FMS inhibitors based on phenoxypyrimidine scaffold as potential treatment for inflammatory disorders

Accepted Manuscript Novel LCK/FMS inhibitors based on phenoxypyrimidine scaffold as potential treatment for inflammatory disorders Ahmed Karam Farag, Ahmed Elkamhawy, Ashwini M. Londhe, Kyung-Tae Lee, Ae Nim Pae, Eun Joo Roh PII:

S0223-5234(17)30795-X

DOI:

10.1016/j.ejmech.2017.10.003

Reference:

EJMECH 9795

To appear in:

European Journal of Medicinal Chemistry

Received Date: 20 January 2017 Revised Date:

19 May 2017

Accepted Date: 2 October 2017

Please cite this article as: A.K. Farag, A. Elkamhawy, A.M. Londhe, K.-T. Lee, A.N. Pae, E.J. Roh, Novel LCK/FMS inhibitors based on phenoxypyrimidine scaffold as potential treatment for inflammatory disorders, European Journal of Medicinal Chemistry (2017), doi: 10.1016/j.ejmech.2017.10.003. 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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Novel LCK/FMS inhibitors based on phenoxypyrimidine scaffold as

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potential treatment for inflammatory disorders

Ahmed Karam Faraga,b, Ahmed Elkamhawya,c, Ashwini M. Londheb,d, Kyung-Tae Leee, Ae

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Nim Paeb,d, Eun Joo Roha,b*

Chemical Kinomics Research Center, Korea Institute of Science and Technology (KIST), Seoul 02792, Republic of Korea.

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Division of Bio-Medical Science &Technology, KIST School, Korea University of Science and Technology, Seoul 02792,

Republic of Korea. c

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Department of Pharmaceutical Organic Chemistry, Faculty of Pharmacy, Mansoura University, Mansoura 35516, Egypt. Convergence Research Center for Diagnosis, Treatment and Care System of Dementia, Korea Institute of Science and

Technology (KIST), Seoul 02792, Republic of Korea.

Department of Life and Nanopharmaceutical Science, Kyung Hee University, Seoul 130-701, Republic of Korea.

*

Corresponding author: Eun Joo Roh

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Email address: [email protected]

Postal address: Korea Institute of Science and Technology, Chemical Kinomics Research Center, Future Convergence Research Division, Hwarang-ro 14-gil 5, Seongbuk-gu, Seoul 02792, Republic of Korea.

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Abstract Tyrosine kinases including LCK and FMS are involved in inflammatory disorders

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as well as many types of cancer. Our team has designed and synthesized thirty novel pyrimidine based inhibitors targeting LCK, classified into four different series (amides, ureas, imines (Schiff base) and benzylamines). Twelve of them

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showed nanomolar IC50 values. Compound 7g showed excellent selectivity profile

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and was selectively potent over FMS kinase (IC50 value of 4.6 nM). Molecular docking study was performed to help us rationalize the obtained results and predict the possible binding mode for our compounds in both LCK and FMS. Based on the obtained biological assay data and modelling results, a detailed SAR study was

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discussed. As a further testing regarding the anti-inflammatory effect of the new compounds, in vitro cellular assay over RAW 264.7 macrophages was performed. Compound 7g exhibited excellent anti-inflammatory effect. Therefore, we report

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the design of novel phenoxypyrimidine derivatives as potent and selective LCK

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inhibitors and the discovery of 7g as potent and selective FMS/ LCK dual inhibitor for the potential application in inflammatory disorders including rheumatoid arthritis (RA).

Keywords

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LCK, FMS, Inflammation, Synthesis, molecular docking and RAW 264.7

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macrophages.

1. Introduction

Inflammatory diseases have been the concern of many research groups and

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pharmaceutical companies over the past decades. Inflammation process results in

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several disorders including multiple sclerosis, RA, inflammatory bowel disease (IBD) and psoriasis [1]. The classical treatment strategy for these diseases depends on the use of NSAIDs and corticosteroids. Despite the success that these old strategies had, the risk of resistance, unaffordable side effects and cost-of-

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treatment make new approaches in this area worthy to be considered [2]. The growing understanding of cellular signaling cascades and immune system offers

RA.

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new therapeutic avenues to a wide diversity of inflammatory disorders including

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Protein kinases covalently modify proteins by phosphorylating specific residues, thus modulating their activity. They are currently the most extensively explored drug targets in cancer research for its importance in regulating several cellular signaling pathways which control cellular proliferation, migration, apoptosis, differentiation and metabolism [3, 4]. Protein tyrosine kinases (PTK) are considered as important molecular targets on the way of treatment of inflammatory

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and autoimmune diseases [5]. To date thirty small molecule inhibitors have been approved by the FDA for treatment of cancer and inflammation disorders with two

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drugs in 2016; namely Cabozantinib and lenvatinib. Amongst these thirty approved inhibitors, only two drugs accounts for inflammatory disorders; namely tofacitinib [6] (JAK-3 selective inhibitor, approved in 2012) and nintedanib [7, 8] (multi-

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kinase inhibitor, approved in 2014) for treatment of RA and idiopathic pulmonary fibrosis respectively. Even though many other drug candidates are currently in

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clinical trials for inflammatory disorders, the needs from this research area were not yet met [9].

Since the first discovery of JAK-STAT signaling pathway and its relation to

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inflammatory cytokines in the 1990s, the search for potential PTK targets for treatment of inflammatory diseases was growing [10, 11]. Numerous kinases are now considered as approved targets for treatment of RA such as CSF1R, LCK, Btk,

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Syk and JAK3 [12]. Many research groups proved that inhibition of CSF1R can

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slow the progression of amyotrophic lateral sclerosis [13], prevent erosions and reduce symptoms of RA and bone osteolysis [14]. Others proved the use of LCK inhibitors in treatment of T cell-mediated disorders, such as organ transplant rejection [15, 16], RA or psoriasis [17], while others proved the usefulness of the dual target inhibition of FMS and KIT in preclinical disease models for inflammation and cancer [18].

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Lymphocyte-specific kinase (LCK) is a 56 kDa protein, a member of the src family of cytoplasmic tyrosine kinases that include 8 more kinases namely; Blk, Fgr, Fyn,

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HCK, Lyn, Src, Yes, Yrk [19]. It’s expressed exclusively in T cells and Natural Killer (NK) cells and it has been proved, through genetic evidences in both mice and human, that LCK is crucial for T cell receptor (TCR) mediated signaling

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required for T cell development and activation [20-24]. Different potent and selective LCK inhibitors have been developed recently [25-28] and some

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representative examples are illustrated in Figure 1.

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(Please insert Figure 1 here)

Colony-stimulating factor receptor (CSF1R) (namely; FMS) is exclusively

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expressed in monocyte-macrophages and belongs to the PDGFR family of type III receptor tyrosine kinase (RTK) which includes FLT-3, KIT, PDGFR α and β and is

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the exclusive receptor for IL-34 and CSF1. Binding of CSF1 to the extracellular domain of CSF1R leads to receptor dimerization and activation of the intracellular FMS kinase domain resulting in phosphorylation and activation of several intracellular signaling molecules ultimately resulting in the survival, proliferation and differentiation of the target cells [29]. Experiments using M-CSF blocking antibodies showed that CSF1-R aggravates collagen-induced arthritis in mice and

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CSF1-R knockout mice were more resistant to this. These experiments and others [30-33] proved that FMS is a suitable target for inflammatory diseases caused by

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(Please insert Figure 2 here)

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macrophages. Examples of known FMS inhibitors [34-36] are depicted in Figure 2.

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1.1. Rational design.

The rational design of the newly synthesized compounds is illustrated in Figure 3. compound V; a potent LCK inhibitor of Roche pharmaceuticals [37, 38], was taken

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as a lead compound.

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(Please insert Figure 3 here)

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Compound V is believed to be type I inhibitor with the 7-anilino NH and N6 of the pyrimidopyrimidine core directed to the adenine-binding pocket as hinge contactor. The side chain on position 7 is to project out of the receptor as solvent exposure allowing possible variations to this moiety. The 2,6-dichlorophenyl on N3 is believed to be directed toward the hydrophobic pocket adjacent to the ATP-binding site.

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Guided by the structural features and binding mode of compound V, we adopted a non-classical bioisosterism strategy through opening of the saturated pyrimidine

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ring of the parent pyrimido[4,5-d]pyrimidine core to get a pyrimidine based type I inhibitors. The pyrimidine was substituted on position 2 with different solvent exposure moieties and on position 4 with different phenoxy moieties to explore the

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effect of substituting this position on binding affinity and selectivity; and on position 5 with different substituted phenyl groups connected through a linker

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which is amide, urea, aminomethylene or aminomethyl groups.

Based on previous findings, our objective was to design and synthesize a novel series of 4-phenoxypyrimidne-2-amine derivatives that could inhibit LCK and/or

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FMS kinases with potential activity against inflammation disorders. Consequently, herein we report the design, synthesis, molecular docking study and SAR analysis of a series of 4-phenoxypyrimidine-2-amine derivatives and the success to obtain

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selectivity for LCK over other Src family members represented by 7j and the

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discovery of 7g as dual selective LCK/FMS inhibitor with anti-inflammatory activity represented by its ability to inhibit FMS and LCK in vitro with low nanomolar IC50 in the enzymatic assay while in the cell-based assay it was able to reduce the production of cytokines including TNF-α, IL-6 and PGE2 as well as inhibit the expression of iNOS, TNF-α and IL-6 mRNA.

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2. Results and discussion 2.1. Chemistry.

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The targeted phenoxypyrimidine-like derivatives were prepared as represented in Scheme 1. The intermediates 4a–f were synthesized in three steps from the commercially available 2,4-dichloro-5-nitropyrimidine which was substituted at

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position 4 with different phenols by SNAr reaction in acetone under basic condition

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of aqueous sodium bicarbonate solution as reported in literature [39, 40] to afford 2a–e. Refluxing compounds 2a–e with different anilines; namely 3,5dimethoxyaniline and 4-morpholinoaniline in THF and pyridine afforded the novel nitro derivatives 3a–f in a good yield as yellow to deep red solids [41]. Compounds

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3a–f were reduced to the corresponding amines by catalytic hydrogenation in

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methanol and DCM at room temperature using Pd/C as a catalyst to obtain 4a–f.

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(Please insert Scheme 1 here)

Compounds 4a–f were then refluxed with: (i) acid chlorides to afford the amides 5a–b; (ii) isocyanates in DCM to afford the corresponding ureas 6a–k; (iii) aldehydes in dry isopropanol (IPA) under anhydrous conditions and under N2 to

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afford the Schiff bases 7a–m; (iv) aldehyde in dry IPA then NaBH3CN to afford the benzylamines 8a–d.

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Several reported solvents and conditions were explored for the preparation of 7a– m and consequently 8a–d, such as MeOH, DCM, toluene and neat reaction with IPA being the best option with the highest yield and to complete reaction in our

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case [42-45]. 7g was taken as illustrative example to confirm the E-configuration

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of the Schiff bases 7a–m. Based on the 3D shape of the imines, we hypothesized that the pyrimidine proton and the imine proton will be close to each other in space, if the imine attain the E-configuration. To confirm that, COSY and NOESY 2D NMR experiments were carried out. The cross peak between the aforementioned

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protons was observed (see supporting material for the NMR charts). Together with the well-known energy difference between the E-isomers and the Z-isomers and the potential steric clash between the pyrimidine proton and the ortho-proton

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on the phenyl ring bearing R3 substituents, confirms the E-configuration of the

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imines 7a–m. All the intermediates and final compounds were characterized by HNMR,

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CNMR and/or HRMS. Previously reported intermediates (2b and 2c)

[39, 40, 46] were assigned by 1HNMR only (see Experimental section and supporting material).

2.2. In vitro enzymatic assay.

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Compound 6b (available in our kinase library) was screened over a panel of 53 kinases as a proof-of-design and showed high selectivity for Src family members

supporting material).

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(Please insert Figure 4 here)

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including c-Src, Lyn and LCK as depicted in Figure 4 (details can be found in

Therefore it was tested in IC50 mode over these 3 kinases (LCK, Lyn and c-Src) and found to be 8-fold more potent for LCK than Lyn and 16-fold more potent than c-Src as shown in Table 1. Therefore our objective was to improve the potency and

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selectivity for LCK by adopting medicinal chemistry strategies.

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(Please insert Table 1 here)

After synthesis of Twenty-nine more derivatives, they were assayed for LCK inhibitory activity and the results are summarized in Table 2 (assay conditions, protocols and materials can be found in the supporting material).

(Please insert Table 2 here)

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2.3. Selectivity profile.

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To evaluate the selectivity of our compounds, the most potent six derivatives belonging to the Schiff base series (7e, 7g, 7h, 7j, 7k and 7l), were assayed as % inhibition at 10 uM against selected kinase panel (Figure 5). All of derivatives

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showed no inhibition against KDR, P38α and Tie2. Compound 7j and 7k showed

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very good selectivity against the closely related Src family members (c-Src, Fyn and Lyn) as well as Abl-1, c-FMS and showed very weak inhibition against JAK3. Compound 7g showed excellent selectivity against all the tested kinases with only strong inhibitory activity against c-FMS. On the other hand, 7h and 7l were the

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over JAK3.

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least selective among the tested compounds and both of them showed high potency

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(Please insert Figure 5 here)

Since 7g is the only derivative that has the 3,5-dimethoxybenzylideneamino moiety on position 5 amongst the tested compounds, we hypothesized that this moiety may be responsible for the high FMS potency. Therefore 7b, 7c, 7f, 7g and 7i were

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tested for IC50 over FMS kinase. 7c, 7f, 7g and 7i showed low nanomolar activity

2.4. SAR analysis for LCK and FMS.

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(Please insert Table 3 here)

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with 7g being the most potent with 4.6 nM IC50 value (Table 3).

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Our SAR study is based on investigating the substitution pattern on the pyrimidine core on positions 2, 4 and 5 as illustrated on compound 6b in Figure 6.

