Discovery of (2 S)-8-[(3 R)-3-Methylmorpholin-4-yl]-1-(3-methyl-2

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Discovery of (2S)-8-[(3R)-3-Methylmorpholin-4-yl]-1-(3-methyl-2-oxo-butyl)-2(trifluoromethyl)-3,4-dihydro-2H-pyrimido[1,2-a]pyrimidin-6-one: a Novel Potent and Selective Inhibitor of Vps34 for the Treatment of Solid Tumors Benoit Pasquier, Youssef El-Ahmad, Bruno Filoche-Rommé, Christine Dureuil-Sizaire, Florence Fassy, Pierre-Yves Abecassis, Magali Mathieu, Thomas Bertrand, Tsiala Benard, Cédric Barrière, Samira El Batti, Jean-Philippe Letallec, Véronique Sonnefraud, Maurice Brollo, Laurence Delbarre, Véronique Loyau, Fabienne Pilorge, Luc Bertin, Patrick Richepin, Jérôme Arigon, Jean-Robert Labrosse, Jacques Clément, Florence Durand, Romain Combet, Pierre Perraut, Vincent Leroy, Frédéric Gay, Dominique Lefrançois, François Bretin, Jean-Pierre Marquette, Nadine Michot, Anne Caron, Christelle Castell, Laurent Schio, Gary McCort, Hélène Goulaouic, Carlos Garcia-Echeverria, and Baptiste Philippe Ronan J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/jm5013352 • Publication Date (Web): 17 Nov 2014 Downloaded from http://pubs.acs.org on November 19, 2014

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Journal of Medicinal Chemistry

Castell, Christelle; Sanofi, Oncology Schio, Laurent; Sanofi, Oncology McCort, Gary; Sanofi, Oncology Goulaouic, Hélène; Sanofi, Oncology Garcia-Echeverria, Carlos; Sanofi Oncology, Ronan, Baptiste; Sanofi, Oncology

ACS Paragon Plus Environment


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Discovery of (2S)-8-[(3R)-3-Methylmorpholin-4yl]-1-(3-methyl-2-oxo-butyl)-2-(trifluoromethyl)3,4-dihydro-2H-pyrimido[1,2-a]pyrimidin-6-one: a Novel Potent and Selective Inhibitor of Vps34 for the Treatment of Solid Tumors Benoit Pasquier,† Youssef El-Ahmad,† Bruno Filoche-Rommé,† Christine Dureuil,† Florence Fassy,† Pierre-Yves Abecassis,# Magali Mathieu,‡ Thomas Bertrand,‡ Tsiala Benard,§ Cédric Barrière,† Samira El Batti,† Jean-Philippe Letallec,† Véronique Sonnefraud,† Maurice Brollo,† Laurence Delbarre,† Véronique Loyau,† Fabienne Pilorge,† Luc Bertin,† Patrick Richepin,† Jérôme Arigon,∆ Jean-Robert Labrosse,∆ Jacques Clément,∆ Florence Durand,∆ Romain Combet,∆ Pierre Perraut,∆ Vincent Leroy,† Frédéric Gay,† Dominique Lefrançois,† François Bretin,† Jean-Pierre Marquette,‡ Nadine Michot,┴ Anne Caron,Ⱶ Christelle Castell,† Laurent Schio,† Gary McCort,† Hélène Goulaouic,† Carlos Garcia-Echeverria,† and Baptiste Ronan*,† †

Oncology Drug Discovery, # Drug Disposition and Safety, ‡ Structure Design Informatics and

Structural Biology, § Pharmaceutical Sciences Operations, ┴ Protein Production, Ⱶ Global Biotherapeutic, Sanofi, 13 quai Jules Guesde, 94403 Vitry-sur-Seine, France.


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∆

Lead Generation Candidate Realization (LGCR C&BD), Sanofi, 371 rue du professeur Joseph

Blayac, 34184 Montpellier, France. ABSTRACT Vps34 (the human class III phosphoinositide 3-kinase) is a lipid kinase involved in vesicle trafficking and autophagy and therefore constitutes an interesting target for cancer treatment. Because of the lack of specific Vps34 kinase inhibitors, we aimed to identify such compounds to further validate the role of this lipid kinase in cancer maintenance and progression. Herein, we report the discovery of a series of tetrahydropyrimido-pyrimidinone derivatives. Starting with hit compound 1a, medicinal chemistry optimization led to compound 31. This molecule displays potent activity, an exquisite selectivity for Vps34 with excellent properties. The X-ray crystal structure of compound 31 in human Vps34 illustrates how the unique molecular features of the morpholine synthon bestows selectivity against class I PI3Ks. This molecule exhibits suitable in vivo mouse PK parameters and induces a sustained inhibition of Vps34 upon acute administration. Compound 31 constitutes an optimized Vps34 inhibitor that could be used to investigate human cancer biology. INTRODUCTION Phosphoinositide 3-kinases (PI3Ks) are lipid kinases that phosphorylate the 3-hydroxyl group of the inositol ring of phosphatidylinositol (PtdIns) lipid substrates. PI3K isoforms have been divided into three classes (class I, class II and class III) based on structural features and lipid substrate preferences.1 Class I uses PtdIns(4,5)P2 as substrate to produce PtdIns(3,4,5)P3.1-3 By contrast, Vps34 (also known as PIK3C3) specifically catalyzes the phosphorylation of PtdIns, generating PtdIns3P. Vps34 catalytic subunit works in concert with the regulatory subunit Vps15


2


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(also known as p150). Vps15 is myristoylated allowing the Vps34 protein complex to be anchored to intracellular membranes. Membrane-bound PtdIns3P binds to proteins containing a FYVE, PX or WD40 domain.1-4 These PtdIns3P-binding proteins are involved in the formation of autophagosomes and participate also in endosomal trafficking from early to late endosomes.4 Genetic alterations of Vps34 are rare events. However, evidences of a potential role of Vps34 in cell proliferation were reported using Vps34 -/- MEF cells and knockdown approaches in U251 glioblastoma cell line.5,6 Moreover, Vps34 biological functions were described to be related to mTOR signaling5,7,8 which is a pathway frequently dysregulated in cancer and metabolic disorders.9 Finally, Vps34 plays an active role in macroautophagy (hereafter referred to as autophagy).10 Autophagy is a conserved process by which cells turn over their own constituents to protect themselves from metabolic stress, including decreased nutrient availability and hypoxic conditions. Autophagy could also help tumor cells to resist to cancer treatments such as chemotherapeutic agents and ionizing radiation.11-12 Thus, the inhibition of the kinase activity of Vps34 might constitute an interesting therapeutic approach for cancer treatment as a single agent or in combination. So far most of the research on this field was done using non-specific compounds such as the pan-PI3K inhibitor, 3-methyladenine.11b We report herein the discovery and optimization of a potent and selective small molecular mass Vps34 kinase inhibitor. We first set out to understand how to address selectivity within a series of tetrahydropyrimido-pyrimidinone analogs with the objective to dial out activity on class I PI3Ks and gain Vps34 inhibitory activity. During the optimization process, we have been able to highlight which parts of the molecule in this series were able to separately fine tune potency, selectivity and in vitro ADME properties and were successful in rationalizing structure-activity relationships by discovering compounds exhibiting drug-like properties. Finally, compound 31


3

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was selected for PK/PD experiments and showed statistically significant in vivo Vps34 target modulation. At the time we first submitted this work, there was no report of a selective inhibitor of Vps34 in the literature. However during the review process, such compound (named VPS34-IN1) was described.13 While it appears to be a potent and selective inhibitor, no information was disclosed regarding its in vitro ADME properties and in vivo target engagement. RESULTS AND DISCUSSION High-throughput phenotypic screening. On the basis of the potential role of autophagy in cancer maintenance and progression, we primarily aimed to identify small molecule autophagy inhibitors. In order to screen multiple biological targets simultaneously, we carried out a cellbased high-throughput phenotypic screening campaign.14 The screening identified, amongst several chemistry classes having non-identified targets, tetrahydropyrimido-pyrimidinone derivatives active against the autophagy-relevant biological target Vsp34, using recombinant Vps34 protein in a TR-FRET format. An additional family was shown to be active against ULK, using the Millipore Kinase ProfilerTM assay panel (result not shown). Representative examples of the validated tetrahydropyrimido-pyrimidinone hits are compounds 1a-e15 (Figure 1). These molecules, bearing a (S)-trifluoromethyl substitution at the 2-position and a morpholine group at the 8-position, inhibited Vps34 and had also cross reactivity against class I PI3Ks (isoforms α, β,

δ and γ) and, at a lower level, mTOR (compounds 1a-e in Table 1). It is worthy to mention that the use of a morpholine group as hinge binder in kinase modulators was already reported to switch off activity on protein kinases for the development of class I PI3Kβ selective inhibitors.1517

In addition, PI3Kβ selective inhibitors from chemotype such as pyrido[1,2-a]pyrimidin-4-one


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(TGX-22118a,b and AZD648218c,d, Figure 1) was reported and investigated as potential antithrombotic agents. These compounds contain a pyrimidinone moiety with a morpholine group as hinge binder similar to that of our hits, but differ from our series by the aromatic pyrido part. Furthermore, the reported binding mode of these compounds shows that the morpholine group likely interacts similarly with Vps34. Our objective being to obtain highly selective Vps34 inhibitors, further comparison with PI3Kβ ligands did not help us in achieving this goal since our compounds already exhibited a profile of activities of interest. To confirm Vps34 inhibition in a cellular setting, we established an assay to measure the production of PtdIns3P using a GFPFYVE transfected Hela cell line as previously described.19 Compound 1a which displayed the higher cellular potency on GFP-FYVE among all compounds tested, was thus selected as advanced hit for backscreening. Its corresponding (2R)-trifluoromethyl isomer 1b was less active against Vps34.


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O

O N

N

N

N

O

N

N

O

NH

NH CO2H

TGX221

AZD6482

O

O 5 N

8 N O

1 N

N HO

O N

2-(S) CF3

N O

N HO

(S)

N

N

CF3

1c (R = CH2CH2Ph) 1d (R = Bn) 1e (R = Ph)

O

O N

O N

N N

N R

O

1b

N

N

(S)

1a

N

N

2-(R) CF3

N

CF3

O

N

N

N

CF3 O

O

N HO

N

CF3

(R) F 2

O

F 3

4

Figure 1. Structures of pyrido-pyrimidinone PI3Kβ selective inhibitors and representative examples of tetrahydropyrimido-pyrimidinone hits. Back screening. We screened additional tetrahydropyrimido-pyrimidinone derivatives from the Sanofi chemical library to set-up an initial structure-activity relationship data focused on potency and selectivity over lipid kinases (thereby, substitutions at the 1-position of the pyrimidinone scaffold were particularly explored at first intent). This led to the identification of compounds 2,15 320 and 415 (Figure 1), which display attractive properties as illustrated in the line chart21 (see


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Figure S1 in Supporting Information). At this early stage, we observed that N-side chains, such as substituted aryls (e.g. compound 2), linear alkyl ethers (e.g. compound 3) and (R)-2-hydroxy2-phenyl-ethyl (e.g. compound 4), exhibited favorable biochemical Vps34 selectivity versus class I PI3Ks and mTOR (Table 1). Interestingly, compound 4 was at least 60-fold more selective than its corresponding (S)-isomer compound 1a based on class I PI3Kβ biochemical activity. However, this biochemical selectivity was not confirmed in cellular settings (see Table 1, inhibition of Akt phosphorylation at Ser473 in U87MG glioblastoma cell line). Table 1. Enzymatic and Cellular Potencies of Compounds 1-4 IC50 (nM)a Biochemical activity

Compd

Vps34 PI3Kα PI3Kβ PI3Kδ

Cellular activity

PI3Kγ

mTOR

GFP-FYVE p-Akt (Ser473)

1a

2

6

16

2

15

1020

1

-

1b

176

10000

10000

4329

10000

10000

-

-

1c

2

17

36

14

107

435

10

-

1d

2

50

99

53

1603

-

64

-

1e

96

10000

93

61

1885

-

258

-

2

7

743

340

270

3514

904

95

192

3

7

567

233

211

1747

> 10000

264

91

4

3

1044

998

752

> 10000 > 10000

71

180

a

IC50 values are reported as the mean from at least 2 independent experiments, see Experimental Methods for assays details.

Compounds 2, 3 and 4 were suitable chemical probes with high Vps34 enzymatic potency, significant GFP-FYVE cellular potency and attractive ligand efficiency (LE)22 and ligand lipophilicity efficiency (LLE) values23,24 (Table 2). Higher solubility was observed for compound


7

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3 which bears a linear alkyl ether N-side chain. In addition, we also identified promising in vitro ADME properties including high permeability and reasonable microsomal stability (Table 2). Table 2. Calculated and Measured In Vitro Properties of Compounds 2-4 Solubilityd Caco-2 Compd MW logD7.4

a

LE

b

c

LLE

M/H

e

LM

f

pH 7.4

Papp

(µM)

(nm/s)

(% lability)

rhCYP3A4g IC50 (µM)

2

416

2.9

0.40

5.26

64

331

10 / 18

40

3

390

2.4

0.44

5.76

5122

254

16 / 17

40

4

424

2.2

0.39

6.32

650

37

43 / 24

40

a

Measured logD. bCalculated ligand efficiency. cCalculated ligand lipophilicity efficiency. d Measured on cristalline material. eMeasured apparent permeability. fMeasured on Mouse / Human Liver Microsomes. gMeasured recombinant human CYP3A4.

Given the preceding profiles, further structure-based chemical exploration was initiated with the goal to increase the GFP-FYVE cellular potency and selectivity over class I PI3Ks (i.e. IC50 > 1 µM, p-Akt (Ser473)). Vps34 X-ray structures of compounds 2, 3 and 4. To guide the optimization chemical program, the X-ray crystal structures of human Vps34 in complex with compounds 2 (3.0 Å), 3 (2.7 Å) and 4 (1.9 Å) were determined (see Table S1 in Supporting Information). All inhibitors of this series adopt a DFG-in conformation. Inhibitors interact with the hinge region of Vps34 via the oxygen atom of the morpholine moiety (Figure 2). This moiety is also involved in favorable Van der Waals interactions with surrounding residues. The pyrimidinone aromatic ring is stacked between Ile634 (P-loop) and Ile760 side-chains while the carbonyl function of this moiety is H-bonded to catalytic Lys636 side-chain via a conserved water molecule that interacts


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with Asp644 and Tyr670 side-chains. The (2S)-trifluoromethyl branched on the tetrahydropyrimidine ring points towards a small hydrophobic pocket under the P-loop which comprises residues Phe612 to Ala619.

a

b

c

d


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Figure 2. (a) Interactions of compound 2 in the active site of Vps34. (b) The same, rotated view 90°. Hydrogen bonds are shown as dashed lines. (c) Surface representation of Vps34 active site with compound 2. (d) Binding mode of compound 4 in complex withVps34. Calculation of surface interactions between compound 2 and Vps34 (Figure 2c) showed a shape complementarity between the trifluoromethyl substituent and the P-loop, suggesting that this molecular interplay could drive potency in a way similar to the one reported recently for PI3K inhibitors.25 Compounds 2-4 differ only by their N-substituent, which points towards the exit of the ATP-binding pocket. The difluoro-phenyl moiety of compound 2 is perpendicular to the bicyclic scaffold (Figure 2 a-c). One of the fluorine atoms points down towards Pro689 creating a favorable Van der Waals interaction, while the other fluorine atom seems constrained between Phe612 and Phe684 side-chains. There is no visible direct or indirect interaction for the flexible isopropyloxyethyl moiety of the N-substituent of compound 3 (data not shown), which makes it difficult to explain the observed discrepancies in activity between Vps34 and class I PI3Ks. Similarly, there are striking differences in the selectivity of the molecules of these series against class I PI3Ks, depending on the stereochemistry of the N-side chain. Indeed, compound 4 with a R-stereochemistry exhibits micromolar activities against all class I PI3Ks, while the S-isomer (compound 1a) has double digit nM affinity against the same enzymes (Table 1). This cannot be explained by the binding mode of these molecules to Vps34 as both stereoisomers are expected to bind to class I PI3Ks in the same way they bind to Vps34. In summary, while the available structures allow us to understand the potency of these Vps34 inhibitors, they cannot explain the striking differences in the selectivity profiles against class I


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PI3Ks based on the binding interactions of the N-substituents of the different stereoisomers. This might be related to the presence of different water networks in the vicinity of these inhibitors,26-30 as observed with compound 4 for which the hydroxyl oxygen makes a water-mediated interaction with the main-chain carbonyl of Asp747 (Figure 2d). Thermodynamic signature. To further understand the nature of its interactions, the thermodynamic signature of compound 2 bound to Vps34 was measured by isothermal titration calorimetry (ITC) (Figure 3). Compound 2 achieved nanomolar binding affinity against Vps34 with a Gibbs free energy value of -11.4 kcal/mol. The ITC measurements indicated that the entropic term (-6.5 kcal/mol) contributed slightly more than the enthalpic term (-4.9 kcal/mol) to the free energy of binding. Starting from a compound that already display high binding affinity to Vps34, we hypothesized that we could increase the selectivity against class I PI3Ks by improving the enthalpy contribution, for instance by introducing extra H-bond interactions.31-33

Figure 3. Thermodynamic signature of compound 2. The examination of the binding mode and properties of compounds 2-4 led to the following conclusions: while it has been difficult to associate the nature of the N-substituent and the selectivity profile of the corresponding molecules, the position which points towards the entrance


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of the binding site constitutes a good target to improve in vitro ADME properties. Modifying the hinge binder moiety15-17 has already resulted in an enhanced selectivity profile and further modifications might bring additional selectivity and potency. Similarly, it might be possible to replace the trifluoromethyl moiety to either gain Vps34 potency and/or lose activity against the other lipid kinases. Thus, further medicinal chemistry optimization of the pyrimidinone series could be guided as illustrated in Figure 4.

Hinge Gate Keeper Selectivity Potency

P-Loop Potency Selectivity

Pro689 ADME/PK Selectivity

Figure 4. Structure of compound 2 with handles for further medicinal chemistry optimization. Optimization phase. In an initial phase of our medicinal chemistry optimization, we focused our attention on the (2S)-trifluoromethyl group. Compounds were designed and synthesized in order to explore its replacement on the tetrahydropyrimidine ring (Figure 5). A gem dimethyl group at the 2-position of the tetrahydropyrimidine moiety led to improvement in selectivity (Table 3). Indeed, dimethyl (compound 5)15 and cyclopropyl (compound 6)15 derivatives retained Vps34 biochemical activity as well as GFP-FYVE cellular potency while selectivity against the four class I PI3K isoforms was improved compared to compound 1a. Compounds 5 and 6 also have improved LLE values (with MW below 400) while retaining similar selectivity compared to compounds 2-4 although their in vitro hepatic microsomal stabilities were reduced (Table 4 vs


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Table 2). Based on the properties of compounds 3 and 5, we made compound 720 with the aim of improving the microsomal stability by introduction of the polar isopropyloxyethyl side chain. Unfortunately, compound 7 led to a decrease of Vps34 inhibition as well as GFP-FYVE cellular potency (Table 3) and was not further pursued. O

O 5 N

8

1 N

N HO

N O

O N

N

2 CF3

N

N

N

CF3

N HO

N O

O

N

O 1a

3

5

O

O N N O

N HO

O N

N

N

N

N N

N

N

O

CF3

O O

6

N

O

7

8

Figure 5. Selection of compounds (1a, 3, 5-8) synthesized to explore the structure-activity relationships of the substituents on the tetrahydropyrimidine ring. Table 3. Enzymatic and Cellular Potencies of Compounds 1a, 3, 5-8 IC50 (nM)a Biochemical activity

Compd Vps34 PI3Kα 1a

2

6

Cellular activity

PI3Kβ

PI3Kδ

PI3Kγ

mTOR

GFP-FYVE

16

2

15

1020

1


13

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3

7

567

233

211

1747

> 10000

264

5

1

1918

4591

1173

7350

10000

21

6

0.8

931

3504

606

2930

4069

26

7

11

7429

> 10000

4132

8

9

88

34

27

> 10000 > 10000 628

1735

596 61

a


Table 4. Calculated and Measured In Vitro Properties of Compounds 5 and 6 Solubilityd Compd

MW

logD7.4

a

LE

b

c

LLE

Caco-2 e

M/H LM

f

pH 7.4

Papp

(µM)

(nm/s)

(% lability)

rhCYP3A4g IC50 (µM)

5

384

1.99

0.44

7.01

1274

153

86 / 57

19

6

382

1.83

0.45

7.27

358

83

57 / 51

15

a

Measured logD. bCalculated ligand efficiency. cCalculated ligand lipophilicity efficiency. d Measured on cristalline material. eMeasured apparent permeability. fMeasured on Mouse / Human Liver Microsomes. gMeasured recombinant human CYP3A4.

