Discovery of in Vivo Chemical Probes for Treating Alzheimer's

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Discovery of in-vivo chemical probes for treating Alzheimer´s disease: Dual phosphodiesterase 5 (PDE5) and class I histone deacetylases-selective inhibitors. Obdulia Rabal, Juan A Sánchez-Arias, Mar Cuadrado-Tejedor, Irene De Miguel, Marta PérezGonzález, Carolina García-Barroso, Ana Ugarte, Ander Estella, Elena Sáez, Maria Espelosin, Susana Ursua, Haizhong Tan, Wei Wu, Musheng Xu, Ana Garcia-Osta, and Julen Oyarzabal ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.8b00648 • Publication Date (Web): 10 Dec 2018 Downloaded from http://pubs.acs.org on December 16, 2018

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ACS Chemical Neuroscience

Discovery of in-vivo chemical probes for treating Alzheimer´s disease: Dual phosphodiesterase 5 (PDE5) and class I histone deacetylasesselective inhibitors. Obdulia Rabal,1,5 Juan A. Sánchez-Arias,1,5 Mar Cuadrado-Tejedor,2,3,5 Irene de Miguel,1 Marta Pérez-González,2 Carolina García-Barroso,2 Ana Ugarte,1 Ander Estella-Hermoso de Mendoza,1 Elena Sáez,1 Maria Espelosin,2 Susana Ursua,2 Tan Haizhong,4 Wu Wei,4 Xu Musheng,4 Ana Garcia-Osta,2,5 and Julen Oyarzabal.1,5,*

1Small

Molecule Discovery Platform, Molecular Therapeutics Program, 2Neurobiology

of Alzheimer’s Disease, Neurosciences Division, Center for Applied Medical Research (CIMA), University of Navarra, Avenida Pio XII 55, E-31008 Pamplona, Spain 3Anatomy

Department, School of Medicine, University of Navarra, Irunlarrea 1, E-31008

Pamplona, Spain 4WuXi

Apptec (Tianjin) Co. Ltd., TEDA, No. 111 HuangHai Road, 4th Avenue, Tianjin

300456, PR China 5These

authors contributed equally to this work.

ABSTRACT In order to determine the contributions of histone deacetylase (HDAC) isoforms to the beneficial effects of dual PDE5 and pan-HDAC inhibitors on in vivo models of Alzheimer’s disease (AD), we have designed, synthesized and tested novel chemical probes with the desired target compound profile of phosphodiesterase 5 (PDE5)- and class I HDAC-selective inhibitors. Compared to previous hydroxamate-based series, these molecules exhibit longer residence times on HDACs. In this scenario, shorter or longer pre-incubation times may have a significant impact on the IC50 values of these compounds

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and therefore on their corresponding selectivity profiles on the different HDAC isoforms. On the other hand, different chemical series have been explored and, as expected, some pairwise comparisons show a clear impact of the scaffold on biological responses (e.g., 35a vs 40a). The lead identification process led to compound 29a, which shows an adequate ADME-Tox profile and in vivo target engagement (histone acetylation and cAMP/cGMP response element-binding (CREB) phosphorylation) in the central nervous system (CNS), suggesting that this compound represents an optimized chemical probe; thus, 29a has been assayed in a mouse model of AD (Tg2576). Keywords -- Chemical probes, Dual inhibitors, PDE5 inhibition, class I HDAC-selective inhibition, Alzheimer, In-vivo test

INTRODUCTION Inhibitors of histone deacetylases (HDACs) and phosphodiesterases (PDEs) have both been identified as alternative treatments for Alzheimer’s disease (AD) [1–7]. As strategies targeting multiple mechanisms are the best option to treat multifactorial diseases such as AD [8,9], we validated the in vivo efficacy of a new systems therapeutics approach based on the dual inhibition of HDACs and PDE5, either using a combination of single therapeutic agents (for example PDE5 inhibitors 1-3 and HDAC inhibitors 4-12, Chart 1; confirmed with 3 and 4) [10] or with our proprietary first-in-class dual inhibitor 13 (CM414) [11,12], Chart 2. PDE5 inhibitors increase cGMP concentrations, which might ultimately promote memory-related gene transcription by directly and/or indirectly activating binding to the cAMP response element (CREB) [13] and decrease levels of phosphorylated Tau (pTau) by favouring the inactive form of GSK3 [14,15]. Treatment of different animal models with specific PDE5 inhibitors (1 sildenafil, 2 vardenafil and 3 tadalafil, Chart 1) reverses cognitive decline and/or enhances synaptic plasticity [14–16]. 2 ACS Paragon Plus Environment

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HDACs are “erasers” that remove acetyl groups from lysine residues in histone and other non-histone substrates and trigger the transcription of transcriptionally silent chromatin. Eighteen HDACs have been identified and are divided into four groups according to phylogenetic sequence and function: class I (HDACs 1, 2, 3, and 8), class IIa (HDACs 4, 5, 7, and 9), class IIb (HDACs 6 and 10) and class IV (HDAC11). Pan-HDAC inhibitors, such as compound 4 (SAHA) [17] and ortho-amino anilides predominantly targeting class I HDAC enzymes (6 entinostat [18], 7 CI-994 [19], 8 RGFP136 [20], 10 BRD4884 [21]), and other HDAC2-selective benzamides [22] have been used to improve cognitive dysfunction. Class I HDACs are ubiquitously expressed, display a predominantly nuclear localization, and function as transcriptional co-repressors of CREB-regulated genes that are important for learning and memory [17,23]. However, a persistent missing link is the identification of the specific HDAC(s) responsible for mediating the pro-cognitive effects [24]. Here, we pursued a strategy to develop first-in-class dual PDE5- and class I HDACselective inhibitors that are able to cross the blood-brain barrier (BBB) as chemical probes to be tested in a mouse model of AD. Using a similar design strategy as reported for our previous hydroxamic derivatives, such as compound 13 [12,25,26], in the present study, ortho-aminoanilides (also referred as benzamides) were incorporated as the zinc-binding group (ZBG) into the appropriate growing vector (guided by structural information) of scaffolds borne by known PDE5 inhibitors to confer them with the additional ability to bind HDACs. This ZBG confers great selectivity for class I HDACs [27] and tends to exhibit longer residence times than hydroxamate-based HDAC inhibitors, a characteristic associated with the time-dependent responses of histone acetylation, cell viability and gene expression [28].



O O

O N

HN

O

N

HN

N O S O N

O H

N

N

N

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N

N

N

O

2 Vardenafil

IC50 = 8.5 nM (vs PDE5A)

O

N

O S O N

1 Sildenafil

N

O

3 Tadalafil IC50 = 9.4 nM (vs PDE5A)

IC50 = 0.89 nM (vs PDE5A)

OH HN O

N N O

NH

N

H N OH O

N

O O

N H

NH H N

N

4

5

SAHA

O

6 Entinostat

Quisinostat

IC50 = 30 nM (vs HDAC1) IC50 = 170 nM (vs HDAC2) IC50 = 100 nM (vs HDAC3) IC50 = 38 nM (vs HDAC6)

N H2

IC50 = 0.11 nM (vs HDAC1) IC50 = 0.33 nM (vs HDAC2) IC50 = 4.86 nM (vs HDAC3) IC50 = 76.8 nM (vs HDAC6)

IC50 = 243 nM (vs HDAC1) IC50 = 453 nM (vs HDAC2) IC50 = 248 nM (vs HDAC3) IC50 > 10000 nM (vs HDAC6)

O NH

F

O

O NH

N H2

H N

H N

N H

O N H2

O

N H2

7

8

CI-994 / Tacedinaline

9

RGFP136

IC50 = 900 nM (vs HDAC1) IC50 = 900 nM (vs HDAC2) IC50 = 1300 nM (vs HDAC3)

IC50 = 1140 nM (vs HDAC1) IC50 = 560 nM (vs HDAC3)

O

IC50 = 900 nM (vs HDAC2)

O NH

O

O N H2

N H

H N

O

H N

N H2

HN

O

S

O

10

F

11

O

12

BRD4884 IC50 = 29 nM (vs HDAC1) IC50 = 62 nM (vs HDAC2) IC50 = 1009 nM (vs HDAC3)

IC50 = 13 nM (vs HDAC1) IC50 = 115 nM (vs HDAC2) IC50 = 22450 nM (vs HDAC3) IC50 > 50000 nM (vs HDAC6)


HDAC2 Kd < 100 nM

N

OH

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Chart 1. Chemical structures of PDE5 inhibitors (compounds 1-3) and HDAC inhibitors (compounds 4-12). The IC50 values against PDE5 for compounds 1, 2 and 3 have been reported [29]. HDAC IC50 values for compounds 4 [30], 5 [31], 6 [28], 7 [32], 8 [33], 9 [34], 10 [21], 11 [30] and 12 [35] have been also described in previous studies.

O O

N

HN

IC50 = 60 nM (vs PDE5A)

N N

13 CM-414 O HN

IC50 = 310 nM (vs HDAC1) IC50 = 490 nM (vs HDAC2) IC50 = 322 nM (vs HDAC3) IC50 = 91 nM (vs HDAC6)

OH

Chart 2. Previously reported data for lead compound 13 (CM-414) [11,12].

RESULTS Rationale and Design The well-established pharmacophore for HDACi comprises a hydrophobic capping group that tightly binds to the rim of the catalytic tunnel (also known as the recognition group), a hydrophobic linker spanning the 11 Å catalytic channel and a ZBG. In our dual inhibitors, the PDE5 inhibitor core serves as the recognition group. Here, we envisioned the attachment of ortho-aminoanilides to the piperazinylsulfonamide group of the sildenafil (compound 18a) or vardenafil (compound 23a) cores through a pyrimidyl linker, similar to compound 5, which is well known for conferring class I selectivity [12,25]. Designed compounds 18a and 23a were docked into the HDAC2 crystal structure complexed with N-(4-aminobiphenyl-3-yl)benzamide inhibitor 9 (PDB entry 3MAX 5 ACS Paragon Plus Environment


[34], Figure 1A). As shown in Figure 1A, the ortho-amidoanilide group of compounds 18a and 23a is predicted to chelate the Zn metal in a bidentate manner, with both the amide oxygen and the amino nitrogen interacting with the Zn atom. Furthermore, both structures superimpose well with the corresponding co-crystallized ligand 9 (pink), particularly regarding the overlap of the pyrimidyl moiety of compounds 18a and 23a with the amide-bonded phenyl ring of compound 9, at the gate of the 11 Å catalytic channel. The other para-phenyl ring of compound 9 occupies the 14 Å long internal cavity adjacent to the Zn binding site that is characteristic of HDAC1 and HDAC2 [28,34,36] (also known as the foot pocket) and is probably involved in the release of the acetate product and ligands [37]. Here, the sildenafil and vardenafil cores of compounds 18a and 23a, respectively, project beyond the 11 Å channel and are oriented towards the loop comprising Tyr206-Tyr209 (HDAC2) and Tyr201-Tyr204 (HDAC1). Potent inhibitors of class I HDACs tend to interact through their capping groups with the L1 loop (Tyr28Pro37 in HDAC2, Tyr23-Pro32 in HDAC1) [38], as we have previously reported for our therapeutic tool 13 [11]. Intrigued by the different predicted positions of the capping groups in these two compounds, and in an attempt to assess the quality of our predictions, we reviewed all the HDAC crystals complexed with a benzamide inhibitor. However, to date, no inhibitor of this chemical family of benzamides contains a bulky capping group (e.g., compound 9 lacks this group), and the largest capping group was observed for 4acetamido-N-[2-amino-5-(2-thienyl)phenyl]benzamide crystallized with HDAC2 (PDB entry 4LY1) [28]. As shown in Figure S1, the orientation of the “capping” group overlaps well with our designed inhibitors (and in opposition to compound 4 crystallized into HDAC2, which projects towards the L1 loop). Taken together, the conformation of the benzamide moiety, which is imposed by the chelation geometry, seems to be crucial for orienting the capping group of this benzamide-based family of inhibitors opposite to the


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L1 loop. Concerning HDAC6 selectivity, an analysis of crystal structures of HDAC6 in complex with several inhibitors [38,39] confirms the hypothesis that the foot-pocket is unusable or closed in class II HDACs [40], establishing the basis for the selectivity of benzamides for class I HDACs over HDAC6. Finally, for PDE5, para derivatization of the ethoxyphenyl group of 1 and 2 projects towards the solvent, thereby predictably accommodating proposed substitutions, as shown in Figure 1B for the structure of compound 18a docked into the PDE5 cavity.