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(Please insert Figure 6 here)

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The 2-aminopyrimidine core in our design was conserved for the essential hinge anchorage. On position 2, the 3,5-dimethoxyaniline moiety was tested as a solvent

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exposure in the amide series (5a) and urea series (6a and 6d), but wasn’t tolerated (5a vs 5b and 6a vs 6b) so its use as solvent exposure moiety was discontinued. Learning the lesson from A-770041 of Abbott, the solvent exposure moiety should be devoid from a basic amine that’s capable of forming a charge-reinforced hydrogen bonding with Asp326 to be able to obtain selectivity amongst other Src family members especially c-Src and Fyn. Based on that, the 4-morpholinoaniline

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moiety was used and showed generally better potency (5b vs 5a) so was conserved in the Schiff base derivatives and the benzylamines.

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All of our derivatives are based on a substituted phenoxy group on position 4. For LCK, the 2-methoxy-4-methyl substituents generally showed optimum activity over other tested substitutions. Introduction of 4-methoxy moiety as in 7g lead to

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increased potency in comparison to 7b by 4-folds. However when 3-methoxy

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moiety was introduced, as in 7c, the activity was less than 7b by 17-folds. Introduction of 4-fluoro or 4-trifluoromethyl moieties, as in 7i and 7f respectively, significantly compromised the activity. However for FMS, the 2-methoxy-4methyl group, represented by 7b wasn’t tolerated. With exception of 7b all the

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other tested compounds namely; 7c, 7f, 7g and 7i, showed high potency. The introduction of a hydrophobic group capable of Hydrogen bond (HB) formation on position 4 was well tolerated in FMS than in LCK (7i and 7f). Therefore we

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concluded that fine tuning of the substitution on position 4 of the pyrimidine ring

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had a significant role in changing the activity for both LCK and FMS kinases. On position 5, we tested the insertion of 4 linkers resulting in amides (5a–b), ureas (6a–k), Schiff bases (7a–m) or benzylamines (8a–d). Amides were synthesized to mimic the 2 atoms linker in compound V, but the activity was unsatisfactory. Urea derivatives were synthesized and generally showed better activity (5b vs 6b). The Schiff bases were prepared to attain the sp2

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configuration of the linker without having the carbonyl O atom (cf. amides) which showed to be effective strategy in our case. In an attempt to improve the

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physicochemical properties of the Schiff base derivatives, the azomethine group was saturated to result in benzylamine derivatives (8a–d). However saturation of this double bond resulted in dramatic decrease in LCK activity as 8a was less

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active than 7m by > 1500-fold while 8d was 5-fold less active than 7h.

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In the urea series, substituting the phenyl ring with hydrophobic groups at 3’,5’positions was not tolerated (6f and 6k), however substituting this position with 3’,5’-dimethoxy was tolerated (6b and 6h) albeit with low potency. 2’,6’-dichloro substituents (6e) and 2’-methoxy (6g) were still tolerated by the receptor.

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In the Schiff base series, hydrophobic substituents were favored over hydrophilic ones (7j vs 7b). Mono-substitution on positions 3’ or 4’ with hydrophobic

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substituents resulted in 2-digit nanomolar active compounds (7e, 7h and 7k) while disubstitution resulted in single digit nanomolar inhibitors (7j and 7m).

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Substitution on position 2’ showed less activity (7d vs 7e or 7h). It should be noted that 7g has 3’,5’-dimethoxy substitution on the phenyl ring yet shows 2-digit nanomolar activity probably because of having different phenoxy part on position 4 of the pyrimidine ring. It was noted that the Schiff base derivatives (7a–m) showed the most potent activity in our series with 7j and 7m being the most potent LCK inhibitors.

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In the benzylamine series, the saturation of the N–C bond aimed at conferring flexibility to this position and it was observed that position 4’ was the best for

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substitution with small hydrophobic group (8b vs 8c and 8d vs 8c). In summary, Schiff bases were the most potent derivatives among the newly synthesized compounds. Compounds 7j and 7m were the most potent LCK

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inhibitors in our series. Compound 7g had the highest potency for FMS, showed

2.5. Molecular docking study.

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high potency for LCK and had excellent selectivity profile.

In the pursuit of understanding the binding mode of our potent compounds with LCK and FMS kinases, we performed a molecular docking study using

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Schrodinger software suit (maestro) [47].

First of all, 7j; the most potent LCK inhibitor in our series, was docked in the

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active crystal structure of LCK kinase domain. By analysis of the obtained docking

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poses, we found that 7j showed 2 possible binding modes: one normal mode that fits our design and the other is a flipped mode where the morpholino part in buried deep inside the receptor and held in that position by a HB contact with Lys273 (see supporting information). As expected from our design, the pyrimidine N3 together with the anilino NH were able to make a pair of HB anchorage to the hinge Met319 (Figure 7A). In the normal binding pose, the 3’,5’-dichlorophenyl was

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buried deep inside the hydrophobic pocket near the ATP binding site and contributed into several van der waal interactions explaining the high potency of 7j.

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Since the normal binding pose accounts for a better docking score of -7.707 compared to -7.298 for the flipped mode, we believe that this mode is more close to the real binding mode and explains the enzyme assay data more clearly. It’s also

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Tyr318 in the hinge region (Figure 7B).

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worth mentioning that the pyrimidine core formed a pi-pi stacking interaction with

(Please insert Figure 7 here)

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On the other hand, 7g showed a flipped binding mode with the 3’,5’dimethoxyphenyl pointing outside of the receptor, yet it was able to make a HB to

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the hinge Met319 along with several van der waal interactions with Tyr318, Val295 and Leu371 and HB with Lys273 probably contributed to its potency

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(Figure 8).

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In an attempt to rationalize the high potency of our FMS inhibitors, Compound 7g was docked in the active crystal structure of FMS kinase domain. In all the docking

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poses, 7g showed a flipped binding mode with the morpholino moiety inserted deep inside the binding pocket and projects into the solvent area interacting with Leu558 with the GLIDE docking score -6.000. Pyrimidine N3 and the aniline NH

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anchored the hinge part with pair of HB with residue TYR665. Another HB was observed between oxygen of 4-methoxy on the phenoxy ring and Lys612 located

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on β3 close to the hinge region of FMS kinase (Figure 9A and 9B). Similarly, 7b was docked into FMS and showed a lower docking score of -5.758. It showed only one HB interaction with residue Tyr665 in the hinge region which explains its

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lower potency (Figure 9C).

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(Please insert Figure 9 here)

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Compound 7g was shown to be more potent than 7b by 190-fold while the only difference is the substitution pattern on the phenoxy moiety. The p-methoxy in 7g was replaced by p-methyl in 7b along with having a methoxy group on the 2position in case of 7b. We believe that having the methoxy group at position 2 lead to steric bulkiness that forced the compound to shift from binding tightly with the receptor along with losing its chance to bind with Lys612 due to having a methyl

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group. This comparison highlights the importance of carefully modulating the substitution pattern on the phenoxy moiety to avoid bulkiness while keeping a

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(Please insert Figure 10 here)

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small HBA in position 4 for potential HB with Lys612 (Figure 10).

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2.6. Cellular assay for anti-inflammatory activity.

Stimulation of CSF1R by CSF1 is known to increase the production LPS as well as other inflammatory cytokines such as TNF-α and IL-1β [48, 49]. It’s also known

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that stimulation of Src family kinases has a significant role in the production of LPS and therefore pro-inflammatory cytokines [50]. Inhibition of CSF1R and LCK by small molecules is known to inhibit the production of these cytokines resulting

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in anti-inflammatory action. Therefore, to test the anti-inflammatory effect of our

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compounds, LPS-induced RAW 264.7 macrophages were used. All the materials and experimental methods can be found in the supporting material. The results for each test are described in details as follow. 2.6.1. Cell viability and NO production in RAW 264.7 macrophages.

Since it’s very important for our compounds to show anti-inflammatory effect without significant cytotoxicity to the macrophages, our compounds were first

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tested for cell viability in vitro in raw 264.7 macrophages side by side to NO production assay as a preliminary evaluation method and the results are shown in

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Figures 11 and 12. A combined results for both tests are tabulated in Table 4. The results showed that with exception of 7k, all of the tested compounds didn’t show significant cytotoxicity to the raw 264.7 macrophages. It was also noted that 7g

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was able to inhibit the LPS-induced NO production in RAW 264.7 macrophages in dose-dependent manner and showed more than 2.5-fold better potency than the

inflammatory screening assay.

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positive control used (L-NIL [51]) therefore it was selected for further anti-

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(Please insert Figure 11 here)

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(Please insert Figure 12 here)

(Please insert Table 4 here)

2.6.2. LPS-induced PGE2 production in RAW 264.7 macrophages.

It’s known that RAW 264.7 macrophages release PGE2 upon stimulation with LPS so 7g was tested for LPS-induced PGE2 production inhibition. As shown in Figure

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13, 7g was able to inhibit the production of PGE2 in dose-dependent manner. NS398, a known selective and potent COX-2 inhibitor [52], was used as a positive

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(Please insert Figure 13 here)

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control agent.

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2.6.3. iNOS and COX-2 proteins and mRNA expressions in LPS-induced RAW 264.7 macrophages.

Compound 7g was then tested to determine whether its anti-inflammatory effect

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was attributed to inhibiting the expression of COX-2 and iNOS proteins. Western plotting technique was used and the results showed that LPS upregulated the levels of COX-2 and iNOS proteins and that pretreatment with 7g was able to inhibit the

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upregulation of iNOS and iNOS mRNA, but not COX-2 enzyme as illustrated in

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Figure 14. This results clarify that the inhibition of PGE2 production is not related to COX-2 inhibition.

(Please insert Figure 14 here)

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2.6.4. Productions of pro-inflammatory cytokines in LPS-induced RAW 264.7 macrophages.

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The effect of 7g on LPS-induced production of TNF-α, IL-1β and IL-6 was explored and the results are shown in Figure 15. 7g was able to inhibit the

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the production of IL-1β at the same concentration.

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production of TNF-α and IL-6 at concentration of 12 uM, but was unable to inhibit

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2.6.5. Expressions of TNF-α and IL-6 mRNA in LPS-induced RAW 264.7 macrophage.

The effect of 7g on the expression of TNF-α and IL-6 mRNA in LPS-induced

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RAW 264.7 macrophages was explored to clarify the reason for the noticed

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inhibition of TNF-α and IL-6. 7g was able to inhibit the expression of TNF-α and IL-6 mRNA at 2–4 h as shown in Figure 16.

(Please insert Figure 16 here)

3. Conclusion

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In conclusion, the search for a potent and selective kinase inhibitors to modulate the immune system and target inflammatory diseases and cancers where immunity

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is involved, is still a growing research area and the interest of several research groups and pharmaceutical companies. In this report we have designed and synthesized four series of a novel inhibitors based on phenoxypyrimidine-like

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scaffold for LCK and FMS kinases in an attempt to target inflammatory disorders. In vitro enzymatic assay for both kinases, selectivity profile as a proof-of-design,

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SAR study, molecular docking study and in vitro cell-based assay over LPSinduced RAW 264.7 macrophages were all performed as part of this study. We identified a novel LCK selective inhibitors (for example; 7j), LCK-FMS dual

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inhibitor (7g) and FMS selective inhibitors (for example; 7f). The SAR and the modelling results helped us to rationalize the obtained results. Hence we introduce 7g as potential dual FMS/LCK inhibitor with excellent selectivity profile, for the

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treatment of inflammatory disorders including RA. We believe that this report can

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be a step forward in the search for dual kinase inhibitors, acting through different mechanisms, to target inflammatory disorders. The obtained results proves the hypothesis that this novel substitution and substitution pattern on the pyrimidine ring worth to be deeply explored with more derivatives and more biological assays.

4. Experimental section

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4.1. Chemistry General: All reactions and manipulations were performed using standard Schlenk

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techniques. Starting materials, reagents and solvents were purchased from commercial suppliers and were used without purification with the following exception. Deuterated chloroform was pretreated according to literature method

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[53] and was only used for the imine samples (7a-m). All melting points were measured by Optimelt Automated Melting Point System (Stanford Research

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Systems) and are uncorrected. Thin-layer chromatography was performed with Merk silica gel 60 F254 pre-coated glass sheets. Column chromatography was performed on Merck Silica Gel 60 (230-400 mesh) for all the samples except the

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imines, and Aluminum oxide (activated, neutral, Brockmann I of Aldrich Co.) for the imines and the eluting solvents are noted as mixed solvent with given volumeto-volume ratios or as percentage. 1H & 13C NMR was measured on a 400 MHz

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Bruker Avance NMR spectrometer. Chemical shifts and coupling constants are

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presented in parts per million (ppm) relative to Me4Si and hertz (Hz), respectively and the following abbreviations are used: s, singlet; d, doublet; dd, doublet of doublets; ddd, doublet of doublets of doublets; t, triplet; m, multiplet. NMR spectra were obtained from DMSO-d6 or CDCl3 solutions as indicated. High-resolution mass spectra were performed on Waters ACQUITY UPLC BEH C18 1.7µ−Q-TOF SYNAPT G2-Si High Definition Mass Spectrometry.

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4.2. General procedure for preparation of 2a-e [39, 40]

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To a solution of 2,4-dichloro-5-nitro-pyrimidine (1.0 g; 5.15 mmol) in acetone (20 mL), cooled to 0° C., was added a solution of appropriate phenol (5.15 mmol) in a mixture of 1 N NaHCO3 (aq) (5 mL) and H2O (2 mL) dropwise. After completion

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of addition, the reaction mixture was allowed to warm to ambient temperature, and

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was then stirred at room temperature for 2 h (monitored by TLC). The resultant mixture was evaporated in vacuo, and the residue was washed sequentially with EtOAc, 1 N NaOH (aq) (3 x 100 mL). The organic phase was washed sequentially with H2O (3 x 200 mL) and brine (3 x 200 mL), and dried over anhydrous MgSO4.

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The mixture was filtered and the solvent was evaporated under reduced pressure to give the crude material. The crude material was purified by flash column

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chromatography using 10 – 20% EtOAc in hexane mixture as the mobile phase to yield 2a-e as yellow oil solidifies on trituration with hexane and drying.