The dimethyl and trifluoromethyl groups at the 2-position occupy the same hydrophobic pocket under the P-loop. The differences in the P-loop structures of the different lipid kinases (Vps34 vs class I PI3Ks, Figure 6) are not sufficient to explain the observed selectivity (Table 3). Based on the properties of compounds 3 and 7, the (S)-2-methyl-2-trifluoromethyl compound 820 was synthesized to maximize interactions with the Vps34 P-loop and likely to improve cellular potency. Although this hypothesis was validated, it resulted in an unexpected loss of selectivity (Table 3).


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Figure 6. Sequence alignment of the catalytic domains of Vps34 (PIK3C3) and class I PI3K α,

β, δ and γ (A, B, D and G). P-loop and hinge segments are underlined, the gate-keeper residue of Vps34 is indicated in red (Met682 vs Ile in the class I PI3K isoforms). Small black triangles indicate residues of interest around the morpholine moiety binding site. At this stage, the combination of the 2,2-dimethyl group and the N-alkyl ether side chain (e.g. compound 7, Figure 7) provided Vps34 selectivity over class I PI3Ks in a cellular setting with less than 50% inhibition of p-Akt (Ser473) at 1 µM (data not shown). However, the available results also indicated that the 2,2-dimethyl group seemed to be less suitable than the (2S)trifluoromethyl group to maintain GFP-FYVE cellular potency whatever the lateral N-side chain structure.


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Figure 7. “On target” (Vps34, GFP-FYVE) and “Off target” (class I PI3Ks) properties are combined to yield compound profiles (pIC50 scale) complemented with in vitro ADME data ((%) lability in human and mouse liver microsomes, divided by 10 to fit in the pIC50 numerical scale). The green dotted line represents targeted level for “On target” activity. The red dotted line represents the highest acceptable level for “Off target” activity. At this stage, compound 7 represents a suitably balanced selectivity for Vps34 & GFP-FYVE vs class I PI3Ks but shows a clear limitation for further N-side chain expansion. Compounds 3 and 6 have very similar profiles. However, preference was given to compound 3 due to better metabolic profile and a higher potential for the side chain exploration while maintaining potency.


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We next turned our attention to the morpholine hinge-binding group while fixing the (2S)trifluoromethyl substitution to maintain GFP-FYVE cellular potency. As depicted in Figure 4, the hinge-binder region is a lever to modulate selectivity against other kinases. The binding mode of ATP-competitive lipid kinase inhibitors has been well documented34 and compounds with different levels of selectivity against protein and lipid kinases have been developed. Furthermore, looking at the sequence of the ATP binding site of Vps34, structural differences (e.g. the hinge region including the gate keeper) exist with class I PI3Ks that could be key to selectivity (Figure 6). To this end, compounds with a variety of substituted morpholine moieties able to interact with the hinge moiety were synthesized. This strategy was previously successfully performed to identify selective ATP-competitive kinase inhibitors of mTOR.35-37 Our results supported the hypothesis that this region is highly sensitive to modification. In order to facilitate the optimization of the morpholine ring, we decided to decorate the bicyclic scaffold with a N-side chain whose condensation on the tetrahydropyrimido moiety was trivial, thus allowing (an easy) access to all targeted compounds. Results obtained during our initial medicinal chemistry optimization led us to select a methyl-(2-chloro)-thiophene-5-yl) chain as we noted the good chemical reactivity of the 2-chloro-5-(chloromethyl)thiophene reagent for the N-alkylation, whatever other decorations on the pyrimidinone part. Therefore, compound 9 which has a selectivity profile similar to that of compound 3, was chosen as reference compound in the optimization of the morpholine ring. A selection of representative molecules (compounds 9-17)38 is shown in Table 5. Among the groups explored, only the 3(R)-methyl morpholine derivate leads to a selectivity improvement against class I PI3Ks while keeping Vps34 enzymatic and cellular potency similar to the one observed for the non-substituted morpholine (compound 9, Table 5). There is also a striking difference between the 3(R) isomer (compound 17) and its


17

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corresponding 3(S) isomer (compound 16), with an increase of more than 10 fold in selectivity and potency. Compound 15 bearing the 3,5(R,R)-dimethyl morpholine displayed comparable selectivity improvement to the 3(R)-methyl morpholine compound 17, but with loss of solubility and microsomal stability (Table 6). Table 5. Enzymatic and Cellular Potencies of Compounds 9-17 O

Activity IC50 (nM)a

N N

R5

N

CF3

Biochemical

S

Cellular

Cl

Compd

R5

Vps34 PI3Kα PI3Kβ PI3Kδ PI3Kγ mTOR GFP-FYVE

9

O

N

3

502

218

169

7169

2038

143

10

O

N

20

3686

10000

3790

10000

694

1132

11

O

N

1367

10000

10000

10000 10000

1220

9485

12

O

N

194

10000

10000

10000 10000

5253

956

13

O

N

3396

10000

10000

10000 10000

10000

10000

14

O

N

27

10000

10000

10000 10000

4873

3055


18


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

15

O

N

16

O

N

O

N

17

1

10000

10000

86

1012

643

364

2

2899

10000

1297

Page 20 of 88

10000 10000

10000

148

1820

1289

3847

10000

2945

110

a


Table 6. Measured In Vitro Properties of Compounds 9, 15 and 17

Compd MW logD7.4

a

Solubilityb pH 7.4 (µM)

Caco-2 Papp

c

M/H LM

d

(nm/s)

(% lability)

rhCYP3A4e IC50 (µM)

9

434

2.94

34

223

41 / 16

40

15

462

3.36

6

276

83 / 88

40

17

448

3.18

24

319

48 / 31

40

a

Measured logD. bMeasured on cristalline material. cMeasured apparent permeability. d Measured on Mouse / Human Liver Microsomes. eMeasured recombinant human CYP3A4.

The X-ray crystal structure of human Vps34 in complex with compound 15 (2.8 Å) was determined (Figure 8a) (see Table S1 in Supporting Information). Compound 15 adopts the same binding mode as compound 2 (Figures 2a-c). One of the methyl substituents of the morpholine moiety is directed towards Leu750 while the second one points towards the vicinity of Met682. Superimposition of a class I PI3Kδ X-ray structure (Figure 8b) illustrates how methylation of


19

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both 3 and 5-positions of the morpholine moiety drives selectivity against class I PI3Ks: Met900 (which corresponds to Leu750 in Vps34) and Ile825 (corresponding to Met682 in Vps34), respectively below and above the morpholine plane, prevent binding to compound 15. These residues are conserved among class I PI3Ks (Figure 6).

a

b

Figure 8. Comparison of compound 15 co-crystallized in the binding site of Vps34 and docked with the published X-ray structure of class I PI3Kδ (2WXL). (a) Surface representation of Vps34 (salmon) around the dimethyl morpholine moiety of compound 15 (surface represented as a green mesh) showing that the shape of the molecule fits well with the frame of the pocket. (b) Surface representation of PIK3δ protein (cyan) after superimposition on the Vps34 complex. The shape of compound 15 (green mesh) can be seen protruding through the protein surface, indicating clashes near Ile825 (Met682 in Vps34) on one side and Met900 (Leu750 in Vps34) on the other. These residues are indicated by small black triangles in the sequence alignment (Fig 6). While the optimization was focused on the biological profile and fully exploited, the in vitro ADME properties of the selective Vps34 kinase modulators remained to be improved. Structural identifications of morpholino-tetrahydropyrimido-pyrimidinone metabolites using in vitro


20


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Page 22 of 88

mouse, human and rat hepatic microsomal fractions as well as human recombinant CYP3A4 and CYP2D6 were carried out in order to investigate putative metabolic pathways and hot spots for this chemical series. Depending on the nature of the N-side chain at the 9-position and the animal and human selected species, the main metabolic pathway could be linked to successive oxidations on the morpholine ring or N-dealkylation as illustrated with compound 9 (Figure 9).

Figure 9. In mouse and human hepatic microsomal fractions, the main metabolic pathway of compound 9 resulted from morpholine oxidation (blue bar chart) while N-dealkylation was observed as the main metabolic pathway in rat microsomal fractions (green bar chart). The metabolite M4, resulting from the morpholine carbonylation, was only observed in rat. With the aim of solving the identified metabolic issues,39 polar groups were introduced into the N-side chain of the tetrahydropyrimido moiety with the objective to compensate the contribution to the metabolic instability of the 3(R)-methyl morpholine and to improve the binding enthalpy by providing one additional hydrogen bond interaction with Vps34. A selection of compounds made to establish a preliminary structure-activity relationship set of data is shown in Table 7. Compounds 18-3438 exhibited Vps34 enzymatic activity and cellular potency (Table 7) with


21

Page 23 of 88

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


selectivity against class I PI3Ks and mTOR kinases (IC50 values > 2 µM on class I PI3Ks and mTOR). This selectivity profile was further confirmed in a cellular setting with less than 50% inhibition of p-Akt (Ser473) at 1 µM (data not shown). Overall, they showed acceptable microsomal stability in all the tested species (Table 8), with a profile superior to those observed with compounds 9, 15 and 17 which contain a non polar chloro-thiophene N-side chain. Depending on the nature of the N-side chain, compounds 18-34 displayed significant concomitant human CYP3A4 induction as well as high intrinsic clearance, and CYP3A4 contribution in human hepatocytes (Table 8). The generated structure-activity relationship results also highlighted that fluoro substitution at the 3-position of the pyrimidinone moiety led to a decrease of human CYP3A4 induction (compounds 19 and 21 vs compounds 18 and 20) and reduced intrinsic clearance on human hepatocytes (compound 24 vs compound 23). Furthermore, the introduction of additional polarity while decreasing pKa at the N-side chain was sufficient to reduce intrinsic clearance and CYP3A4 contribution in human hepatocytes. The nitrogen atom of the pyridine moiety of compounds 22-25 and 28-30, although not involved in interaction with either the protein or water molecule in the active site, contributed to the permeability (in agreement with its calculated pKa value) and microsomal stability. In addition and as expected, increasing the polarity of the N-side chain led to an improvement of solubility, compared to reference compounds 2, 9, 15, 17 while maintaining good permeability. Recently,40 we investigated in depth the biology of the Vps34 protein in cancer cell lines using compound 22. Based on its limited microsomal stabilities, we assumed that compound 22 would be suitable only for in vitro settings. In consequence, and according to CYP3A liabilities (e.g. induction and contribution), N-methyl-oxadiazole (e.g. compounds 33-34), N-methyl-pyridines (e.g. compound 24) and N-methyl-ketones (e.g. compounds 29, 30, 31-32) were preferred to N-methyl-oxazoles


22


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 24 of 88

(e.g. compounds 18, 19, 20 and 21) and N-alkyl-alcohols/ethers (e.g. compound 26-27) derivatives. Finally, compounds 35 and 36,38 which bear a dimethyl substitution on their tetrahydropyrimidinone moiety, lost cellular potency and microsomal stability as already observed with non-substituted morpholine compounds. Table 7. Enzymatic and Cellular Potencies of a Selection of Pyrimidinones (Compounds 1836) Made to Explore the N-Side Chain Activitiy

O R4

IC50 (nM)a

N R3

N

N

N R2 R1

O

Compd

R1

18

Biochemical

Cellular

R2

R3

R4

Vps34

GFP-FYVE

CF3

H

H

4

325

CF3

H

F

3

208

CF3

H

H

7

145

CF3

H

F

3

68

CF3

H

H

2

31

O N

19 O N

20 N O

21 N O

22

N

Cl


23

Page 25 of 88

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


23

N

CF3

H

H

3

72

CF3

H

F

2

44

CF3

H

F

2

110

CF3

H

F

3

20

CF3

H

F

2

73

CF3

H

H

2

27

CF3

H

H

2

50

CF3

H

F

2

26

F

24

N F

25

N OMe

26 OH

27 O

O

28

N

O

29 N

O

30 N


24


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

O

31

O

32

N

33

Page 26 of 88

CF3

H

H

2

82

CF3

H

F

1

32

CF3

H

F

1

42

CF3

H

F

1

31

Me

Me

F

64

1528

Me

Me

F

8

208

O N

N

34

N O

35 O N

O

36

N

a


Table 8. Measured In Vitro Properties of Compounds 18-36 rhCYP

Compd MW

logD7.4a

e

HCYP 3A4

f

H hepatocytesg

Solubility

Caco-2

M/R/H

3A4

pH 7.4b

Pappc

LMd

IC50

(Induc/

CLint

(µM)

(nm/s)

(% lability)

(µM)

Emax)

(ml/h/ 106 cells)

18

399

1.73

547

63

8/23/8

25

61/30

0.04


25

Fm (%) CYP3A 88

Page 27 of 88

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


19

417

1.94

284

120

38/43/15

40

35.6/10

0.05

88

20

399

1.66

987

48

6/37/5

23

51/30

0.01

87

21

417

1.86

683

87

14/30/5

26

17.5/10

0.04

92

22

443

2.01

732

98

72/44/63

40

-

-

-

23

427

1.86

2129

45

19/37/18

40

12.4/30

0.257

63

24

445

2.08

2820

87

23/18/22

40

14.7/10

0.043

53

25

443

2.33

3448

219

38/31/24

40

49.3/10

0.29

97

26

408

1.84

3080

103

18/27/12

40

9.2/10

0.068

96

27

394

2.18

5071

246

21/38/26

40

1.4/3

0.141

62

28

437

2.09

321

86

44/29/8

40

23.8/30

0.22

25

29

437

1.37

580

18

29/28/10

40

7.2/1

0.03

14

30

455

1.26

39

31

13/24/8

25

3/30

0.036

63

31

402

2.17

1205

146

17/13/1

40

4.5/30

0.055

53

32

420

2.44

264

207

33/19/12

18

7.5/10

0.03

65

33

432

1.80

4626

84

24/18/5

40

-0.2/30

0.019

63

34

432

1.75

4626

151

11/14/2

40

-0.8/1

0.046

73

35

377

1.63

1735

79

38/48/20

40

-

-

-

36

415

2.00

525

79

68/23/47

40

-

-

-

a

Measured logD. bMeasured on cristalline material. cMeasured apparent permeability. d Measured on Mouse / Human Liver Microsomes. eMeasured recombinant human CYP3A4. f Measured Induction human CYP3A4. gMeasured Intrinsic clearance (CLint) using human hepatocytes and contribution (Fm) CYP3A.

The selectivity profile observed with the 3(R)-methyl morpholine moiety was reproduced with other R1 groups (Figure 10). A Molecular Matching Pairs analysis clearly confirmed that improvement of selectivity was correlated with a decrease of affinity for class I PI3Ks while Vps34 affinity and GFP-FYVE potency were unchanged with 3(R)-methyl substitution of the


26


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Page 28 of 88

morpholine residue (see Figure S2 in Supporting Information). This decrease in “off-target” binding affinity was confirmed in a cellular setting. Thus we observed less than 50% inhibition of p-Akt (Ser473) at 1 µM (data not shown). O

O N

N O

N

N R1

S CF3

N R N O

N

N R1

S CF3

Figure 10. Impact of systematic exchange between non-substituted morpholine and 3(R)-methyl morpholine synthons on pyrimidinone scaffold using Molecular Matching Pairs Analysis.21 The potency variation ∆ (pIC50 parameter) is computed for each molecular pair and each pharmacological parameter and represented on the Y axis. The transformation does not have a strong effect on the “On target” parameters (average value 0.29 and 0.38 log units for ∆ (Vps34) and ∆ (GFP-FYVE) respectively). However, it significantly impacts all the “Off target” parameters with loss of 1.05, 1.42, 0.55 and 1.32 log in potency vs class I PI3K α, β, γ and δ, respectively (p 40 µM), which was further confirmed using human liver microsomes with midazolam or testosterone as CYP3A substrates. Moreover, they were not human CYP3A4 inducers. Intrinsic clearance in human hepatocytes was low with a moderate CYP3A4 contribution below 73%. Thereby, and on the basis of its in vitro ADME properties, compound 31 was selected for in vivo pharmacological studies. In vitro studies. Compound 31 displayed an IC50 value of 2 nM and 82 nM on the Vps34 enzymatic assay and the GFP-FYVE cellular assay respectively. This compound was also characterized using immobilized protein and surface plasmon resonance technology. It showed a binding equilibrium constant KD of 2.59 ± 2.27 nM and a dissociation rate constant koff of 2.15 ± 0.95 10-3 s-1, corresponding to a residence half-life, t1/2 of 5.4 min. Moreover, it has also good indicators of drug-likeness (LE = 0.41 and LLE = 6.22). The thermodynamic signature of compound 31 was measured by isothermal titration calorimetry (ITC) (Figure 11). This molecule achieved nanomolar binding affinity against Vps34 (KD = 2.7 ± 0.9 nM confirming the KD obtained by SPR) corresponding to a Gibbs free energy of -11.9 kcal/mol. The ITC measurements indicated that the enthalpic contribution (-13 kcal/mol ) was


28


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Page 30 of 88

the main driver of the thermodynamic signature probably resulting from additional H-bond interactions compared to compound 2. Thus, the enthalpy gain was well enhanced although compensated by some entropy loss which could be the cost for stabilizing the inhibitor in the binding pocket, resulting in only minor affinity change.