Figure 1. (A) Docking 18a (green) and 23a (orange) into the three-dimensional structure of HDAC2 (PDB entry 3MAX). The co-crystallized benzamide 9 of 3MAX is shown in pink sticks, with the para-phenyl ring occupying the 14 Å cavity characteristic of HDAC1 and HDAC2 [36]. (B) Docking 18a (green) into the PDE5 binding site (PDB entry 1TBF) [41].

SAR and Biochemical profiling The synthesis of both compounds 18a and 23a is reported in the CHEMISTRY section (Schemes 1 and 2, respectively). Both compounds were potent, low nanomolar PDE5 7 ACS Paragon Plus Environment


inhibitors, with the vardenafil-based derivative 23a being a log unit more potent than its corresponding matched pair 18a (IC50 values of 4 and 41 nM, respectively, Table 1), and this finding was consistent with the expected trend for both chemical series [25]. Concerning HDACs, compounds were routinely screened versus HDAC1, HDAC2 and HDAC6 isoforms for various pre-incubation times, 30 min (standard period used for hydroxamic derivatives) [12,25,26] and 4 hours (HDAC1 and HDAC2), as an accurate determination of IC50 values for extremely slow HDAC binders remains challenging [28]. Compound 23a exhibited time-dependent HDAC2 inhibition, with increasing inhibition observed when the pre-incubation time was prolonged from 30 minutes to 4 hours (the IC50 decreased from 2620 nM to 467 nM, Table 1). Then, compound 23a achieved midnanomolar class I HDAC inhibition, with excellent selectivity over HDAC6 (IC50 > 20 µM). Strikingly, its sildenafil-based matched pair 18a was a weaker HDAC1 inhibitor and had no effect on HDAC2 activity at the assayed conditions. A set of close analogues was prepared to examine SAR (Table 1). Other sulfamoyl-linked analogues listed in Table 1 (compounds 18b, 23b, 18c and 23c, synthesis shown in Schemes 1 to 2) did not show improved HDAC binding affinities, although we partially confirmed the observed impact of the pre-incubation time on inhibitor potency and the variability of this response to different HDAC isoforms (e.g., for compounds 18c and 23c). Replacement of this linker by a methylene group afforded anilides 29a (phenyl), 29b (thiophene) and 29c-34c (cyclobutane) (synthesis shown in Scheme 3). The phenyl linker of compound 29a conferred the highest activity against HDAC1 and HDAC2 in the mid-nanomolar range (IC50 = 180 and 668 nM at 4 h), although PDE5 activity was attenuated (IC50 = 114 nM). Bioisosteric substitution of the phenyl group of the anilide by a thiophene (derivatives 35a, 40a and 35b, synthesis shown in Scheme 4) retained the HDAC1 and HDAC2 inhibitory activity, particularly for the vardenafil analogue 40a. Among all compounds


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listed in Table 1, analogue 29a showed a significant time-dependent effect on class I HDAC inhibition, particularly towards HDAC2. For HDAC1, its inhibitory potency also increased significantly (~1 log unit) with the pre-incubation time: 673 nM (30 min), 180 nM (4 hours) and 69 nM (6 hours). This finding is consistent with its residence time (347 min, as determined by the jump dilution approach [26]). Given its interesting biochemical profile, the HDAC3 inhibitory activity of compound 29a was further determined, with similar findings obtained as for HDAC2; HDAC3 IC50 > 20 µM and 777 nM after 30 min and 4 hours of compound pre-incubation, respectively. As expected, all compounds listed in Table 1 were inactive against HDAC6 (IC50 > 20 µM), even if the pre-incubation time was extended to 4 hours, as assayed for compound 29a. Table 1. Dual PDE5 and HDAC inhibitors with slow binding kinetics and structures based on sildenafil and vardenafil scaffolds.

O

O N

O HN

N

O HN

N R1

N

N

R1

A

B


N

Cpd

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

CORE

R1

PDE5A

HDAC1

HDAC1

IC50 nM

IC50 nM

t


=

4

HDAC2 IC50 nM

hours

A

O O S N

N

H 2N H N

N

O

N

B O O S N

A

O S O N H

B

O S O N H

A

N

H 2N H N

N

O

N

N

H 2N H N

N

O

N

H 2N H N

N

O

N

N

H 2N H N

H S N O

B

O

t

=

HDAC6

Page 10 of 66

4 IC50 nMa

hours

41

3300

1290

>20000

>20000

>20000

4

393

331

2620

467

>20000

24

>20000

>20000

>20000

>20000

>20000

6

2650

3280

>10000

>10000

>20000

49

1760

7350

>20000

6420

>20000

8

1880

3520

>20000

3580

>20000

114

673

180

>20000

668

>20000

O

H 2N H N

H S N O O

A

HDAC2

O

H 2N H N O


Page 11 of 66 1 2 3 29b 4 5 6 7 8 9 10 29c 11 12 13 14 15 16 17 34c 18 19 20 21 22 23 35a 24 25 26 27 28 29 40a 30 31 32 33 34 35 36 35b 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


A

H 2N

160

3560

1040

>20000

1670

>20000

74

>20000

>20000

>20000

>20000

>20000

5

7780

>20000

14200

4860

>20000

1080

4170

831

>14000

11500

>14000

26

661

97

>20000

406

>20000

498

1850

2390

12400

3100

>20000

HN S

A

O

H 2N H N O

B

H 2N H N O

A

H 2N H N

S

O

B

H 2N H N

S

O

A

H 2N H N

S

O

Biochemical data in Tables 1-3 is the average of at least two assays performed on different days. In cases in which pIC50 difference was greater than 0.3 log units, in absolute terms, additional assays were carried out until a satisfactory error was obtained (individual results greater than 2 MADs of the average are discarded). aCompound 29a was also pre-incubated with HDAC6 for 4 hours, and its IC50 was constant at a value greater than 20 M.

In parallel with the sildenafil- and vardenafil-based analogues shown in Table 1, we examined whether the incorporation of an ortho-amino anilide moiety into the tadalafil core would result in dual PDE5 and HDAC inhibitors, as previously observed for hydroxamic-based derivatives [25]. Thus, compounds 50a-50b, with linker moieties 11 ACS Paragon Plus Environment


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connecting the scaffold to the chelating group similar to those in compounds in Table 1, were designed and synthetized as described in Scheme 5. Despite moderate class I HDAC inhibitory activity (Table 2), particularly for compound 50b, the poor activity against PDE5 (e.g., 50b IC50 > 10 µM) prevented us from further developing this chemical series. Table 2. Dual PDE5 and HDAC inhibitors with slow binding kinetics and a structure based on the tadalafil scaffold.

H

Cpd

R1

H O

O

PDE5A HDAC IC50 nM

R1

N

N

N H

O

O

1 IC50

HDAC1 HDAC2 HDAC2 t

=

hours

4

IC50 nM

t

=

HDAC6

4 IC50 nM

hours

nM

50a N

H 2N H N

N

O

N

50b

H 2N

5950

>20000

5810

>20000

12900

>20000

>10000

2300

516

12600

1930

>20000

HN O

N


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By revisiting the sildenafil analogues and after having identified the benzyl linker of compound 29a as an optimal linker for class I HDAC activity, we expanded our exploration to include 2-aminoanilides substitutions in position 5 to produce compounds that are able to occupy the foot pocket of the active site of class I HDACs (compounds 52a-52b, Table 3 and synthesis shown in Scheme 6); therefore, we aimed to develop HDAC1- and HDAC2-selective inhibitors that did not target HDAC3 [21,30] – as additional chemical probes to determine the contribution of just two class I HDAC isoforms. Compared to compound 29a, derivatization with a thiophene ring (52a) or a fluorine atom (52b) was detrimental for PDE5 and HDAC activity (low micromolar range). Next, we turned our attention to other alternative Zn-chelating groups, such as the sulphur-bonded 4-hydroxy-1H-pyrimidin-6-one of compound 54 (synthesis in Scheme 7). Disappointingly, although compound 54 was a potent nanomolar PDE5 inhibitor, only slight activity was detected towards HDAC6 (IC50 = 7.7 µM) and no activity was observed towards class I HDACs (IC50 > 20 M). Table 3. SAR exploration of sildenafil-based analogues occupying the class I HDAC foot pocket or containing alternative zinc-binding groups. O N

O HN

N N

R1



Cpd

R1

PDE5A HDAC IC50 nM

1 IC50

Page 14 of 66

HDAC1 HDAC2 HDAC2 t

=

hours

4

IC50 nM

t

=

HDAC6

4 IC50 nM

hours

nM

52a

N H2 H N

3640

>20000

833

>20000

14500

>14000

1860

>20000

2470

>20000

9110

>20000

5

40% at 40% at 25% at 25%

O S

52b

N H2 H N O

54

F

O

at 7670

HN S

N

20 µM

OH

20 µM

20 µM

20 µM

O

Cellular Response: From functional impact on H3K9 acetylation and CREB phosphorylation To cytotoxicity. Selected dual inhibitors with promising biochemical profiles were progressed into cellular assays, including the functional responses in the induction of histone acetylation (AcH3K9) and CREB phosphorylation (pCREB), determined in SH-SY5Y cells, and cytotoxicity towards the healthy hepatic cell line THLE-2. As shown in Table 4, our anilides generally exerted minor effects on THLE-2 cell proliferation (LC50 > 10 µM, 72 h incubation) compared with our hydroxamic-based lead compound 13 (LC50 = 7 M) or 14 ACS Paragon Plus Environment

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the reference compound 6 (LC50 = 159 nM, transgenic APP/PS1 mice tolerate the administration of this compound for 10 days[18]). Compound 29a, which was further progressed to in vivo testing, showed similar low cytotoxicity in THLE-2 cells after 24 and 48 h of incubation (LC50 = 15.1 and 14.5 µM, respectively) and in PBMCs (LC50 = 14 µM at 72 hours and > 100 µM at 24 h), although a greater anti-proliferative effect was observed on neurons and glial cells from wild type (WT) mice (LC50 = 4.8 µM after 72 hours). Changes in the AcH3K9 mark induced by these molecules were routinely determined after treatment for 24 hours (optimal read-out), as a delayed induction of H3K9 acetylation was observed for reference compound 6 (described in Table S1, supporting information), aligned with the results obtained for other marks (H2BK5 and H4K12) by Lauffer et al.[28] Values presented in Table 4 are reported as the mean fold change versus control vehicle-treated cultures, with values greater than 1 illustrating the induction of acetylation (or phosphorylation, in the case of the pCREB mark). Consistent with the biochemical profile, compounds 23a and 29a strongly increased the level of this mark at a dose of 1 µM (5.1 and 2.9 fold change compared with the basal level, respectively), similar to reference compound 6 (5.1 fold change) under the same conditions. A dose-response evaluation of the effects of 29a on the acetylation of H3K9 after a 24 h incubation was performed (Figure 2). At concentrations exceeding 26 nM, compound 29a elicited a significant (1.5-fold change, p value < 0.05) increase in the AcH3K9 mark, with a maximum response of 7.9-fold at 1 µM. Thus, based on its IC50 values for class I HDACs (leading to low toxicity) and previously reported data,[10,11,12,25,26] this level of AcH3K9 induced by the compound at concentrations exceeding 26 nM corroborates the functional synergistic effect induced by the simultaneous inhibition of PDE5 and HDAC class I enzymes.