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4.2.1. 2-chloro-4-(2-methoxy-4-methylphenoxy)-5-nitropyrimidine (2a)

Yellowish white solid, yield 85%, mp: 118.8–119.6 °C. 1H NMR (400 MHz, CDCl3): δ 9.18 (s, 1H), 7.07 (d, 1H, J = 7.8 Hz), 6.86–6.84 (m, 2H), 3.78 (s, 3H), 2.43 (s, 3H);

13

C NMR (100 MHz, CDCl3): δ 162.99, 162.14, 157.09, 150.07,

138.07, 137.81, 131.22, 121.59, 121.51, 113.78, 55.90, 21.56.

ACCEPTED MANUSCRIPT

4.2.2. 2-chloro-4-(3-methoxyphenoxy)-5-nitropyrimidine (2b) (Reported [46])

RI PT

Yellowish white solid, yield 59%, mp: 213.3–214.9 °C. 1H NMR (400 MHz, CDCl3): δ 9.16 (s, 1H), 7.37 (t, 1H, J = 8.3 Hz), 6.89 (ddd, 1H, J = 8.4, 2.4, 0.7 Hz), 6.78 (ddd, 1H, J = 8.1, 2.3, 0.7 Hz), 7.73 (t, 1H, J = 2.3 Hz), 3.84 (s, 3H).

SC

4.2.3. 2-chloro-4-(4-methoxyphenoxy)-5-nitropyrimidine (2c) (Reported

M AN U

[39, 40])

Yellowish white solid, yield 89%, mp: 106.1–108.1 °C. 1H NMR (400 MHz, DMSO-d6): δ 9.10 (s, 1H), 7.16–7.12 (m, 2H), 7.02–6.99 (m, 2H), 3.78 (s, 3H).

TE D

4.2.4. 2-chloro-4-(4-(trifluoromethyl)phenoxy)-5-nitropyrimidine (2d) Yellowish white solid, yield 48%. 1H NMR (400 MHz, DMSO-d6): δ 9.47 (s, 1H), 7.95 (d, 2H, J = 8.6 Hz), 7.61 (d, 2H, J = 8.4 Hz); 13C NMR (100 MHz, CDCl3): δ

EP

160.72, 155.43, 149.69, 147.80, 126.88, 125.05, 119.53, 115.65.

AC C

4.2.5. 2-chloro-4-(4-fluorophenoxy)-5-nitropyrimidine (2e) Yellowish white solid, yield 82%, mp: 238.1–240.0 °C. 1H NMR (400 MHz, CDCl3): δ 9.17 (s, 1H), 7.18–7.16 (m, 4H); 13C NMR (100 MHz, CDCl3): δ 161.90, 161.28, 160.98, 158.86, 158.21, 147.07, 132.25, 123.46, 123.37, 116.82, 116.59.

ACCEPTED MANUSCRIPT

4.3. General procedure for preparation of 3a-f [41] To a solution of compounds 2a-e (1 mmol) in THF (5 mL), cooled to 0° C.,

RI PT

was added a solution of appropriate aniline (1 mmol) in THF (3 mL) and pyridine (79.1 mg, 1 mmol). The reaction mixture was allowed to warm to ambient temperature, and was then refluxed at 80 °C for 4 h (monitored by TLC). The

SC

resultant mixture was evaporated in vacuo to give the crude material which was

the mobile phase to yield 3a-f.

M AN U

purified by column chromatography using 30 – 50% EtOAc in hexane mixtures as

4.3.1. 4-(2-methoxy-4-methylphenoxy)-N-(4-morpholinophenyl)-5nitropyrimidin-2-amine (3a)

TE D

Red orange solid, yield 73%, mp: 184.1–185.9 °C. 1H NMR (400 MHz, DMSO-d6): δ 10.63 (s, 1H), 9.13 (s, 1H), 7.13–7.08 (m, 3H), 6.87 (d, 2H, J = 8.0 Hz), 6.57 (d,

EP

2H, J = 8.7 Hz), 3.72 (t, 4H, J = 4.6 Hz), 3.69 (s, 3H), 3.00 (t, 4H, J = 4.4 Hz), 2.43 (s, 3H). HRMS (ES+): m/z calculated for C22H23N5O5: 460.1597 [M+Na]+.

AC C

Found 460.1603.

4.3.2. 4-(3-methoxyphenoxy)-N-(4-morpholinophenyl)-5-nitropyrimidin-2amine (3b)

Red solid, yield 61%. 1H NMR (400 MHz, DMSO-d6): δ 10.65 (s, 1H), 9.14 (s, 1H), 7.47–7.43 (m, 1H), 7.17 (d, 2H, J = 8.9 Hz), 7.01–6.87 (m, 3H), 6.58 (d, 2H,

ACCEPTED MANUSCRIPT

J = 9.0 Hz), 3.77 (s, 3H), 3.72 (t, 4H, J = 4.3 Hz), 2.99 (t, 4H, J = 4.1 Hz); 13C NMR (100 MHz, DMSO-d6): δ 165.51, 159.36, 156.79, 153.72, 148.37, 145.76,

RI PT

127.61, 124.24, 124.14, 122.54, 114.60, 114.46, 55.33, 54.39, 47.90, 45.63.

4.3.3. 4-(4-methoxyphenoxy)-N-(4-morpholinophenyl)-5-nitropyrimidin-2-

SC

amine (3c)

M AN U

Orange solid, yield 51%, mp: 226.0–227.1 °C. 1H NMR (400 MHz, DMSO-d6): δ 10.56 (s, 1H), 9.11 (s, 1H), 7.21 (d, 2H, J = 8.1 Hz), 7.13 (d, 2H, J = 7.8 Hz), 7.07 (d, 2H, J = 8.0 Hz), 6.57 (d, 2H, J = 7.9 Hz), 3.83 (s, 3H), 3.72 (s, 4H), 2.99 (s, 4H); 13C NMR (100 MHz, DMSO-d6): δ 163.14, 158.83, 158.11, 157.21, 147.17,

TE D

145.24, 130.08, 123.02, 122.69, 120.73, 114.69, 114.60, 65.94, 55.45, 48.54. HRMS (ES+): m/z calculated for C21H21N5O5: 446.1441 [M+Na]+. Found 446.1442.

EP

4.3.4. 4-(4-(trifluoromethyl)phenoxy)-N-(4-morpholinophenyl)-5-

AC C

nitropyrimidin-2-amine (3d) Red orange solid, yield 53%, mp: 220.1–221.0 °C. 1H NMR (400 MHz, DMSO-d6): δ 10.68 (s, 1H), 9.17 (s, 1H), 7.95 (d, 2H, J = 8.6 Hz), 7.58 (d, 2H, J = 8.4 Hz), 7.03 (d, 2H, J = 9.0 Hz), 6.54 (d, 2H, J = 9.1 Hz), 3.71 (t, 4H, J = 4.4 Hz), 2.95 (t, 4H, J = 4.6 Hz);

13

C NMR (100 MHz, DMSO-d6): δ 163.12, 159.32, 158.91,

155.62, 147.86, 130.33, 127.77, 127.52, 124.13, 123.48, 121.35, 115.56, 115.00,

ACCEPTED MANUSCRIPT

66.44, 48.93. HRMS (ES+): m/z calculated for C21H18F3N5O4: 484.1209 [M+Na]+. Found 484.1208.

RI PT

4.3.5. 4-(4-fluorophenoxy)-N-(4-morpholinophenyl)-5-nitropyrimidin-2amine (3e)

Red orange solid, yield 73%. 1H NMR (400 MHz, DMSO-d6): δ 10.65 (s, 1H),

SC

9.14 (s, 1H), 7.41–7.37 (m, 4H), 7.11 (d, 2H, J = 9.0 Hz), 6.61 (d, 2H, J = 9.0 Hz),

M AN U

3.72 (t, 4H, J = 4.8 Hz), 2.98 (t, 4H, J = 4.7 Hz); 13C NMR (100 MHz, DMSO-d6): δ 163.38, 159.33, 158.78, 148.47, 147.93, 130.36, 124.66, 124.58, 123.52, 121.43, 121.34,

117.01,

116.77,

115.56,

115.20,

66.48,

49.09.

HRMS

(ES+): m/z calculated for C20H18FN5O4: 434.1241 [M+Na]+. Found 434.1241.

TE D

4.3.6. N-(3,5-dimethoxyphenyl)-4-(2-methoxy-4-methylphenoxy)-5nitropyrimidin-2-amine (3f)

EP

Yellow solid, yield 65%, mp: 131.6–133.5 °C. 1H NMR (400 MHz, DMSO-d6): δ 10.59 (s, 1H), 9.19 (s, 1H), 7.13 (d, 1H, J = 8.0 Hz), 7.04 (s, 1H), 6.83 (d, 1H, J =

AC C

7.9 Hz), 6.57 (s, 1H), 6.18 (s, 1H), 3.71 (s, 3H), 3.60 (s, 6H), 2.38 (s, 3H);

13

C

NMR (100 MHz, CDCl3): δ 161.15, 161.07, 159.82, 158.06, 150.48, 138.99, 138.50, 137.33, 122.03, 121.35, 113.85, 101.78, 98.28, 95.96, 55.87, 55.38, 55.30, 21.53.

ACCEPTED MANUSCRIPT

4.4. General procedure for preparation of 4a-f To a solution of compounds 3a-f (1 mmol) in methanol (50 mL) and DCM

RI PT

(5 mL), was added 10% Pd/C (0.1 mmol). The reaction mixture was stirred at room temperature under hydrogen atmosphere for 6 h. After completion of the reaction, the resulting mixture was filtered through Celite. The resultant mixture was

SC

evaporated in vacuo to give the desired amine derivative 4a-f as yellow solid turns

M AN U

to deep blue on standing.

4.4.1. 4-(2-methoxy-4-methylphenoxy)-N2-(4-

morpholinophenyl)pyrimidine-2,5-diamine (4a) Yellow solid, yield 90%, mp: 130.6–132.6 °C. 1H NMR (400 MHz, DMSO-d6): δ

TE D

8.59 (s, 1H), 7.79 (s, 1H), 7.21 (d, 2H, J = 9.0 Hz), 7.04–7.01 (m, 2H), 6.81 (dd, 1H, J = 8.0, 1.2 Hz), 6.62 (d, 2H, J = 9.0 Hz), 4.79 (s, 2H), 3.72–3.68 (m, 7H),

EP

2.93 (t, 4H, J = 4.8 Hz), 2.39 (s, 3H); 13C NMR (100 MHz, DMSO-d6): δ 158.47, 152.22, 151.61, 145.15, 143.03, 139.56, 136.24, 134.84, 123.38 121.82, 121.51,

AC C

118.69, 116.07, 114.22, 66.65, 56.05, 50.08, 21.45. HRMS (ES+): m/z calculated for C22H25N5O3: 430.1855 [M+Na]+. Found 430.1855. 4.4.2. 4-(3-methoxyphenoxy)-N2-(4-morpholinophenyl)pyrimidine-2,5diamine (4b)

ACCEPTED MANUSCRIPT

Yellow solid, yield 85%, mp: 84.7–86.7 °C. 1H NMR (400 MHz, DMSO-d6): δ 8.64 (s, 1H), 7.85 (s, 1H), 7.36 (t, 1H, J = 8.1 Hz), 7.31 (d, 1H, J = 9.1 Hz), 6.87

RI PT

(dd, 1H, J = 8.3, 2.4 Hz), 6.83–6.79 (m, 2H), 6.65 (d, 1H, J = 9.0 Hz), 4.54 (s, 2H), 3.76 (s, 3H), 3.71 (d, 4H, J = 4.7 Hz), 2.94 (d, 4H, J = 5.1 Hz);

13

C NMR (100

MHz, DMSO-d6): δ 160.27, 158.54, 157.54, 153.94, 151.66, 144.86, 143.20,

SC

134.32, 129.91, 129.78, 122.38, 118.37, 115.64, 113.98, 110.72, 107.91, 107.76,

[M+Na]+. Found 416.1699.

M AN U

66.17, 55.33, 49.60. HRMS (ES+): m/z calculated for C21H23N5O3: 416.1699

4.4.3. 4-(4-methoxyphenoxy)-N2-(4-morpholinophenyl)pyrimidine-2,5diamine (4c)

TE D

Yellow solid, yield 85%, mp: 190.7–192.0 °C. 1H NMR (400 MHz, DMSO-d6): δ 8.58 (s, 1H), 7.82 (s, 1H), 7.28 (d, 2H, J = 9.0 Hz), 7.15 (d, 2H, J = 9.0 Hz), 7.02

EP

(d, 2H, J = 9.0 Hz), 6.64 (d, 2H, J = 9.0 Hz), 4.52 (s, 2H), 3.80 (s, 3H), 3.71 (t, 4H, J = 4.5 Hz), 2.93 (t, 4H, J = 4.5 Hz); 13C NMR (100 MHz, DMSO-d6): δ 158.86,

AC C

156.98, 152.24, 146.57, 145.30, 143.32, 134.71, 123.49, 122.15, 118.98, 116.13, 115.04, 114.98, 66.63, 55.93, 50.08. HRMS (ES+): m/z calculated for C21H23N5O3: 416.1699 [M+Na]+. Found 416.1700. 4.4.4. N2-(4-morpholinophenyl)-4-(4(trifluoromethyl)phenoxy)pyrimidine-2,5-diamine (4d)

ACCEPTED MANUSCRIPT

Yellow solid, yield 88%, mp: > 260.0 °C. 1H NMR (400 MHz, DMSO-d6): δ 8.70 (s, 1H), 7.91 (s, 1H), 7.85 (d, 2H, J = 8.6 Hz), 7.46 (d, 2H, J = 8.6 Hz), 7.21 (d, 2H,

4H, J = 4.8 Hz);

RI PT

J = 8.9 Hz), 6.61 (d, 2H, J = 8.8 Hz), 4.63 (s, 2H), 3.70 (t, 4H, J = 4.3 Hz), 2.91 (t, 13

C NMR (100 MHz, DMSO-d6): δ 157.61, 156.69, 152.00,

145.39, 144.50, 134.56, 127.44, 126.15, 123.42, 122.85, 118.91, 116.01, 66.62,

SC

49.97. HRMS (ES+): m/z calculated for C21H20F3N5O2: 454.1467 [M+Na]+. Found

M AN U

454.1467.