Figure 11. Thermodynamic signature of compound 31. The X-ray crystal structure of human Vps34 in complex with compound 31 (2.8 Å) was determined (see Table S1 in Supporting Information). The methyl residue of the morpholine moiety points towards Met682. This orientation provides selectivity against class I PI3Ks as shown with compound 15 (Figure 8). Higher resolution of analogue structures with other polar carbonyl substituents (data not shown) displayed water-mediated interactions between the carbonyl oxygen and Asp747, similar to those already observed with compound 4 (Figure 2d) and which might contribute to the observed enhanced enthalpy for compound 31 compared to compound 2. Indeed, only strong hydrogen bonds are able to contribute favorably to the binding enthalpy. Based on p-Akt cellular activities of compounds 2 and 4 (Table 1), this approach of


29

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maximizing binding affinity was not sufficient to improve selectivity. Furthermore, crystallographic and thermodynamic analysis of human Vps34 in complex with compound 2 and 31 indicate that the structuring induced by the polar N-side chain is mainly responsible for the entropy loss which neutralizes the enthalpy gain (Figures 3 and 11). Although the introduction of the nonpolar methyl group on the morpholine moiety does not have a strong effect in its binding target (Figure 10), it is mainly responsible of the large selectivity by making the interaction of the compound with the off-target cavity unfavorable (Figure 8b). Compound 31 exhibited selectivity against mTOR (IC50 > 10 µM) and class I PI3Ks (IC50 values of 2.7, 4.5, 2.5 and > 10 µM on PI3K α, β, δ, γ isoforms respectively). This selectivity was confirmed in a cellular setting with an IC50 = 8.5 µM on p-Akt (Ser473). We also tested selectivity of compound 31 against class II PI3Ks, PIK3C2A and PIK3C2G. This molecule was found inactive (less than 50% inhibition at 10 µM in corresponding enzymatic assay) (data not shown). Its profiling was then extended to a panel of more than 200 endogenous kinases.41 Jurkat and Ramos cells were selected because they contain a large number of lipid kinases at a detectable level. Binding of compound 31 to the ATP site of protein and lipid kinases from cell lysates was measured after incubation at 1 µM. Results confirmed binding to endogenous Vps34 (>95% binding) (see Figures S3 and S2 in Supporting Information). Among the other lipid and protein kinases tested, moderate binding was found on class I PI3Kα, β, δ isoforms at 1µM (~40%, ~70%, ~60%, respectively) and SMG1 (~60% binding at 1µM) (see Figures S4 and S2 in Supporting Information). In vivo pharmacological studies. Compound 31 was evaluated in a pharmacokinetic (PK) study in male Sprague Dawley rats (Table 9 and Figure 12). After administration by the intravenous


30


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Page 32 of 88

(iv) route (Table 9a), compound 31 concentrations were quantifiable up to 6, 8 and 24 h (last sampling time) depending on animals. The total systemic clearance was moderate (i.e. 1.46 L/h/kg) corresponding to 35% of hepatic blood flow in this species. After oral administration (po) (Table 9b), compound 31 was rapidly absorbed with maximal plasma concentrations observed at 0.5 h and a bioavailability of 85%. Slight rebounds of concentrations were observed at 4 and 8 h post-oral dosing with no obvious explanation. Nevertheless several hypotheses can be postulated, in particular enterohepatic circulation via direct biliary elimination of the parent compound followed by excretion in the intestinal track and subsequent absorption may occur and/or an additional site of absorption in the gastro-intestinal track is also likely to be considered. In vivo PK properties are consistent with the measured eADME in vitro data. Table 9. Pharmacokinetic Parameters of Compound 31 in Male Sprague Dawley Rats: (a) After a Single Intravenous Administration at 3 mg/kg of the Compound in a SBEβ βCD 40% Water Solution (b) After a Single Oral Administration at 10 mg/kg of the Compound in a PEG200 98%/ PS80 2% Solution. (a) Compound 31 iv (3 mg/kg) Co (ng/ml)

Tlast (h)

4030

[6-24]

AUC(0-24 h) (h.ng/ml) AUC(0-inf) (h.ng/ml) 2060

2060

T1/2 (h)

Cl (L/h/kg)

[0.88-11.5]

1.46

(b) Compound 31 po (10 mg/kg) Co (ng/mL) Tmax (h) AUC(0-24 h) (h.ng/ml) AUC(0-inf) (h.ng/ml) 1520

0.5

5840

5920

F (%) 85.0


31

Page 33 of 88

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Figure 12. Plasma concentration profiles following a single intravenous (3 mg/kg) and oral (10 mg/kg) administration of compound 31 to Sprague Dawley male rats (representation in semilogarithmic scale). Compound 31 was also evaluated in a pharmacokinetic (PK) study in female SCID (Severe Combined Immune Deficient) mice (Table 10 and Figure 13). After iv injection at 3 mg/kg, plasma clearance was found moderate (i.e. 2.3 L/h/kg), corresponding to 44% of hepatic blood flow in this species, volume of distribution at steady state was moderate and terminal elimination half-life was short. Table 10. Pharmacokinetic Parameters of Compound 31 in Female SCID (Severe Combined Immune Deficient) Mice: (a) After a Single Intravenous Administration at 3 mg/kg in a SBEβ βCD 40% Water Solution. (a) Compound 31 iv (3 mg/kg) Co

Tlast AUC(0-24 h) AUC(0-inf)

T1/2

Cl

Vss


32


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

(ng/ml)

(h)

(h.ng/ml)

(h.ng/ml)

(h)

5530

2

1290

1310

0.38

Page 34 of 88

(L/h/kg) (L/kg) 2.28

0.78

Figure 13. Plasma concentration profile following a single intravenous (3 mg/kg) administration to female SCID mice (representation in semi-logarithmic scale). Compound 31 was next investigated in an acute PK/PD experiment (Figure 14a) using GFPFYVE H1299 tumors xenografted in SCID mice. Target engagement was measured using anti GFP Antibody and IHC as a pharmacodynamic read-out. Oral administration of compound 31 at 100 mg/kg revealed a sustained inhibition (>80%) of granular staining up to 24 h (Figure 14a,b). Oral administration of compound 31 (50 mg/kg and 100 mg/kg) revealed a dose-dependent target modulation (Figure 14b, Table 11). Correlation between Vps34 modulation and compound 31 concentration in plasma was observed, fitting with an ordinary response model revealing a direct PK-PD response system (Figure 14c).


33

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a

b

c

Figure 14. Effect of compound 31 on granular staining of H1299-GFP-FYVE xenografted in SCID mice model. (a) Immunohistochemistry using anti-GFP Ab on H1299-GFP-FYVE xenograft. Tumor cells from mice treated with vehicle (control) displayed cytoplasmic granular staining (brown) while this staining is significantly reduced after compound 31 administration from 1 to 24 h. (b) Semi-quantitive evaluation allows to determine the percentage of inhibition of PD biomarker. Y axis represents the % of inhibition of granular staining for each time point at the 2 doses (50 mg/kg po and 100 mg/kg po). (c) PK/PD relationship. The graph represents the plasma concentration of compound 31 vs the inhibitory effect on granular staining.


34


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Page 36 of 88

Equilibrium between both concentrations is assumed to be rapidly achieved by a very rapid distribution from plasma to the tumor compartments. Hence, the measured concentration in plasma can directly serve as input function in the pharmacodynamic model component, thereby directly linking measured concentration to the Vps34 inhibition effect. According to the PK/PD model, the in vivo effective plasma concentration to achieve 50% PD modulation (EC50) was estimated at 0.37 µM (Figure 14c). Table 11: Compound 31 PK exposure from PK/PD in H1299-GFP-FYVE s.c. tumors xenografted in SCID mice 50 mg/kg po

Dose 1

Time (h)

8

16

100 mg/kg po 24

1

8

16

24

Plasma concentration (µM)

9.12 0.53 0.27 0.21 24.68 1.54 2.88 1.40

Plasma concentration (SD)

1.54

0.06

0.11

0.06

7.28

0.60

1.45

0.43

99

50

39

30

99

88

97

90

H1299-GFP-FYVE % Inhibition of granular staining

CHEMISTRY A general 5 step synthesis of compounds 1-3415, 20, 38 was developped and is shown in Schemes 1-6. Compounds 1-4,15, 20 8-27,15, 20, 38 30-3438 were synthesized as shown in Scheme 5 by standard N-alkylation of intermediates 57-67 with the appropriate alkyl halide, alkyl tosylate or epoxide in the presence of a base such as cesium carbonate, sodium hydride or potassium phosphate tribasic, or by Ullmann arylation with aryl iodide or heteroaryl iodide catalyzed by copper iodide. Compounds 5-7,15, 20 28-29,38 35-3638 were prepared as shown in Scheme 6 by


35

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substitution of the 8-chloro group in intermediates 68-74 with morpholine or (R)-3methylmorpholine. Intermediates 57-67 were obtained as shown in Schemes 2 and 3 by substitution of the 8-chloro group in intermediates 51-53 with the corresponding morpholine. Intermediates 68-74 were synthesized as shown in Schemes 4 by standard N-alkylation of intermediates 51 or 54-56 with alkyl halide, alkyl mesylate or epoxide in the presence of a base such as cesium carbonate. The 8-chloro-1,2,3,4-tetrahydropyrimido[1,2-a]pyrimidin-6-one key intermediates 51-56 were prepared as shown in Scheme 1 by reaction of cyclic guanidines 41-44 with malonate followed by chlorination of the 8-hydroxy group in intermediates 45-50 with phosphoryl chloride.15,20 The guanidine intermediates 41-44 were obtained either by direct hydrogenation16,36 of the 2-amino-4-trifluoromethyl pyrimidine 37 or by reaction of diamines 3840 with cyanogen bromide.42-43 The diamines 38-40 were either commercially available or synthesized using standard literature methods.20,38,44-46 Scheme 1. Synthesis of intermediates 41-56a


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N 37 H2N

CF3

N a

O HN H2N

c or d R3 R2

N

R4 HO

41 R2 = CF3, R3 = H 42 R2 = CF3, R3 = Me 43 R2 = R3 = Me 44 R2-R3 = CH2-CH2

O e, f

N N

N H

R3 R2

R4 Cl

45 R2 = CF3, R3 = R4 = H 46 R2 = CF3, R3 = H, R4 = F 47 R2 = CF3, R3 = Me, R4 = H 48 R2 = R3 = Me, R4 = H 49 R2 = R3 = Me, R4 = F 50 R2-R3 = CH2-CH2, R4 = H

N N

N H

R3 R2

51 R2 = CF3, R3 = R4 = H 52 R2 = CF3, R3 = H, R4 = F 53 R2 = CF3, R3 = Me, R4 = H 54 R2 = R3 = Me, R4 = H 55 R2 = R3 = Me, R4 = F 56 R2-R3 = CH2-CH2, R4 = H

b

H2N R3 R2

H2N

38 R2 = CF3, R3 = Me 39 R2 = R3 = Me 40 R2-R3 = CH2-CH2

a

Reagents and conditions: (a) H2 4 bar, Pd/C, H2O, 12 N HCl, 40°C; (b) BrCN, MeCN, rfx; c) diethyl malonate, MeONa, MeOH, 55°C; d) dimethyl fluoromalonate, MeONa, MeOH, 55°C; e) 1,2-dichloroethane, POCl3, 65°C; f) chiral chromatographic separation as required.

Scheme 2. Synthesis of intermediates 57-65a R5 N O R5H

N

a Cl

57 O

O

N

N H

CF3

N

58 O N

N R5

N

N H

CF3

60 O

51

59 O N

N 62 O

61 O

N 63 O

N

N 64 O

N 65 O


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a

Reagents and conditions: (a) morpholine or substituted morpholine; compounds 57-59: 80100°C; compounds 60-62: TEA, microwave, 140-180°C; compounds 63-65: TEA, microwave, 240°C.

Scheme 3. Synthesis of intermediates 66-67a R

R4

O

NH

O

R4 R

O N

Cl

N

N R3

N H

a

R3 R2

N

N

O

52 R2 = CF3, R3 = H, R4 = F 53 R2 = CF3, R3 = Me, R4 = H

N H

R2

66 R = H, R2 = CF3, R3 = Me, R4 = H 67 R = Me, R2 = CF3, R3 = H, R4 = F

a

Reagents and conditions: (a) compound 66: morpholine, 80°C; compound 67: (R)-3methylmorpholine, 100°C.

Scheme 4. Synthesis of intermediates 68-74a O

O R4 Cl

N N

N H

R3 R2

51 R2 = CF3, R3 = R4 = H 54 R2 = R3 = Me, R4 = H 55 R2 = R3 = Me, R4 = F 56 R2-R3 = CH2-CH2, R4 = H

a

R1X

R4

a

Cl

N N

R3 N R2 R1

68 R1 = CH2CO(pyridin-2-yl), R2 = CF3, R3 = R4 = H 69 R1 = CH2CO(pyridin 3-yl), R2 = CF3, R3 = R4 = H 70 R1 = (S)-CH2CH(OH)C6H5, R2 = R3 = Me, R4 = H 71 R1 = CH2CH2OiPr, R2 = R3 = Me, R4 = H 72 R1 = CH2(isoxazol-3-yl), R2 = R3 = Me, R4 = F 73 R1 = CH2CO(pyridin-2-yl), R2 = R3 = Me, R4 = F 74 R1 = (S)-CH2CH(OH)C6H5, R2-R3 = CH2-CH2, R4 = H

Reagents and conditions: (a) electrophile R1X, base, solvent, ∆(°C).


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Scheme 5. Synthesis of compounds 1-4, 8-27, 30-34a from Figures 1 and 5 and Tables 5 and 7 O

O R1X

R4

R3 R5

N

N H

a

N

R5

N

R2

57-67 a

R4

N

R3 N R2 R1

1-4, 8-27, 30-34

Reagents and conditions: (a) electrophile R1X, base, solvent, ∆(°C).

Scheme 6. Synthesis of compounds 5-7, 28-29, 35-36a from Figure 5 and Table 7 R

R4 N

R4 R

O

N R3

Cl

O

NH

O

N R2 R1

68-74

a

N O

N N

R3 N R2 R1

5-7, 28-29, 35-36

a

Reagents and conditions: (a) compounds 5-7: morpholine, 80°C; compounds 28-29, 35-36: (R)-3-methylmorpholine, 100°C.

CONCLUSION Starting from a high-throughput phenotypic screening campaign, we have identified and developed a novel series of tetrahydropyrimido-pyrimidinone derivatives as Vps34 kinase inhibitors. The multiparametric chemical optimization program resulted in compound 31, which can be readily obtained in a 5 step synthesis and has appropriate physicochemical and in vitro


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ADME properties. Compound 31 was found to exhibit nanomolar biochemical and cellular activities against Vps34 and exquisite selectivity against protein and other lipid kinases. The original off-target lipid kinase activities of initial hits were dialed out by using a methyl morpholine moiety as hinge binder. This was confirmed by solving the X-ray crystal structure of human Vps34 in complex with compound 31. This molecule exhibited suitable mouse PK and showed significant and sustained pathway modulation in a H1299-GFP-FYVE s.c. tumor xenografted mechanistic model in mice. Target engagement was correlated with plasma concentrations of compound 31, revealing a direct PK-PD response. Our group recently described in vitro application for a Vps34 inhibitor in cancer.40 Compound 31 is a novel optimized small molecular mass Vps34 kinase inhibitor with the required potency, selectivity, and drug-like properties to further explore the effects of Vps34 kinase modulation in cancer therapy, either as single agent or in combination. EXPERIMENTAL SECTION Binding equilibrium and rate constants by Surface Plasmon Resonance (SPR). The affinity of compound 31 was evaluated by Surface Plasmon Resonance (SPR) on ProteOn (Biorad). Human Vps34 protein, 2-887 construct with N terminal GST tag was produced in-house in insect cells and purified to 98% purity by chromatography on GSTrap 4B and Superdex 200. The protein was diluted at 16 µg/ml in 10 mM MES pH 6.0 and was immobilized using standard amine coupling procedure in 0.005% Tween-20 PBS buffer, in the presence of an active site ligand. The compound was tested at 6 concentrations in 8 experiments. Analyte injections were performed at a flow rate of 100 µL/min for a 1 min association phase, followed by a 5 min dissociation phase, in HEPES-NaOH pH 7.0, 5 mM MgCl2, 150 mM NaCl, 2 mM TCEP, 2% DMSO. From the association (‘on rate', kon) and dissociation rates (‘off rate’, koff), the


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equilibrium dissociation constant (‘binding constant', Kd) were calculated using a Langmuir 1:1 binding model. t1/2, half-life of the complex, is defined as ln2/koff. KiNativTM profiling of compound 31 added to Jurkat and Ramos cell lysates at ActivX Biosciences Inc. The B lymphocyte cell line, Ramos (CRL-1596TM), and the T-lymphocyte cell line, Jurkat (TIB-152™), were acquired from American Type Culture Collection (ATCC). Cells were cultured in RPMI-1640 media with 10% fetal bovine serum. Cells were lysed by sonication in a detergent containing lysis buffer, cleared by centrifugation, and the resulting supernatant was collected for compound treatment. Final protein concentration of lysates was 4 mg/ml. 5 µL of compound 31 was added from 100X stock solutions in DMSO to 445 µL of lysate in duplicate. 5 µL of DMSO was added to 445 µL of lysate in quadruplicate for controls. After 15 minute incubation, 5 µL of a 100X aqueous solution of the ATP probe I (desthiobiotin-adenosine triphosphate-acylphosphate probe linker I) was added to each sample (final concentration of ATP probe I was 0.5 µM). After five minutes, 50 µL of a 10X aqueous solution of the ATP probe II (desthiobiotin-adenosine triphosphate-acylphosphate probe linker II) was added to each sample (final concentration of ATP probe II was 20 µM). All samples were then incubated for an additional 10 minutes. The final DMSO concentration was 1%. Following the probe reaction samples were prepared for targeted MS analysis using the ActivX standard protocol 41. Briefly, samples were prepared for trypsin digestion (denature, reduce alkylate), digested with trypsin, and desthiobiotinylated peptides were enriched on streptavidin resin. Enriched probe-labeled peptides were analyzed by liquid chromatography tandem mass spectrometry (LC-MS/MS) on a Thermo-LTQ ion trap mass spectrometer using proprietary data collection methodology customized for Ramos cells, as previously described.41 All quantitation was performed by extracting characteristic fragment ion signals from targeted MS/MS spectra and comparing


41

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signals in control and treated samples. Proprietary software is used for this purpose with manual validation/visual inspection performed as needed based on data flagging/filtering measures. For each peptide quantitated, the change in MS signal for the treated samples relative to the MS signal for the control samples was expressed as percentage inhibition using the following equation:

Inhibition (%) = 1 − × 100 All inhibited data points were visually verified, as were all datapoints showing variability outside of normal limits. Significance of datapoints showing >35% inhibition was determined according to the Student T-test (Excel 2010): Array1: MS signals from control samples Array2: MS signals from treated samples Tails = 1 (one-tailed distribution) Type = 2 (two-sample equal variance) In cases where scores 4 per cell, in a total of 25 fields acquired. The percentage inhibition in reference to untreated cells was calculated for each concentration of compound. The IC50 value was defined as the concentration of compound where percent inhibition is equal to 50.


