and WO02004/014382) [77, 78]. Three more patents were

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filed by Rigel later on with similar structures in WO2005/012294, WO2006/068770 and WO2007/120980 [79-81]. Later on this research group published structure of Fostamatinib, with

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its salt and method of preparation in patent literature [82]. More patents were filed describing the specific uses of Fostamatinib in WO2007/124221, WO2008/061201 and WO2008/064274 [8385].

Another Patent claiming the use of inhaled formulation of xinafoate salt of N4-[(2, 2-

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diflouro-4H-benzo [1, 4] oxazin-3-one)-6-yl]-5-flouro-N2-[3-(methylaminocarbonylmethylene oxy) phenyl]-2, 4-pyrimidine diamine in the treatment of asthma was also filed by Pfizer in 2009 [86].

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In 2001, imidazo [1, 2-c] pyrimidine (BAY 61-3606) derivatives were claimed to have Syk

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inhibitory activity by Bayer [87, 88]. In the same patent another series triazolopyrimidine derivatives was described to have Syk inhibitory activity. In 2003, another research group i. e. Boehringer Pharmaceutical also published a series of Syk inhibitors i. e. naphthyridines (WO2003/057695) [89]. Anilinopyrimidine was also claimed to be Syk inhibitors by Astellas [36].

Vertex

pharmaceuticals

also

published

a

novel

class

of

4-thiazolyl-2-

phenylaminopyrimidines as potent and selective Syk inhibitors in patent literature in 2002 [90].

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Kissei research group filed a patent of 1,2,4-triazolo[4,3-c]pyrimidine and 1,2,4triazolo[1,5-c]pyrimidine exhibiting strong inhibition of syk and ZAP-70 kinase [91]. Later on same research group filed patent of another series i.e. imidazo[1, 2-c] pyrimidine by optimizing

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its bioavailability by decreasing the size of polar group [92].

Novartis published two classes inluding purine derivatives and 2, 4, di (hetero)-

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arylammino-pyrimidine derivatives as Syk and ZAP-70 inhibitors respectively in patent literature [93, 94]. In 2007, phthalazin-1(2H)-one was also found to have Syk inhibitory activity by

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Hoffmann-La Roche research group [95]. Another pharmaceutical company Glaxo Group Limited filed a patent in 2007 showing pyrrolopyrimidine derivatives and 1H-indazol-4-yl-2, 4pyrimidine diammino derivatives as Syk inhibitors [96-98].

2.7 Kinetics of enzyme inhibition using Real Time Fluorescence Assay

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A real time fluorescence kinase assay was employed for investigating steady state kinetics and activation mechanism of Syk [99]. This kinase assay utilizes a nonnatural amino acid, the Sox amino acid, in the peptide substrate which undergoes an enhancement in fluorescence following

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phosphorylation. The experiment revealed the formation of ternary complex of kinase domain of Syk (360-635), ATP, and peptide substrate. From experimental evidences a little role of

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activation loop autophosphorylation in regulation of activity of Syk kinase was found. 10 fold greater activity of kinase domain was observed when compared with activity of full length Syk kinase that suggested negative role of SH2 and linker domain on the regulation of activity. Substrate either binds randomly or orderly with ATP binding first. R112, R406, R788 and R343 structurally related pyrimidine analogues

compete with ATP while binding. Also

imidazopyrimidine analogue BAY 61-3606 is orally selective ATP competitive Syk inhibitor.

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3. Conclusion Syk is principally involved in cell signaling in B-cells, mast cells, macrophages and neutrophils.