4.4.5. 4-(4-fluorophenoxy)-N2-(4-morpholinophenyl)pyrimidine-2,5diamine (4e)

Yellow solid, yield 91%, mp: > 260.0 °C. 1H NMR (400 MHz, DMSO-d6): δ 8.93

TE D

(s, 1H), 7.92 (s, 1H), 7.35–7.26 (m, 6H), 6.71 (d, 2H, J = 9.0 Hz), 3.72 (t, 4H, J = 4.3 Hz), 2.98 (t, 4H, J = 4.3 Hz);

13

C NMR (100 MHz, DMSO-d6): δ 161.09,

EP

159.47, 158.69, 152.89, 149.04, 145.39, 144.27, 134.16, 124.38, 124.29, 119.57, 116.73, 116.49, 116.32, 116.15, 66.48, 50.13. HRMS (ES+): m/z calculated for

AC C

C20H20FN5O2: 404.1499 [M+Na]+. Found 404.1500. 4.4.6. N2-(3,5-dimethoxyphenyl)-4-(2-methoxy-4methylphenoxy)pyrimidine-2,5-diamine (4f)

Purple solid, yield 90%, mp: 82.9–84.9 °C. 1H NMR (400 MHz, DMSO-d6): δ 8.70 (s, 1H), 7.83 (s, 1H), 7.03 (d, 1H, J = 7.9 Hz), 6.98 (s, 1H), 6.78 (d, 1H, J = 7.9

ACCEPTED MANUSCRIPT

Hz), 6.71 (s, 2H), 5.90 (s, 1H), 4.59 (s, 2H), 3.69 (s, 3H), 3.54 (s, 6H), 2.34 (s, 3H); 13

C NMR (100 MHz, DMSO-d6): δ 160.79, 158.01, 151.57, 151.33, 143.53, 142.39,

RI PT

139.45, 136.19, 123.32, 123.00, 121.57, 114.52, 95.99, 92.33, 56.06, 55.18, 21.44.

SC

HRMS (ES+): m/z calculated for C20H22N4O4: 405.1539 [M+Na]+. Found 405.1537.

M AN U

4.5. General procedure for preparation of 5a-b

To an ice-cold solution of the amines 4a-f (0.1 mmol) in DCM (5 mL) was added the benzoyl chloride derivative (1.1 eq). The mixture allowed to warm to room temperature and then refluxed at 50 °C for 12h. After completion of the reaction,

TE D

the solvent was evaporated and the residue was purified by flash column chromatography using EtOAc in hexane mixtures (1:3) as mobile phase to obtain

EP

the amides 5a-b as white solids.

AC C

4.5.1. N-(2-(3,5-dimethoxyphenylamino)-4-(2-methoxy-4methylphenoxy)pyrimidin-5-yl)-3,5-dimethoxybenzamide (5a)

White solid, yield 53%, mp: 161.8–162.8 °C. 1H NMR (400 MHz, DMSO-d6): δ 9.89 (s, 1H), 9.43 (s, 1H), 8.41 (s, 1H), 7.17 (d, 2H, J = 2.2 Hz), 7.01–6.99 (m, 2H), 6.79–6.75 (m, 3H), 6.71 (t, 1H, J = 2.2 Hz), 6.02 (t, 1H, J = 2.2 Hz), 3.81 (s, 6H), 3.68 (s, 3H), 3.56 (s, 6H), 2.34 (s, 3H); 13C NMR (100 MHz, DMSO-d6): δ 165.89,

ACCEPTED MANUSCRIPT

163.91, 160.87, 160.82, 157.60, 156.88, 151.22, 142.24, 139.23, 137.83, 136.59, 136.27, 122.70, 121.62, 114.66, 111.43, 106.13, 104.18, 97.39, 93.71, 56.07, 55.98,

Found 569.2010. 4.5.2. N-(4-(2-methoxy-4-methylphenoxy)-2-(4-

RI PT

55.31, 21.44. HRMS (ES+): m/z calculated for C29H30N4O7: 569.2012 [M+Na]+.

SC

morpholinophenylamino)pyrimidin-5-yl)-3,5-dimethoxybenzamide (5b)

M AN U

White solid, yield 67%, mp: 180.7–182.0 °C. 1H NMR (400 MHz, CDCl3): δ 9.23 (s, 1H), 8.01 (s, 1H), 7.14 (d, 2H, J = 8.9 Hz), 7.01 (d, 1H, J = 7.7 Hz), 6.97 (d, 2H, J = 2.2 Hz), 6.78–6.76 (m, 3H), 6.64 (d, 2H, J = 9.0 Hz), 6.56 (t, 1H, J = 2.2 Hz), 3.80–3.77 (m, 10H), 3.66 (s, 3H), 2.99 (t, 4H, J = 4.8 Hz), 2.37 (s, 3H); 13C NMR

TE D

(100 MHz, CDCl3): δ 165.51, 163.68, 160.46, 157.33, 151.15, 145.88, 138.99, 136.20, 135.88, 132.80, 122.71, 121.18, 119.59, 115.35, 113.91, 105.69, 103.78,

EP

66.21, 55.68, 55.57, 49.30, 21.02. HRMS (ES+): m/z calculated for C31H33N5O6:

AC C

594.2329 [M+Na]+. Found 594.2334.

4.6. General procedure for preparation of 6a-k To an ice-cold solution of the amines 4a-f (0.1 mmol) in DCM (5 mL) was added appropriate phenyl isocyanate derivative (1.1 eq). The mixture allowed to warm to room temperature and then refluxed at 50 °C for 12h. After completion of the

ACCEPTED MANUSCRIPT

reaction, the solvent was evaporated and the residue was purified by flash column chromatography using EtOAc-hexane mixtures (1:3 to 1:1) to obtain the urea 6a-k

RI PT

as white to yellowish white solids. 4.6.1. 1-(3,5-dimethoxyphenyl)-3-(2-(3,5-dimethoxyphenylamino)-4-(2methoxy-4-methylphenoxy)pyrimidin-5-yl)urea (6a)

SC

White solid, yield 92%, mp: 174.2–175.0 °C. 1H NMR (400 MHz, DMSO-d6): δ

M AN U

9.24 (s, 1H), 9.12 (s, 1H), 8.88 (s, 1H), 8.31 (s, 1H), 7.12 (d, 1H, J = 8.0 Hz), 7.02 (d, 1H, J = 1.5 Hz), 6.81 (dd, 1H, J = 7.5, 1.5 Hz), 6.72 (d, 2H, J = 2.1 Hz), 6.67– 6.66 (m, 2H), 6.15 (t, 1H, J = 2.2 Hz), 5.97 (t, 1H, J = 2.2 Hz), 3.72 (s, 6H), 3.71 (s, 3H), 3.55 (s, 6H), 2.36 (s, 3H); 13C NMR (100 MHz, DMSO-d6): δ 160.60, 160.26,

TE D

159.44, 154.26, 152.39, 150.58, 149.05, 142.09, 141.24, 138.46, 136.24, 122.39, 121.17, 114.04, 113.36, 96.53, 96.29, 96.23, 93.96, 92.88, 55.60, 54.96, 54.71,

584.2117.

EP

20.90. HRMS (ES+): m/z calculated for C29H31N5O7: 584.2121 [M+Na]+. Found

AC C

4.6.2. 1-(3,5-dimethoxyphenyl)-3-(4-(2-methoxy-4-methylphenoxy)-2-(4morpholinophenylamino)pyrimidin-5-yl)urea (6b)

White solid, yield 85.6%, mp: 193.4–195.2 °C. 1H NMR (400 MHz, DMSO-d6): δ 9.11 (s, 1H), 9.08 (s, 1H), 8.76 (s, 1H), 8.25 (s, 1H), 7.22 (d, 2H, J = 9.0 Hz), 7.10 (d, 1H, J = 7.9 Hz), 7.07 (d, 1H, J = 1.6 Hz), 6.84 (dd, 1H, J = 8.0, 1.2 Hz), 6.66 (d,

ACCEPTED MANUSCRIPT

2H, J = 2.2 Hz), 6.62 (d, 2H, J = 9.1 Hz), 6.14 (t, 1H, J = 2.2 Hz), 3.72–3.71 (m, 13H), 2.95 (t, 4H, J = 4.6 Hz), 2.40 (s, 3H); 13C NMR (100 MHz, DMSO-d6): δ

RI PT

160.56, 159.84, 154.62, 152.49, 150.87, 149.95, 145.34, 141.29, 138.56, 136.18, 133.16, 122.79, 121.07, 118.91, 115.27, 113.71, 112.06, 96.16, 93.82, 66.06, 55.55, 54.92, 49.24, 20.89. HRMS (ES+): m/z calculated for C31H34N6O6: 609.2438

SC

[M+Na]+. Found 609.2440.

M AN U

4.6.3. 1-(2-(3,5-dimethoxyphenylamino)-4-(2-methoxy-4-

methylphenoxy)pyrimidin-5-yl)-3-(4-methoxyphenyl)urea (6c) White solid, yield 75%. 1H NMR (400 MHz, DMSO-d6): δ 9.21 (s, 1H), 8.92 (s, 1H), 8.89 (s, 1H), 8.24 (s, 1H), 7.35 (d, 2H, J = 9.0 Hz), 7.12 (d, 1H, J = 8.0 Hz),

TE D

7.01 (d, 1H, J = 1.5 Hz), 6.87 (d, 2H, J = 9.0 Hz), 6.81 (dd, 1H, J = 8.0, 1.2 Hz), 6.73 (d, 2H, J = 2.2 Hz), 5.97 (d, 1H, J = 2.2 Hz), 3.72 (s, 3H), 3.71 (s, 3H), 3.55 (s, 13

C NMR (100 MHz, DMSO-d6): δ 160.82, 159.85, 154.99,

EP

6H), 2.36 (s, 3H);

154.62, 153.22, 151.16, 149.33, 142.70, 139.04, 136.76, 133.17, 122.98, 121.72,

AC C

120.19, 114.56, 114.28, 96.83, 93.37, 56.16, 55.68, 55.27, 21.47. HRMS (ES+): m/z calculated for C28H29N5O6: 554.2016 [M+Na]+. Found 554.2016.

4.6.4. 1-(2,6-dichlorophenyl)-3-(2-(3,5-dimethoxyphenylamino)-4-(2methoxy-4-methylphenoxy)pyrimidin-5-yl)urea (6d)

ACCEPTED MANUSCRIPT

White solid, yield 90%, mp: 209.3–209.5 °C. 1H NMR (400 MHz, DMSO-d6): δ 9.23 (s, 1H), 8.88 (s, 1H), 8.71 (s, 1H), 8.64 (s, 1H), 7.54 (d, 2H, J = 8.0 Hz), 7.32

RI PT

(t, 1H, J = 7.9 Hz), 7.15 (d, 1H, J = 8.0 Hz), 7.03 (d, 1H, J = 1.6 Hz), 6.82 (dd, 1H, J = 8.8, 1.2 Hz), 6.72 (d, 2H, J = 2.2 Hz), 5.97 (t, 1H, J = 2.2 Hz), 3.73 (s, 3H), 3.55 (s, 6H), 2.36 (s, 3H);

13

C NMR (100 MHz, DMSO-d6): δ 160.80, 154.72,

121.73,

114.59,

114.26,

96.81,

SC

152.78, 151.14, 142.65, 138.97, 136.82, 134.12, 133.58, 128.97, 128.88, 123.02, 93.34,

56.14,

55.26,

21.48.

HRMS

M AN U

(ES+): m/z calculated for C27H25Cl2N5O5: 592.1130 [M+Na]+. Found 592.1135. 4.6.5. 1-(2,6-dichlorophenyl)-3-(4-(2-methoxy-4-methylphenoxy)-2-(4morpholinophenylamino)pyrimidin-5-yl)urea (6e)

TE D

White solid, yield 93%, mp: 233.2–234.3 °C. 1H NMR (400 MHz, DMSO-d6): δ 9.09 (s, 1H), 8.77 (s, 1H), 8.65 (s, 1H), 8.56 (s, 1H), 7.54 (d, 2H, J = 8.1 Hz), 7.31

EP

(t, 1H, J = 7.8 Hz), 7.22 (d, 2H, J = 9.0 Hz), 7.13 (d, 1H, J = 7.9 Hz), 7.09 (d, 1H, J = 1.5 Hz), 6.85 (dd, 1H, J = 8.0, 1.2 Hz), 6.62 (d, 2H, J = 9.1 Hz), 3.73–3.73 (m,

AC C

7H), 2.95 (t, 4H, J = 4.6 Hz), 2.40 (s, 3H);

13

C NMR (100 MHz, DMSO-d6): δ

155.09, 152.87, 151.46, 145.90, 139.15, 136.77, 134.14, 133.75, 133.66, 128.96, 128.83, 123.42, 121.65, 119.47, 115.85, 114.30, 113.06, 66.63, 56.13, 49.82, 21.47. HRMS (ES+): m/z calculated for C29H28Cl2N6O4: 617.1447 [M+Na]+. Found 617.1450.

ACCEPTED MANUSCRIPT

4.6.6. 1-(3,5-dichlorophenyl)-3-(4-(2-methoxy-4-methylphenoxy)-2-((4morpholinophenyl)amino)pyrimidin-5-yl)urea (6f)

RI PT

White solid, yield 79%, mp: 210.1–212.1 °C. 1H NMR (400 MHz, DMSO-d6): δ 9.39 (s, 1H), 9.18 (s, 1H), 8.71 (s, 1H), 8.43 (s, 1H), 7.52 (d, 2H, J = 1.8 Hz), 7.23 (d, 2H, J = 8.8 Hz), 7.17 (t, 1H, J = 1.8 Hz), 7.10–7.08 (m, 2H), 6.84 (dd, 1H, J =

SC

8.0, 1.1 Hz), 6.63 (d, 2H, J = 9.0 Hz), 3.73–3.70 (m, 7H), 2.96 (t, 4H, J =4.6 Hz),

M AN U

2.40 (s, 3H); 13C NMR (100 MHz, DMSO-d6): δ 155.15, 152.55, 150.91, 145.46, 142.32, 138.58, 136.21, 134.05, 133.07, 122.80, 121.09, 120.72, 119.06, 115.98, 115.28, 113.74, 111.47, 66.09, 55.57, 49.25, 20.93. HRMS (ES+): m/z calculated for C29H28Cl2N6O4: 617.1447 [M+Na]+. Found 617.1447.