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IC50 values were obtained from a dose−response curve with 10 concentrations tested in single replicate and fitted with XLFit 4 software using a 4-parameter logistic model. p-Akt cellular assay. U87MG tumor cell line was treated with compounds for 1.5 h. Cells were lysed according to manufacturer instructions (Phospho-Akt Ser 473 whole cell lysate kit, Meso Scale). The IC50 value was defined as the concentration of compound where percent inhibition is equal to 50. IC50 values were obtained from a dose−response curve with 10 concentrations tested in single replicate and fitted with XLFit 4 software using a 4-parameter logistic model. Pharmacodynamics (PD) study in tumor tissues. For PK/PD studies, 3 × 106 H1299-GFPFYVE tumor cells with 50% Matrigel were subcutaneously injected on the dorsal side of SCID mice from Charles River, one tumor per mouse. When xenografted tumors reached a range of ~200-400 mm3, mice were treated with vehicle (98% PEG200: 2% PS80) or a single dose of compound 31 at 100 and 50 mg/kg via oral gavage. Three mice treated with vehicle alone and three mice treated with compound 31 were sacrificed at each time-point described in the results section; tumor tissues were harvested for IHC analysis and plasma samples were collected to determine the concentration of compound 31. The tumor samples were fixed in formol (4%) and embedded in paraffin (FFPE format). For each tumor block, 4 sections were performed at 4 different levels and immunohistochemistry anti GFP was done on Ventana Discovery XT automated system. Evaluation of compound activity was done by semi-quantitative assessment of the % of H1299-GFP-FYVE tumor cells with granular cytoplasmic GFP detection. Determination of thermodynamic parameters by Isothermal Titration Calorimetry (ITC). ITC experiments were completed with VP-ITC or AUTO iTC200 calorimeters (GE Healthcare -


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MicroCal) at 25°C. Compounds 2 or 31 and in-house produced Vps34 were prepared in 50 mM HEPES pH 7.5, 150 mM NaCl, 1 mM TCEP, 5 mM MgCl2, pH 7.5 and 5% DMSO. The syringe contained the compound; the sample cell was filled with protein and reference cell with water. A control experiment for each compound was also performed, and the heat of dilution was measured by titrating each compound into buffer alone. The heat of dilution was subtracted, and the binding isotherms were plotted and analyzed using Origin Software. The ITC measurements were fit to a one-site binding model that yields ∆H (enthalpy of binding) and KD (dissociation constant). Cocrystallization. Crystallization assays were performed on purchased Vps34 from Sprint Bioscience which was produced according to the Structural Genomics Consortium (SGC) protocol (EXP-09-AE9801). The protein was concentrated to 10 mg/ml in 20 mM Hepes pH7.5, 100 mM NaCl, 1 mM TCEP and incubated overnight with 1 mM ligand. It crystallized by the hanging drop method in a range of conditions: 1.7-2M ammonium sulfate, 100 mM Hepes pH 7.5 or Tris pH8.5 (compounds 3, 7, 15, 24, 31); 1M NaCitrate pH 8 (compound 4); 1.4M NaMalonate Tris pH 8.0 (compound 2). Multiple clustered needle-like crystals rapidly grew and were tested for X-ray diffraction using glycerol as cryoprotectant. Data were processed using automated methods implemented by GlobalPhasing.47 The apostructure of Vps34 solved by the SGC (PDB code 3IHY) was initially used as a model for Molecular Replacement. The structures were refined and corrected using Buster (BUSTER-TNT 2.11.5, GlobalPhasing Ltd) and COOT.48 Final quality checks were done with MolProbity.49 Data collection and data processing statistics can be found in Supporting Information Table S1


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Chemistry. The nomenclature of the compounds was carried out with the ACDLABS software, version 11.01. All solvents and reagents obtained from commercial sources were used without further purification. Thin layer chromatography was carried out on Merck silica gel 60 F254 glass plates. Silica gel chromatography was performed using prepacked Merck silica gel cartridges (15−40 µm). The microwave oven used was a Biotage, InitiatorTM 2.0, 400 W max, 2450 MHz instrument. The 1H NMR spectra at 400 MHz was performed on a Bruker Avance DRX-400 spectrometer, with the chemical shifts (δ in ppm) in the solvent dimethyl sulfoxide-d6 (d6-DMSO) referenced at 2.5 ppm at a temperature of 303 K, and coupling constants (J) are given in hertz. The mass spectra (MS) were obtained by methods A and B. Method A: Waters UPLC-SQD instrument; ionization, positive or negative mode electrospray (ES+ and ES−) or both; chromatographic conditions, column Acquity BEH C18 1.7 µM, 2.1 mm × 50 mm; solvents (A) water (0.1% formic acid), (B) MeCN (0.1% formic acid); column temperature 50 °C; flow rate 1 ml/min; gradient (2 min) from 5% to 50% of B in 0.8 min; 1.2 min 100% of B; 1.85 min 100% of B; 1.95 min 5% of B; retention time = Tr (min). Method B: Waters ZQ instrument; ionization, positive or negative mode electrospray (ES+ or ES−) or both; chromatographic conditions, column XBridge C18 2.5 µM, 3 mm × 50 mm; solvents (A) water (0.1% formic acid), (B) MeCN (0.1% formic acid); column temperature 70 °C; flow rate 0.9 ml/min; gradient (7 min) from 5% to 100%. Purities for final compounds were measured using UV detection at 220 nm and are ≥95.0%.


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1-[(2S)-2-Hydroxy-2-phenyl-ethyl]-2,2-dimethyl-8-morpholino-3,4-dihydropyrimido[1,2a]pyrimidin-6-one (5). A mixture of 70 (265 mg, 0.79 mmol) in morpholine (6.5 ml, 75 mmol) was stirred at 50°C overnight. The reaction mixture was concentrated under reduced pressure. The crude residue was dissolved in EtOAc (20 ml) and washed with water (3 x 10 ml). The organic layer was dried over magnesium sulfate and concentrated under reduced pressure. The crude residue was purified by recrystallization in isopropyl ether to give compound 5 (219 mg, 72% yield) as a white solid. 1H NMR (400 MHz): 0.99 (s, 3 H), 1.33 (s, 3 H), 1.68 – 1.77 (m, 1 H), 1.83 -1.92 (m, 1 H), 3.28 – 3.81 (m partially hidden, 12 H), 4.94 (s, 1 H), 5.04 (m, 1 H), 5.42 (d, J=3.3 Hz, 1 H), 7.20-7.36 (m, 5 H). Mass Spectrometry: method A: Tr (min)=1.01; [M+H]+ m/z 385, [M+HCOOH-H]- m/z 429; GC-Tof - EI: M+. m/z 384. 1-[(2S)-2-Hydroxy-2-phenyl-ethyl]-8-morpholino-spiro[3,4-dihydropyrimido[1,2a]pyrimidine-2,1'-cyclopropane]-6-one (6). To a solution of 74 (360 mg, 1.18 mmol) in MeCN (10 ml) were added cesium carbonate (50 mg, 0.15 mmol) and morpholine (262 mg, 3.01 mmol). The reaction mixture was stirred at 80°C under microwave irradiation for 2 h, then allowed to cool to room temperature and concentrated under reduced pressure. The crude residue was dissolved in EtOAc (20 ml), washed with water (3 x 10 ml) and brine (10 ml). The organic layer was dried over magnesium sulfate and concentrated under reduced pressure. The crude residue was purified by silica gel chromatography (CH2Cl2/MeOH, 95:5%) to give compound 6 (45 mg, 79% yield) as a white solid. [α]D 25 = - 23.9 (2.412 mg / 0.5 ml DMSO). 1H NMR (400 MHz): 0.50 - 0.74 (m, 2 H), 0.96 - 1.21 (m, 2 H), 1.75 (t, 2 H), 3.31 - 3.47 (m, 5 H), 3.48 - 3.67 (m, 5 H), 3.67 - 3.86 (m, 2 H), 4.85 (m, 1 H), 4.92 (s, 1 H), 5.40 (d, 1 H), 7.19- 7.39 (m, 5 H). Mass spectrometry: method A; Tr (min) = 1.38, [M+H]+ m/z 383.


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1-(2-Isopropoxyethyl)-2,2-dimethyl-8-morpholino-3,4-dihydropyrimido[1,2-a]pyrimidin-6one (7). A solution of 71 (187 mg, 0.62 mmol) in morpholine (2.5 ml, 28.67 mmol) was stirred at 80°C under microwave irradiation for 2 h, then allowed to cool to room temperature and concentrated under reduced pressure. The crude residue was dissolved in CH2Cl2 (20 ml), washed with water (3 x 10 ml) and brine (10 ml). The organic layer was dried over magnesium sulfate and concentrated under reduced pressure. The crude residue was purified by silica gel chromatography (CH2Cl2/MeOH, 90:10%) to give compound 7 (60 mg, 27% yield) as a white solid. [α]D 25 = - 12.2 (1.837 mg / 0.5 ml DMSO). 1H NMR (400 MHz): 1.07 (d, 6 H), 1.28 (s, 6 H), 1.83 (t, 2 H), 3.37 (t, 4 H), 3.47 - 3.57 (m, 5 H), 3.60 (t, 4 H), 3.71 (t, 2 H), 4.89 (s, 1 H). Mass spectrometry: method A; Tr (min) = 1.43, [M+H]+ m/z 351. (2S)-1-(2-Isopropoxyethyl)-2-methyl-8-morpholino-2-(trifluoromethyl)-3,4dihydropyrimido[1,2-a]pyrimidin-6-one (8). To a solution of 66 (95 mg, 0.3 mmol) in DMF (4 ml) were added cesium carbonate (126 mg, 0.39 mmol) and 2-isopropoxyethyl 4methylbenzenesulfonate (100 mg, 0.39 mmol). The reaction mixture was stirred at 90°C for 17 h and then allowed to cool to room temperature. The solid was filtered and the filtrate was diluted with EtOAc (20 ml) and washed with water (3 x 10 ml). The organic layer was dried over magnesium sulfate and concentrated under reduced pressure. The crude residue was purified by silica gel chromatography (CH2Cl2/MeOH/MeCN, 90:5:5%) to compound 8 (53 mg, 43% yield) as a yellow solid. Mp: 139 °C. [α]D 25 = + 32.0 (1.277 mg / 0.5 ml DMSO). 1H NMR (400 MHz): 1.05 (d, J=6.8 Hz, 3 H), 1,06 (d, J=6.8 Hz, 3 H), 1.61 (s, 3 H), 2.00 – 2.13 (m, 1 H), 2.36 (dt, J=4.1 and 14.7 Hz, 1 H), 3.34 – 3.43 (m, 5 H), 3.47 – 3.57 (m, 4 H), 3.59 – 3.64 (m, 4 H), 3.79 3.89 (m, 1 H), 3.91 – 3.97 (m, 1 H), 5.00 (s, 1 H). Mass spectrometry: method A; Tr (min) = 0.88, [M+H]+ m/z 405.


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(2S)-1-[(5-Chloro-2-thienyl)methyl]-8-morpholino-2-(trifluoromethyl)-3,4-dihydro-2Hpyrimido[1,2-a]pyrimidin-6-one (9). To a solution of 59 (360 mg, 1.18 mmol) in MeCN (10 ml) were added cesium carbonate (460 mg, 1.42 mmol) and 2-chloro-5-(chloromethyl)thiophene (197 mg, 1.18 mmol). The reaction mixture was stirred at 130°C under microwave irradiation for 1.5 h, then allowed to cool to room temperature and concentrated under reduced pressure. The crude residue was dissolved in EtOAc (20 ml) and washed with water (3 x 10 ml). The organic layer was dried over magnesium sulfate and concentrated under reduced pressure. The crude residue was purified by silica gel chromatography (CH2Cl2/MeOH/MeCN, 96:2:2%) to give compound 9 (245 mg, 48% yield) as a white solid. Mp: 169.8 °C. [α]D 25 = + 65.5 (2.289 mg / 0.5 ml DMSO). 1H NMR (400 MHz): 1.87 to 2.00 (m, 1 H), 2.31 to 2.39 (m, 1 H), 3.12 to 3.22 (m, 1 H), 3.42 to 3.49 (m, 4 H), 3.59 to 3.64 (m, 4 H), 4.19 (dd, J=5.5 et 14.1 Hz, 1 H), 4.62 (d, J=15.7 Hz, 1 H), 4.65 (m, 1 H), 5.03 (s, 1 H), 5.21 (d, J=15.7 Hz, 1 H), 6.96 (d, J=3.9 Hz, 1 H), 7.04 (d, J=3.9 Hz, 1 H). Mass spectrometry: method A; Tr (min) = 0.95, [M+H]+ m/z 435. (2S)-1-[(5-Chloro-2-thienyl)methyl]-8-(8-oxa-3-azabicyclo[3.2.1]octan-3-yl)-2(trifluoromethyl)-3,4-dihydro-2H-pyrimido[1,2-a]pyrimidin-6-one (10). To a solution of 60 (128 mg, 0.387 mmol) in DMF (2 ml) were added sodium hydride 60% in mineral oil (46 mg, 0.96 mmol) and 2-chloro-5-(chloromethyl)thiophene (88 mg, 0.53 mmol). The reaction mixture was stirred at room temperature for 2.5 h and concentrated under reduced pressure. The crude residue was purified by silica gel chromatography (CH2Cl2/MeOH, 99:1%) to give compound 10 (86 mg, 48% yield) as a white solid. [α]D 25 = + 43.3 (1.281 mg / 0.5 ml DMSO). 1H NMR (400 MHz): 1.65 - 1.85 (m, 4 H); 1.93 (m, 1 H); 2.35 (m, 1 H); 2.95 (d broad, J=13.3 Hz, 2 H); 3.16 (dt, J=5.4 and 14.2 Hz, 1 H); 3.67 - 3.87 (m broad, 2 H); 4.19 (dd, J=5.4 et 14.2 Hz, 1 H); 4.36 (m, 2 H); 4.63 (d broad, J=15.4 Hz, 2 H); 4.92 (s, 1 H); 5.18 (d, J=15.4 Hz, 1 H); 6.96 (d, J=3.9


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Hz, 1 H); 7.04 (d, J=3.9 Hz, 1 H). Mass spectrometry: method A; Tr (min) = 0.99; [M+H]+ 461, [M-H+HCO2H]- 505. (2S)-1-[(5-Chloro-2-thienyl)methyl]-8-(3-oxa-8-azabicyclo[3.2.1]octan-8-yl)-2(trifluoromethyl)-3,4-dihydro-2H-pyrimido[1,2-a]pyrimidin-6-one (11). By a similar procedure to that described for the synthesis of 10, 61 (128 mg, 0.39 mmol), sodium hydride 60% in mineral oil (46 mg, 0.96 mmol) and 2-chloro-5-(chloromethyl)thiophene (88 mg, 0.53 mmol) were stirred in DMF (2 ml) at room temperature for 2.5 h. Compound 11 (44 mg, 25% yield) was obtained after chromatographic purification as a white solid. [α]D 25 = + 64.7 (1.661 mg / 0.5 ml DMSO). Mp: 184°C. 1H NMR (400 MHz): 1.80 – 2.05 (m, 5 H), 2.31 -2.40 (m, 1 H), 3.12 – 3.21 (m, 1 H), 3.45 – 3.60 (m, 4 H), 4.20 (dd, J=5.6 and 14.2 Hz, 1 H), 4.37 (m broad, 2 H), 4.60 – 4.70 (m, 2 H), 5.01 (s, 1 H), 5.18 (d, J=16.8 Hz, 1 H), 6.95 (d, J=3.8 Hz, 1 H), 7.01 (d, J=3.8 Hz, 1 H). Mass spectrometry: method A: Tr (min)=1.01; [M+H]+ m/z 461 (2S)-1-[(5-Chloro-2-thienyl)methyl]-8-[(1S,4S)-2-oxa-5-azabicyclo[2.2.1]heptan-5-yl]-2(trifluoromethyl)-3,4-dihydro-2H-pyrimido[1,2-a]pyrimidin-6-one (12). By a similar procedure to that described for the synthesis of 10, 62 (127 mg, 0.4 mmol), sodium hydride 60% in mineral oil (47 mg, 0.96 mmol) and 2-chloro-5-(chloromethyl)thiophene (91 mg, 0.54 mmol) were stirred in DMF (2 ml) at room temperature for 2.5 h. Compound 12 (68 mg, 38% yield) was obtained after chromatographic purification as a white solid. [α]D 25 = + 27.4 (2.136 mg / 0.5 ml DMSO). 1H NMR (400 MHz): 1.79 – 2.00 (m, 3 H), 2.31 – 2. 40 (m, 1 H), 3.08 (m broad, 1 H), 3.17 (dt, J=5.1 and 14.2 Hz, 1 H), 3.32 – 3.40 (m, 1 H), 3.60 (m broad, 1 H), 3.72 (dd, J=1.0 and 7.2 Hz, 1 H), 4.19 (dd, J=5.5 and 14.2 Hz, 1 H), 4.56 (d, J=15.8 Hz, 1 H), 4.60 – 5.10 (m broad, 4 H), 5.29 (d, J=15.8 Hz, 1 H), 6.97 (d, J=3.8 Hz, 1 H), 7.03 (d, J=3.8 Hz, 1 H). Mass spectrometry: method A: Tr (min)=0.93; [M+H]+ m/z 447, [M+HCOOH-H]- m/z 491.


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(2S)-1-[(5-Chloro-2-thienyl)methyl]-8-[(3S,5S)-3,5-dimethylmorpholin-4-yl]-2(trifluoromethyl)-3,4-dihydro-2H-pyrimido[1,2-a]pyrimidin-6-one (13). By a similar procedure to that described for the synthesis of 10, 63 (76 mg, 0.23 mmol), sodium hydride 60% in mineral oil (25 mg, 0.52 mmol) and 2-chloro-5-(chloromethyl)thiophene (47 mg, 0.28 mmol) were stirred in DMF (1 ml) at room temperature for 2.5 h. Compound 13 (61 mg, 57% yield) was obtained after chromatographic purification as a white solid. [α]D 25 = + 87.5 (2.483 mg / 0.5 ml DMSO). Mp: 90°C. 1H NMR (400 MHz): 1.19 (d, J=6.8 Hz, 6 H), 1.88 – 2.00 (m, 1 H), 2.31 – 2. 40 (m, 1 H), 3.16 – 3.25 (m, 1 H), 3.52 – 3.65 (m, 2 H), 3.93 – 4.03 (m, 4 H), 4.20 (dd, J=5.5 and 14.2 Hz, 1 H), 4.58 – 4.67 (m, 2 H), 4.91 (s, 1 H), 5.23 (d, J=15.7 Hz, 1 H), 6.96 (d, J=3.9 Hz, 1 H), 7.02 (d, J=3.9 Hz, 1 H). Mass spectrometry: method A: Tr (min)=1.03; [M+H]+ m/z 463. (2S)-1-[(5-Chloro-2-thienyl)methyl]-8-[(3S,5R)-3,5-dimethylmorpholin-4-yl]-2(trifluoromethyl)-3,4-dihydro-2H-pyrimido[1,2-a]pyrimidin-6-one (14). By a similar procedure to that described for the synthesis of 10, 64 (79 mg, 0.24 mmol), sodium hydride 60% in mineral oil (25 mg, 0.52 mmol) and 2-chloro-5-(chloromethyl)thiophene (47 mg, 0.28 mmol) were stirred in DMF (1 ml) at room temperature for 2.5 h. Compound 14 (57 mg, 52% yield) was obtained after chromatographic purification as a white solid. [α]D 25 = + 67.4 (1.756 mg / 0.5 ml DMSO). Mp: 94°C. 1H NMR (400 MHz): 1.15 (d, J=6.8 Hz, 3 H); 1.20 (d, J=6.6 Hz, 3 H); 1.99 (m, 1 H); 2.37 (m, 1 H); 3.19 (dt, J=5.4 and 14.4 Hz, 1 H); 3.52 (d broad, J=11.5 Hz, 2 H); 3.70 (dd, J=5.4 et 11.5 Hz, 2 H); 4.04 (m broad, 1 H); 4.14 (m broad, 1 H); 4.21 (dd, J=5.4 et 14.4 Hz, 1 H); 4.63 (d broad, J=15.9 Hz, 2 H); 4.90 (s, 1 H); 5.25 (d, J=15.9 Hz, 1 H); 6.96 (d, J=3.9 Hz, 1 H); 6.98 (d, J=3.9 Hz, 1 H). Mass spectrometry: method B; Tr (min) = 1.05; [M+H]+ 463.