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The level of CREB phosphorylation has little room for improvement and is substantially biased by the incubation time, as shown in Table 4 for well-known PDE5 inhibitors 1-3. Here, the incubation time was also required to be extended to 4 hours to achieve a significant response (greater than 1.4 fold change compared to vehicle-treated cells),[26] and only compound 29a elicited a significant response: 2.8-fold change at 500 nM compared with the basal level. A detailed dose-response assay showed that compound 29a increased the level of the pCREB mark by 47% (1.47-fold) at a concentration of 100 nM, with the maximum response of 5.11-fold observed for a 1 M treatment (described in Table S2, supporting information). Compared to the other more potent PDE5 inhibitors reported in Table 4 (e.g., 18a, 23a, and 40a), a possible explanation for the gain in CREB phosphorylation by 29a is its high PAMPA permeability (Pe = 82.9 nm/s). Table 4. Cellular response of prioritized dual PDE5 and class I HDAC inhibitors.

Cpd

THLE-2 LC50 nM

AcH3K9

levels

(fold pCREB levels

change compared with the basal level (1))

(fold

PAMPA

change Pe (nm/s)

compared with the

after treatment with 1 basal level (1)) µM for 24 hours

after treatment with 500 nM for 4 hours

1

>100000

N.D.

1.9 (30 min) 0.6 (2 hours)


27.5

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1.1 (4 hours) 2

>100000

N.D.

0.9 (30 min)

21.4

1.2 (2 hours) 2.2 (4 hours) 3

60300

N.D.

1.3 (30 min)

26.8

1.4 (2 hours) 1.0 (4 hours) 6

159

5.1

N.D.

0.59

13 [12]

7200

N.D.

2.0

15.7

18a

23000

2.3

0.5

29.5

23a

21300

5.1

0.9

10.3

29a

11700

2.9

2.8

82.9[a]

35a

42700

1.4

0.7

18.0

40a

7990

1.6

0.5

21.4

Quantification of the AcH3K9, AcTub and pCREB levels on Western blots is reported as the mean value of at least three experiments. Each reported permeability rate corresponds to the mean value of three assays. Cytotoxicity results correspond to the mean value of at least two tests performed on different days. If the difference in pLC50 was greater than 1 log unit, in absolute terms, new experiments were carried out until a satisfactory error was obtained (results exceeding 3 MADs of the mean were discarded). N.D. = not determined; [a]the concentration of each compound was measured by LC-MS (standard approach involves UV spectroscopy, see methods).



Figure 2. Levels of H3K9 acetylation were measured in SH-SY5Y cells, by AlphaLisa approach, after their treatment with 29a for 24 hours (*p ≤0.05, **p≤0.005, ***p≤ 0.001).

Functional response of Tg2576 neurons to compound 29a and ADME properties Next, we concentrated on compound 29a (Mw = 537 Da, TPSA = 124 Å) as other potent class I HDAC inhibitor that substantially increased the acetylation of H3K9; compound 23a exhibited poorer permeability and a clearly undesirable Mw (673 Da) and TPSA (198 Å). The impact of compound 29a on AD-related markers was determined in Tg2576 neurons using western blot assays. After exposure to compound 29a (50 nM) for 48 hours, the expression of the Aβ42 precursor 99-amino-acid-long APP-carboxy-terminal fragments (C99), a marker of hAPP processing, was reduced by 20%. Under the same assay conditions, the levels of phosphorylated Tau (pTau) were decreased by 32% compared to untreated cultures.


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A first in vitro evaluation of the ADME properties of compound 29a was performed. This compound displayed minimal inhibition, less than 20% at 10 µM, towards five of the cytochrome P450 isoforms (1A2, 2C19, 2C9, 2D6, and 3A4). Compound 29a exhibited moderate metabolic stability in human and mouse liver microsomes, with >55% of the compound remaining after its incubation (at 1 M) with the microsomes for 20 min, and estimated half-lives of 39.8 min (human) and 55.9 min (mouse). The good permeability obtained using our PAMPA assay (Pe = 82.9 nm/s) was replicated using CACO-2 cells (Pe = 1.4 × 10-6 cm/s), without efflux (efflux ratio of 0.86). Finally, a patch-clamp electrophysiology assay (using CHO cells with stable expression of hERG potassium channels) yielded an IC50 value > 30 μM, suggesting that the compound did not present cardiovascular safety issues. Based on these data, compound 29a exhibited a promising in vitro ADME profile that was only slightly limited by its low solubility (2.14 µg/mL) and high plasma protein binding activity in human and mouse (0.2% of the unbound fraction in both species). In terms of phosphodiesterase selectivity, IC50 values of compound 29a towards PDE9, PDE3A and PDE6 are > 10 µM, > 10 µM and 7.6 nM, respectively. PDE9 and PDE6 are cGMP-specific enzymes, while PDE3A also hydrolyses cAMP. PDE3A is involved in cardiac contractility and is associated with undesired cardiac side-effects [42]; therefore, the lack of activity of compound 29a towards PDE3A was regarded as a positive feature.

In vivo efficacy tests with 29a: From target engagement To impact on memory A pharmacokinetic (PK) study of the effects of compound 29a on C57BL/6J mice was performed after an intraperitoneal (i.p.) injection of a 20 mg/kg dose, and the results showed that despite its moderate in vitro microsomal stability, compound 29a has a short half-life in vivo (t1/2 = 7.5 h), reaching a Cmax of 934 nM at 15 min (plasma concentrations



are shown in Figure 3A and additional PK parameters are shown in the supporting information). Nevertheless, compound 29a has a low, but acceptable brain penetration (LogBB = -0.98; monitored at Tmax) that exceeds the dose required to achieve the aforementioned functional cellular response (26 nM for AcH3K9 in SH-SY5Y cells and 50 nM for AD-related biomarkers in primary cultures). The functional in vivo efficacy and brain accessibility of compound 29a were confirmed by monitoring the levels of the AcH3K9 and pCREB marks over time after administering this dose to WT mice (n=3). The level of AcH3K9 increased from 1.4 (30 min) to 1.9 (60 min). For pCREB, a stronger induction was detected from 1.4 (15 min) to 2.3 (30 min) and 2.4 (60 min). Thus, target engagement was confirmed in the hippocampus after the administration of compound 29a (20 mg/Kg, i.p.), as shown in Figure 3B. The compound (at 20 mg/kg, i.p.) was administered to 16-month-old Tg2576 mice for two weeks and contextual fear conditioning was assessed to determine if compound 29a reverses cognitive impairments. As depicted in Figure 3C, 16-month-old Tg2576 mice showed a significant decrease in freezing behaviour (37.9 ± 9.5 % of mice displayed freezing behaviours) compared to wild type (WT) mice (71.31±6.2 % of mice displayed freezing behaviours). Although Tg2576 mice treated with compound 29a showed a partial restoration of memory function (59.65 ±9.2 % of mice displayed freezing behaviours), the effect was not significant (p value = 0.17).


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Figure 3. (A) Plasma concentrations of 29a after i.p. administration (20 mg/kg) (n=5). (B) Western blots showing histone 3 acetylation at lys 9 (AcH3K9) and pCREB levels in the hippocampus of wild type mice 15, 30 min and 1 hour after the intraperitoneal (i.p.)



injection of compound 29a (20 mg/kg) or vehicle (n = 3). Actin was used as a loading control. (C) Freezing behaviours of 16-month-old WT (n=8) or Tg2576 mice treated (i.p.) with vehicle (n=10) or 20 mg/kg compound 29a (n=11) for 2 weeks. Data are presented as the percent of time spent freezing during a 2 min test, (* p ≤ 0.05).

DISCUSSION

Based on our investigations of a novel therapeutic approach for treating AD based on dual inhibition of PDEs and HDACs [11,12,25,26], we describe a novel chemical probe, 29a (CM-675), with class I HDAC selectivity over HDAC6 (>1 log unit) in the present study. Compared to our hydroxamic acid-based lead compound 13 [11], compound 29a has a 10 times longer residence time on class I HDACs; e.g., 35 versus 347 min on HDAC1, respectively. The slow binding kinetics of compound 29a emphasize the importance of screening HDAC inhibitors under adequate experimental enzymatic assay conditions (i.e., pre-incubation time) to prevent an underestimation of their potency and avoid discarding them early in the drug discovery process. Other authors have also reported similar findings for this family of ZBG: compounds 6 [28] and 9 [34] (whose HDAC2 IC50 decreased from 900 nM to 27 nM when delaying the compound incubation time from 1 h to 24 h). Thus, the use of longer pre-incubation times with compounds exhibiting long residence times (or are expected to exhibit long residence times, according to their structural characteristics), as reported here, is key to obtain precise IC50 values and therefore selectivity profiles for HDAC isoforms. Together with setting adequate experimental assay conditions, kinetic binding profiling of HDAC inhibitors (i.e., structural-kinetic relationships, SKR) is becoming recognized


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as an increasingly important method to discern kinetic selectivity between highly homologous HDAC isoforms belonging to the same family (e.g., HDAC1 and HDAC2), thus complementing thermodynamic profiling (i.e., classical structure-activity relationships, SAR) [22]. Here, the kinetic selectivity profile of compound 29a merits further investigation, specifically its residence time on HDAC2 and HDAC3 isoforms, as a pre-requisite to associate its biochemical profile with the observed functional response and mode of action (MoA). Functionally, compound 29a is able to induce significant cellular responses (AcH3K9 and pCREB) at low concentrations (26-100 nM), while its worst cytotoxicity (towards neurons and glia cells) is in the micromolar range. This finding endows compound 29a with a therapeutic window of around one log unit, suggesting (together with the results from the patch-clamp electrophysiology assay) that compound 29a is safe for in vivo administration. Moreover, compound 29a reaches the brain at a dose sufficient to exert in vivo effects on functional hallmarks in the mouse hippocampus, as confirmed following acute i.p. administration of a 20 mg/kg dose. Disappointingly, we did not observe a significant improvement in memory restoration after two weeks of treatment with compound 29a, although a certain trend is acknowledged. From the compound optimization perspective, compound 29a contains the sildenafil scaffold. Based on the SAR results presented in Table 1, vardenafil-based analogues seem to be more potent at inhibiting class I HDACs than compounds based on the sildenafil core, suggesting that the vardenafil-matched pair of 29a might be a good alternative. However, during the course of our investigations with hydroxamic acid-based derivatives, the vardenafil matched pair of our sildenafil-based lead compound 13 exhibited a narrower therapeutic range and poorer in vivo pCREB efficacy that prevented its progression to investigations



using in vivo models of AD. Therefore, sildenafil-based compounds were prioritized over compounds with the vardenafil chemotype in the present study. Additional studies are underway to identify the relationships among chemical structures, residence times, binding kinetics, binding sites, biochemical inhibition and functional responses. These studies may provide researchers new opportunities to develop HDAC isoform-selective molecules, based on their binding kinetics, and then to obtain dual phosphodiesterase and HDAC inhibitors with the required target compound profile to treat AD. In addition, the kinetic profiling of these molecules is also very important to define the optimal dosing scheme for in vivo testing in terms of determining the efficacy and safety.