TE D

4.6.7. 1-(4-(2-methoxy-4-methylphenoxy)-2-((4-

morpholinophenyl)amino)pyrimidin-5-yl)-3-(2-methoxyphenyl)urea

EP

(6g)

White solid, yield 81%, mp: 140.0–141.0 °C. 1H NMR (400 MHz, DMSO-d6): δ

AC C

9.09 (s, 1H), 8.99 (s, 1H), 8.79–8.76 (m, 2H), 8.13 (dd, 1H, J = 7.8, 1.7 Hz), 7.22 (d, 2H, J = 8.9 Hz), 7.11–7.08 (m, 2H), 7.01 (dd, 1H, J = 8.1, 1.6 Hz), 6.96–6.83 (m, 3H), 6.62 (d, 2H, J = 9.1 Hz), 3.86 (s, 3H), 3.74–3.71 (m, 7H), 2.95 (t, 4H, J =4.6 Hz), 2.40 (s, 3H); 13C NMR (100 MHz, DMSO-d6): δ 160.36, 155.04, 153.30, 151.47, 150.60, 148.32, 145.87, 139.19, 136.69, 133.80, 129.25, 123.39, 122.30, 121.64, 120.97, 119.43, 119.01, 115.85, 114.28, 112.88, 111.27, 66.63, 56.21,

ACCEPTED MANUSCRIPT

56.11, 49.83, 21.47. HRMS (ES+): m/z calculated for C30H32N6O5: 579.2332 [M+Na]+. Found 579.2336.

RI PT

4.6.8. 1-(3,5-dimethoxyphenyl)-3-(4-(3-methoxyphenoxy)-2-((4morpholinophenyl)amino) pyrimidin-5-yl)urea (6h)

White solid, yield 82%, mp: 205.9–207.9 °C. 1H NMR (400 MHz, DMSO-d6): δ

SC

9.20 (s, 1H), 9.13 (s, 1H), 8.74 (s, 1H), 8.21 (s, 1H), 7.40 (t, 1H, J = 8.9 Hz), 7.32

M AN U

(d, 2H, J = 8.9 Hz), 6.93–6.90 (m, 2H), 6.86 (dd, 1H, J = 7.5, 1.6 Hz), 6.68–6.65 (m, 4H), 6.14 (t, 1H, J = 2.1 Hz), 3.77 (s, 3H), 3.72–3.71 (m, 10H), 2.95 (t, 4H, J = 4.5 Hz);

13

C NMR (100 MHz, DMSO-d6): δ 161.12, 160.86, 155.43, 153.95,

153.20, 151.54, 146.13, 141.90, 133.61, 130.58, 119.75, 115.89, 114.68, 112.97, 108.66,

96.75,

94.38,

66.63,

TE D

111.78,

55.89,

55.49,

49.82.

HRMS

(ES+): m/z calculated for C30H32N6O6: 595.2281 [M+Na]+. Found 595.2281.

EP

4.6.9. 1-(3,5-dimethoxyphenyl)-3-(2-(4-morpholinophenylamino)-4-(4(trifluoromethyl)phenoxy) pyrimidin-5-yl)urea (6i)

AC C

White solid, yield 75%, mp: 246.5–247.5 °C. 1H NMR (400 MHz, DMSO-d6): δ 9.27 (s, 1H), 9.11 (s, 1H), 8.76 (s, 1H), 8.28 (s, 1H), 7.90 (d, 2H, J = 8.6 Hz), 7.54 (d, 2H, J = 8.4 Hz), 7.19 (d, 2H, J = 8.5 Hz), 6.68 (d, 2H, J = 2.1 Hz), 6.62 (d, 2H, J = 8.9 Hz), 6.14 (t, 1H, J = 2.1 Hz), 3.71–3.69 (m, 10H), 2.93 (t, 4H, J = 4.4 Hz); 13

C NMR (100 MHz, DMSO-d6): δ 161.11, 161.02, 156.17, 155.48, 153.29, 152.68,

ACCEPTED MANUSCRIPT

146.22, 141.84, 133.24, 127.60, 127.57, 126.79, 126.47, 126.03, 123.87, 123.33, 119.91,

115.74,

112.68,

96.84,

94.40,

66.56,

55.49,

49.68.

HRMS

4.6.10.

RI PT

(ES+): m/z calculated for C30H29F3N6O5: 633.2049 [M+Na]+. Found 633.2045. 1-(3,5-dimethoxyphenyl)-3-(4-(4-fluorophenoxy)-2-(4-

morpholinophenylamino)pyrimidin-5-yl)urea (6j)

SC

White solid, yield 80%, mp: 248.0–250.0 °C. 1H NMR (400 MHz, DMSO-d6): δ

M AN U

9.18 (s, 1H), 9.08 (s, 1H), 8.74 (s, 1H), 8.21 (s, 1H), 7.34 (d, 4H, J = 6.5 Hz), 7.27 (d, 2H, J = 8.9 Hz), 6.68–6.66 (m, 4H), 6.14 (t, 1H, J = 2.2 Hz), 3.72–3.70 (m, 10H), 2.95 (t, 4H, J = 4.6 Hz); 13C NMR (100 MHz, DMSO-d6): δ 161.12, 158.87, 155.39, 153.18, 151.68, 148.97, 148.17, 146.18, 144.61, 141.88, 140.97, 135.24,

TE D

133.50, 124.60, 124.51, 119.81, 116.84, 116.61, 115.89, 96.77, 94.39, 66.61, 55.49,

583.2078. 4.6.11.

EP

49.81. HRMS (ES+): m/z calculated for C29H29FN6O5: 583.2081 [M+Na]+. Found

1-(3,5-bis(trifluoromethyl)phenyl)-3-(4-(2-methoxy-4-

AC C

methylphenoxy)-2-((4-morpholinophenyl)amino)pyrimidin-5-yl)urea (6k)

White solid, yield 85%, mp: 216.8–218.2 °C. 1H NMR (400 MHz, DMSO-d6): δ 9.72 (s, 1H), 9.21 (s, 1H), 8.69 (s, 1H), 8.50 (s, 1H), 8.12 (s, 2H), 7.65 (s, 1H), 7.23 (d, 2H, J = 8.8 Hz), 7.09–7.07 (m, 2H), 6.84 (dd, 1H, J = 8.0, 1.0 Hz), 6.63 (d, 2H,

ACCEPTED MANUSCRIPT

J = 9.0 Hz), 3.73–3.70 (m, 7H), 2.96 (t, 4H, J = 4.6 Hz), 2.40 (s, 3H); 13C NMR (100 MHz, DMSO-d6): δ 160.71, 155.37, 152.68, 150.86, 145.47, 141.72, 138.52,

115.23,

114.26,

113.72,

111.07,

66.06,

55.54,

RI PT

136.21, 132.96, 130.82, 130.50, 124.56, 122.74, 121.85, 121.05, 119.07, 117.62, 49.20,

20.89.

HRMS

SC

(ES+): m/z calculated for C31H28F6N6O4: 685.1974 [M+Na]+. Found 685.2001.

M AN U

4.7. General procedure for preparation of 7a-m

To a solution of compounds 4a-f (0.2 mmol) in anhydrous IPA (5 mL), was added catalytic amount of TFA then suitable benzaldehyde derivative (0.22 mmol). The reaction mixture was refluxed at 80 °C for 6 – 12h, the resultant mixture was

TE D

neutralized by 2 drops of TEA and evaporated in vacuo to give the crude material which was purified by column chromatography on neutral alumina as stationary

EP

phase as fast as possible and using 30–50% EtOAc in hexane mixtures as the

AC C

eluent to yield 7a-m as yellow solids. 4.7.1. (E)-5-((2,5-dimethoxybenzylidene)amino)-4-(2-methoxy-4methylphenoxy)-N-(4-morpholinophenyl)pyrimidin-2-amine (7a)

Yellow solid, yield 26%, mp: 183.6–185.6 °C. 1H NMR (400 MHz, CDCl3): δ 9.22 (s, 1H), 8.21 (s, 1H), 7.74 (d, 1H, J = 3.2 Hz), 7.19 (d, 2H, J = 8.9 Hz), 7.08 (d, 1H, J = 7.8 Hz), 6.99 (dd, 1H, J = 9.0, 3.2 Hz), 6.92–6.82 (m, 4H), 6.69 (d, 2H, J = 9.0

ACCEPTED MANUSCRIPT

Hz), 3.87–3.85 (m, 10H), 3.73 (s, 3H), 3.06 (t, 4H, J = 4.8 Hz), 2.44 (s, 3H); 13C NMR (100 MHz, CDCl3): δ 162.73, 156.87, 156.38, 154.13, 153.86, 151.67,

RI PT

151.46, 146.47, 139.82, 136.11, 132.86, 125.75, 125.52, 123.11, 121.19 119.68, 119.60, 116.45, 113.56, 112.88, 110.47, 66.99, 56.39, 55.94, 55.91, 50.21, 21.54. HRMS (ES+): m/z calculated for C31H33N5O5: 578.2379 [M+Na]+. Found 578.2379.

SC

4.7.2. (E)-5-((3,5-dimethoxybenzylidene)amino)-4-(2-methoxy-4-

M AN U

methylphenoxy)-N-(4-morpholinophenyl)pyrimidin-2-amine (7b) Yellow solid, yield 20%, mp: 169.5–170.0 °C. 1H NMR (400 MHz, DMSO-d6): δ 9.39 (s, 1H), 8.81 (s, 1H), 8.32 (s, 1H), 7.22 (d, 2H, J = 8.5 Hz), 7.11–7.08 (m, 4H), 6.84 (d, 1H, J = 8.1 Hz), 6.65–6.61 (m, 3H), 3.81 (s, 6H), 3.72 (t, 4H, J = 4.7 Hz),

TE D

3.70 (s, 3H), 2.97 (t, 4H, J = 4.6 Hz), 2.40 (s, 3H); 13C NMR (100 MHz, CDCl3): δ 162.49, 160.99, 160.43, 156.89, 153.82, 151.37, 146.60, 139.54, 138.99, 136.29,

EP

132.65, 123.95, 123.06, 121.20, 119.72, 116.42, 113.49, 106.18, 104.05, 66.98, 55.87, 55.58, 50.16, 21.55. HRMS (ES+): m/z calculated for C31H33N5O5: 578.2379

AC C

[M+Na]+. Found 578.2374.

4.7.3. (E)-5-((3,5-dimethoxybenzylidene)amino)-4-(3-methoxyphenoxy)-N(4-morpholinophenyl)pyrimidin-2-amine (7c)

Yellow solid, yield 27%, mp: 183.6–185.5 °C. 1H NMR (400 MHz, DMSO-d6): δ 9.46 (s, 1H), 8.80 (s, 1H), 8.36 (s, 1H), 7.40 (t, 1H, J = 8.6 Hz), 7.32 (d, 2H, J =

ACCEPTED MANUSCRIPT

8.0 Hz), 7.08 (d, 2H, J = 2.3 Hz), 6.93–6.91 (m, 2H), 6.87 (dd, 1H, J = 7.6, 1.5 Hz), 6.68–6.64 (m, 3H), 3.81 (s, 6H), 3.77 (s, 3H), 3.72 (t, 4H, J = 4.4 Hz), 2.96 (t, 4H,

RI PT

J = 4.8 Hz); 13C NMR (100 MHz, DMSO-d6): δ 162.23, 160.56, 160.32, 159.05, 156.98, 153.65, 145.87, 138.40, 132.49, 129.94, 119.58, 115.18, 114.20, 111.13, 108.15, 106.09, 105.88, 103.40, 66.03, 55.30, 49.11. HRMS (ES+): m/z calculated

SC

for C30H31N5O5: 564.2223 [M+Na]+. Found 564.2217.

M AN U

4.7.4. (E)-5-((2-chlorobenzylidene)amino)-4-(2-methoxy-4methylphenoxy)-N-(4-morpholinophenyl)pyrimidin-2-amine (7d) Yellow solid, yield 25%, mp: 224.6–226.0 °C. 1H NMR (400 MHz, CDCl3): δ 9.40 (s, 1H), 8.32–8.29 (m, 2H), 7.42–7.34 (m, 3H), 7.19 (d, 2H, J = 8.7 Hz), 7.09 (d,

TE D

1H, J = 7.8 Hz), 6.97 (s, 1H), 6.86–6.83 (m, 2H), 6.69 (d, 2H, J = 8.9 Hz), 3.86 (t, 4H, J = 4.6 Hz), 3.73 (s, 3H), 3.06 (t, 4H, J = 4.8 Hz), 2.45 (s, 3H); 13C NMR (100

EP

MHz, CDCl3): δ 162.66, 157.11, 156.79, 153.35, 151.34, 146.66, 139.54, 136.29, 135.85, 133.95, 132.55, 131.79, 129.85, 128.37, 127.02, 123.06, 121.18, 119.80,

AC C

116.40, 113.51, 66.97, 55.90, 50.13, 21.55. HRMS (ES+): m/z calculated for C29H28ClN5O3: 552.1778 [M+Na]+. Found 552.1773. 4.7.5. (E)-5-((3-chlorobenzylidene)amino)-4-(2-methoxy-4methylphenoxy)-N-(4-morpholinophenyl)pyrimidin-2-amine (7e)

ACCEPTED MANUSCRIPT

Yellow solid, yield 30%, mp: 201.0–203.0 °C. 1H NMR (400 MHz, DMSO-d6): δ 9.46 (s, 1H), 8.91 (s, 1H), 8.38 (s, 1H), 7.96 (s, 1H), 7.87 (d, 1H, J = 7.1 Hz), 7.59–

RI PT

7.53 (m, 2H), 7.22 (d, 2H, J = 8.0 Hz), 7.11–7.08 (m, 2H), 6.84 (d, 1H, J = 8.2 Hz), 6.62 (d, 2H, J = 8.2 Hz), 3.73–3.69 (m, 7H), 2.97 (t, 4H, J = 4.8 Hz), 2.41 (s, 3H); 13

C NMR (100 MHz, DMSO-d6): δ 162.90, 157.71, 157.56, 151.98, 151.43, 146.33,

121.58,

120.00,

115.68,

114.23,

66.61,

SC

139.45, 139.11, 136.58, 134.92, 134.16, 133.05, 131.25, 127.89, 127.51, 123.29, 56.12,

49.65,

21.48.