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(2S)-1-[(5-Chloro-2-thienyl)methyl]-8-[(3R,5R)-3,5-dimethylmorpholin-4-yl]-2(trifluoromethyl)-3,4-dihydro-2H-pyrimido[1,2-a]pyrimidin-6-one (15). By a similar procedure to that described for the synthesis of 10, 65 (76 mg, 0.24 mmol), sodium hydride 60% in mineral oil (25 mg, 0.52 mmol) and 2-chloro-5-(chloromethyl)thiophene (47 mg, 0.28 mmol) were stirred in DMF (1 ml) at room temperature for 2.5 h. Compound 15 (73 mg, 66% yield) was obtained after chromatographic purification as a white solid. [α]D 25 = + 22.2 (1.903 mg / 0.5 ml DMSO). Mp: 140°C. 1H NMR (400 MHz): 1.15 (d, J=6.4 Hz, 6 H); 2.05 (m, 1 H); 2.38 (m, 1 H); 3.21 (dt, J=5.6 et 14.4 Hz, 1 H); 3.56 (m, 2 H); 3.92 – 4.03 (m, 4 H); 4.24 (dd, J=5.6 et 14.4 Hz, 1 H); 4.62 (d large, J=16.1 Hz, 2 H); 4.89 (s, 1 H); 5.34 (d, J=16.1 Hz, 1 H); 6.95 (d, J=3.9 Hz, 1 H); 6.96 (d, J=3.9 Hz, 1 H). Mass spectrometry: method B; Tr (min) = 1.03; [M+H]+ 463. (2S)-1-[(5-Chloro-2-thienyl)methyl]-8-[(3S)-3-methylmorpholin-4-yl]-2-(trifluoromethyl)3,4-dihydro-2H-pyrimido[1,2-a]pyrimidin-6-one (16). By a similar procedure to that described for the synthesis of 9, 58 (101 mg, 0.31 mmol), cesium carbonate (315 mg, 0.96 mmol) and 2chloro-5-(chloromethyl)thiophene (57 mg, 0.34 mmol) were stirred in MeCN (5 ml) at room temperature for 18 h. Compound 16 (54 mg, 39% yield) was obtained after chromatographic purification as a white solid. [α]D 25 = + 101.0 (1.700 mg / 0.5 ml DMSO). Mp: 119°C. 1H NMR (400 MHz): 1.13 (d, J=6.6 Hz, 3 H); 1.93 (m, 1 H); 2.35 (m, 1 H); 3.05 (dt, J=3.8 et 12.8 Hz, 1 H); 3.17 (dt, J=5.2 et 14.7 Hz, 1 H); 3.39 (dt, J=3.2 et 11.9 Hz, 1 H); 3.53 (dd, J=3.1 et 11.6 Hz, 1 H); 3.66 (d, J=11.6 Hz, 1 H); 3.81 -3.89 (m, 2 H); 4.15 - 4.23 (m, 2 H); 4.64 (d large, J=15.6 Hz, 2 H); 4.97 (s, 1 H); 5.18 (d, J=15.6 Hz, 1 H); 6.96 (d, J=3.9 Hz, 1 H); 7.03 (d, J=3.9 Hz, 1 H). Mass spectrometry: method A; Tr (min) = 1; [M+H]+ 449. (2S)-1-[(5-Chloro-2-thienyl)methyl]-8-[(3R)-3-methylmorpholin-4-yl]-2-(trifluoromethyl)3,4-dihydro-2H-pyrimido[1,2-a]pyrimidin-6-one (17). By a similar procedure to that described


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for the synthesis of 9, 59 (102 mg, 0.31 mmol), cesium carbonate (315 mg, 0.96 mmol) and 2chloro-5-(chloromethyl)thiophene (57 mg, 0.34 mmol) were stirred in MeCN (2.5 ml) at room temperature for 18 h. Compound 17 (29 mg, 21% yield) was obtained after chromatographic purification as a white solid. Mp: 188°C. 1H NMR (400 MHz): 1.10 (d, J=6.8 Hz, 3 H); 1.97 (m, 1 H); 2.36 (m, 1 H); 3.06 (dt, J=3.5 and 12.8 Hz, 1 H); 3.18 (dt, J=3.9 and 13.9 Hz, 1 H); 3.38 (dt, J=3.4 and 11.6 Hz, 1 H); 3.53 (dd, J=2.5 and 11.5 Hz, 1 H); 3.65 (d, J=11.5 Hz, 1 H); 3.77 (d broad, J=12.8 Hz, 1 H); 3.87 (dd, J=3.4 and 11.6 Hz, 1 H); 4.14 - 4.26 (m, 2 H); 4.61 (d, J=15.9 Hz, 1 H); 4.65 (m, 1 H); 4.96 (s, 1 H); 5.24 (d, J=15.9 Hz, 1 H); 6.96 (d, J=3.9 Hz, 1 H); 7.01 (d, J=3.9 Hz, 1 H). Mass spectrometry: method A; Tr (min) = 1; [M+H]+ 449. (2S)-1-(Isoxazol-3-ylmethyl)-8-[(3R)-3-methylmorpholin-4-yl]-2-(trifluoromethyl)-3,4dihydro-2H-pyrimido[1,2-a]pyrimidin-6-one (18). By a similar procedure to that described for the synthesis of 9, 59 (80 mg, 0.25 mmol), cesium carbonate (409 mg, 1.26 mmol) and 3(chloromethyl)isoxazole (35 mg, 0.30 mmol) were stirred in MeCN (2.5 ml) at 50°C for 2 h. Compound 18 (50 mg, 50% yield) was obtained after chromatographic purification as a white solid. [α]D 25 = - 42.2 (2.087 mg / 0.5 ml DMSO). 1H NMR (400 MHz): 0.87 (d, J=6.8 Hz, 3 H); 2.23 (m, 1 H); 2.40 (m, 1 H); 2.92 (dt, J=3.8 and 13.0 Hz, 1 H); 3.18 - 3.33 (m partially masqued, 2 H); 3.46 (dd, J=2.9 et 11.6 Hz, 1 H); 3.53 (d broad, J=13.0 Hz, 1 H); 3.58 (d, J=11.6 Hz, 1 H); 3.80 (dd, J=3.5 and 11.6 Hz, 1 H); 4.09 (m, 1 H); 4.25 (dd, J=5.4 and 13.9 Hz, 1 H); 4.60 (d, J=16.4 Hz, 1 H); 4.76 (m, 1 H); 4.91 (s, 1 H); 5.20 (d, J=16.4 Hz, 1 H); 6.49 (d, J=1.7 Hz, 1 H); 8.82 (d, J=1.7 Hz, 1 H). Mass spectrometry: method A; Tr (min) = 0.94; [M+H]+ 400. (2S)-7-Fluoro-1-(isoxazol-3-ylmethyl)-8-[(3R)-3-methylmorpholin-4-yl]-2-(trifluoromethyl)3,4-dihydro-2H-pyrimido[1,2-a]pyrimidin-6-one (19). By a similar procedure to that described for the synthesis of 9, 67 (125 mg, 0.37 mmol), cesium carbonate (505 mg, 1.55 mmol) and 3-


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(chloromethyl)isoxazole (43 mg, 0.37 mmol) were stirred in MeCN (2 ml) at 50°C for 18 h. Compound 19 (100 mg, 64% yield) was obtained after chromatographic purification as a white solid. [α]D 25 = - 55.5 (1.589 mg / 0.5 ml DMSO). 1H NMR (400 MHz): 1.04 (d, J=6.8 Hz, 3 H), 2.16 – 2.29 (m, 1 H), 2.38 – 2.46 (m, 1 H), 3.18 (dt, J=4.0 and 12.7 Hz, 1 H), 3.27 – 3.37 (m partially hidden, 2 H), 3.50 (dd, J=2.9 – 11.5 Hz, 1 H), 3.57 (d, J=11,5 Hz, 1 H), 3.71 – 3.79 (m, 2 H), 4.13 (q broad, J=6.8 Hz, 1 H), 4.23 (dd, J=5.6 and 14.2 Hz, 1 H), 4.64 (d, J=16.6 Hz, 1 H), 4.71 – 4.80 (m, 1 H), 5.14 (d, J=16.6 Hz, 1 H), 6.49 (d, J=1.7 Hz, 1 H), 8.83 (d, J=1.7 Hz, 1 H). Mass spectrometry: method B: Tr (min)= 1.04; [M+H]+ m/z 418, [M+HCOOH-H]- m/z 462. (2S)-1-(Isoxazol-5-ylmethyl)-8-[(3R)-3-methylmorpholin-4-yl]-2-(trifluoromethyl)-3,4dihydro-2H-pyrimido[1,2-a]pyrimidin-6-one (20). By a similar procedure to that described for the synthesis of 9, 59 (80 mg, 0.25 mmol), cesium carbonate (409 mg, 1.26 mmol) and 5bromomethyl-isoxazole (49 mg, 0.30 mmol) were stirred in MeCN (2 ml) at 50°C for 2 h. Compound 20 (45 mg, 45% yield) was obtained after chromatographic purification as a white solid. [α]D 25 = - 26.3 (1.584 mg / 0.5 ml DMSO). 1H NMR (400 MHz): 0.91 (d, J=6.8 Hz, 3 H); 2.24 (m, 1 H); 2.41 (m, 1 H); 2.92 (dt, J=4.2 and 12.9 Hz, 1 H); 3.17 - 3.34 (m partially masqued, 2 H); 3.45 (dd, J=3.1 and 11.4 Hz, 1 H); 3.53 (d broad, J=13.0 Hz, 1 H); 3.59 (d, J=11.4 Hz, 1 H); 3.80 (dd, J=3.5 and 11.4 Hz, 1 H); 4.09 (m, 1 H); 4.25 (dd, J=5.4 and 14.2 Hz, 1 H); 4.73 (d, J=16.9 Hz, 1 H); 4.81 (m, 1 H); 4.92 (s, 1 H); 5.21 (d, J=16.9 Hz, 1 H); 6.36 (d, J=1.7 Hz, 1 H); 8.48 (d, J=1.7 Hz, 1 H). Mass spectrometry: method B; Tr (min) = 0.97; [M+H]+ 418, [M-H]416, [M-H+HCO2H]- 462. (2S)-7-Fluoro-1-(isoxazol-5-ylmethyl)-8-[(3R)-3-methylmorpholin-4-yl]-2-(trifluoromethyl)3,4-dihydro-2H-pyrimido[1,2-a]pyrimidin-6-one (21). By a similar procedure to that described for the synthesis of 9, 67 (100 mg, 0.30 mmol), cesium carbonate (380 mg, 1.2 mmol) and 5-


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bromomethyl-isoxazole (60 mg, 0.37 mmol) were stirred in MeCN (2 ml) at 50°C for 18 h. Compound 21 (68 mg, 56% yield) was obtained after chromatographic purification as a white solid. [α]D 25 = - 34.2 (1.714 mg / 0.5 ml DMSO). 1H NMR (400 MHz): 1.07 (d, J=6.8 Hz, 3 H), 2.17 – 2.30 (m, 1 H), 2.40 – 2.47 (m, 1 H), 3.18 (dt, J=5.1 and 14.2 Hz, 1 H), 3.30 – 3.38 (m partially hidden, 2 H), 3.50 (dd, J=2.7 et 11.5 Hz, 1 H), 3.57 (d, J=11.5 Hz, 1 H), 3.70 – 3.80 (m, 2 H), 4.11 (q broad, J=6.8 Hz, 1 H), 4.21 (dd, J=5.1 and 14.2 Hz, 1 H), 4.73 – 4.83 (m, 2 H), 5.15 (d, J=16.8 Hz, 1 H), 6.35 (d, J=1.5 Hz, 1 H), 8.48 (d, J=1.5 Hz, 1 H). Mass spectrometry: method B: Tr (min)= 0.97; [M+H]+ m/z 418, [M-H]- m/z 416, [M+HCOOH-H]- m/z 462. (2S)-1-[(5-Chloro-3-pyridyl)methyl]-8-[(3R)-3-methylmorpholin-4-yl]-2-(trifluoromethyl)3,4-dihydro-2H-pyrimido[1,2-a]pyrimidin-6-one (22). By a similar procedure to that described for the synthesis of 9, 59 (400 mg, 1.26 mmol), cesium carbonate (1.64 g, 5.03 mmol) and 3chloro-5-(chloromethyl)pyridine (244 mg, 1.51 mmol) were stirred in DMF (2.5 ml) at room temperature for 17 h. Compound 22 (195 mg, 35% yield) was obtained after chromatographic purification as a white solid. Mp: 202.4 °C. [α]D 25 = - 38.9 (2.575 mg / 0.5 ml DMSO). 1H NMR (400 MHz): 0.71 (d, J=6.6 Hz, 3 H), 2.34-2.45 (m, 2 H), 2.89 (dt, J=4.2 et 13.2 Hz, 1 H), 3.183.31 (m, 2 H), 3.38-3.46 (m, 2 H), 3.52 (d, J=11.6 Hz, 1 H), 3.76 (dd, J=3.2 et 11.0 Hz, 1 H), 4.01 (m, 1 H), 4.21 (m, 1 H), 4.60 (d, J=16.4 Hz, 1 H), 4.86 (m, 1 H), 4.89 (s, 1 H), 5.11 (d, J=16.4 Hz, 1 H), 7.82 (s broad, 1 H), 8.45 (s broad, 1 H), 8.49 (d, J=2.0 Hz, 1 H). Mass spectrometry: method B; Tr (min) = 1.00; (m/z): [M-H+HCO2H]- 488. (2S)-1-[(2-Fluoro-4-pyridyl)methyl]-8-[(3R)-3-methylmorpholin-4-yl]-2-(trifluoromethyl)3,4-dihydro-2H-pyrimido[1,2-a]pyrimidin-6-one (23). By a similar procedure to that described for the synthesis of 9, 59 (150 mg, 0.47 mmol), 4-(chloromethyl)-2-fluoropyridine (83 mg, 0.57 mmol) and cesium carbonate (460 mg, 1.41 mmol) were stirred in MeCN (5 ml) at room


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temperature for 24 h. Compound 23 (150 mg, 74% yield) was obtained after chromatographic purification as a white solid. [α]D 25 = - 52.6 (2.126 mg / 0.5 ml DMSO). 1H NMR (400 MHz): 0.63 (d, J=6.5 Hz, 3 H); 2.42 (m partially masqued, 2 H); 2.85 (m, 1 H); 3.18 – 3.43 (m, 4 H); 3.50 (d, J=11.5 Hz, 1 H); 3.74 (m, 1 H); 3.94 (m, 1 H); 4.25 (m, 1 H); 4.64 (d, J=16.9 Hz, 1 H); 4.82 (m, 1 H); 4.89 (s, 1 H); 5.10 (d, J=16.9 Hz, 1 H); 7.04 (s, 1 H); 7.22 (s, 1 H); 8.14 (d, J=4.8 Hz, 1 H). Mass spectrometry: method B; Tr (min) = 0.97; [M+H]+ 428, [M-H]- 426, [MH+HCO2H]- 472. (2S)-7-Fluoro-1-[(2-fluoro-4-pyridyl)methyl]-8-[(3R)-3-methylmorpholin-4-yl]-2(trifluoromethyl)-3,4-dihydro-2H-pyrimido[1,2-a]pyrimidin-6-one (24). By a similar procedure to that described for the synthesis of 9, 67 (1.4 g, 4.16 mmol), cesium carbonate (4.07 g, 12.49 mmol) and 4-(chloromethyl)-2-fluoropyridine (0.76 g, 5 mmol) were stirred in MeCN (50 ml) at room temperature for 20 h. Compound 24 (1.2 g, 64% yield) was obtained after chromatographic purification as a white solid. [α]D 25 = - 63.2 (2.193 mg / 0.5 ml DMSO). 1H NMR (400 MHz): 0.85 (d, J=6.8 Hz, 3 H); 2.43 (m, 2 H); 3.10 (m, 1 H); 3.19 (m, 1 H); 3.32 (m, 1 H); 3.41 (dd, J=2.9 and 11.5 Hz, 1 H); 3.48 (d, J=11.5 Hz, 1 H); 3.58 (m, 1 H); 3.68 (m, 1 H); 3.94 (q broad, J=6.8 Hz, 1 H); 4.24 (m, 1 H); 4.68 (d, J=17.1 Hz, 1 H); 4.79 (m, 1 H); 5.05 (d, J=17.1 Hz, 1 H); 7.02 (s broad, 1 H); 7.21 (d broad, J=5.1 Hz, 1 H); 8.14 (d, J=5.1 Hz, 1 H). Mass spectrometry: method B; Tr (min) = 1.03; [M+H]+ 446, [M-H]- 444, [M-H+HCO2H]- 490. (2S)-7-Fluoro-1-(2-methoxy-4-pyridyl)-8-[(3R)-3-methylmorpholin-4-yl]-2(trifluoromethyl)-3,4-dihydro-2H-pyrimido[1,2-a]pyrimidin-6-one (25). To a solution of 67 (175 mg, 0.52 mmol) in DMF (2 ml) were added Copper iodide (148 mg, 0.78 mmol), potassium phosphate tribasic (165 mg, 0.78 mmol) and 4-bromo-2-methoxypyridine (97 mg, 0.52 mmol). The reaction mixture was heated at 160°C under microwave irradiation for 3 h and concentrated


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under reduced pressure. The crude residue was purified by silica gel chromatography (CH2Cl2/MeOH, 90:10%) to give compound 25 (98 mg, 43% yield) as a white solid. [α]D 25 = 92.7 (2.441 mg / 0.5 ml DMSO). 1H NMR (400 MHz): 1.01 (d, J=6.7 Hz, 3 H); 2.40 – 2.47 (m, 2 H); 3.12 (dt, J=3.5 and 12.8 Hz, 1 H); 3.25 - 3.52 (m partially masqued, 4 H); 3.55 (d broad, J=12.8 Hz, 1 H); 3.70 (dd, J=2.8 and 11.4 Hz, 1 H); 3.87 (s broad, 4 H); 4.28 (m, 1 H); 5.08 (m, 1 H); 6.90 (d, J=1,7 Hz, 1 H); 7.04 (dd, J=1.7 and 5.6 Hz, 1 H); 8.17 (d, J=5.6 Hz, 1 H). Mass spectrometry: method B; Tr (min) = 1.16; [M+H]+ 444. (2S)-7-Fluoro-1-(2-hydroxy-2-methyl-propyl)-8-[(3R)-3-methylmorpholin-4-yl]-2(trifluoromethyl)-3,4-dihydro-2H-pyrimido[1,2-a]pyrimidin-6-one (26). To a solution of 67 (100 mg, 0.297 mmol) in DMF (2 ml) were added 2,2-dimethyloxirane (21.4 mg, 0.297 mmol) and potassium phosphate tribasic (260 mg, 1.19 mmol). The reaction mixture was heated at 60°C overnight and concentrated under reduced pressure. The crude residue was purified by silica gel chromatography (CH2Cl2/MeOH, 90:10%) to give compound 26 (45 mg, 37% yield) as a white solid. [α]D 25 = + 3.6 (1.865 mg / 0.5 ml DMSO). 1H NMR (400 MHz): 1.03 (s, 3 H); 1.14 (s, 3 H); 1.21 (d, J=6.8 Hz, 3 H); 2.24 (m, 1 H); 2.41 (m, 1 H); 3.02 (d, J=14.7 Hz, 1 H); 3.25 - 3.36 (m partially masqued, 2 H); 3.43 (dt, J=2.6 et 11.7 Hz, 1 H); 3.55 - 3.66 (m, 2 H); 3.77 - 3.89 (m, 2 H); 4.14 (dd, J=6.6 and 14.4 Hz, 1 H); 4.26 (m, 1 H); 4.45 (d, J=14.7 Hz, 1 H); 4.75 (s, 1 H); 4.93 (m, 1 H). Mass spectrometry: method B; Tr (min) = 0.99; [M+H]+ 409, [M-H+HCO2H]453. (2S)-7-Fluoro-1-(2-methoxyethyl)-8-[(3R)-3-methylmorpholin-4-yl]-2-(trifluoromethyl)-3,4dihydro-2H-pyrimido[1,2-a]pyrimidin-6-one (27). By a similar procedure to that described for the synthesis of 9, 67 (80 mg, 0.24 mmol), cesium carbonate (310 mg, 95 mmol) and 1-bromo-2methoxyethane (40 mg, 0.28 mmol) were stirred in MeCN (2 ml) at room temperature for 18 h.