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CHEMISTRY Scheme 1.

O

N

HN

O

O

O N

i)

O

N

HN

14

15

SO2 Cl

S

N N

O

16a-c, R = CH3 or CH2CH3

OR

A

O

17a-c

O

N

HN

iv)

A = a)

N N

O OH

S

O

O

A

iii)

O

N

O

N N

O

O HN

N

HN

N

N

O

O

ii)

N

S

O

A

b)

NH

N N

H N

N H2

18a-c

N

c) H N

N N

N

N

O

Conditions: i) ClSO3H, 0 ºC, then rt, 2 h; ii) corresponding amine, Et3N, EtOH, 100 ºC, MW, 1 h; iii) LiOH·H2O, THF/MeOH/H2O (10:1:5 or 15:1:5), rt, overnight; iv) EDC·HCl, HOBt, benzene-1,2-diamine, NMM, DMF, rt, overnight.

Sildenafil derivatives 18a-c were prepared as described in Scheme 1 from commercially available

5-(2-ethoxyphenyl)-1-methyl-3-propyl-6H-pyrazolo[4,3-d]pyrimidin-7-one

(14). This compound was first transformed into key intermediate 15 by sulphonylation at 5’-position of the phenyl ring and then esters 16a-c were synthesized after reaction with the convenient amine under microwave irradiation. Then, desired amido anilines 18a-c were obtained through hydrolysis and reaction with benzene-1,2-diamine using EDC/HOBt/NMM as coupling system.



Page 26 of 66

Scheme 2.

O

HN N

O

O

O N

N

O

i)

HN N

19

N

O

S

N

N

O

A

S

O

22a-c

O

OR

O

A

N

N


N

A = a)

N

b)

NH

N N

H N

iii)

O

HN N

S

O

A

iv)

O OH

O

O

HN

HN N

20

SO2 Cl

O O

O

ii)

N

N

N H2

23a-c

N

c) H N

N N

N

N

O

Conditions: i) ClSO3H, 0 ºC, then rt, 2 h; ii) corresponding amine, Et3N, EtOH, 100 ºC, MW, 1 h; iii) LiOH·H2O, THF/MeOH/H2O (10:1:5 or 30:1:10), rt, overnight; iv) EDC·HCl, HOBt, benzene-1,2diamine, NMM, DMF, rt, overnight.

Vardenafil analogues 23a-c were synthesized following the same method described for sildenafil derivatives 18a-c (Scheme 2).

Scheme 3.


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iv) and v)

A) O

i)

14

O

O

HN

N

HN

ii)

N

O

N

N

HN

iii)

N

N

O

O

O

O N

viii)

N

24

O

I

B

O

25

vi)

H N

A

OR O

N N

N A

N

HN

N H2

O


29a-c

27a-c, R = H vii)

28a-c, R = PFP

O

B)

O

19

i)

O

HN N

I

O N

N

iv) and v)

N

OR

A

30

O

HN

O

vi)

N

N

31c, R = CH2CH3 32c, R = H 33c, R = PFP

a)

b)

HN

viii)

vii) A=

O

N H N

A

N

N

N H2

O

34c

c) S

Conditions: i) NIS, TFA, 0 ºC, then rt, overnight; ii) 4,4,5,5-tetramethyl-2-(4,4,5,5-tetramethyl-1,3,2dioxaborolan-2-yl)-1,3,2-dioxaborolane, KOAc, Pd(dppf)Cl2, 1,4-dioxane, 80-100 ºC, 48 h; iii) corresponding bromide, Pd(PPh3)4, K2CO3, 1,4-dioxane/H2O (5:2 or 16:1), 85 ºC, MW, 1 h; iv) ethyl 3methylenecyclobutanecarboxylate, 9-BBN, THF, reflux, 4 h; v) xantphos or X-Phos, Pd2(dba)3, Na2CO3, 1,4-dioxane/H2O (35:6), reflux, overnight; vi) LiOH·H2O, THF/MeOH/H2O (3:3:2 or 1:3:1), rt, overnight; vii) 2,3,4,5,6-pentafluorophenol, DIC, CH2Cl2, 0 ºC, then rt, 6 h or overnight; viii) benzene-1,2-diamine, DIEA, DMAP (optional), DMF, 60-80 ºC, overnight.

Synthesis of compounds 29a-c is shown in Scheme 3A. In this case intermediate 14 was transformed into iodide 24 and then esters 26a-c were prepared through Suzuki coupling. Subsequent hydrolysis with LiOH led us to corresponding carboxylic acids and desired amido anilines 29a-c were obtained after preparation of pentafluorophenyl active esters 28a-c and reaction with benzene-1,2-diamine. On the other hand, vardenafile analogue 34c was synthesized by a similar strategy as used for ester 29c starting from compound 19 (Scheme 3B).



Page 28 of 66

Scheme 4. A) O

O O

N

HN

O

i)

N

H N

A

28a

O

i)

N N

N

O

O N

HN

O PFP

N N

N H2 O

S

35a-b

O

N

HN

PFP

28c O

B) 30

O

HN N

B O

O

O

O

O

ii)

N

N

36

iii)

O

HN N

N

N

i)

O

iv)

37a, R = CH3

A=

a)

N

N

N H2

H N

A

38a, R = H v)

HN N

OR

A

O

O

S

40a

39a, R = PFP

b)

Conditions: i) thiophene-1,2-diamine, DIEA, DMAP, DMF, 60 ºC, overnight; ii) 4,4,5,5-tetramethyl-2(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1,3,2-dioxaborolane, KOAc, Pd(dppf)Cl2, 1,4-dioxane, 90 ºC, overnight; iii) methyl 4-(bromomethyl)benzoate, Na2CO3, Pd(PPh3)4, 1,4-dioxane/H2O (10:1), 85 ºC, MW, 1 h; iv) LiOH·H2O, THF/MeOH/H2O (3:3:2), rt, overnight; v) 2,3,4,5,6-pentafluorophenol, DIC, CH2Cl2, 0 ºC, then rt, overnight.

Synthesis of thiophene derivatives 35a-b and 40a is shown in Scheme 4. Compounds 35a-b were simple obtained from described intermediates 28a and 28c by reaction with thiophene-1,2-diamine. A similar synthetic strategy was followed to afford vardenafil analogue 40a from iodide 30.

Scheme 5.


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O HO

i)

ii)

O

O

O

H

O

H

Cl

iii)

NH

N O

O

41

O

O

42

N H H

O

N H

43

44

O

O

N N H

N

45, R = BOC O

v)

N

O

ix)

viii)

n B

O

47a, n = 0, A = B = N, R = CH2CH3 47b, n = 1, A = B = CH, R = CH3

O

O

O OR

48a, n = 0, A = B = N, R = OH 48b, n = 1, A = B = CH, R = OH

A

N

x)

N

N

N H

n B

O

O

N

O A

N N H

N H

OR

A

N N

O

46, R = H

O

N

R

vi) or vii)

O

O

O

O

N

O

O

O

iv)

H

n B

N H

N H2

O

O O

50a, n = 0, A = B = N 50b, n = 1, A = B = CH

49a, n = 0, A = B = N, R = PFP 49b, n = 1, A = B = CH, R = PFP

Conditions: i) MnO2, CH2Cl2, rt, overnight; ii) methyl (2R)-2-amino-3-(1H-indol-3-yl)propanoate, propoan-2-ol, 80-100 ºC, overnight; iii) 2-chloroacetyl chloride, Et3N, CH2Cl2, 0 ºC, then rt, 3 h; iv) tertbutyl 4-(aminomethyl)piperidine-1-carboxylate, MeOH, 80-100 ºC, overnight; v) HCl/EtOAc (1.0 M), 0 ºC, then rt, 3 h; vi) ethyl 2-chloropyrimidine-5-carboxylate, K2CO3, DMF, 80 ºC, overnight; vii) methyl 4formylbenzoate, CH3COOH, CH2Cl2, rt, 1 h, then NaBH3CN, rt, overnight; viii) LiOH·H2O, THF/MeOH/H2O (10:3:2 or 3:2:2), rt, overnight; ix) 2,3,4,5,6-pentafluorophenol, DIC, CH2Cl2, 0 ºC, then rt, overnight; x) benzene-1,2-diamine, DIEA, DMAP, DMF, 60 ºC, overnight.

Tadalafil-based amides 50a-b were prepared following the synthetic route outlined in Scheme 5 from commercially available 1,3-benzodioxol-5-ylmethanol (41). Oxidation of this alcohol with MnO2 led us to piperonal and then compound 44 was synthesized following a known synthetic procedure [43]. The stereochemistry of this intermediate has been previously studied and it was confirmed to be the 6R/12aR cis isomer. Nevertheless the subsequent transformations have not been described before and as it is known that both stereogenic centers of tadalafil core can epimerize and their stereochemistry have 29 ACS Paragon Plus Environment


Page 30 of 66

not been studied, compounds are described as mixture of diastereomers. Reaction of this intermediate 44 with tert-butyl 4-(aminomethyl)piperidine-1-carboxylate and elimination of BOC-protecting group in acidic media led us to amine 46. Then, esters 47a and 47b were prepared by alkylation or reductive amination respectively. Hydrolysis of these esters using LiOH afforded carboxylic acids 48a-b which were activated with pentafluorophenol to finally obtain amido anilines 50a and 50b.

Scheme 6.

O

O

O

O O

N

HN

N

i) and ii)

N

iii)

N

N

H N

OH

R=

a)

51a-b

S

b)

N

HN

N N

NHBOC

H N

N H2

O

O

O

27a

O

N

HN

R

52a-b

R

F

Conditions: i) SOCl2, 80 ºC, 2 h; ii) corresponding amine, Et3N, CH2Cl2, rt; iii) HCl/EtOAc (4.0 N), 25 ºC, 16 h.