HRMS

M AN U

(ES+): m/z calculated for C29H28ClN5O3: 552.1778 [M+Na]+. Found 552.1785. 4.7.6. (E)-4-(4-(trifluoromethyl)phenoxy)-N-(4-morpholinophenyl)-5((3,5-dimethoxybenzylidene)amino)pyrimidin-2-amine (7f)

TE D

Yellow solid, yield 35%, mp: 222.2–223.0 °C. 1H NMR (400 MHz, DMSO-d6): δ 9.53 (s, 1H), 8.82 (s, 1H), 8.42 (s, 1H), 7.89 (d, 2H, J = 8.6 Hz), 7.55 (d, 2H, J =

EP

8.4 Hz), 7.20 (s, 2H), 7.08 (d, 2H, J = 2.2 Hz), 6.65–6.61 (m, 3H), 3.80 (s, 6H), 3.71 (t, 4H, J = 4.3 Hz), 2.94 (t, 4H, J = 4.5 Hz); 13C NMR (100 MHz, CDCl3): δ

AC C

162.47, 161.09, 160.16, 156.93, 155.63, 152.54, 146.99, 138.42, 131.80, 127.91, 127.59, 126.83, 126.79, 125.46, 124.35, 123.24, 122.76, 120.04, 116.08, 106.34, 104.29, 66.92, 55.59, 49.79. HRMS (ES+): m/z calculated for C30H28F3N5O4: 602.1991 [M+Na]+. Found 602.1995. 4.7.7. (E)-5-((3,5-dimethoxybenzylidene)amino)-4-(4-methoxyphenoxy)-N(4-morpholinophenyl)pyrimidin-2-amine (7g)

ACCEPTED MANUSCRIPT

Greenish yellow solid, yield 40%, mp: 226.1–227.3 °C. 1H NMR (400 MHz, CDCl3): δ 8.79 (s, 1H), 8.25 (s, 1H), 7.21 (d, 2H, J = 8.8 Hz), 7.14 (d, 2H, J = 9.0

RI PT

Hz), 7.07 (d, 2H, J = 2.2 Hz), 6.97 (d, 2H, J = 9.0 Hz), 6.92 (s, 1H), 6.71 (d, 2H, J = 8.9 Hz), 6.57 (t, 1H, J = 2.2 Hz), 3.86–3.84 (m, 13H), 3.06 (t, 4H, J = 4.8 Hz); 13

C NMR (100 MHz, CDCl3): δ 163.09, 161.05, 159.98, 157.15, 157.05, 152.87,

SC

146.85, 146.22, 138.74, 132.34, 124.39, 123.29, 120.07, 116.42, 114.48, 106.23, 104.18, 66.97, 55.68, 55.59, 50.09. HRMS (ES+): m/z calculated for C30H31N5O5:

M AN U

564.2223 [M+Na]+. Found 564.2222.

4.7.8. (E)-5-((4-chlorobenzylidene)amino)-4-(2-methoxy-4methylphenoxy)-N-(4-morpholinophenyl)pyrimidin-2-amine (7h)

TE D

Yellow solid, yield 35%, mp: 198.5–199.0 °C. 1H NMR (400 MHz, DMSO-d6): δ 9.43 (s, 1H), 8.90 (s, 1H), 8.37 (s, 1H), 7.94 (d, 2H, J = 8.6 Hz), 7.58 (d, 2H, J =

EP

8.5 Hz), 7.22 (d, 2H, J = 8.0 Hz), 7.10–7.07 (m, 2H), 6.84 (d, 1H, J = 7.0 Hz), 6.62 (d, 2H, J = 8.6 Hz), 3.72 (t, 4H, J = 4.5 Hz), 3.69 (s, 3H), 2.97 (t, 4H, J = 4.9 Hz); C NMR (100 MHz, DMSO-d6): δ 162.90, 157.88, 157.61, 151.44, 146.28, 139.46,

AC C

13

136.55, 136.08, 135.85, 133.11, 130.41, 129.43, 123.29, 121.58, 119.95, 115.69, 114.23, 66.61, 56.11, 49.66, 21.48. HRMS (ES+): m/z calculated for C29H28ClN5O3: 552.1778 [M+Na]+. Found 552.1776. 4.7.9. (E)-5-((3,5-dimethoxybenzylidene)amino)-4-(4-fluorophenoxy)-N(4-morpholinophenyl)pyrimidin-2-amine (7i)

ACCEPTED MANUSCRIPT

Yellow solid, yield 26%, mp: 202.5–203.3 °C. 1H NMR (400 MHz, DMSO-d6): δ 9.46 (s, 1H), 8.82 (s, 1H), 8.38 (s, 1H), 7.37–7.35 (m, 4H), 7.28 (d, 2H, J = 7.8 Hz),

2.98 (t, 4H, J = 4.6 Hz);

13

RI PT

7.10 (d, 2H, J = 2.3 Hz), 6.69–6.65 (m, 3H), 3.82 (s, 6H), 3.73 (t, 4H, J = 4.5 Hz), C NMR (100 MHz, DMSO-d6): δ 162.45, 160.64,

159.21, 158.36, 156.98, 151.39, 148.73, 146.00, 138.46, 132.47, 123.33, 119.72, 116.04,

115.26,

105.96,

103.50,

66.09,

55.38,

49.17.

HRMS

SC

116.28,

4.7.10.

M AN U

(ES+): m/z calculated for C29H28FN5O4: 552.2023 [M+Na]+. Found 552.2023. (E)-4-(2-methoxy-4-methylphenoxy)-N-(4-

morpholinophenyl)-5-((3,5-dichlorobenzylidene)amino)pyrimidin-2amine (7j)

TE D

Yellow solid, yield 51%, mp: 233.7–235.2 °C. 1H NMR (400 MHz, CDCl3): δ 8.89 (s, 1H), 8.26 (s, 1H), 7.72 (d, 2H, J = 1.8 Hz), 7.34 (t, 1H, J = 1.8 Hz), 7.10 (d, 2H,

EP

J = 8.0 Hz), 6.99 (d, 1H, J = 7.8 Hz), 6.79–6.77 (m, 2H), 6.63 (d, 2H, J = 7.2 Hz), 3.80 (t, 4H, J = 4.4 Hz), 3.66 (s, 3H), 3.00 (s, 4H), 2.39 (s, 3H); 13C NMR (100

AC C

MHz, CDCl3): δ 165.91, 162.71, 156.96, 154.63, 151.16, 139.97, 139.15, 136.63, 135.44, 130.47, 126.61, 122.90, 121.23, 119.99, 116.41, 113.40, 66.88, 55.83, 50.15, 21.59. HRMS (ES+): m/z calculated for C29H27Cl2N5O3: 586.1389 [M+Na]+. Found 586.1392.

ACCEPTED MANUSCRIPT

4.7.11.

(E)-4-(2-methoxy-4-methylphenoxy)-N-(4-

morpholinophenyl)-5-((3-(trifluoromethoxy)

RI PT

benzylidene)amino)pyrimidin-2-amine (7k) Yellow solid, yield 34%, mp: 205.3–206.4 °C. 1H NMR (400 MHz, CDCl3): δ 9.06 (s, 1H), 8.37 (s, 1H), 7.86–7.84 (m, 2H), 7.50 (t, 1H, J = 8.0 Hz), 7.34–7.31 (m,

SC

1H), 7.21 (d, 2H, J = 8.7 Hz), 7.10 (d, 1H, J = 7.8 Hz), 7.05 (s, 1H), 6.89–6.87 (m,

4.7 Hz), 2.48 (s, 3H);

13

M AN U

2H), 6.72 (d, 2H, J = 8.9 Hz), 3.89 (t, 4H, J = 4.6 Hz), 3.76 (s, 3H), 3.09 (t, 4H, J = C NMR (100 MHz, CDCl3): δ 162.48, 158.25, 157.06,

154.95, 151.30, 149.71, 146.70, 139.36, 139.25, 136.41, 132.47, 130.00, 127.05, 123.20, 123.11, 123.02, 121.20, 120.29, 119.81, 116.37, 113.43, 66.97, 55.84,

Found 602.1990.

(E)-5-((3-isopropoxybenzylidene)amino)-4-(2-methoxy-4-

EP

4.7.12.

TE D

50.11, 21.56. HRMS (ES+): m/z calculated for C30H28F3N5O4: 602.1991 [M+Na]+.

methylphenoxy)-N-(4-morpholinophenyl)pyrimidin-2-amine (7l)

AC C

Yellow solid, yield 30%, mp: 158.3–159.3 °C. 1H NMR (400 MHz, CDCl3): δ 8.91 (s, 1H), 8.27 (s, 1H), 7.51 (s, 1H), 7.42 (d, 1H, J = 7.6 Hz), 7.34 (t, 1H, J = 7.9 Hz), 7.17 (d, 2H, J = 8.7 Hz), 7.07 (d, 1H, J = 7.8 Hz), 7.02 (s, 1H), 6.98 (dd, 1H, J = 8.0, 1.7 Hz), 6.85–6.83 (m, 2H), 6.68 (d, 2H, J = 8.8 Hz), 4.70–4.63 (m, 1H), 3.86 (t, 4H, J = 4.5 Hz), 3.72 (s, 3H), 3.06 (t, 4H, J = 4.7 Hz), 2.44 (s, 3H), 1.36 (d, 6H, J = 6.1 Hz);

13

C NMR (100 MHz, CDCl3): δ 162.52, 160.53, 158.28, 156.85,

ACCEPTED MANUSCRIPT

153.46, 151.38, 146.56, 139.58, 138.35, 136.24, 132.72, 129.65, 124.18, 123.07, 121.72, 121.20, 119.70, 119.48, 116.42, 114.41, 113.49, 70.03, 66.98, 55.87, 50.17,

RI PT

22.07, 21.55. HRMS (ES+): m/z calculated for C32H35N5O4: 576.2587 [M+Na]+. Found 576.2584. 4.7.13.

(E)-5-((3,5-bis(trifluoromethyl)benzylidene)amino)-4-(2-

SC

methoxy-4-methylphenoxy)-N-(4-morpholinophenyl)pyrimidin-2-

M AN U

amine (7m)

Yellow solid, yield 53%, mp: 259.0–259.6 °C. 1H NMR (400 MHz, CDCl3): δ 9.23 (s, 1H), 8.47 (s, 1H), 8.39 (s, 2H), 7.95 (s, 1H), 7.21 (d, 2H, J = 8.4 Hz), 7.11 (d, 1H, J = 7.8 Hz), 7.08 (s, 1H), 6.91–6.88 (m, 2H), 6.72 (d, 2H, J = 8.8 Hz), 3.89 (t,

TE D

4H, J = 4.6 Hz), 3.77 (s, 3H), 3.10 (t, 4H, J = 4.8 Hz), 2.49 (s, 3H); 13C NMR (100 MHz, CDCl3): δ 162.45, 157.28, 156.90, 155.97, 151.20, 146.90, 139.29, 139.10,

EP

136.64, 132.26, 132.15, 131.93, 128.07, 124.60, 123.70, 122.95, 122.16, 121.89, 121.24, 120.74, 119.99, 116.31, 113.41, 66.95, 55.82, 50.04, 21.57. HRMS

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(ES+): m/z calculated for C31H27F6N5O3: 654.1916 [M+Na]+. Found 654.1915.

4.8. General procedure for preparation of 8a-e To a solution of compounds 4a-f (0.2 mmol) in anhydrous IPA (5 mL), was added catalytic amount of TFA then suitable benzaldehyde derivative (0.22 mmol). The

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reaction mixture was refluxed at 80 °C for 6 – 12 h, and then allowed to cool to room temperature followed by addition of (0.4 mmol) of sodium cyanoborohydride

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and stirred at room temperature for 12 h. After competition of the reaction, the resultant mixture was neutralized by TEA (0.1 mL) and evaporated in vacuo to give the crude material which was purified by column chromatography using 30–

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50% EtOAc in hexane mixtures as the eluent to yield 8a-e as white solids.

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4.8.1. N5-(3,5-bis(trifluoromethyl)benzyl)-4-(2-methoxy-4methylphenoxy)-N2-(4-morpholinophenyl)pyrimidine-2,5-diamine (8a) White solid, yield 54%, mp: 148.7–149.6 °C. 1H NMR (400 MHz, CDCl3): δ 7.89 (s, 2H), 7.80 (s, 1H), 7.51 (s, 1H), 7.25 (s, 1H), 7.18 (d, 2H, J = 9.0 Hz), 7.07 (d,

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1H, J = 7.8 Hz), 6.85–6.83 (m, 2H), 6.70 (d, 2H, J = 9.0 Hz), 6.59 (s, 1H), 4.50 (s, 2H), 4.31 (s, 1H), 3.83 (t, 4H, J = 4.6 Hz), 3.73 (s, 3H), 3.02 (t, 4H, J = 4.7 Hz), 13

C NMR (100 MHz, CDCl3): δ 159.32, 152.66, 151.18, 146.05,

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2.43 (s, 3H);

141.93, 139.16, 139.11, 136.53, 133.68, 132.52, 132.19, 131.86, 131.53, 127.39,

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124.67, 123.00, 122.45, 121.95, 121.48, 121.23, 119.22, 116.70, 113.42, 67.00, 55.79, 50.39, 48.16, 21.55. HRMS (ES+): m/z calculated for C31H29F6N5O3: 634.2253 [M+H]+. Found 634.2253. 4.8.2. 4-(2-methoxy-4-methylphenoxy)-N2-(4-morpholinophenyl)-N5-(3(trifluoromethoxy)benzyl)pyrimidine-2,5-diamine (8b)

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White solid, yield 52%, mp: 194.7–195.8 °C. 1H NMR (400 MHz, CDCl3): δ 7.57 (s, 1H), 7.40–7.34 (m, 2H), 7.28 (s, 1H), 7.18 (d, 2H, J = 8.9 Hz), 7.13 (d, 1H, J =

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7.2 Hz), 7.05 (d, 1H, J = 7.8 Hz), 6.84–6.82 (m, 2H), 6.69 (d, 2H, J = 8.9 Hz), 6.62 (s, 1H), 4.41 (s, 2H), 3.84 (t, 4H, J = 4.6 Hz), 3.73 (s, 3H), 3.02 (t, 4H, J = 4.7 Hz), 2.43 (s, 3H);

13

C NMR (100 MHz, CDCl3): δ 159.13, 152.24, 151.24, 149.69,

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145.89, 141.49, 139.25, 138.90, 136.40, 133.92, 130.03, 125.63, 123.04, 122.95, 121.21, 119.81, 119.69, 119.03, 116.74, 113.42,67.02, 55.80, 50.45, 48.19, 21.55.