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Compound 27 (38 mg, 48% yield) was obtained after chromatographic purification as a white solid. [α]D 25 = + 3.6 (2.454 mg / 0.5 ml DMSO). 1H NMR (400 MHz): 1.23 (d, J=6.6 Hz, 3 H); 2.08 (m, 1 H); 2.37 (m, 1 H); 3.19 - 3.52 (m partially masqued, 4 H); 3.26 (s, 3 H); 3.57 - 3.68 (m, 3 H); 3.85 (d broad, J=14 Hz, 2 H); 4.16 (m, 2 H); 4.26 (q broad, J=6.6 Hz, 1 H); 4.59 (m, 1 H). Mass spectrometry: method B; Tr (min) = 1.05; [M+H]+ 395. (2S)-8-[(3R)-3-Methylmorpholin-4-yl]-1-[2-oxo-2-(2-pyridyl)ethyl]-2-(trifluoromethyl)-3,4dihydro-2H-pyrimido[1,2-a]pyrimidin-6-one (28). By a similar procedure to that described for the synthesis of 5, 68 (2.36 g, 6.3 mmol) and (R)-3-methylmorpholine (2.44 g, 23 mmol) were stirred at 80°C for 18 h. Compound 28 (1.58 g, 57% yield) was obtained after chromatographic purification as a white solid. Mp: 198°C. [α]D 25 = - 114.3 (2.701 mg / 0.5 ml DMSO). 1H NMR (400 MHz): 0.67 (d, J=6.8 Hz, 3 H); 2.22 (m, 1 H); 2.44 (m, 1 H); 2.81 (dt, J=3.4 and 12.7 Hz, 1 H); 3.06 - 3.34 (m partially masqued, 4 H); 3.59 (m, 1 H); 3.84 (q broad, J=6.8 Hz, 1 H); 4.36 (m, 1 H); 4.70 (m, 1 H); 4.83 (d, J=18.1 Hz, 1 H); 4.87 (s, 1 H); 5.69 (d, J=18.1 Hz, 1 H); 7.72 (m, 1 H); 7.97(d, J=7.8 Hz, 1 H); 8.04 (dt, J=1.5 and 7.8 Hz, 1 H); 8.76 (d broad, J=5.0 Hz, 1 H). Mass spectrometry: method B; Tr (min) = 1.03; [M+H]+ 438. (2S)-8-[(3R)-3-Methylmorpholin-4-yl]-1-[2-oxo-2-(3-pyridyl)ethyl]-2-(trifluoromethyl)-3,4dihydro-2H-pyrimido[1,2-a]pyrimidin-6-one (29). By a similar procedure to that described for the synthesis of 5, 68 (109 mg, 0.29 mmol), TEA (0.16 ml, 1.18 mmol) and R-methylmorpholine (79 mg, 0.79 mmol) were stirred in NMP (2 ml) at 170°C under microwave irradiation for 1 h. Compound 29 (35 mg, 27% yield) was obtained after chromatographic purification as a white solid. Mp: 270°C. 1H NMR (400 MHz): 0.65 (d, J=6.4 Hz, 3 H); 2.23 (m, 1 H); 2.44 (m, 1 H); 2.85 (m, 1 H); 3.11 – 3.41 (m partially masqued, 4 H); 3.63 (m, 1 H); 3.96 (q broad, J=6.4 Hz, 1 H); 4.37 (m, 1 H); 4.63 (m, 1 H); 4.73 (d, J=17.9 Hz, 1 H); 4.90 (s, 1 H); 5.68 (d, J=17.9 Hz, 1


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H); 7.60 (dd, J=5.1 et 7.8 Hz, 1 H); 8.38 (d, J=7.8 Hz, 1 H); 8.84 (d, J=5.1 Hz, 1 H); 9.21 (s, 1 H); NH not localized. Mass spectrometry: method B; Tr (min) = 0.85; [M+H]+ 438, [M-H]- 436. (2S)-7-Fluoro-8-[(3R)-3-methylmorpholin-4-yl]-1-[2-oxo-2-(3-pyridyl)ethyl]-2(trifluoromethyl)-3,4-dihydro-2H-pyrimido[1,2-a]pyrimidin-6-one (30). By a similar procedure to that described for the synthesis of 9, 67 (80 mg, 0.24 mmol), 2-bromo-1-(pyridin-3yl)ethanone (71 mg, 0.36 mmol) and cesium carbonate (310 mg, 0.95 mmol) were stirred in MeCN (5 ml) at 60°C for 2 h. Compound 30 (17 mg, 17% yield) was obtained after chromatographic purification as a white solid. [α]D 25 = - 142.7 (1.950 mg / 0.5 ml DMSO). 1H NMR (400 MHz): 0.82 (d, J=6.7 Hz, 3 H); 2.24 (m, 1 H); 2.47 (m partially masqued, 1 H); 3.05 – 3.23 (m, 2 H); 3.30 – 3.39 (m, 2 H); 3.43 (dd, J=2.7 and 11.4 Hz, 1 H); 3.53 (d broad, J=11.0 Hz, 1 H); 3.60 (d broad, J=12.7 Hz, 1 H); 4.01 (m, 1 H); 4.34 (dd, J=5.3 et 14.4 Hz, 1 H); 4.62 (m, 1 H); 4.78 (d, J=18.1 Hz, 1 H); 5.65 (d, J=18.1 Hz, 1 H); 7.61 (dd, J=4.9 and 8.1 Hz, 1 H); 8.38 (td, J=1.5 and 8.1 Hz, 1 H); 8.85 (dd, J=1.5 and 4.9 Hz, 1 H); 9.21 (d, J=1.5 Hz, 1 H). Mass spectrometry: method B; Tr (min) = 0.9; [M+H]+ 456, [M-H]- 454. (2S)-8-[(3R)-3-Methylmorpholin-4-yl]-1-(3-methyl-2-oxo-butyl)-2-(trifluoromethyl)-3,4dihydro-2H-pyrimido[1,2-a]pyrimidin-6-one (31). By a similar procedure to that described for the synthesis of 9, 59 (1.5 g, 4.71 mmol), cesium carbonate (4.61 g, 14.14 mmol) and 1-bromo-3methylbutan-2-one (933 mg, 5.66 mmol) were stirred in MeCN (37 ml) at room temperature for 17 h. Compound 31 (1.44 g, 76% yield) was obtained after chromatographic purification as a white solid. Mp: 217.3 °C. [α]D 25 = - 51.4 (2.440 mg / 0.5 ml DMSO). 1H NMR (400 MHz): 1.03 (m, 9 H); 2.16 (m, 1 H); 2.37 (m, 1 H); 2.69 (m, 1 H); 2.97 (m, 1 H); 3.15 to 3.36 (m partially masqued, 2 H); 3.45 - 3.65 (m, 3 H); 3.82 (m, 1 H); 4.09 (q broad, J=6.6 Hz, 1 H); 4.23


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(d, J=18.3 Hz, 1 H); 4.30 (m, 1 H); 4.47 (m broad, 1 H); 4.90 (s, 1 H); 4.99 (d, J=18.3 Hz, 1 H). Mass spectrometry: method A; Tr (min) = 1.05; (m/z): [M+H]+ 403, (m/z): [M-H]- 401. (2S)-7-Fluoro-8-[(3R)-3-methylmorpholin-4-yl]-1-(3-methyl-2-oxo-butyl)-2(trifluoromethyl)-3,4-dihydro-2H-pyrimido[1,2-a]pyrimidin-6-one (32). By a similar procedure to that described for the synthesis of 9, 67 (1.7 g, 5.06 mmol), cesium carbonate (8.07 g, 24.77 mmol) and 1-bromo-3-methylbutan-2-one (1.25 g, 7.58 mmol) were stirred in MeCN (50 ml) at room temperature for 4 h. Compound 32 (1.4 g, 66% yield) was obtained after chromatographic purification as a white solid. Mp: 201 °C. [α]D 25 = - 63.5 (1.858 mg / 0.5 ml DMSO). 1H NMR (400 MHz): 1,03 (d, J=6.8 Hz, 3 H); 1,04 (d, J=6.8 Hz, 3 H); 1.15 (d, J=6.6 Hz, 3 H); 2.16 (m, 1 H); 2.39 (m, 1 H); 2.69 (quin, J=6.8 Hz, 1 H); 3.17 - 3.42 (m partially masqued, 3 H); 3.55 (dd, J=2.9 et 11.5 Hz, 1 H); 3.60 (d, J=11.5 Hz, 1 H); 3.72 – 3.84 (m, 2 H); 4.16 (m, 1 H); 4.27 (d broad, J=18.6 Hz, 2 H); 4.47 (m, 1 H); 4.94 (d, J=18.6 Hz, 1 H). Mass spectrometry: method A; Tr (min) = 1.11; (m/z): [M+H]+ 421, (m/z): [M-H]- 419. (2S)-7-Fluoro-8-[(3R)-3-methylmorpholin-4-yl]-1-[(5-methyl-1,2,4-oxadiazol-3-yl)methyl]2-(trifluoromethyl)-3,4-dihydro-2H-pyrimido[1,2-a]pyrimidin-6-one (33). By a similar procedure to that described for the synthesis of 9, 67 (80 mg, 0.24 mmol), cesium carbonate (310 mg, 0.95 mmol) and 3-(chloromethyl)-5-methyl-1,2,4-oxadiazole (37 mg, 0.29 mmol) were stirred in MeCN (2 ml) at room temperature for 18 h. Compound 33 (75 mg, 73% yield) was obtained after chromatographic purification as a white solid. [α]D 25 = - 37.0 (2.276 mg / 0.5 ml DMSO). 1H NMR (400 MHz): 1.03 (d, J=6.8 Hz, 3 H); 2.17 (m, 1 H); 2.45 (m partially masqued, 1 H); 2.56 (s, 3 H); 3.13 – 3.38 (m partially masqued, 3 H); 3.51 (dd, J=2.9 and 11.5 Hz, 1 H); 3.58 (d, J=11.5 Hz, 1 H); 3.72 – 3.81 (m, 2 H); 4.14 (q broad, J=6.8 Hz, 1 H); 4.26 (dd,


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J=5.5 and 14.8 Hz, 1 H); 4.61 (d, J=16.9 Hz, 1 H); 4.78 (m, 1 H); 5.17 (d, J=16.9 Hz, 1 H). Mass spectrometry: method B; Tr (min) = 0.98; (m/z): [M+H]+ 433. (2S)-7-Fluoro-8-[(3R)-3-methylmorpholin-4-yl]-1-[(3-methyl-1,2,4-oxadiazol-5-yl)methyl]2-(trifluoromethyl)-3,4-dihydro-2H-pyrimido[1,2-a]pyrimidin-6-one (34). By a similar procedure to that described for the synthesis of 9, 67 (100 mg, 0.3 mmol), 5-(chloromethyl)-3methyl-1,2,4-oxadiazole (43 mg, 0.33 mmol) and cesium carbonate (194 mg, 0.59 mmol) were stirred in MeCN (5 ml) at 75°C for 3 h. Compound 34 (95 mg, 73% yield) was obtained after chromatographic purification as a white solid. [α]D 25 = - 36.1 (2.581 mg / 0.5 ml DMSO) 1H NMR (400 MHz): 1.02 (d, J=6.8 Hz, 3 H); 2.22 (m, 1 H); 2.30 (s, 3 H); 2.47 (m partially masqued, 1 H); 3.15 (m, 1 H); 3.25 – 3.38 (m, 2 H); 3.47 (dd, J=2.9 and 11.5 Hz, 1 H); 3.57 (d, J=11.5 Hz, 1 H); 3.67 (m, 1 H); 3.76 (m, 1 H); 4.05 (q large, J=6.8 Hz, 1 H); 4.29 (dd, J=5.0 and 14.3 Hz, 1 H); 4.83 (m, 1 H); 4.89 (d, J=17.4 Hz, 1 H); 5.18 (d, J=17.4 Hz, 1 H). Mass spectrometry: method B; Tr (min) = 0.96; (m/z): [M+H]+ 433, (m/z): [M-H]- 431. 7-Fluoro-1-(isoxazol-3-ylmethyl)-2,2-dimethyl-8-[(3R)-3-methylmorpholin-4-yl]-3,4dihydropyrimido[1,2-a]pyrimidin-6-one (35). By a similar procedure to that described for the synthesis of 5, 72 (100 mg, 0.32 mmol) and R-3-methylmorpholine (99 mg, 0.98 mmol) were stirred at 100°C for 18 h. Compound 35 (78 mg, 65% yield) was obtained after chromatographic purification as a white solid. [α]D 25 = - 56.8 (1.144 mg / 0.5 ml DMSO). 1H NMR (400 MHz): 1.04 (d, J=6.7 Hz, 3 H); 1.31 (s, 6 H); 1.95 (t, J=6.1 Hz, 2 H); 3.10 (dt, J=3.3 and 13.3 Hz, 1 H); 3.30 (m masqued, 1 H); 3.46 (dd, J=3.1 and 11.5 Hz, 1 H); 3.53 (d, J=11.5 Hz, 1 H); 3.64 (d broad, J=13.3 Hz, 1 H); 3.73 (dd, J=2.6 and 11.5 Hz, 1 H); 3.76 - 3.85 (m, 2 H); 4.06 (m, 1 H); 4.71 (d, J=16.8 Hz, 1 H); 4.77 (d, J=16.8 Hz, 1 H); 6.45 (d, J=1.6 Hz, 1 H); 8.77 (d, J=1.6 Hz, 1 H). Mass spectrometry: method B; Tr (min) = 0.95; (m/z): [M+H]+ 378.


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7-Fluoro-2,2-dimethyl-8-[(3R)-3-methylmorpholin-4-yl]-1-[2-oxo-2-(2-pyridyl)ethyl]-3,4dihydropyrimido[1,2-a]pyrimidin-6-one (36). By a similar procedure to that described for the synthesis of 5, 73 (110 mg, 0.31 mmol) and R-3-methylmorpholine (32 mg, 0.31 mmol) were stirred at 60°C for 18 h. Compound 36 (53 mg, 41% yield) was obtained after chromatographic purification as a white solid. [α]D 25 = - 67.9 (1.604 mg / 0.5 ml DMSO). 1H NMR (400 MHz): 0.82 (d, J=6.8 Hz, 3 H); 1.32 (s, 3 H); 1.33 (s, 3 H); 1.97 (t, J=6.2 Hz, 2 H); 2.86 (dt, J=3.6 and 12.7 Hz, 1 H); 3.09 (dt, J=3.0 and 12.0 Hz, 1 H); 3.16 (d broad, J=11.4 Hz, 1 H); 3.20 - 3.42 (m partially masqued, 3H); 3.78 (m, 1 H); 3.82 (t, J=6.2 Hz, 2 H); 5.08 (d broad, J=18.7 Hz, 1 H); 5.18 (d broad, J=18.7 Hz, 1 H); 7.72 (ddd, J=1.3 and 4.8 et 7.6 Hz, 1 H); 7.98 (d broad, J=7.6 Hz, 1 H); 8.06 (dt, J=1.7 and 7.6 Hz, 1 H); 8.76 (d broad, J=4.8 Hz, 1 H). Mass spectrometry: method B; Tr (min) = 1.09; (m/z): [M+H]+ 416. (4R,4S)-4-(Trifluoromethyl)-1,4,5,6-tetrahydropyrimidin-2-ylamine hydrochloride (41). To a solution of 37 (22 g, 135 mmol) in water (200 ml) and MeOH (50 ml) were added 12N HCl (aq) (50 ml) and 10% Pd/C (1.1 g). The reaction mixture was hydrogenated under 3 bar of H2 at room temperature for 24 h. The reaction mixture was filtered and the filtrate concentrated under reduced pressure. The crude residue was dried under reduced pressure in presence of P2O5 to give compound 41 (27 g, 99% yield) as a grey solid. 1H NMR (400 MHz): 8.96 (s, 1H), 8.58 (s, 1H), 7.42 (s, 2H), 4.41 (m, 1H), 3.37 (m, 1H), 3.20 (m, 1H), 2.12 – 1.98 (m, 2H). Mass spectrometry: method A; Tr (min) = 0.17; [M+H]+: m/z 168. (4R,4S)-4-Methyl-4-(trifluoromethyl)-5,6-dihydro-1H-pyrimidin-2-ylamine hydrobromide (42). To a solution of 38 (458 mg, 2.93 mmol) in MeCN (16 ml) was added cyanogen bromide (311 mg, 2.93 mmol). The reaction mixture was heated under reflux for 2.5 h, then allowed to cool to room temperature and concentrated under reduced pressure to give compound 42 (695


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mg, quantitative) as a yellow solid. 1H NMR (300 MHz): 1.45 (s, 3 H); 1.94 (m, 1 H); 2.17 (m, 1 H); 3.11 to 3.44 (m partially masked, 2 H); 6.97 (s b, 2 H); 8.17 (s broad, 1 H); 8.60 (s broad, 1 H). Mass spectrometry: method A; Tr (min) = 0.24; [M+H]+: m/z 182. 4,4-Dimethyl-5,6-dihydro-1H-pyrimidin-2-ylamine hydrobromide (43). By a similar procedure to that described for the synthesis of 42, 39 (3.9 g, 38 mmol) and cyanogen bromide (4.25 g 40 mmol) were stirred in MeCN (30 ml) under reflux for 2.5 h. Compound 43 (6 g, 76% yield) was obtained after workup. 1H NMR (400 MHz): 1.13 (s, 6 H), 1.53 (t, J=6.6 Hz, 2 H), 3.19 (t, J=6.6 Hz, 2 H). Mass spectrometry: method A; Tr (min)=0.18; [M+H]+ m/z 128. 4,6-Diazaspiro[2.5]oct-4-en-5-amine hydrobromide (44). By a similar procedure to that described for the synthesis of 42, 40 (1.4 g, 5.34 mmol), cyanogen bromide (566 mg, 5.34 mmol) were stirred in MeCN (50 ml) under reflux for 2.5 h. Compound 44 (652 mg, quantitative) was obtained after workup as a yellow solid and used without any further purification in the next step of the synthesis. (2R,2S)-8-Hydroxy-2-(trifluoromethyl)-1,2,3,4-tetrahydropyrimido[1,2-a]pyrimidin-6-one (45). Sodium methoxide (10 g, 185 mmol) was added to a mixture of 41 (10 g, 49 mmol) and diethyl malonate (50 ml). The reaction mixture was heated at 100°C for 75 min, then cooled to room temperature and concentrated under reduced pressure. The crude residue was triturated in diethyl ether (50 ml), filtered and stirred in cold water (20 ml). The aqueous layer was acidified to pH 5-6 by addition of 12N HCl (aq). The suspension was filtered and the crude residue was washed with diethyl ether to give compound 45 (11.5 g, 100% yield) as a white solid. 1H NMR (400 MHz): 10.8 (m broad, 1H), 8.48 (m broad, 1H), 4.81 (s, 1H), 4.29 (m, 1H), 4.12 (m, 1H),