Synthesis of compounds 52a-b is shown in Scheme 6 starting from previously described 27a. This intermediate was first transformed into its corresponding acyl chloride and then desired amides 52a and 52b were isolated after reaction with appropriated amines and acidic deprotection.

Scheme 7.


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O O O

N

HN

O

O N

i)

O

N

HN

N

N

N

HN

N N

ii)

N O

14

53

O Br

H N

S N

OH

O

54

Conditions: i) 2-bromoacetyl bromide, AlCl3, CH2Cl2, 0 ºC, 30 min, then 15 ºC, 15.5 h ii) 4-hydroxy-2sulfanyl-1H-pyrimidin-6-one, NaOH/H2O, EtOH, 20 ºC, 2 h.

Synthesis of compound 54 is outlined in Scheme 7. Intermediate compound 53 was obtained by reacting intermediate 14 with 2-bromoacetyl bromide. Then, nucleophilic substitution of 53 with 4-hydroxy-2-sulfanyl-1H-pyrimidin-6-one in basic media afforded compound 54.



MATERIALS AND METHODS Chemistry. General Procedure. Unless otherwise noted, all reagents and solvents were of the highest commercial quality and used without further purification. All experiments dealing with moisture sensitive compounds were conducted under N2. As standard extraction procedure, mixtures were extracted with EtOAc and the organic layers were washed with brine, dried over anhydrous Na2SO4, filtered and concentrated. Flash column chromatography was performed on silica gel Merck 60 (particle size 40-63 µm) under standard techniques. Automated flash column chromatography was performed using ready-to-connect cartridges from Varian, on irregular silica gel, particle size 15-40 µm (normal phase disposable flash columns) on a Biotage SPX flash purification system. Microwaveassisted reactions were performed in a Biotage Smith Synthesis microwave reactor. The NMR spectroscopic data were recorded on a Bruker AV400 or VARIAN 400MR spectrometer with standard pulse sequences, operating at 400 MHz. Chemical shifts (δ) are reported in parts per million (ppm) downfield from tetramethylsilane (TMS), which was used as internal standard. The abbreviations used to explain multiplicities are s = singlet, d = doublet, t = triplet, m = multiplet. Coupling constants (J) are in hertz. HPLCanalysis was performed using a Shimadzu LC-20AB or LC-20AD with a Luna-C18(2), 5 µm, 2.0*50mm column at 40 ºC and UV detection at 215, 220 and 254 nm. Flow from the column was split to a MS spectrometer. The MS detector (Agilent 1200, 6110MS or Agilent 1200, 6120MS Quadropole) was configured with an electrospray source or API/APCI. N2 was used as the nebulizer gas. The source temperature was maintained at 50 ºC. Data acquisition was accomplished with ChemStation LC/MSD quad software. All tested compounds possessed a purity of at least 95% established by HPLC. Reported yields were not optimized, the emphasis being on purity of product rather than quantity. 32 ACS Paragon Plus Environment

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Some intermediate compounds, to achieve those novel molecules described in Tables 1, 2 and 3, had been previously reported by our group: a) 15, 16a, 16b, 17a, 17b, 24, 25, 26c and 27c [12], b) 20, 21a, 22a, 30, 31c, 32c, 36, 42, 43, 44, 45, 46, 47a and 48a [25], and c) 26a, 26b, 27a, 27b, 37a and 38a [26].

Methyl

4-[[[4-ethoxy-3-(1-methyl-7-oxo-3-propyl-6H-pyrazolo[4,3-d]pyrimidin-5-

yl)phenyl]sulfonylamino]methyl]benzoate (16c) To a solution of compound 15 (410 mg, 1.0 mmol) in EtOH (15 mL) was added methyl 4-(aminomethyl)benzoate (0.202 g, 1.2 mmol) and Et3N (303 mg, 3.0 mmol) and the reaction mixture was stirred at 100 ºC under MW for 1 hour. Then, the reaction mixture was concentrated under vacuum to give the desired compound 16c (0.4 g, 74%). ESI-MS m/z 540 [M+H]+ calc. for C26H29N5O6S. This intermediate was used in the next step without further characterization.

4-[[[4-Ethoxy-3-(1-methyl-7-oxo-3-propyl-6H-pyrazolo[4,3-d]pyrimidin-5yl)phenyl]sulfonylamino]methyl]benzoic acid (17c) To a solution of compound 16c (0.2 g, 0.37 mmol) in THF/MeOH/H2O (10:1:5, 16 mL) was added LiOH·H2O (82 mg, 1.90 mmol) and the resulting mixture was stirred at room temperature overnight. Then, the mixture was diluted with water and adjusted pH to 2-3 with 1.0 N HCl. Then, the standard extraction procedure was carried out to give compound 17c (0.15 g, 77%). ESI-MS m/z 526 [M+H]+ calc. for C25H27N5O6S. This intermediate was used in the next step without further characterization.



N-(2-aminophenyl)-2-[4-[4-ethoxy-3-(1-methyl-7-oxo-3-propyl-6H-pyrazolo[4,3d]pyrimidin-5-yl)phenyl]sulfonylpiperazin-1-yl]pyrimidine-5-carboxamide (18a) To a solution of compound 17a (0.583 g, 1.0 mmol) in DMF (20 mL) was added EDC·HCl (230 mg, 1.2 mmol), HOBt (160 mg, 1.2 mmol), benzene-1,2-diamine (108 mg, 1.0 mmol) and NMM (505 mg, 5 mmol) and the mixture was stirred at room temperature overnight. Then, the mixture was diluted with water and the standard extraction procedure was carried out to give the crude product which was purified by prep-HPLC (method 1 described in supporting information) to obtain pure compound 18a (400 mg, 59%) as a white solid; m.p. 233-234 ºC. 1H NMR (DMSO-d6, 400 MHz): δ 9.51 (s, 1H), 8.86 (s, 2H), 7.85-7.81 (m, 2H), 7.36-7.32 (m, 1H), 7.11-7.09 (m, 1H), 6.97-6.93 (m, 1H), 6.76-6.73 (m, 1H), 6.57-6.54 (m, 1H), 4.21-4.13 (m, 5H), 3.95-3.93 (m, 4H), 3.01-2.98 (m, 4H), 2.77-2.73 (m, 2H), 1.75-1.69 (m, 2H), 1.26 (t, J = 6.8 Hz, 3H), 0.90 (t, J = 6.8 Hz, 3H). ESI-MS m/z 673.1 [M+H]+ calc. for C32H36N10O5S.

N-(2-aminophenyl)-2-[4-[[4-ethoxy-3-(1-methyl-7-oxo-3-propyl-6H-pyrazolo[4,3d]pyrimidin-5-yl)phenyl]sulfonylamino]-1-piperidyl]pyrimidine-5-carboxamide (18b) To a solution of compound 17b (0.598 g, 1.0 mmol) in DMF (30 mL) was added EDC·HCl (230 mg, 1.2 mmol), HOBt (162 mg, 1.2 mmol), benzene-1,2-diamine (216 mg, 2.0 mmol) and NMM (303 mg, 3.0 mmol) and the mixture was stirred at room temperature overnight. Then, the mixture was diluted with water and the standard extraction procedure was carried out to give the crude product which was purified by prep-HPLC (method 1 described in supporting information) to obtain pure compound 18b (500 mg, 73%) as a white solid; m.p. 240-241 ºC. 1H NMR (DMSO-d6, 400 MHz): δ 9.46 34 ACS Paragon Plus Environment

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(s, 1H), 8.84 (s, 2H), 8.01 (s, 1H), 7.93-7.92 (m, 1H), 7.82-7.80 (m, 1H), 7.33-7.31 (m, 1H), 7.11-7.09 (m, 1H), 6.94-6.93 (m, 1H), 6.75-6.73 (m, 1H), 6.58-6.54 (m, 1H), 4.94 (br s, 2H), 4.47-4.42 (m, 2H), 4.24-4.10 (m, 5H), 3.36 (s, 1H), 3.20-3.10 (m, 2H), 2.802.74 (m, 2H), 1.75-1.65 (m, 4H), 1.35-1.30 (m, 5H), 0.91 (t, J = 7.2 Hz, 3H). ESI-MS m/z 687.3 [M+H]+ calc. for C33H38N10O5S.

N-(2-aminophenyl)-4-[[[4-ethoxy-3-(1-methyl-7-oxo-3-propyl-6H-pyrazolo[4,3d]pyrimidin-5-yl)phenyl]sulfonylamino]methyl]benzamide (18c) To a solution of compound 17c (0.532 g, 1.0 mmol) in DMF (30 mL) was added EDC·HCl (230 mg, 1.2 mmol), HOBt (162 mg, 1.2 mmol), benzene-1,2-diamine (108 mg, 1.0 mmol) and NMM (505 mg, 5.0 mmol) and the mixture was stirred at room temperature overnight. Then, the mixture was diluted with water and the standard extraction procedure was carried out to give the crude product which was purified by prep-HPLC (method 2 described in supporting information) to obtain pure compound 18c (0.4 g, 65%) as a white solid; m.p. 157-158 ºC. 1H NMR (DMSO-d6, 400 MHz): δ 9.69 (s, 1H), 8.25-8.23 (m, 1H), 8.01-8.00 (m, 1H), 7.91-7.87 (m, 2H), 7.40-7.38 (m, 2H), 7.32-7.30 (m, 1H), 7.17-7.16 (m, 1H), 7.01-6.97 (m, 1H), 6.84-6.82 (m, 1H), 6.69-6.67 (m, 1H), 4.22-4.13 (m, 5H), 4.07-4.04 (m, 2H), 2.80-2.75 (m, 2H), 1.76-1.70 (m, 2H), 1.32 (t, J = 7.2 Hz, 3H), 0.92 (t, J = 7.2 Hz, 3H). ESI-MS m/z 616.1 [M+H]+ calc. for C31H33N7O5S.



Ethyl 2-[4-[[4-ethoxy-3-(5-methyl-4-oxo-7-propyl-3H-imidazo[5,1-f][1,2,4]triazin-2yl)phenyl]sulfonylamino]-1-piperidyl]pyrimidine-5-carboxylate (21b) To a solution of compound 20 (410 mg, 1.0 mmol) in EtOH (15 mL) was added ethyl 2(4-amino-1-piperidyl)pyrimidine-5-carboxylate (Int. 2, synthesis described in supporting information) (0.250 g, 1.0 mmol) and Et3N (303 mg, 3.0 mmol) and the reaction mixture was stirred at 100 ºC under MW for 1 hour. Then, the reaction mixture was concentrated under vacuum to give the desired product 21b (0.4 g, 64%). ESI-MS m/z 625 [M+H]+ calc. for C29H36N8O6S. This intermediate was used in the next step without further characterization.

Methyl 4-[[[4-ethoxy-3-(5-methyl-4-oxo-7-propyl-3H-imidazo[5,1-f][1,2,4]triazin-2yl)phenyl]sulfonylamino]methyl]benzoate (21c) To a solution of compound 20 (410 mg, 1.0 mmol) in EtOH (15 mL) was added methyl 4-(aminomethyl)benzoate (0.21 g, 1.3 mmol) and Et3N (303 mg, 3.0 mmol) and the reaction mixture was stirred at 100 ºC under MW for 1 hour. Then, the reaction mixture was concentrated under vacuum to give the desired product compound 21c (0.4 g, 74%). ESI-MS m/z 540 [M+H]+ calc. for C26H29N5O6S. This intermediate was used in the next step without further characterization.