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HRMS (ES+): m/z calculated for C30H30F3N5O4: 604.2148 [M+Na]+. Found 604.2148.

4.8.3. 4-(2-methoxy-4-methylphenoxy)-N2-(4-morpholinophenyl)-N5-(4-

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(trifluoromethoxy)benzyl)pyrimidine-2,5-diamine (8c) White solid, yield 27%, mp: 174.3–175.0 °C. 1H NMR (400 MHz, CDCl3): δ 7.60

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(s, 1H), 7.44 (d, 2H, J = 8.6 Hz), 7.21–7.17 (m, 4H), 7.04 (d, 1H, J = 7.8 Hz), 6.84–6.81 (m, 2H), 6.70 (d, 2H, J = 9.0 Hz), 6.49 (s, 1H), 4.38 (s, 2H), 4.18 (s, 1H),

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3.84 (t, 4H, J = 4.6 Hz), 3.73 (s, 3H), 3.03 (t, 4H, J = 4.7 Hz), 2.42 (s, 3H); 13C NMR (100 MHz, CDCl3): δ 159.08, 152.21, 151.24, 148.46, 145.90, 139.24, 138.98, 137.67, 136.39, 133.92, 128.78, 123.11, 123.03, 121.22, 119.02, 116.75, 113.43, 67.02, 55.83, 50.45, 48.06, 21.55. HRMS (ES+): m/z calculated for C30H30F3N5O4: 604.2148 [M+Na]+. Found 604.2149.

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4.8.4. N5-(4-chlorobenzyl)-4-(2-methoxy-4-methylphenoxy)-N2-(4morpholinophenyl)pyrimidine-2,5-diamine (8d)

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White solid, yield 28%, mp: 160.3–161.7 °C. 1H NMR (400 MHz, CDCl3): δ 7.58 (s, 1H), 7.36–7.30 (m, 4H), 7.17 (d, 2H, J = 8.9 Hz), 7.04 (d, 1H, J = 7.8 Hz), 6.84–6.81 (m, 2H), 6.69 (d, 2H, J = 9.0 Hz), 6.60 (s, 1H), 4.35 (s, 2H), 3.84 (t, 4H,

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J = 4.7 Hz), 3.73 (s, 3H), 3.02 (t, 4H, J = 4.7 Hz), 2.43 (s, 3H);

13

C NMR (100

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MHz, CDCl3): δ 159.09, 152.16, 151.24, 145.85, 139.25, 138.95, 137.40, 136.38, 133.96, 133.06, 123.09, 123.04, 121.22, 118.99, 116.74, 113.43, 67.02, 55.83, 50.46, 48.11, 21.55. HRMS (ES+): m/z calculated for C29H30ClN5O3: 554.1935

Conflict of interest

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[M+Na]+. Found 554.1938.

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The authors declare no conflict of interest.

Acknowledgments

This work was supported by the Creative Fusion Research Program through the Creative Allied Project funded by the National Research Council of Science & Technology (CAP-12-01-KIST) and by the KIST Institutional programs (Grant Nos. 2E26090) from Korea Institute of Science and Technology. Also by the

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National Research Council of Science & Technology (NST) grant by the Korea

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government (MSIP) (No. CRC-15-04-KIST).

Abbreviations used

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PDGFR, Platelet-derived growth factor receptor; NSAIDs, Non-Steroidal AntiInflammatory Drugs; RA, Rheumatoid Arthritis; SAR, Structure-Activity

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relationship; HB, Hydrogen Bond; HBA, Hydrogen Bond Acceptor; DCM, Dichloromethane; TFA, Trifluoroacetic acid; NO, Nitric Oxide; iNOS, inducible Nitric Oxide Synthase ; TNF-α, Tumor Necrosis Factor-Alpha; PGE2, Prostaglandin E2; HRMS, High Resolution Mass Spectrometry; NMR, Nuclear

References and notes

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Magnetic Resonance; IL, Interleukin.

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[48] I.K. Campbell, M.J. Rich, R.J. Bischof, J.A. Hamilton, The colony-stimulating factors and collageninduced arthritis: exacerbation of disease by M-CSF and G-CSF and requirement for endogenous M-CSF, J. LEUKOCYTE BIOL., 68 (2000) 144-150. [49] T. Hanamura, E. Asakura, T. Tanabe, Macrophage colony-stimulating factor (M-CSF) augments cytokine induction by lipopolysaccharide (LPS)-stimulation and by bacterial infections in mice, Immunopharmacology, 37 (1997) 15-23. [50] F. Meng, C.A. Lowell, Lipopolysaccharide (LPS)-induced Macrophage Activation and Signal Transduction in the Absence of Src-Family Kinases Hck, Fgr, and Lyn, The Journal of Experimental Medicine, 185 (1997) 1661-1670. [51] W.M. Moore, R.K. Webber, G.M. Jerome, F.S. Tjoeng, T.P. Misko, M.G. Currie, L-N6-(1Iminoethyl)lysine: A Selective Inhibitor of Inducible Nitric Oxide Synthase, Journal of Medicinal Chemistry, 37 (1994) 3886-3888. [52] N. Futaki, S. Takahashi, M. Yokoyama, I. Arai, S. Higuchi, S. Otomo, NS-398, a new anti-inflammatory agent, selectively inhibits prostaglandin G/H synthase/cyclooxygenase (COX-2) activity in vitro, Prostaglandins, 47 (1994) 55-59. [53] P.A. Wender, N. Buschmann, N.B. Cardin, L.R. Jones, C. Kan, J.-M. Kee, J.A. Kowalski, K.E. Longcore, Gateway synthesis of daphnane congeners and their protein kinase C affinities and cell-growth activities, Nat Chem, 3 (2011) 615-619.

Scheme 1. Synthesis of compounds 5a,b, 6a–k, 7a–m and 8a–d. Figure 1. Representative examples of type I and type II LCK inhibitors. Figure 2. Representative examples of diverse scaffolds for FMS inhibitors. Figure 3. Schematic diagram showing the rational design for type-I LCK inhibitors belonging to the phenoxypyrimidine-like scaffold by non-classical bioisosterism and scaffold hopping of compound V of Roche pharmaceuticals.

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Figure 4. % Enzyme Inhibition (relative to DMSO controls) of 6b (10 uM) on selected panel of 53 kinases.

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Figure 5. Selectivity profile for 7e, 7g, 7h, 7j, 7k and 7l over a selected panel of protein kinases. % remaining enzyme activity is plotted against the compound label for every kinase. Figure 6. SAR for phenoxypyrimidine-like derivatives over FMS and LCK represented on 6b.

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Figure 7. Docking of 7j into LCK (A) 3D representation of 7j (carbons in cyan) in the ATP-binding site of LCK (PDB: 3LCK). Hydrogen bonds with the Met319 are shown as brown dashed lines. (B) 2D schematic representation of the binding mode of 7j in LCK. Figure 8. Docking of 7g into LCK (A) 3D representation of 7g (carbons in orange) in the ATP-binding site of LCK (PDB: 3LCK). Hydrogen bonds with the Met319 and Lys273 are shown as green dashed lines. (B) 2D schematic representation of the binding mode of 7g in LCK.

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Figure 9. Docking of 7g into FMS kinase (A) 3D representation of 7g (carbons in teal color) in the ATP-binding site of FMS (PDB: 3LCD). Hydrogen bonds are shown as red dashed lines. (B) 2D schematic representation of the binding mode of 7g in FMS. (C) 3D representation of 7b (carbons in salmon color) in the ATPbinding site of FMS (PDB: 3LCD); hydrogen bond is shown in green dashed line.

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Figure 10. Comparison between the binding pose of 7g (carbon in green color) and 7b (carbon in raspberry color) inside FMS protein (PDB: 3LCD). Hydrogen bonds are shown in yellow color. Figure 11. Effect of selected compounds on the cell viability of LPS-induced RAW 264.7 macrophages. Figure 12. Effect of selected compounds on the LPS-induced NO production in RAW 264.7 macrophages. L-NIL; a known potent and selective iNOS inhibitor was used as positive control agent.

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Figure 13. Effect of 7g on LPS-induced PGE2 production in RAW 264.7 macrophages. NS-398 was used as a positive control agent.

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Figure 14. Effect of 7g on LPS-induced iNOS and COX-2 protein and mRNA expressions in RAW 264.7 macrophages. (A) Western blot assay showing that 7g inhibited the production of iNOS protein at 6–24h, but not COX-2 protein. (B) Western blot assay showing that 7g inhibited the production of iNOS protein at concentration of 6–12 uM, but had no effect on COX-2 up to 12 uM. (C) Relative iNOS mRNA expression was inhibited by 7g at 2–12h while had no effect on COX-2 mRNA expression.

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Figure 15. Effect of 7g on productions of pro-inflammatory cytokines (TNF-α, IL1β and IL-6) in LPS-induced RAW 264.7 macrophages. Figure 16. Effect of 7g on the expressions of TNF-α and IL-6 mRNA in LPSinduced RAW 264.7 macrophage. Table 1. In vitro enzyme assay data (as IC50 (uM) values) for 6b over LCK, Lyn and c-Src kinases.

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Table 2. IC50 ± SD (uM) values for the newly synthesized compounds over LCK. Table 3. Comparative IC50 ± SD (nM) values for selected compounds over LCK and FMS.

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Table 4. Effects of selected compounds on cell viability (as IC80 (uM) values), NO production and PGE2 inhibition (as IC50 (uM) values) in RAW 264.7 macrophages.

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Novel LCK/FMS inhibitors based on phenoxypyrimidine scaffold as

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potential treatment for inflammatory disorders

Ahmed Karam Faraga,b, Ahmed Elkamhawya,c, Ashwini M. Londheb,d, Kyung-Tae Leee, Ae

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Nim Paeb,d, Eun Joo Roha,b*

Chemical Kinomics Research Center, Korea Institute of Science and Technology (KIST), Seoul 02792, Republic of Korea.

b

Division of Bio-Medical Science &Technology, KIST School, Korea University of Science and Technology, Seoul 02792,

Republic of Korea. c

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Department of Pharmaceutical Organic Chemistry, Faculty of Pharmacy, Mansoura University, Mansoura 35516, Egypt. Convergence Research Center for Diagnosis, Treatment and Care System of Dementia, Korea Institute of Science and

Technology (KIST), Seoul 02792, Republic of Korea.

Department of Life and Nanopharmaceutical Science, Kyung Hee University, Seoul 130-701, Republic of Korea.

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Corresponding author: Eun Joo Roh

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Email address: [email protected]

Postal address: Korea Institute of Science and Technology, Chemical Kinomics Research Center, Future Convergence Research Division, Hwarang-ro 14-gil 5, Seongbuk-gu, Seoul 02792, Republic of Korea.

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List of tables

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Table 1. In vitro enzyme assay data (as IC50 (uM) values) for 6b over LCK, Lyn and c-Src kinases. Table 2. IC50 ± SD (uM) values for the newly synthesized compounds over LCK.

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Table 3. Comparative IC50 ± SD (nM) values for selected compounds over LCK and FMS.

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Table 4. Effects of selected compounds on cell viability (as IC80 (uM) values), NO production and PGE2 inhibition (as IC50 (uM) values) in RAW 264.7 macrophages.

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Table 1. In vitro enzyme assay data (as IC50 (uM) values) for 6b over LCK, Lyn and c-Src kinases.

c-Src LCK LYN

Compound IC50 (uM) a Staurosporine b 6b 8.54 1.29 0.502 1.01 4.20 0.473

Compound 6b was tested in 10-dose singlicate IC50 mode.

b

Staurosporine IC50 values are given in nanomolar.

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Kinase

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Table 2. IC50 ± SD (uM) values for the newly synthesized Twenty-nine compounds over LCK.