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3.31 (m, 1H), 2.20 (m, 1H), 2.08 (m, 1H). Mass spectrometry: method A; Tr (min) = 0.26; [M+H]+: m/z 236, [M-H]-: m/z 234. (2R,2S)-7-Fluoro-8-hydroxy-2-(trifluoromethyl)-1,2,3,4-tetrahydropyrimido[1,2a]pyrimidin-6-one (46). By a similar procedure to that described for the synthesis of 45, dimethylfluoromalonate (57 g, 0.38 mol), sodium methoxide (6.18 g, 0.12 mol) and 41 (10 g, 38 mmol) were stirred at 100°C for 3 h. Compound 46. (9.5 g, 99% yield) was obtained after workup as a brown gum. 1H NMR (400 MHz): 2.00 – 2.12 (m, 2 H), 3.43 (m partially hidden, 1 H), 3.97 (m, 1 H), 4.22 (m, 1 H), 7.28 (m broad, 1 H). Mass spectrometry: method A: Tr (min)=0.28; [M+H]+ m/z 254; [M-H]+ m/z 252. (2R,2S)-8-Hydroxy-2-methyl-2-(trifluoromethyl)-3,4-dihydro-1H-pyrimido[1,2a]pyrimidin-6-one (47). To a solution of sodium (44 mg, 1.93 mmol) in MeOH (4 ml) at room temperature were added a solution of 42 (101 mg, 0.39 mmol) in MeOH (2 ml) and diethyl malonate (0.264 ml, 2.31 mmol). The reaction mixture was heated under reflux for 6 h, then cooled to room temperature and concentrated under reduced pressure. The crude residue was stirred with cold water and the aqueous layer was acidified to pH 5 by addition of 8N HCl (aq). After stirring for 15 minutes, diethyl ether (3 ml) was added and the suspension was filtered and dried under reduced pressure to give compound 47 (92 mg, 95% yield) was obtained as a beige solid. 1H NMR (300 MHz,): 1.40 (s, 3 H); 1.92 (m, 1 H); 2.22 (m, 1 H); 3.45 (m, 1 H); 3.95 (m, 1 H); 4.61 (s broad, 1 H); 10.30 (m, 2 H). Mass spectrometry: method B; Tr (min) = 0.5, [M+H]+: m/z 250, [M-H]-: m/z 248. 8-Hydroxy-2,2-dimethyl-3,4-dihydro-1H-pyrimido[1,2-a]pyrimidin-6-one (48). By a similar procedure to that described for the synthesis of 45, dimethylmalonate (20 g, 0.151 mol), sodium


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methoxide (3.0 g, 57 mmol) and 43 (3.9 g, 19 mmol) were stirred at 100°C for 3 h. Compound 48 (2.7 g, 73% yield) was obtained after workup as a white solid. 1H NMR (400 MHz): 1.24 (s, 6 H), 1.75 (t, J=6.6 Hz, 2 H), 3.73 (t, J=6.6 Hz, 2 H), 4.59 (s, 1 H), 7.67 (s broad, 1 H), 10.18 (s broad, 1 H). Mass spectrometry: method B: Tr (min)=0.46; [M+H]+ m/z 196; [M-H]- m/z 194. 7-Fluoro-8-hydroxy-2,2-dimethyl-3,4-dihydro-1H-pyrimido[1,2-a]pyrimidin-6-one (49). By a similar procedure to that described for the synthesis of 45, dimethylfluoromalonate (28.9 g, 192 mmol), sodium methoxide (3.9 g, 72 mmol) and 43 (5.0 g, 24 mmol) were stirred at 100°C for 3 h. Compound 49 (3.15 g, 61% yield) was obtained after workup as brown solid. 1H NMR (400 MHz): 1.21 (s, 6 H), 1.76 (t, J=6.6 Hz, 2 H), 3.75 (t, J=6.6 Hz, 2 H), 7.89 (s broad, 1 H). Mass spectrometry: method B: Tr (min)=0.42; [M+H]+ m/z 214; [M-H]- m/z 212. 8-Hydroxy-spiro[3,4-dihydro-1H-pyrimido[1,2-a]pyrimidine-2,1'-cyclopropane]-6-one (50). By a similar procedure to that described for the synthesis of 45, 44 (1.2 g, 9.59 mmol), sodium methoxide (815 mg, 35.45 mmol) and diethyl malonate (6 ml) were stirred in MeCN (50 ml) under reflux for 15 h. Compound 50 (320 mg, 17% yield) was obtained after workup as a white solid. Mass spectrometry: method A; Tr (min) = 0.47; [M+H]+: m/z 194. (2S)-8-Chloro-2-(trifluoromethyl)-1,2,3,4-tetrahydropyrimido[1,2-a]pyrimidin-6-one (51). To a suspension of 45 (34 g, 145 mmol) in 1,2-dichloroethane (500 ml) was added phosphorus oxychloride (60 ml) at room temperature. The reaction mixture was heated at 65°C for 3 h, then cooled to room temperature and concentrated under reduced pressure. The crude residue was diluted with cold water (100 ml) and EtOAc (400 ml). The pH of the aqueous layer was adjusted to 6 by addition of 32% NaOH (aq). The layers were separated and the organic layer was dried over magnesium sulfate and concentrated under reduced pressure. The crude orange residue was


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purified by silica gel chromatography (CH2Cl2/MeOH, 97:3%) to give (R,S)-8-chloro-2(trifluoromethyl)-1,2,3,4-tetrahydropyrimido[1,2-a]pyrimidin-6-one (20 g, 55% yield) as a white solid: Mass spectrometry: method A; Tr (min) = 0.51; [M+H]+: m/z 254; [M-H]-: m/z 252. Separation of two enantiomers of (R,S)-8-chloro-2-(trifluoromethyl)-1,2,3,4tetrahydropyrimido[1,2-a]pyrimidin-6-one (17 g) was made by chiral chromatography, with Chiralpak AD as stationary phase and EtOH / heptane (20:80%) as mobile phase. The levogyre (in MeOH) enantiomer was concentrated to give 8.52 g (23% yield) of (2R)-8-chloro-2(trifluoromethyl)-1,2,3,4-tetrahydropyrimido[1,2-a]pyrimidin-6-one as a white solid. The dextrogyre (in MeOH) enantiomer was concentrated to give 8.21 g (22% yield) of (2S)-8-chloro2-(trifluoromethyl)-1,2,3,4-tetrahydropyrimido[1,2-a]pyrimidin-6-one 51, as a white solid. 1H NMR (400 MHz): 9.15 (s, 1H), 5.80 (s, 1H), 4.36 (m, 1H), 4.11 (m, 1H), 3.46 (m, 1H), 2.27 2.08 (m, 2H). Mass spectrometry: method A; Tr (min) = 0.51, [M+H]+: m/z 254; [M-H]-: m/z 252; [α]D 25 nm = + 21.3 (2.462 mg / 0.5 ml MeOH); [α]D 25 = - 21.8 (2.327 mg / 0.5 ml DMSO). The absolute configuration of 51 (2S) was unambiguously assigned according to X-ray crystal structure of human Vps34 in complex with analogs obtained from 51 (e.g. compound 2 and 4 in Figure 8). Morever, in this series, corresponding (2R)-trifluoromethyl isomer led to less active compounds against Vps34 (e.g. compounds 1a vs 1b in Table 1). (2S)-8-Chloro-7-fluoro-2-(trifluoromethyl)-1,2,3,4-tetrahydropyrimido[1,2-a]pyrimidin-6one (52). By a similar procedure to that described for the synthesis of 51, 46 (9.5 g, 37 mmol), phosphorus oxychloride (30 g, 191 mmol) were stirred in 1,2-dichloroethane (50 ml) at 65°C for 2 h. (R,S)-8-Chloro-7-fluoro-2-(trifluoromethyl)-1,2,3,4-tetrahydropyrimido[1,2-a]pyrimidin-6one (6.3 g, 63% yield) was obtained after chromatographic purification as a a brown solid. Separation of two enantiomers of (R,S)-8-chloro-7-fluoro-2-(trifluoromethyl)-1,2,3,4-


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tetrahydropyrimido[1,2-a]pyrimidin-6-one (14 g) was made by chiral chromatography, with Chiralpak AD as stationary phase and EtOH / heptane (30:70%) as mobile phase. The dextrogyre ([α]D 25 = + 19.1 (2.695 mg / 0.5 ml DMSO)) enantiomer was concentrated to give 2.85 g (28% yield) of (2R)-8-chloro-7-fluoro-2-(trifluoromethyl)-1,2,3,4-tetrahydropyrimido[1,2-a]pyrimidin6-one as a white solid. The levogyre ([α]D 25 = - 18.7 (2.960 mg / 0.5 ml DMSO)) enantiomer was concentrated to give 2.51 g (25% yield) of (2S)-8-chloro-7-fluoro-2-(trifluoromethyl)1,2,3,4-tetrahydropyrimido[1,2-a]pyrimidin-6-one 52 as a white solid. 1H NMR (400 MHz): 2.07 – 2.25 (m, 2 H), 3.52 (ddd, J=4.6 - 10.5 and 14.5 Hz, 1 H), 4.13 (td, J=4.6 and 14.5 Hz, 1 H), 4.31 – 4.39 (m, 1 H), 9.12 (s, 1 H). Mass spectrometry: method B: Tr (min)=0.95; [M+H]+ m/z 272; [M-H]- m/z 270. The absolute configuration of 52 (2S) was unambiguously assigned according to X-ray crystal structure of human Vps34 in complex with analogs obtained from 52 (data not shown). (2S)-8-Chloro-2-methyl-2-(trifluoromethyl)-3,4-dihydro-1H-pyrimido[1,2-a]pyrimidin-6one (53). By a similar procedure to that described for the synthesis of 51, 47 (91 mg, 0.37 mmol), phosphorus oxychloride (0.17 ml, 1.83 mmol) were stirred in 1,2-dichloroethane ( 3 ml) at 65°C for 1.5 h. (R,S)-8-Chloro-2-methyl-2-(trifluoromethyl)-3,4-dihydro-1H-pyrimido[1,2a]pyrimidin-6-one (102 mg, 100% yield) was obtained after chromatographic purification as a a brown solid. Separation of two enantiomers of (R,S)-8-chloro-2-methyl-2-(trifluoromethyl)-3,4dihydro-1H-pyrimido[1,2-a]pyrimidin-6-one (14 g) was made by chiral chromatography, with Chiralpak AD as stationary phase and EtOH / heptane (20/80%) as mobile phase. The dextrogyre ([α]D 25 = + 53.6 (1.781 mg / 0.5 ml DMSO)) enantiomer was concentrated to give 38 mg (39% yield) of (2R)-8-chloro-2-methyl-2-(trifluoromethyl)-3,4-dihydro-1H-pyrimido[1,2-a]pyrimidin6-one as a white solid. The levogyre ([α]D 25 = - 52.0 (2.716 mg / 0.5 ml DMSO)) enantiomer


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was concentrated to give 39 mg (40% yield) of (2S)-8-chloro-2-methyl-2-(trifluoromethyl)-3,4dihydro-1H-pyrimido[1,2-a]pyrimidin-6-one 53 as a white solid. 1H NMR (400 MHz): 1.45 (s, 3 H), 1.91 - 2.07 (m, 1 H), 2.35 (dt, J=4.4 and 14.7 Hz, 1 H), 3.38-3.51 (m, 1 H), 4.05 - 4.18 (m, 1 H), 5.81 (s, 1 H), 9.16 (s, 1 H). Mass spectrometry: method B; Tr (min) = 0.84, [M+H]+ m/z 268, [M-H]- m/z 266. The absolute configuration of 53 (2S) was unambiguously assigned according to X-ray crystal structure of human Vps34 in complex with analogs obtained from 53 (data not shown). 8-Chloro-2,2-dimethyl-3,4-dihydro-1H-pyrimido[1,2-a]pyrimidin-6-one (54). By a similar procedure to that described for the synthesis of 51, 48 (2.7 g, 14 mmol), 1,2-dichloroethane (40 ml) and phosphorus oxychloride (6.3 g, 42 mmol) were stirred at 65°C for 2 h. Compound 54 (1.76 g, 60% yield) was obtained after chromatographic purification as a white solid. 1H NMR (400 MHz): 1.23 (s, 6 H), 1.78 (t, J=6.6 Hz, 2 H), 3.79 (t, J=6.6 Hz, 2 H), 5.60 (s, 1 H), 8.51 (s, 1 H). Mass spectrometry: method B: Tr (min)=0.78; [M+H]+ m/z 214; [M-H]- m/z 212. 8-Chloro-7-fluoro-2,2-dimethyl-3,4-dihydro-1H-pyrimido[1,2-a]pyrimidin-6-one (55). By a similar procedure to that described for the synthesis of 51, 49 (3.15 g, 14 mmol), 1,2dichloroethane (40 ml) and phosphorus oxychloride (6.7 g, 44 mmol) were stirred at 65°C for 2 h. Compound 55 (2.2 g, 65% yield) was obtained after chromatographic purification as a brown solid. 1H NMR (400 MHz): 1.22 (s, 6 H), 1.79 (t, J=6.6 Hz, 2 H), 3.83 (t, J=6.6 Hz, 2 H), 8.41 (s, 1 H). Mass spectrometry: method B: Tr (min)=0.86; [M+H]+ m/z 232; [M-H]- m/z 230. 8-Chloro-spiro[3,4-dihydro-1H-pyrimido[1,2-a]pyrimidine-2,1'-cyclopropane]-6-one (56). By a similar procedure to that described for the synthesis of 51, 50 (320 mg, 1.66 mmol) and phosphorus oxychloride (0.76 ml) were stirred in 1,2-dichloroethane (25 ml) at 65°C for 3 h.


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Compound 56 (350 mg, quantitative) was obtained after chromatographic purification as a white solid. Mass spectrometry: method A; Tr (min) = 1.88, [M+H]+ m/z 212. (2S)-8-Morpholino-2-(trifluoromethyl)-1,2,3,4-tetrahydropyrimido[1,2-a]pyrimidin-6-one (57). A mixture of 51 (10 g, 39.43 mmol) and morpholine (43.50 g, 499.31 mmol) was heated at 110°C for 1.5 h. The reaction mixture was cooled to room temperature and the crude residue was dissolved in EtOAc (50 ml) and washed with water (50 ml). The solid was filtered, washed with diethyl ether (2 x 30 ml) and dried under reduced pressure to give compound 57 (10 g, 83% yield) as a white solid. 1H NMR (400 MHz): 2.01 – 2.23 (m, 2 H), 3.31 – 3.35 (m, 1 H), 3.36 – 3.40 (m, 4 H), 3.58 – 3.62 (m, 4 H), 4.07 – 4.14 (m, 1 H), 4.19 – 4.29 (m, 1 H), 4.92 (s, 1 H), 8.14 (m, 1 H). Mass spectrometry: method A; Tr (min) 0.69; [M+H]+ (m/z) 305, [M-H]- (m/z) 303; [α]D 25 = +14.2 (2.045 mg / 0.5 ml MeOH). (2S)-8-[(3S)-3-Methylmorpholin-4-yl]-2-(trifluoromethyl)-1,2,3,4-tetrahydropyrimido[1,2a]pyrimidin-6-one (58). By a similar procedure to that described for the synthesis of 57, 51 (1.04 g, 4.14 mmol), S-3-methyl-morpholine (500 mg, 4.94 mmol) and TEA (1.7 ml) were stirred at 180°C under microwave irradiation for 1.5 h. Compound 58 (950 mg, 73% yield) was obtained after chromatographic purification as a white solid. 1H NMR (400 MHz): 1.12 (d, J=6.8 Hz, 3 H), 2.00 – 2.22 (m, 2 H), 2.98 (dt, J=3.8 and 12.8 Hz, 1 H), 3.34 – 3.40 (m, 2 H), 3.50 (dd, J=2.9 and 11.5 Hz, 1 H), 3.64 (d, J=11.5 Hz, 1 H), 3.75 – 3.88 (m, 2 H), 4.06 – 4.29 (m, 3 H), 4.87 (s, 1 H), 8.11 (d, J=3.9 Hz, 1 H). Mass spectrometry: method A: Tr (min)= 0.55; [M+H]+ m/z 319; [M-H]- m/z 317. (2S)-8-[(3R)-3-Methylmorpholin-4-yl]-2-(trifluoromethyl)-1,2,3,4-tetrahydropyrimido[1,2a]pyrimidin-6-one (59). By a similar procedure to that described for the synthesis of 57, 51


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(2.08 g, 8.2 mmol), R-3-methyl-morpholine (1.36 g, 9.88 mmol) and TEA (3.4 ml) were stirred at 180°C under microwave irradiation for 1.5 h. Compound 59 (2.08 g, 70% yield) was obtained after chromatographic purification as a white solid. 1H NMR (400 MHz): 1.11 (d, J=6.8 Hz, 3 H), 2.01 – 2.22 (m, 2 H), 2.98 (dt, J=3.9 and 12.9 Hz, 1 H), 3.32 – 3.39 (m, 2 H), 3.51 (dd, J=2.9 and 11.5 Hz, 1 H), 3.64 (d, J=11.5 Hz, 1 H), 3.76 (d broad, J=12.9 Hz, 1 H), 3.85 (dd, J=3.5 and 11.4 Hz, 1 H), 4.11 (dm, J=14.4 Hz, 1 H), 4.15 – 4.30 (m, 2 H), 4.86 (s, 1 H), 8.12 (d, J=3.9 Hz, 1 H). Mass spectrometry: method A: Tr (min)= 0.55; [M+H]+ m/z 319; [M-H]- m/z 317. (2S)-8-(8-Oxa-3-azabicyclo[3.2.1]octan-3-yl)-2-(trifluoromethyl)-1,2,3,4tetrahydropyrimido[1,2-a]pyrimidin-6-one (60). By a similar procedure to that described for the synthesis of 57, 51 (420 mg, 1.66 mmol), 8-oxa-3-aza-bicyclo[3.2.1]oct-3-yl (280 mg, 2.47 mmol) and TEA (0.8 ml) were stirred at 160°C under microwave irradiation for 0.5 h. Compound 60 (547 mg, 100% yield) was obtained after chromatographic purification as a white solid. 1H NMR (400 MHz): 1.58 – 1.85 (m, 4 H), 2.00 – 2.21 (m, 2 H), 2.88 (d, J=12.7 Hz, 2 H), 3.30 (m partially hidden, 1 H), 3.73 (t broad, J=12.7 Hz, 2 H), 4.11 (dm, J=13.9 Hz, 1 H), 4.16 – 4.27 (m, 1 H), 4.34 (m, 2 H), 4.82 (s, 1 H), 8.11 (s broad, 1 H). Mass spectrometry: method A: Tr (min)= 0.55; [M+H]+ m/z 331; [M-H]- m/z 329. (2S)-8-(3-Oxa-8-azabicyclo[3.2.1]octan-8-yl)-2-(trifluoromethyl)-1,2,3,4tetrahydropyrimido[1,2-a]pyrimidin-6-one (61). By a similar procedure to that described for the synthesis of 57, 51 (100 mg, 0.39 mmol), 3-oxa-8-aza-bicyclo[3.2.1]oct-8-yl (71 mg, 0.47 mmol) and TEA (0.25 ml) were stirred at 180°C under microwave irradiation for 1 h. Compound 61 (120 mg, 93% yield) was obtained after chromatographic purification as a white solid. 1H NMR (400 MHz): 1.79 – 1.94 (m, 4 H), 2.02 – 2.20 (m, 2 H), 3.31 – 3.32 (m, 1 H), 3.47 (d broad, J=10.8 Hz, 2 H), 3.57 (d broad, J=10.8 Hz, 2 H), 4.06 – 4.16 (m, 1 H), 4.23 (m, 1 H), 4.30