2-[4-[[4-Ethoxy-3-(5-methyl-4-oxo-7-propyl-3H-imidazo[5,1-f][1,2,4]triazin-2yl)phenyl]sulfonylamino]-1-piperidyl]pyrimidine-5-carboxylic acid (22b) To a solution of compound 21b (0.7 g, 1.12 mmol) in THF/MeOH/H2O (30:1:10, 41 mL) was added LiOH·H2O (250 mg, 5.96 mmol) and the resulting mixture was stirred at room 36 ACS Paragon Plus Environment

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temperature overnight. The mixture was diluted with water and adjusted pH to 2-3 with 1.0 N HCl. Then, the standard extraction procedure was carried out to give compound 22b (0.5 g, 75%). ESI-MS m/z 597 [M+H]+ calc. for C27H32N8O6S. This intermediate was used in the next step without further characterization.

4-[[[4-Ethoxy-3-(5-methyl-4-oxo-7-propyl-3H-imidazo[5,1-f][1,2,4]triazin-2yl)phenyl]sulfonylamino]methyl]benzoic acid (22c) To a solution of compound 21c (0.5 g, 0.93 mmol) in THF/MeOH/H2O (10:1:5, 16 mL) was added LiOH·H2O (204 mg, 4.86 mmol) and the resulting mixture was stirred at room temperature overnight. The mixture was diluted with water and adjusted pH to 2-3 with 1.0 N HCl. Then, the standard extraction procedure was carried out to give compound 22c (0.35 g, 72%). ESI-MS m/z 526 [M+H]+ calc. for C25H27N5O6S. This intermediate was used in the next step without further characterization.

N-(2-aminophenyl)-2-[4-[4-ethoxy-3-(5-methyl-4-oxo-7-propyl-3H-imidazo[5,1f][1,2,4]triazin-2-yl)phenyl]sulfonylpiperazin-1-yl]pyrimidine-5-carboxamide (23a) To a solution of compound 22a (0.15 g, 0.26 mmol) in DMF (10 mL) was added EDC·HCl (60 mg, 0.31 mmol), HOBt (42 mg, 0.31 mmol), benzene-1,2-diamine (33 mg, 0.31 mmol) and NMM (130 mg, 1.29 mmol) and the mixture was stirred at room temperature overnight. The mixture was diluted with water and then the standard extraction procedure was carried out to give the crude product which was purified by prep-HPLC (method 3 described in supporting information) to obtain pure compound 23a (100 mg, 57%) as a white solid; m.p. 261-262 ºC. 1H NMR (DMSO-d6, 400 MHz): δ



11.66 (s, 1H), 9.54 (s, 1H), 8.89 (s, 2H), 7.91-7.88 (m, 2H), 7.39-7.37 (m, 1H), 7.12-7.10 (m, 1H), 6.99-6.95 (m, 1H), 6.78-6.76 (m, 1H), 6.61-6.58 (m, 1H), 4.22-4.17 (m, 2H), 3.97 (s, 4H), 3.03 (s, 4H), 2.85-2.81 (m, 2H), 2.48 (s, 3H), 1.76-1.71 (m, 2H), 1.31 (t, J = 7.2 Hz, 3H), 0.93 (t, J = 7.6 Hz, 3H). ESI-MS m/z 673.1 [M+H]+ calc. for C32H36N10O5S.

N-(2-aminophenyl)-2-[4-[[4-ethoxy-3-(5-methyl-4-oxo-7-propyl-3H-imidazo[5,1f][1,2,4]triazin-2-yl)phenyl]sulfonylamino]-1-piperidyl]pyrimidine-5-carboxamide (23b) To a solution of compound 22b (0.2 g, 0.34 mmol) in DMF (20 mL) was added EDC·HCl (78 mg, 0.4 mmol), HOBt (55 mg, 0.4 mmol), benzene-1,2-diamine (44 mg, 0.41 mmol) and NMM (172 mg, 1.7 mmol) and the mixture was stirred at room temperature overnight. The mixture was diluted with water and then the standard extraction procedure was carried out to give the crude product which was purified by prep-HPLC (method 4 described in supporting information) to obtain pure compound 23b (150 mg, 64%) as a white solid; m.p. 148-149 ºC. 1H NMR (MeOD, 400 MHz): δ 8.88 (s, 2H), 8.19 (s, 1H), 8.10-8.07 (m, 1H), 7.38-7.35 (m, 1H), 7.21-7.13 (m, 2H), 7.01-6.99 (m, 1H), 6.91-6.89 (m, 1H), 4.68-4.64 (m, 2H), 4.34-4.28 (m, 2H), 3.48-3.43 (m, 1H), 3.21-3.15 (m, 2H), 3.04-3.00 (m, 2H), 2.62 (s, 3H), 1.89-1.84 (m, 4H), 1.51-1.47 (m, 5H), 1.01 (t, J = 7.1 Hz, 3H). ESI-MS m/z 687.2 [M+H]+ calc. for C33H38N10O5S.

N-(2-aminophenyl)-4-[[[4-ethoxy-3-(5-methyl-4-oxo-7-propyl-3H-imidazo[5,1f][1,2,4]triazin-2-yl)phenyl]sulfonylamino]methyl]benzamide (23c)


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To a solution of compound 22c (0.15 g, 0.28 mmol) in DMF (10 mL) was added EDC·HCl (66 mg, 0.34 mmol), HOBt (461 mg, 0.34 mmol), benzene-1,2-diamine (46 mg, 0.43 mmol) and NMM (144 mg, 1.43 mmol) and the mixture was stirred at room temperature overnight. The mixture was diluted with water and then the standard extraction procedure was carried out to give the crude product which was purified by prep-HPLC (method 5 described in supporting information) to obtain pure compound 23c (0.1 g, 58%) as a pale red solid; m.p. 119-120 ºC. 1H NMR (MeOD, 400 MHz): δ 8.078.01 (m, 2H), 7.96-7.94 (m, 2H), 7.46-7.44 (m, 2H), 7.38-7.31 (m, 5H), 4.45-7.40 (m, 4H), 3.16-3.12 (m, 2H), 2.68 (s, 3H), 1.93-1.87 (m, 2H), 1.46-1.42 (m, 3H), 1.07-1.03 (m, 3H). ESI-MS m/z 616.1 [M+H]+ calc. for C31H33N7O5S.

(2,3,4,5,6-Pentafluorophenyl)

4-[[4-ethoxy-3-(1-methyl-7-oxo-3-propyl-6H-

pyrazolo[4,3-d]pyrimidin-5-yl)phenyl]methyl]benzoate (28a) To a solution of compound 27a (170 mg, 0.38 mmol) and 2,3,4,5,6-pentafluorophenol (77 mg, 0.42 mmol) in CH2Cl2 (20 mL) was added DIC (76 mg, 0.61 mmol) at 0 ºC under N2. The mixture was stirred at room temperature for 6 hours. Then, the standard extraction procedure was carried out to give the crude compound which was purified by prep-TLC to obtain pure compound 28a (200 mg, 86%). ESI-MS m/z 613.2 [M+H]+ calc. for C31H25F5N4O4. This intermediate was used in the next step without further characterization.



pyrazolo[4,3-d]pyrimidin-5-yl)phenyl]methyl]thiophene-2-carboxylate (28b)



To a solution of compound 27b (100 mg, 0.22 mmol) and DIC (44 mg, 0.35 mmol) in CH2Cl2 (10 mL) was added 2,3,4,5,6-pentafluorophenol (44 mg, 0.24 mmol) at 0 ºC under N2 and the mixture was stirred at room temperature overnight. Then, the standard extraction procedure was carried out to give the crude product which was purified by prep-TLC to obtain pure compound 28b (70 mg, 51%) as a yellow solid. ESI-MS m/z 618.1 [M+H]+ calc. for C29H23F5N4O4S. This intermediate was used in the next step without further characterization.

(2,3,4,5,6-pentafluorophenyl)


pyrazolo[4,3-d]pyrimidin-5-yl)phenyl]methyl]cyclobutanecarboxylate (28c) To a solution of compound 27c (850 mg, 2.0 mmol) and DIC (404 mg, 3.2 mmol) in CH2Cl2 (40 mL) was added 2,3,4,5,6-pentafluorophenol (386 mg, 2.1 mmol) at 0 oC under N2 and the mixture was stirred at room temperature overnight. Then, the standard extraction procedure was carried out to give the crude product which was purified by column chromatography to afford pure compound 28c (720 mg, 61%) as a yellow solid. ESI-MS m/z 591.3 [M+H]+ calc. for C29H27F5N4O4. This intermediate was used in the next step without further characterization.

N-(2-aminophenyl)-4-[[4-ethoxy-3-(1-methyl-7-oxo-3-propyl-6H-pyrazolo[4,3d]pyrimidin-5-yl)phenyl]methyl]benzamide (29a) To a solution of compound 28a (200 mg, 0.32 mmol) in DMF (5 mL) was added DMAP (44 mg, 0.36 mmol), benzene-1,2-diamine (71 mg, 0.66 mmol) and DIEA (52 mg, 0.40 mmol) and the mixture was stirred at 60 ºC overnight. The mixture was quenched with


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water and then the standard extraction procedure was carried out to give the crude product which was purified by prep-HPLC (method 6 described in supporting information) to obtain pure compound 29a (36.7 mg, 21%) as a pale yellow solid; m.p. 198-199 ºC. 1H NMR (DMSO-d6, 400 MHz): δ 11.96 (s, 1H), 9.58 (s, 1H), 7.92-7.90 (m, 2H), 7.52 (s, 1H), 7.40-7.35 (m, 3H), 7.15-7.09 (m, 2H), 6.96-6.90 (m, 1H), 6.78-6.76 (m, 1H), 6.586.55 (m, 1H), 4.87 (s, 2H), 4.14-4.03 (m, 7H), 2.78-2.74 (m, 2H), 1.76-1.70 (m, 2H), 1.32-1.28 (m, 3H), 0.95-0.91 (m, 3H). 13C NMR (DMSO-d6, 100 MHz): δ 165.1, 154.8, 153.6, 149.5, 145.0, 144.7, 143.1, 137.9, 132.8, 132.4, 131.9, 130.4, 128.5 (2C), 128.0 (2C), 126.6, 126.4, 124.1, 123.3, 122.6, 116.2, 116.0, 112.9, 64.0, 39.7, 37.8, 27.1, 21.7, 14.5, 13.8. ESI-MS m/z 537.2 [M+H]+ calc. for C31H32N6O3.