R2

R3

5a 5b

2-methoxy-4-methyl 2-methoxy-4-methyl

3,5-dimethoxy 4-morpholino

3,5-dimethoxy 3,5-dimethoxy

> 100 10.3 ± 0.24

6a 6c 6d 6e 6f 6g 6h 6i 6j 6k 7a 7b 7c 7d 7e 7f 7g 7h 7i 7j 7k 7l 7m 8a 8b 8c 8d Staurosporine

2-methoxy-4-methyl 2-methoxy-4-methyl 2-methoxy-4-methyl 2-methoxy-4-methyl 2-methoxy-4-methyl 2-methoxy-4-methyl 3-methoxy 4-trifluoromethyl 4-fluoro 2-methoxy-4-methyl 2-methoxy-4-methyl 2-methoxy-4-methyl 3-methoxy 2-methoxy-4-methyl 2-methoxy-4-methyl 4-trifluoromethyl 4-methoxy 2-methoxy-4-methyl 4-fluoro 2-methoxy-4-methyl 2-methoxy-4-methyl 2-methoxy-4-methyl 2-methoxy-4-methyl 2-methoxy-4-methyl 2-methoxy-4-methyl 2-methoxy-4-methyl 2-methoxy-4-methyl

3,5-dimethoxy 3,5-dimethoxy 3,5-dimethoxy 4-morpholino 4-morpholino 4-morpholino 4-morpholino 4-morpholino 4-morpholino 4-morpholino 4-morpholino 4-morpholino 4-morpholino 4-morpholino 4-morpholino 4-morpholino 4-morpholino 4-morpholino 4-morpholino 4-morpholino 4-morpholino 4-morpholino 4-morpholino 4-morpholino 4-morpholino 4-morpholino 4-morpholino

3,5-dimethoxy 4-methoxy 2,6-dichloro 2,6-dichloro 3,5-dichloro 2-methoxy 3,5-dimethoxy 3,5-dimethoxy 3,5-dimethoxy 3,5-bis(trifluoromethyl) 2,5-dimethoxy 3,5-dimethoxy 3,5-dimethoxy 2-chloro 3-chloro 3,5-dimethoxy 3,5-dimethoxy 4-chloro 3,5-dimethoxy 3,5-dichloro 3-triflouromethoxy 3-isopropoxy 3,5-bis(trifluoromethyl) 3,5-bis(trifluoromethyl) 3-triflouromethoxy 4-triflouromethoxy 4-chloro

41.4 ± 2.58 > 100 58.4 ± 2.81 17.45 ± 1.22 > 100 5.48 ± 0.095 7.97 ± 4.15 > 100 71.15 ± 22.1 > 100 0.11 ± 0.009 0.106 ± 0.056 1.86 ± 0.22 0.225 ± 0.055 0.062 ± 0.004 12.13 ± 3.35 0.022 ± 0.010 0.054 ± 0.020 3.17 ± 0.387 0.0065 ± 0.002 0.0335 ± 0.013 0.039 ± 0.020 0.006 ± 0.0005 11.60 ± 1.26 22.15 ± 0.742 3.30 ± 0.068 0.312 ± 0.031 0.0019 ± 0.00005

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IC50 values (uM)*

compound

IC50 ± SD values are the mean of two independent experiments.

Table 3. Comparative IC50 ± SD (nM) values for selected compounds over LCK and FMS.

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IC50 (nM) * LCK enzyme 106.0 ± 56.0 1860.0 ± 220.0 12130.0 ± 3350.0 22.0 ± 10.0 3170.0 ± 387.0

7b 7c 7f 7g 7i

IC50 ± SD values are the mean of two independent experiments.

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FMS enzyme 883.0 ± 58.0 8.6 ± 1.4 14.0 ± 0.6 4.6 ± 0.05 21.0 ± 4.0

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Table 4. Effects of selected compounds on cell viability (as IC80 (uM) values), NO production and PGE2 inhibition (as IC50 (uM) values) in RAW 264.7 macrophages.

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NT, Not tested.

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IC50 (µM) PGE2 inhibition NT* NT* NT* NT* NT* 5.22 NT* NT* NT* NT* NT* 0.00723

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IC50 (µM) NO inhibition > 80 >1 >1 >1 >1 10.27 >5 >1 > 20 >5 28.29 NT*

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6b 7a 7b 7d 7e 7g 7h 7j 7k 7l -NIL L

IC80 (µM) Cell viability > 80 >1 >1 >1 >1 > 12 >6 0.987 <5 >6 NT* NT*

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Compound

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Novel LCK/FMS inhibitors based on phenoxypyrimidine scaffold as

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potential treatment for inflammatory disorders

Ahmed Karam Faraga,b, Ahmed Elkamhawya,c, Ashwini M. Londheb,d, Kyung-Tae Leee, Ae

SC

Nim Paeb,d, Eun Joo Roha,b*

Chemical Kinomics Research Center, Korea Institute of Science and Technology (KIST), Seoul 02792, Republic of Korea.

b

Division of Bio-Medical Science &Technology, KIST School, Korea University of Science and Technology, Seoul 02792,

Republic of Korea. c

d

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Department of Pharmaceutical Organic Chemistry, Faculty of Pharmacy, Mansoura University, Mansoura 35516, Egypt. Convergence Research Center for Diagnosis, Treatment and Care System of Dementia, Korea Institute of Science and

Technology (KIST), Seoul 02792, Republic of Korea.

Department of Life and Nanopharmaceutical Science, Kyung Hee University, Seoul 130-701, Republic of Korea.

*

Corresponding author: Eun Joo Roh

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Email address: [email protected]

Postal address: Korea Institute of Science and Technology, Chemical Kinomics Research Center, Future Convergence Research Division, Hwarang-ro 14-gil 5, Seongbuk-gu, Seoul 02792, Republic of Korea.

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List of scheme and figures Scheme 1. Synthesis of compounds 5a,b, 6a–k, 7a–m and 8a–d.

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Figure 1. Representative examples of type I and type II LCK inhibitors. Figure 2. Representative examples of diverse scaffolds for FMS inhibitors.

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Figure 3. Schematic diagram showing the rational design for type-I LCK inhibitors belonging to the phenoxypyrimidine-like scaffold by non-classical bioisosterism and scaffold hopping of compound V of Roche pharmaceuticals.

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Figure 4. % Enzyme Inhibition (relative to DMSO controls) of 6b (10 uM) on selected panel of 53 kinases. Figure 5. Selectivity profile for 7e, 7g, 7h, 7j, 7k and 7l over a selected panel of protein kinases. % remaining enzyme activity is plotted against the compound label for every kinase.

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Figure 6. SAR for phenoxypyrimidine-like derivatives over FMS and LCK represented on 6b.

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Figure 7. Docking of 7j into LCK (A) 3D representation of 7j (carbons in cyan) in the ATP-binding site of LCK (PDB: 3LCK). Hydrogen bonds with the Met319 are shown as brown dashed lines. (B) 2D schematic representation of the binding mode of 7j in LCK.

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Figure 8. Docking of 7g into LCK (A) 3D representation of 7g (carbons in orange) in the ATP-binding site of LCK (PDB: 3LCK). Hydrogen bonds with the Met319 and Lys273 are shown as green dashed lines. (B) 2D schematic representation of the binding mode of 7g in LCK. Figure 9. Docking of 7g into FMS kinase (A) 3D representation of 7g (carbons in teal color) in the ATP-binding site of FMS (PDB: 3LCD). Hydrogen bonds are shown as red dashed lines. (B) 2D schematic representation of the binding mode of 7g in FMS. (C) 3D representation of 7b (carbons in salmon color) in the ATPbinding site of FMS (PDB: 3LCD); hydrogen bond is shown in green dashed line.

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Figure 10. Comparison between the binding pose of 7g (carbon in green color) and 7b (carbon in raspberry color) inside FMS protein (PDB: 3LCD). Hydrogen bonds are shown in yellow color.

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Figure 11. Effect of selected compounds on the cell viability of LPS-induced RAW 264.7 macrophages.

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Figure 12. Effect of selected compounds on the LPS-induced NO production in RAW 264.7 macrophages. L-NIL; a known potent and selective iNOS inhibitor was used as positive control agent.

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Figure 13. Effect of 7g on LPS-induced PGE2 production in RAW 264.7 macrophages. NS-398 was used as a positive control agent.

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Figure 14. Effect of 7g on LPS-induced iNOS and COX-2 protein and mRNA expressions in RAW 264.7 macrophages. (A) Western blot assay showing that 7g inhibited the production of iNOS protein at 6–24h, but not COX-2 protein. (B) Western blot assay showing that 7g inhibited the production of iNOS protein at concentration of 6–12 uM, but had no effect on COX-2 up to 12 uM. (C) Relative iNOS mRNA expression was inhibited by 7g at 2–12h while had no effect on COX-2 mRNA expression.

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Figure 15. Effect of 7g on productions of pro-inflammatory cytokines (TNF-α, IL1β and IL-6) in LPS-induced RAW 264.7 macrophages.

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Figure 16. Effect of 7g on the expressions of TNF-α and IL-6 mRNA in LPSinduced RAW 264.7 macrophage.

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Scheme 1.

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Scheme 1. Synthesis of compounds 5a,b, 6a–k, 7a–m and 8a–d. Reagents and conditions: (a) appropriate phenol derivatives, NaHCO3, Acetone, water, rt, 2 h; (b) appropriate aniline derivatives, pyridine, THF, 80 °C, 4 h; (c) H2, 10% Pd/C, MeOH, rt, 12 h; (d) appropriate benzoyl chloride derivatives, DCM, 0–50 °C, 12 h; (e) appropriate phenyl isocyanate reagent, DCM, 0–50 °C, 12 h; (f) appropriate benzaldehyde derivative, anhydrous IPA, TFA, 80 °C, 6–12 h; (g): (i) appropriate benzaldehyde derivative, IPA, TFA, 80 °C, 6–12 h; (ii) NaBH3CN, rt, 12 h.

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Figure 1. Representative examples of type I and type II LCK inhibitors.

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Figure 2. Representative examples of diverse scaffolds for FMS inhibitors.

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Figure 3. Schematic diagram showing the rational design for type-I LCK inhibitors belonging to the phenoxypyrimidine-like scaffold by non-classical bioisosterism and scaffold hopping of compound V of Roche pharmaceuticals.

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% E n z y me i n h i b i t i o n o f 6 b ( 1 0 u M ) o v e r 5 3 k i n a s e s p a n e l

Figure 4. % Enzyme inhibition of 6b (10 uM) on selected panel of 53 kinases.

ABL1 AKT1 AURORA A BRAF BTK C-MET C-SRC CAMK2B CDK1/CYCLIN B CDK1/CYCLIN E CDK2/CYCLIN A CHK1 CHK2 DAPK1 EPHA3 EPHB4 ERK1 ERK2/MAPK1 ERK5/MAPK7 ERK5/MAPK7 (CD) ERK7/MAPK15 FGFR3 FLT1/VEGFR1 FLT3 FLT4/VEGFR3 GSK3B HIPK1 IR IRAK4 JAK3 KDR/VEGFR2 LCK LCK2/ICK LYN MINK/MINK1 MNK2 M ST2/STK3 P38Α/MAPK14 PAK1 PAK2 PDGFRA PHKG2 PIM1 PKA PKCA PLK2 RET ROCK1 RSK1 SYK TAK1 TIE2/TEK TRKA

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Selectivity profile ABL1

c-Src

FMS

FYN

JAK3

LYN

KDR/VEGFR2

P38a/MAPK14

TIE2/TEK

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160.00 140.00 120.00

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100.00 80.00

40.00 20.00 0.00 (7g)

(7h)

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(7e)

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(7j)

(7k)

(7l)

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Figure 5. Selectivity profile for 7e, 7g, 7h, 7j, 7k and 7l over a selected panel of protein kinases. % remaining enzyme activity is plotted against the compound label for every kinase.

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Hydrophilic substitution essential for LCK activity;Ortho- or metasubstituent that can make HB is preferred for FMS activity

Azomethine linker accounted for the highest potent compounds

O 3'

2'

H N

O

H N

4

N

5

O

4' 5'

2

N

N

3,5-dimethoxyaniline moiety wasn't tolerated but 4-morpholinoaniline moiety was optimum

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O

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3,5-disubstitution with lipophilic small group was essential for LCK activity; while 3,5-dimethoxy substitution was optimum for FMS activity

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Figure 6. SAR for phenoxypyrimidine-like derivatives over FMS and LCK represented on 6b.

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Figure 7. Docking of 7j into LCK (A) 3D representation of 7j (carbons in cyan) in the ATP-binding site of LCK (PDB: 3LCK). Hydrogen bonds with the Met319 are shown as brown dashed lines. (B) 2D schematic representation of the binding mode of 7j in LCK.

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Figure 8. Docking of 7g into LCK (A) 3D representation of 7g (carbons in orange) in the ATP-binding site of LCK (PDB: 3LCK). Hydrogen bonds with the Met319 and Lys273 are shown as green dashed lines. (B) 2D schematic representation of the binding mode of 7g in LCK.

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Figure 9. Docking of 7g into FMS kinase (A) 3D representation of 7g (carbons in teal color) in the ATP-binding site of FMS (PDB: 3LCD). Hydrogen bonds are shown as red dashed lines. (B) 2D schematic representation of the binding mode of 7g in FMS. (C) 3D representation of 7b (carbons in salmon color) in the ATPbinding site of FMS (PDB: 3LCD); hydrogen bond is shown in green dashed line.

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Figure 10. Comparison between the binding pose of 7g (carbon in green color) and 7b (carbon in raspberry color) inside FMS protein (PDB: 3LCD). Hydrogen bonds are shown in yellow color.

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Figure 11. Effect of selected compounds on the cell viability of LPS-induced RAW 264.7 macrophages.

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Figure 12. Effect of selected compounds on the LPS-induced NO production in RAW 264.7 macrophages. L-NIL; a known potent and selective iNOS inhibitor was used as positive control agent.

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Figure 13. Effect of 7g on LPS-induced PGE2 production in RAW 264.7 macrophages. NS-398 was used as a positive control agent.

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Figure 14. Effect of 7g on LPS-induced iNOS and COX-2 proteins and mRNA expressions in RAW 264.7 macrophages. (A) Western blot assay showing that 7g inhibited the production of iNOS protein at 6–24h, but not COX-2 protein. (B) Western blot assay showing that 7g inhibited the production of iNOS protein at concentration of 6–12 uM, but had no effect on COX-2 up to 12 uM. (C) Relative iNOS mRNA expression was inhibited by 7g at 2–12h while had no effect on COX-2 mRNA expression.

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Figure 15. Effect of 7g on productions of pro-inflammatory cytokines (TNF-α, IL1β and IL-6) in LPS-induced RAW 264.7 macrophages.

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Figure 16. Effect of 7g on the expressions of TNF-α and IL-6 mRNA in LPSinduced RAW 264.7 macrophage.

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Potent LCK/ FMS inhibitors have been designed and synthesized.

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Nanomolar IC50 values over both kinases were obtained.

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Excellent selectivity for LCK/FMS over closely related kinases was found.

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7g showed excellent anti-inflammatory activity on RAW 264.7 macrophages cell line.

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