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(m broad, 2 H), 4.92 (s, 1 H), 8.13 (d, J=3.7 Hz, 1 H). Mass spectrometry: method A: Tr (min)=0.54; [M+H]+ m/z 331; [M-H]- m/z 329. (2S)-8-[(1S,4S)-2-oxa-5-azabicyclo[2.2.1]heptan-5-yl]-2-(trifluoromethyl)-1,2,3,4tetrahydropyrimido[1,2-a]pyrimidin-6-one (62). By a similar procedure to that described for the synthesis of 57, 51 (508 mg, 2 mmol), (1S,4S)-2-oxa-5-azabicyclo[2.2.1]heptan-5-yl (246 mg, 2.49 mmol) and TEA (1 ml) were stirred at 150°C under microwave irradiation for 0.5 h. Compound 62 (120 mg, 93% yield) was obtained after chromatographic purification as a white solid. 1H NMR (400 MHz): 1.80 (m, 2 H), 1.99 – 2.22 (m, 2 H), 3.10 (m, 1 H), 3.30 (m partially hidden, 2 H), 3.61 (d, J=7.1 Hz, 1 H), 3.71 (dd, J=1.2 and 7.1 Hz, 1 H), 4.12 (dm, J=14.1 Hz, 1 H), 4.17 – 4.28 (m, 1 H), 4.60 (s, 1 H), 4.65 (m broad, 1 H), 4.80 (m broad, 1 H), 8.15 (d, J=3.9 Hz, 1 H). Mass spectrometry: method A: Tr (min)= 0.47; [M+H]+ m/z 317; [M-H]- m/z 315. (2S)-8-[(3S,5S)-3,5-Dimethylmorpholin-4-yl]-2-(trifluoromethyl)-1,2,3,4tetrahydropyrimido[1,2-a]pyrimidin-6-one (63). By a similar procedure to that described for the synthesis of 57, 51 (330 mg, 1.30 mmol) and 3S,5S-dimethyl-morpholine (450 mg, 3.90 mmol) were stirred at 240°C under microwave irradiation for 3 h. Compound 63 (233 mg, 54% yield) was obtained after chromatographic purification as a white solid. 1H NMR (400 MHz): 1.20 (d, J=6.6 Hz, 6 H), 2.04 – 2.21 (m, 2 H), 3.27 – 3.38 (m partially hidden, 1 H), 3.56 (dd, J=3.1 and 11.1 Hz, 2 H), 3.89 – 4.02 (m, 4 H), 4.12 (m, 1 H), 4.24 (m, 1 H), 4.81 (s, 1 H), 8.12 (d, J=3.6 Hz, 1 H); Mass spectrometry: method A: Tr (min)= 0.60; [M+H]+ m/z 333, [M-H]- m/z 331. (2S)-8-[(3S,5R)-3,5-Dimethylmorpholin-4-yl]-2-(trifluoromethyl)-1,2,3,4tetrahydropyrimido[1,2-a]pyrimidin-6-one (64). By a similar procedure to that described for


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the synthesis of 57, 51 (330 mg, 1.30 mmol) and 3S,5R-dimethyl-morpholine (450 mg, 3.90 mmol) were stirred at 240°C under microwave irradiation for 3 h. Compound 64 (226 mg, 52% yield) was obtained after chromatographic purification as a white solid. 1H NMR (400 MHz): 1.20 (d, J=6.6 Hz, 6 H), 2.02 – 2.12 (m, 1 H), 2.14 – 2.21 (m, 1 H), 3.27 – 3.36 (m partially hidden, 1 H), 3.49 (td, J=3.2 and 11.5 Hz, 2 H), 3.70 (d, J=11.5 Hz, 2 H), 4.01 – 4.16 (m, 3 H), 4.23 (m, 1 H), 4.81 (s, 1 H), 8.08 (d, J=3.9 Hz, 1 H); Mass spectrometry: method A: Tr (min)= 0.62; [M+H]+ m/z 333, [M-H]- m/z 331. (2S)-8-[(3R,5R)-3,5-Dimethylmorpholin-4-yl]-2-(trifluoromethyl)-1,2,3,4tetrahydropyrimido[1,2-a]pyrimidin-6-one (65). By a similar procedure to that described for the synthesis of 57, 51 (330 mg, 1.30 mmol), and 3R,5R-dimethyl-morpholine (450 mg, 3.90 mmol) were stirred at 240°C under microwave irradiation for 3 h. Compound 65 (205 mg, 47% yield) was obtained after chromatographic purification as a white solid. 1H NMR (400 MHz): 1.18 (d, J=6.6 Hz, 6 H), 2.01 – 2.11 (m, 1 H), 2.14 – 2.22 (m, 1 H), 3.27 – 3.36 (m partially hidden, 1 H), 3.55 (dd, J=3.2 and 11.0 Hz, 2 H), 3.88 – 4.01 (m, 4 H), 4.13 (m, 1 H), 4.23 (m, 1 H), 4.83 (s, 1 H), 8.12 (d, J=3.7 Hz, 1 H); Mass spectrometry: method A: Tr (min)= 0.59; [M+H]+ m/z 333, [M-H]- m/z 331. (2S)-2-Methyl-8-morpholino-2-(trifluoromethyl)-3,4-dihydro-1H-pyrimido[1,2a]pyrimidin-6-one (66). By a similar procedure to that described for the synthesis of 57, 53 (100 mg, 0.37 mmol) and morpholine (0.987 ml, 11.21 mmol) were stirred in MeCN (4 ml) under reflux for 4.5 h. Compound 66 (102 mg, 86% yield) was obtained after chromatographic purification as a white solid. 1H NMR (400 MHz): 1.43 (s, 3 H), 1.84 – 2.00 (m, 1 H), 2.25 – 2.34 (m, 1 H), 3.32 – 3.44 (m partially masqued, 1 H), 3.38 – 3.42 (m, 4 H), 3.58 – 3.62 (m, 4


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Page 76 of 88

H), 4.00 – 4.10 (m, 1 H), 4.92 (s, 1 H), 8.12 (s, 1 H). Mass spectrometry: method A; Tr (min) = 0.54, [M+H]+ m/z 319, [M-H]- m/z 317. (2S)-7-Fluoro-8-[(3R)-3-methylmorpholin-4-yl]-2-(trifluoromethyl)-1,2,3,4tetrahydropyrimido[1,2-a]pyrimidin-6-one (67). By a similar procedure to that described for the synthesis of 57, 52 (400 mg, 1.47 mmol), and R-3-methylmorpholine (400 mg, 3.9 mmol) were stirred at 100°C for 18 h. Compound 67 (450 mg, 91% yield) was obtained after chromatographic purification as a beige solid. 1H NMR (400 MHz): 1.23 (d, J=6.8 Hz, 3 H), 2.01 – 2.21 (m, 2 H), 3.19 – 3.29 (m partially hidden, 1 H), 3.34- 3.48 (m, 2 H), 3.59 (dd, J=2.9 and 11.5 Hz, 1 H), 3.63 (d, J=11.5 Hz, 1 H), 3.84 (dd, J=3.4 and 11.5 Hz, 1 H), 3.89 (d broad, J=13.5 Hz, 1 H), 4.09 (dm, J=14.4 Hz, 1 H), 4.18 – 4.33 (m, 2 H), 8.18 (s broad, 1 H). Mass spectrometry: method B: Tr (min)= 0.84; [M+H]+ m/z 337; [M-H]- m/z 335. (2S)-8-Chloro-1-[2-oxo-2-(2-pyridyl)ethyl]-2-(trifluoromethyl)-3,4-dihydro-2Hpyrimido[1,2-a]pyrimidin-6-one (68). To a solution of 51 (2.28 g, 9 mmol) in MeCN (50 ml) were added cesium carbonate (8.8 g, 27 mmol) and 2-(bromoacetyl)pyridine hydrobromide (3.79 g, 13.5 mmol). After 20 h at room temperature, the reaction mixture was dissolved in EtOAc (50 ml) and washed with water (50 ml). The aqueous layer was extracted with EtOAc (2 x 25 ml). The combined organic layers were dried over magnesium sulfate and concentrated under reduced pressure. The crude residue (4.5 g) was purified by silica gel chromatography (CH2Cl2) to give compound 68 ( 1.5 g, 45% yield) as a yellow solid: Mass spectrometry: method A; Tr (min) 0.87, [M+H]+ m/z 373, [M-H]- m/z 371. (2S)-8-Chloro-1-[2-oxo-2-(3-pyridyl)ethyl]-2-(trifluoromethyl)-3,4-dihydro-2Hpyrimido[1,2-a]pyrimidin-6-one (69). By a similar procedure to that described for the synthesis


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of 68, 51 (500 mg, 1.97 mmol), bromo-(2-oxo-2-pyridin-3-yl-ethyl (634 mg, 2.25 mmol) and cesium carbonate (1.9 g 5.8 mmol) were stirred in MeCN (10 ml) at room temperature for 3 h. Compound 69 (659 mg, 90% yield) was obtained after chromatographic purification as a white solid. . 1H NMR (300 MHz, δ in ppm, CDCl3): 1.66 (s, 1H) 2.3-2.52 (m, 2H) 3.48 (m, 1H) 4(m, 1H) 4.37 (d, 1H) 4.56 (m, 1H) 5.92 (s, 1H) 7.45 (m, 1H) 8.22 (m, 1H) 8.81 (s, 1H) 9.15 (s, 1H); Mass spectrometry: methode A: [M+H]+: m/z 373 tr(min) = 1.76. 8-Chloro-1-[(2S)-2-hydroxy-2-phenyl-ethyl]-2,2-dimethyl-3,4-dihydropyrimido[1,2a]pyrimidin-6-one (70). By a similar procedure to that described for the synthesis of 68, 54 (401 mg, 1.88 mmol), (S)-styrene oxide (451 mg, 3.75 mmol) and cesium carbonate (1.22 g 3.75 mmol) were stirred in MeCN (12 ml) at 80°C for 24 h. Compound 70 (265 mg, 42% yield) was obtained after chromatographic purification as a yellow solid. 1H NMR (400 MHz): 1.12 (s, 3 H), 1.40 (s, 3 H), 1.76 – 2.00 (m, 2 H), 3.41 (dd, J=8.6 and 14.2 Hz, 1 H), 3.59 (dd, J=2.7 and 14.2 Hz, 1 H), 3.68 – 3.91 (m, 2 H), 5.07 – 5.13 (m, 1 H), 5.44 (d, J=3.9 Hz, 1 H), 5.79 (s, 1 H), 7.25 (t, J=7.5 Hz, 1 H), 7.35 (t, J=7.5 Hz, 2 H), 7.43 (d, J=7.5 Hz, 2 H). Mass spectrometry: method A: Tr (min)=1.17; [M+H]+ m/z 334; [M+HCOOH-H]- m/z 378. 8-Chloro-1-(2-isopropoxyethyl)-2,2-dimethyl-3,4-dihydropyrimido[1,2-a]pyrimidin-6-one (71). By a similar procedure to that described for the synthesis of 68, 54 (200 mg, 0.94 mmol), cesium carbonate (610 mg, 1.87 mmol) and methanesulfonic acid 2-isopropoxy-ethyl ester (341 mg, 1.87 mmol) were stirred in MeCN (7.2 ml) at 65°C for 12 h. Compound 71 (187 mg, 67% yield) was obtained after chromatographic purification as a yellow solid. Mass spectrometry: method B; Tr (min) = 2.60, [M+H]+ m/z 300.


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8-Chloro-7-fluoro-1-(isoxazol-3-ylmethyl)-2,2-dimethyl-3,4-dihydropyrimido[1,2a]pyrimidin-6-one (72). By a similar procedure to that described for the synthesis of 68, 55 (300 mg, 1.29 mmol), 3-(chloromethyl)isoxazole (182 mg, 1.55 mmol), cesium carbonate (2.11 g, 6.47 mmol) were stirred in MeCN (5 ml) at 50°C for 3 h. Compound 72 (320 mg, 79% yield) was obtained after chromatographic purification as a white solid. 1H NMR (400 MHz): 1.31 (s, 6 H), 1.98 (t, J=6.6 Hz, 2 H), 3.89 (t, J=6.6 Hz, 2 H), 4.83 (s, 2 H), 6.54 (d, J=1.5 Hz, 1 H), 8.80 (d, J=1.5 Hz, 1 H). Mass spectrometry: method B: Tr (min)=1.07; [M+H]+ m/z 313. 8-Chloro-7-fluoro-2,2-dimethyl-1-[2-oxo-2-(2-pyridyl)ethyl]-3,4-dihydropyrimido[1,2a]pyrimidin-6-one (73). Compound 73 was prepared by reaction of 55 (800 mg, 3.45 mmol) with 2-(2-oxiranil)-pyridine (837 mg, 6.9 mmol) in the presence of cesium carbonate (2.25 g, 6.9 mmol) in MeCN at 100°C for 3.5 h. The obtained intermediate alcohol (164 mg, 0.46 mmol) was oxidized with Dess-Martin Periodinane (394 mg, 0.93 mmol) in CH2Cl2 (6 ml) at room temperature for 3 h. Compound 73 (160 mg, 98% yield) was obtained as a white solid. 1H NMR (400 MHz): 1.32 (s, 6 H), 2.02 (t, J=6.6 Hz, 2 H), 3.93 (t, J=6.6 Hz, 2 H), 5.19 (s, 2 H), 7.73 (dd, J=5.3 and 7.8 Hz, 1 H), 7.98 (d, J=7.8 Hz, 1 H), 8.06 (dt, J=2.0 and 7.8 Hz, 1 H), 8.78 (d broad, J=5.3 Hz, 1 H). Mass spectrometry: method B: Tr (min)=1.21; [M+H]+ m/z 351. 8-Chloro-1-[(2S)-2-hydroxy-2-phenyl-ethyl]spiro[3,4-dihydropyrimido[1,2-a]pyrimidine2,1'-cyclopropane]-6-one (74). To a solution of 56 (100 mg, 0.47 mmol) in DMF (10 ml) was added sodium hydride 60% in mineral oil (25 mg, 0.61 mmol). The reaction mixture was heated at 50°C for 1 h. (S)-Styrene oxide (73.8 mg, 0.61 mmol) was added and the mixture was heated at 100°C for 15 h. The reaction mixture was diluted with cold water (20 ml) and EtOAc (40 ml). The aqueous layer was extracted with EtOAc (2 x 25 ml). The combined organic layers were dried over magnesium sulfate and concentrated under reduced pressure. The crude residue was


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purified by silica gel chromatography (CH2Cl2/MeOH, 90:10%) to give compound 74 (50 mg, 32% yield) as a white solid. Mass spectrometry: method B; Tr (min) 2.48, [M+H]+ m/z 332. ASSOCIATED CONTENT Supporting Information Available Line chart of an initial set of pyrimidinone derivatives in Figure S1; Impact of exchange between non-substituted morpholine and 3(R)-methyl morpholine residue in Figure S2; Kinativ® profile of compound 31 in Ramos cells in Figure S3; Kinativ® profile of compound 31 in Jurkat cells in Figure S4; Crystallographic data in Table S1. This material is available free of charge via the Internet at http://pubs.acs.org. PDB ID Codes Coordinates and structure factors for VPS34 in complex with compounds 2, 3, 4, 15 and 31 have been deposited in the Protein Data Bank (PDB) with accession codes 4UWF, 4UWG, 4UWH, 4UWK and 4UWL, respectively. AUTHOR INFORMATION Corresponding Author *Phone: +33(0)1 58 93 80 72. Fax: +33(0)1 58 93 80 14. E-mail: [email protected]. Notes The authors declare no competing financial interest. ACKNOWLEDGMENTS


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We acknowledge Odile Flamand (cellular assays), Lionel Vescovi (cellular assays), Hugues Robbe (cellular assays),Christine Valtre (Protein Production), Sophie plantard (Protein Production), Cecile Ducelier (Pharmacokinetic), Sandrine Descloux (Pharmacokinetic), Alain Krick (Metabolite Identification and all DSAR Paris Operational Center), Valérie Czepczor (in vitro metabolism), Laurence Durand (cellular assays), Nadia Yonis (Pharmaceutical Sciences Operations), Marie-Pascale Court (Pharmaceutical Sciences Operations), Anne Thomas (in vivo studies), Emerson Serres (in vivo studies), Isabelle Sanchez (in vivo studies), Françoise le-Gall (Immunohistochemistry), Frédéric Foucault (ITC), Cécile Delorme (ITC), Valérie Bazin (biochemical assays), Marie-France Bachelot (biochemical assays), Françoise Begassat (biochemical assays), Véronique Lalleman (biochemical assays), Serge Sable (and all structural analysis team), Eric Brohan team (and all preparative chromatography team), Tyzoon Nomanbhoy (ActivX), for their technical assistance. Additionally, we thank Axel Ganzhorn and Bertrand Vivet for fruitful discussions. ABBREVIATIONS USED ADME, absorption distribution metabolism excretion; ATP, adenosine triphosphate; CYP, cytochrome P450; CLint, intrinsic clearance; DMF, N,N-dimethylmethanamide; DMSO, dimethyl sulfoxide; GFP, Green fluorescent protein; HLM, human liver microsomes; HCYP, human cytochrome P450; IC50, inhibitory concentration at half-maximal effect; IHC, Immunohistochemistry; ITC, isothermal titration calorimetry; iv, intravenous; LE, ligand efficiency; LLE, ligand lipophilicity efficiency; MLM, mouse liver microsomes; MEF, mouse embryonic fibroblasts; mTOR, mammalian target of rapamycin; MW, molecular weight; p-Akt, phospho-Akt; PD, pharmacodynamic; PI3K, Phosphoinositide 3-kinases; PK, pharmacokinetic; po, per os; PtdIns, phosphatidylinositol; PtdIns3P, phosphatidylinositol 3-phosphate;


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Table of Contents graphic

Selectivity

O N

N O

N O

N

31

CF3 IC50 Vps34 = 2 nM IC50 PI3K α, β, δ, γ >2 µM IC50 mTOR > 10 µM


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Discovery of (2 S)-8-[(3 R)-3-Methylmorpholin-4-yl]-1-(3-methyl-2

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