N-(2-aminophenyl)-5-[[4-ethoxy-3-(1-methyl-7-oxo-3-propyl-6H-pyrazolo[4,3d]pyrimidin-5-yl)phenyl]methyl]thiophene-2-carboxamide (29b) To a solution of compound 28b (70 mg, 0.11 mmol) in DMF (10 mL) was benzene-1,2diamine (24 mg, 0.22 mmol), DMAP (15 mg, 0.12 mmol) and DIEA (26 mg, 0.2 mmol) and the mixture was stirred at 60 ºC overnight. The mixture was quenched with water and then the standard extraction procedure was carried out to give the crude product which was purified by prep-HPLC (method 7 described in supporting information) to obtain pure compound 29b (12 mg, 20%) as a yellow solid; m.p. 195-196 ºC. 1H NMR (DMSOd6, 400 MHz): δ 11.95 (s, 1H), 9.58 (s, 1H), 7.80 (s, 1H), 7.56 (s, 1H), 7.41-7.40 (m, 1H), 7.13-7.00 (m, 2H), 6.97-6.95 (m, 2H), 6.76-6.74 (m, 1H), 6.58-6.56 (m, 1H), 4.86 (s, 2H), 4.19-4.01 (m, 7H), 2.78-2.75 (m, 2H), 1.76-1.71 (m, 2H), 1.33-1.30 (m, 3H), 0.95-0.91 (m, 3H). ESI-MS m/z 543.2 [M+H]+ calc. for C29H30N6O3S



N-(2-aminophenyl)-3-[[4-ethoxy-3-(1-methyl-7-oxo-3-propyl-6H-pyrazolo[4,3d]pyrimidin-5-yl)phenyl]methyl]cyclobutanecarboxamide (29c) To a solution of compound 28c (720 mg, 1.22 mmol) in DMF (40 mL) was added DIEA (348 mg, 2.7 mmol) and benzene-1,2-diamine (158 mg, 1.46 mmol) and the mixture was stirred at 80 ºC overnight. The reaction was quenched with water and then the standard extraction procedure was carried out to give the crude product which was purified by prep-HPLC (method 8 described in supporting information) to obtain pure compound 29c (19.8 mg) as white solid; m.p. 173-174 ºC. 1H NMR (MeOD, 400 MHz): δ 7.78-7.76 (m, 1H), 7.34-7.32 (m, 1H), 7.10-7.01 (m, 3H), 6.85-6.83 (m, 1H), 6.73-6.71 (m, 1H), 4.23 (s, 3H), 4.21-4.17 (m, 2H), 3.20-3.12 (m, 1H), 2.91-2.84 (m, 2H), 2.77-2.75 (m, 1H), 2.60-2.52 (m, 1H), 2.46-2.35 (m, 1H), 2.31 (m, 2H), 2.10-2.03 (m, 2H), 1.85-1.76 (m, 2H), 1.45 (t, J = 6.8 Hz, 3H), 1.01 (t, J = 7.6 Hz, 3H). ESI-MS m/z 515.3 [M+H]+ calc. for C29H34N6O3.



imidazo[5,1-f][1,2,4]triazin-2-yl)phenyl]methyl]cyclobutanecarboxylate (33c) To a solution of compound 32c (980 mg, 2.31 mmol) and DIC (466 mg, 3.7 mmol) in CH2Cl2 (50 mL) was added 2,3,4,5,6-pentafluorophenol (450 mg, 2.45 mmol) at 0 ºC under N2 and the mixture was stirred at room temperature overnight. Then, the standard extraction procedure was carried out to give the crude product which was purified by column chromatography to obtain pure compound 33c (1.15 g, 84%) as a yellow solid. ESI-MS m/z 591.3 [M+H]+ calc. for C29H27F5N4O4. This intermediate was used in the next step without further characterization.


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N-(2-aminophenyl)-3-[[4-ethoxy-3-(5-methyl-4-oxo-7-propyl-3H-imidazo[5,1f][1,2,4]triazin-2-yl)phenyl]methyl]cyclobutanecarboxamide (34c) To a solution of compound 33c (1.15 g, 1.95 mmol) in DMF (40 mL) was added DIEA (413 mg, 3.2 mmol) and benzene-1,2-diamine (238 mg, 2.2 mmol) and the mixture was stirred at 80 ºC overnight. The reaction was quenched with water and then the standard extraction procedure was carried out to give the crude product which was purified by prep-HPLC (method 8 described in supporting information) to obtain pure compound 34c (7.3 mg, 1%) as a yellow solid; m.p. 160-161 ºC. 1H NMR (MeOD, 400 MHz): δ 7.577.55 (m, 1H), 7.40-7.33 (m, 1H), 7.09-7.07 (m, 2H), 7.06-7.01 (m, 1H), 6.85-6.83 (m, 1H), 6.75-6.68 (m, 1H), 4.21-4.14 (m, 2H), 3.18-3.16 (m, 1H), 2.99-2.95 (m, 2H), 2.842.74 (m, 2H), 2.57 (s, 3H), 2.46-2.25 (m, 3H), 2.10-2.04 (m, 2H), 1.86-1.77 (m, 2H), 1.43 (t, J = 6.8 Hz, 3H), 0.97 (t, J = 7.6 Hz, 3H). ESI-MS m/z 515.3 [M+H]+ calc. for C29H34N6O3.

N-(4-amino-3-thienyl)-4-[[4-ethoxy-3-(1-methyl-7-oxo-3-propyl-6H-pyrazolo[4,3d]pyrimidin-5-yl)phenyl]methyl]benzamide (35a) To a solution of compound 28a (110 mg, 0.18 mmol) in DMF (10 mL) was added thiophene-3,4-diamine (25 mg, 0.22 mmol), DMAP (2 mg, 0.018 mmol) and DIEA (75 mg, 0.58 mmol) at 25 ºC and the mixture was stirred at 60 ºC overnight. Then, the mixture was concentrated to give the crude product which was purified by prep-HPLC (method 8 described in supporting information) to obtain pure compound 35a (4.5 mg, 5%). 1H NMR (MeOD, 400 MHz): δ 7.92-7.90 (m, 2H), 7.80 (s, 1H), 7.43-7.37 (m, 3H), 7.157.13 (m, 2H), 6.41-6.40 (m, 1H), 4.24-4.20 (m, 5H), 4.11 (s, 2H), 2.91-2.87 (m, 2H),



1.85-1.79 (m, 2H), 1.48-1.45 (m, 3H), 1.03-0.99 (m, 3H). ESI-MS m/z 543.3 [M+H]+ calc. for C29H30N6O3S.

N-(4-amino-3-thienyl)-3-[[4-ethoxy-3-(1-methyl-7-oxo-3-propyl-6H-pyrazolo[4,3d]pyrimidin-5-yl)phenyl]methyl]cyclobutanecarboxamide (35b) To a solution of compound 28c (100 mg, 0.17 mmol) in DMF (10 mL) was added thiophene-3,4-diamine (23 mg, 0.20 mmol), DMAP (2 mg, 0.017 mmol) and DIEA (44 mg, 0.34 mmol) and the mixture was stirred at 60 ºC overnight. Then, the mixture was concentrated to give the crude product which was purified by prep-HPLC (method 8 described in supporting information) to obtain pure compound 35b (10 mg, 11%) as a yellow solid; m.p. 157-158 ºC. 1H NMR (MeOD, 400 MHz): δ 7.77 (s, 1H), 7.36-7.28 (m, 2H), 7.11-7.09 (m, 1H), 6.32-6.31 (m, 1H), 4.25-4.19 (m, 5H), 3.17-3.14 (m, 1H), 2.93-2.89 (m, 2H), 2.79-2.77 (m, 1H), 2.59-2.34 (m, 2H), 2.33-2.31 (m, 2H), 2.11-2.08 (m, 2H), 1.87-1.84 (m, 2H), 1.49-1.45 (m, 3H), 1.05-1.01 (m, 3H). ESI-MS m/z 521.3 [M+H]+ calc. for C27H32N6O3S.

(2,3,4,5,6-pentafluorophenyl)


imidazo[5,1-f][1,2,4]triazin-2-yl)phenyl]methyl]benzoate (39a) To a solution of compound 38a (180 mg, 0.4 mmol) and DIC (153 mg, 1.21 mmol) in CH2Cl2 (10 mL) was added 2,3,4,5,6-pentafluorophenol (111 mg, 0.6 mmol) at 0 ºC under N2 and the mixture was stirred at room temperature overnight. Then, the mixture was concentrated to give the crude product which was purified by prep-TLC to obtain pure compound 39a (180 mg, 73%) as a pale yellow solid. ESI-MS m/z 613.2 [M+H]+ calc. 44 ACS Paragon Plus Environment

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for C31H25F5N4O4. This intermediate was used in the next step without further characterization.

N-(4-amino-3-thienyl)-4-[[4-ethoxy-3-(5-methyl-4-oxo-7-propyl-3H-imidazo[5,1f][1,2,4]triazin-2-yl)phenyl]methyl]benzamide (40a) To a solution of compound 39a (180 mg, 0.29 mmol) in DMF (10 mL) was added thiophene-3,4-diamine (61 mg, 0.53 mmol), DMAP (1.5 mg, 0.012 mmol) and DIEA (75 mg, 0.58 mmol) and the mixture was stirred at 60 ºC overnight. Then, the mixture was concentrated to give the crude product which was purified by prep-HPLC (method 8 described in supporting information) to obtain pure compound 40a (6 mg, 4%) as a yellow solid; m.p. 213-214 ºC. 1H NMR (MeOD, 400 MHz): δ 7.95-7.93 (m, 2H), 7.63-7.62 (m, 1H), 7.45-7.39 (m, 3H), 7.16-7.14 (m, 2H), 6.43-6.42 (m, 1H), 4.25-4.19 (m, 2H), 4.144.12 (m, 2H), 3.00-2.96 (m, 2H), 2.61 (s, 3H), 1.87-1.81 (m, 2H), 1.49-1.45 (m, 3H), 1.02-0.99 (m, 3H). ESI-MS m/z 543.3 [M+H]+ calc. for C29H30N6O3S.

Methyl

4-((4-((6-(benzo[d][1,3]dioxol-5-yl)-1,4-dioxo-3,4,6,7,12,12a-

hexahydropyrazino[1',2':1,6]pyrido[3,4-b]indol-2(1H)-yl)methyl)piperidin-1yl)methyl)benzoate (47b) To a solution of compound 46 (750 mg, 1.59 mmol) in CH2Cl2 (100 mL) were added methyl 4-formylbenzoate (313 mg, 1.91 mmol) and CH3COOH (1.00 mL) at room temperature. After stirring for 1 hour, NaBH3CN (200 mg, 2.90 mmol) was added and the mixture was stirred overnight. Then, the mixture was poured into water and extracted with CH2Cl2. The combined organic phase was washed with saturated brine, dried with 45 ACS Paragon Plus Environment


anhydrous Na2SO4, filtered, concentrated and purified by prep-TLC to obtain compound 47b (360 mg, 36%) as a white solid. ESI-MS m/z 621 [M+H]+ calc. for C36H36N4O6. This intermediate was used in the next step without further characterization.