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It is liable for the pathogenesis of various autoimmune and inflammatory disorders like rheumatoid arthritis, asthma, allergic rhinitis etc. Thus, inhibiting this kinase can be therapeutically beneficial in treatment of above mentioned diseases. Numerous Syk inhibitors

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has been synthesized and proved therapeutically significant. Some of them have entered later phases of clinical trials. Autoimmune and inflammatory diseases are complex in nature with

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multiple pathways drawn in their pathogenesis. Targeting multiple biological pathways initiated by a single upstream receptor like Syk can be a suitable treatment strategy in future. Recently, Syk has been described as a “ switch ” kinase as opposed to Src family kinases, which are akin to “ rheostat ” kinases. The above findings emphasize to develop new Syk inhibitors for the

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treatment of several inflammatory and autoimmune conditions.

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Thoma, M. Van Eis, A. Von Matt, L. Walliser, G. Zenke, 2,4 Di (hetero)-arylamino-pyrimidine derivatives as ZAP-70 and/or Syk inhibitors. U.S. Patent 7671063, March 16, 2006. [95] D.M. Goldstein, M. Rueth, Methods of inhibiting BTK and SYK protein kinases. U.S. Patent 7501410 March 20, 2009. [96] R.A. Ancliff, L.F. Atkinson, D.M. Barker, C.P. Box, C. Daniel, M.P. Gore, B.S. Guntrip, M. Hasegawa, A.G.G. Inglis, K. Kano, Y. Miyazaki, K.V. Patel, J.T. Ritchie, S. Swanson, L.A. 34

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Walker, R.C. Wellaway, M. Woodrow, Pyrrolopyrimidine derivatives as Syk inhibitors. EP1948659 A1, October 11, 2006.

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[97] P.M. Gore, K.V. Patel, L.A. Walker, M. Woodrow, Pyrrolopyrimidine derivatives as Syk inhibitors. WO2007/042298, October 11, 2006.

[98] F.L. Atkinson, S.A. Campos, L.A. Harrison, N.J. Parr, V.K. Patel, G. Vitulli, 1H-indazol-4-

SC

yl-2,4-pyrimidinediamine derivatives. WO2007/085540, January 12, 2007.

[99] E. Papp, J.K.Y. Tse, H. Ho, S. Wang, D. Shaw, S. Lee, J. Barnett, D.C. Swinney, J.M.

M AN U

Bradshaw, Steady State Kinetics of Spleen Tyrosine Kinase Investigated by a Real Time

AC C

EP

TE D

Fluorescence Assay, Biochem. 46 (2007) 15103-15114.

35

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FIGURE CAPTIONS Figure 1. Domain structure of Syk and ZAP-70 kinase

RI PT

Figure 2. Syk regulated B-cell signaling: BCR- B-cell receptor, ; Syk- Spleen tyrosine kinase, ; Lck-Lymphocyte specific protein tyrosine kinase, ; ITAMs- Intracellular tyrosine activation motifs, ; LAT-Linker of activated T-cells, ; GADS, ; SLP65- SH2 domain containing leukocyte

SC

phosphoprotein of 65 kilodaltons, ; ITK-Interleukin-II inducible T-cell kinase, ; PLCγ1Phospholipase C γ1, ; PIP2-Phosphatidyl inositol 4,5-biphosphate, ; IP3- Inositol triphosphate, ;

M AN U

DAG- Diacyl glycerol, ; NFAT-Nuclear factor of activated T-cells, ; PKC-θ- Protein Kinase C Theta, ; IKK- IκB kinase, ; RasGRP- Ras guanyl releasing protein, ; JNK- jun-N-terminal kinase, ; c-Jun, ; c-Fos, ; AP-1-Activating protein-1.

Figure 3. General structures of different classes of Syk inhibitors: Naphthyridines (A);

TE D

Pyrimidine-5-carboxamide (B); Imidazo[1,2-c]pyrimidine (C); 1,2,4-triazolo[4,3-c]pyrimidine (D); 4- thiazolyl-2-phenylaminopyrimidines (E); Oxindoles (F).

AC C

EP

Figure 4. Structures of Syk inhibitors in clinical trials.

36

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Table 1. Molecular structures and biological activity values of different classes of Syk inhibitors. Structure

1

HN

IC50 (µM) 0.41

NH2

Compound No. Naphthyridines 10

N

HN

25

NH2 N

Br

3

HN

TE D

N

1.5

NH2 S

N

NH2 N

N

AC C

HN

EP

N

4

M AN U

2

SC

N

Structure

RI PT

Compound No.

0.90

N

IC50 (µM) 0.89

N H N

N N

11

HN

1.7

N N

N N

12

N

8.6

N N

N N

13

O

OH N

N N

0.20

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5

HN

0.42

NH2

14

NH2

O

N

0.16

N

N

RI PT

N

N HN

0.008

NH2 N

N N O

HN

0.21

NH2 N

N N

NH2 N

N

AC C

8

EP

N

N

0.072

N

OH

HN

0.20

N N N

16

TE D

7

15

M AN U

6

SC

N

HN Br

0.12

NH2 N

N O

17

HN

NH2 N

N O

3.6

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9

HN

0.078

N H N

RI PT

N N

0.041

NH O NH2

N H2N

N H

N CF3

0.047

NH

NH2

N H

EP

N H2N

O

N

CF3

0.75

NH

H2 N

N H

N

N H

0.15 NH

O NH2

N H2 N

N H

N

Br

26

0.023

NH

H2 N

N H

O NH2

N

NH2

N

NH2 N

NH O N

O

N

AC C

20

0.23

25

TE D

19

SC

18

Pyrimidine-5-carboxamide 24

M AN U

CF3

N

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CF3

21

>10

CF3

27

NH2

N N

H 2N

22

>10

NH O NH2

N H2 N

N H

N

0.03 NH O NH2

N N

EP

N H NH2 N

N

O HN

O

0.23

N

N H

AC C

29

NH2

NH

N H

N N N H CF3

NH

N H

>10

O N

N H 2N

O

N

TE D

23

H2N

28

SC

N

M AN U

N

RI PT

NH O

>10

Imidazo[1,2-c]pyrimidine 33

O

0.092

N

H2N

N

HN

O

O

N

N H

NH2

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O

N

O

34

HN

N H

N

O

NH2

NH2 N N

HN

N

O

O

NH2

O

0.030

N

H2N

N

HN

N H

N

O

N H

N

NH2

0.006

N

H2 N

N

HN

O

N H

N

NH2

O

TE D

32

35

SC

O

7.1

0.006 N

HN

O

31

N

H2 N

N

RI PT

H2 N

1.5

M AN U

30

NH2

36

O

N N

H2N HN

O

N N

O

N H

AC C

EP

O

1,2,4-triazolo[4,3-c]pyrimidine 0.009 39

O

0.004

N N

H2 N

NH2

HN

O

N N

O

N H

NH2

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N N

O H2N

7.7

40

O

0.12

N

HN

N N

O H2N

0.009

NH2

O

TE D

O

N H

N

41

M AN U

N

HN

HN N S N

AC C

EP

42

N H

N

O

NH2

O

0.083 O

N N

H2 N

N

HN

N H

N

O

4- thiazolyl-2-phenylaminopyrimidines 0.008(Ki) 46

N

N N

HN

O

38

N

H2 N

SC

O

O

NH2

N H

N

RI PT

37

NH2

O

CF3

HO

0.044(Ki)

NH N

N S N

ACCEPTED MANUSCRIPT

43

1.817(Ki)

47

0.004(Ki)

NH NH

N

RI PT

N

N

S

44

0.082(Ki)

48

N

N S N

CF3

NH N

N S

AC C

N

49

S N

OH

0.032(Ki) NH

N

N S N

OH

TE D

O

0.028(Ki)

EP

45

M AN U

NH

SC

N

N

0.005 µM

Oxi-indoles 51

0.937 µM

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8.1 µM

OH

Piceatannol HO

10200µM

Natural inhibitors Curcumin

AC C

EP

TE D

OH

RI PT

Inactive

M AN U

OH

52

SC

50

_

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

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