4-((4-((6-(Benzo[d][1,3]dioxol-5-yl)-1,4-dioxo-3,4,6,7,12,12ahexahydropyrazino[1',2':1,6]pyrido[3,4-b]indol-2(1H)-yl)methyl)piperidin-1yl)methyl)benzoic acid (48b) To a mixture of compound 47b (360 mg, 0.58 mmol) in THF/MeOH/H2O (3:2:2, 60 mL) was added LiOH·H2O (59 mg, 1.41 mmol) at room temperature and the mixture was stirred overnight. The mixture was poured into water and then the standard extraction procedure was carried out to afford compound 48b (150 mg, 43%) as a white solid. ESIMS m/z 607 [M+H]+ calc. for C35H34N4O6. This intermediate was used in the next step without further characterization.

Perfluorophenyl

2-(4-((6-(benzo[d][1,3]dioxol-5-yl)-1,4-dioxo-3,4,6,7,12,12a-

hexahydropyrazino[1',2':1,6]pyrido[3,4-b]indol-2(1H)-yl)methyl)piperidin-1yl)pyrimidine-5-carboxylate (49a) To a mixture of compound 48a (450 mg, 0.76 mmol) and DIC (617 mg, 4.89 mmol) in CH2Cl2 (50 mL) was added 2,3,4,5,6-pentafluorophenol (167 mg, 0.91 mmol) at 0 °C under N2 and the mixture was stirred at room temperature overnight. Then, then the mixture was concentrated and purified by prep-HPLC (method 9 described in supporting information) to obtain pure compound 49a (300 mg, 52%) as a white solid. ESI-MS m/z


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761 [M+H]+ calc. for C38H29N6O6F5. This intermediate was used in the next step without further characterization.

Perfluorophenyl

4-((4-((6-(benzo[d][1,3]dioxol-5-yl)-1,4-dioxo-3,4,6,7,12,12a-

hexahydropyrazino[1',2':1,6]pyrido[3,4-b]indol-2(1H)-yl)methyl)piperidin-1yl)methyl)benzoate (49b) To a mixture of compound 48b (150 mg, 0.25 mmol) and DIC (47 mg, 0.37 mmol) in CH2Cl2 (20 mL) was added 2,3,4,5,6-pentafluorophenol (55 mg, 0.30 mmol) at 0 °C under N2 and the mixture was stirred at room temperature overnight. Then, the mixture was concentrated and purified by prep-HPLC (method 9 described in supporting information) to afford compound 49b (30 mg, 15%) as a white solid. ESI-MS m/z 773 [M+H]+ calc. for C41H33F5N4O6. This intermediate was used in the next step without further characterization.

N-(2-aminophenyl)-2-(4-((6-(benzo[d][1,3]dioxol-5-yl)-1,4-dioxo-3,4,6,7,12,12ahexahydropyrazino[1',2':1,6]pyrido[3,4-b]indol-2(1H)-yl)methyl)piperidin-1yl)pyrimidine-5-carboxamide (50a) To a solution of compound 49a (100 mg, 0.13 mmol) in DMF (20 mL) were added benzene-1,2-diamine (21.3 mg, 0.19 mmol), DIEA (3.4 mg, 0.026 mmol) and DMAP (1.6 mg, 0.013 mmol) and the mixture was stirred at 60 °C under N2 overnight. Then, the mixture was cooled to 25 °C and quenched with water. Then, the standard extraction procedure was carried out to give a residue which was purified by prep-HPLC (method 10 described in supporting information) to obtain pure compound 50a (12 mg, 13%) as a 47 ACS Paragon Plus Environment


white solid; m.p. 211-212 ºC. 1H NMR (CD3OD, 400 MHz): δ 8.87 (s, 2H), 7.53-7.51 (m, 1H), 7.32-7.30 (m, 1H), 7.17-7.10 (m, 2H), 7.08-7.04 (m, 2H), 6.97 (s, 1H), 6.90 (dd, J = 7.9, 1.3 Hz, 1H), 6.80 (d, J = 1.8 Hz, 1H), 6.78-6.76 (m, 2H), 6.70 (dd, J = 7.9, 1.3 Hz, 1H), 5.94 (s, 2H), 4.58 (s, 2H), 4.41 (d, J = 17.6 Hz, 1H), 4.31 (dd, J = 11.7, 4.2 Hz, 1H), 4.10 (d, J = 17.6 Hz, 1H), 3.55-3.44 (m, 1H), 3.40 (dd, J = 15.2, 4.2 Hz, 1H), 3.19 (dd, J = 13.7, 6.6 Hz, 1H), 3.03-3.00 (m, 3H), 2.15-2.08 (m, 1H), 1.79-1.75 (m, 2H), 1.29-1.23 (m, 2H). ESI-MS m/z 685.4 [M+H]+ calc. for C38H36N8O5.

N-(2-aminophenyl)-4-((4-((6-(benzo[d][1,3]dioxol-5-yl)-1,4-dioxo-3,4,6,7,12,12ahexahydropyrazino[1',2':1,6]pyrido[3,4-b]indol-2(1H)-yl)methyl)piperidin-1yl)methyl)benzamide (50b) To a solution of compound 49b (30 mg, 0.04 mmol) in DMF (5.00 mL) were added benzene-1,2-diamine (6.3 mg, 0.06 mmol), DMAP (0.5 mg, 4.0 µmol) and DIEA (10 mg, 0.08 mmol) and the mixture was stirred at 60 °C overnight. Then, the mixture was concentrated under vacuum to give the crude product which was purified by prep-HPLC (method 11 described in supporting information) to give pure compound 50b (4.6 mg, 17%). 1H NMR (CD3OD, 400 MHz): δ 8.04 (d, J = 7.28 Hz, 2H), 7.59 (d, J = 6.53 Hz, 2H), 7.49 (d, J = 7.53 Hz, 1H), 7.31 (d, J = 8.28 Hz, 1H), 7.21-7.13 (m, 2H), 7.11-7.02 (m, 2H), 6.96 (s, 1H), 6.92 (d, J = 8.03 Hz, 1H), 6.82-6.75 (m, 3H), 6.67 (d, J = 8.28 Hz, 1H), 5.93 (d, J = 1.51 Hz, 2H), 4.58 (br s, 2H), 4.32 (s, 1H), 4.11-4.05 (m, 2H), 3.42 (br s, 1H), 3.39 (br s, 1H), 3.06 (d, J = 11.29 Hz, 1H), 2.70-2.60 (m, 3H), 2.00 (d, J = 12.05 Hz, 1H), 1.84 (br s, 4H), 1.49 (br s, 2H). ESI-MS m/z 697.5 [M+H]+ calc. for C41H40N6O5.


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Tert-butyl

N-[2-[[4-[[4-ethoxy-3-(1-methyl-7-oxo-3-propyl-6H-pyrazolo[4,3-

d]pyrimidin-5-yl)phenyl]methyl]benzoyl]amino]-4-(2-thienyl)phenyl]carbamate (51a) A mixture of 27a (150 mg, 0.34 mmol) in SOCl2 (400 mg, 3.36 mmol) was stirred at 80 ºC for 2 hours and then the mixture was concentrated in reduced pressure at 45 ºC. The residue was dissolved in CH2Cl2 (5 mL) and added dropwise to a solution of tert-butyl N[2-amino-4-(2-thienyl)phenyl]carbamate (Int. 4, synthesis described in supporting information) (98 mg, 0.34 mmol) and TEA (68 mg, 0.67 mmol) in CH2Cl2 (5 mL) at room temperature. The mixture was concentrated and purified by prep-TLC to obtain crude compound 51a (200 mg, 82%) which was used in the next step without further purification. ESI-MS m/z 719.3 [M+H]+ calc. for C40H42N6O5S. This intermediate was used in the next step without further characterization.

Tert-butyl

N-[2-[[4-[[4-ethoxy-3-(1-methyl-7-oxo-3-propyl-6H-pyrazolo[4,3-

d]pyrimidin-5-yl)phenyl]methyl]benzoyl]amino]-4-fluoro-phenyl]carbamate (51b) A mixture of 27a (150 mg, 0.34 mmol) in SOCl2 (400 mg, 3.36 mmol) was stirred at 80 ºC for 2 hours and then the mixture was concentrated in reduced pressure at 45 ºC. The residue was dissolved in CH2Cl2 (5 mL) and added dropwise to a solution of tert-butyl N(2-amino-4-fluoro-phenyl)carbamate (Int. 5, synthesis described in supporting information) (75 mg, 0.33 mmol) and TEA (67 mg, 0.66 mmol) in CH2Cl2 (5 mL) at room temperature. The mixture was concentrated and purified by prep-TLC to obtain crude compound 51b (100 mg, 45%) which was used in the next step without further purification. ESI-MS m/z 655.3 [M+H]+ calc. for C36H39FN6O5. This intermediate was used in the next step without further characterization. 49 ACS Paragon Plus Environment


N-[2-amino-5-(2-thienyl)phenyl]-4-[[4-ethoxy-3-(1-methyl-7-oxo-3-propyl-6Hpyrazolo[4,3-d]pyrimidin-5-yl)phenyl]methyl]benzamide (52a) A mixture of 51a (200 mg, 0.28 mmol) in HCl/EtOAc (4 N, 5mL) was stirred at 25 ºC for 16 hours and then concentrated in reduced pressure at 40 ºC. The residue was then purified by prep-HPLC (method 8 described in supporting information) to obtain pure compound 52a (12 mg, 7%) as a white solid; m.p. 134-135 ºC. 1H NMR (CDCl3, 400 MHz): δ 11.11 (br s, 1H), 8.33 (d, J = 2.2 Hz, 1H), 7.89 (d, J = 8.4 Hz, 2H), 7.87 (br s, 1H), 7.60 (s, 1H), 7.38-7.36 (m, 3H), 7.27 (dd, J = 8.4, 2.2 Hz, 1H), 7.20-7.18 (m, 2H), 7.05 (dd, J = 5.1, 3.7 Hz, 1H), 7.04-7.00 (m, 1H), 6.87 (d, J = 8.4 Hz, 1H), 4.30-4.25 (m, 5H), 4.11 (s, 2H), 4.02-3.96 (m, 2H), 2.96-2.92 (m, 2H), 1.91-1.85 (m, 2H), 1.61-1.50 (m, 3H), 1.05 (t, J = 7.3 Hz, 3H). ESI-MS m/z 619.3 [M+H]+ calc. for C35H34N6O3S.

N-(2-amino-5-fluoro-phenyl)-4-[[4-ethoxy-3-(1-methyl-7-oxo-3-propyl-6Hpyrazolo[4,3-d]pyrimidin-5-yl)phenyl]methyl]benzamide (52b) A mixture of 51b (100 mg, 0.15 mmol) in HCl/EtOAc (4.0 N, 5mL) was stirred at 25 ºC for 16 hours and then concentrated in reduced pressure at 40 ºC. The residue was then purified by prep-HPLC (method 8 described in supporting information) to obtain pure compound 52b (9 mg, 11%) as a white solid; m.p. 157-158 ºC. 1H NMR (MeOD, 400 MHz): δ 7.94 (d, J = 8.4 Hz, 2H), 7.80 (s, 1H), 7.43-7.38 (m, 3H), 7.15-7.14 (m, 1H), 7.13-7.09 (m, 1H), 6.90-6.89 (m, 1H), 6.87-6.83 (m, 1H), 4.25-4.20 (m, 5H), 4.12 (s, 2H), 2.91-2.87 (t, J =14.8 Hz, 2H), 1.85-1.75 (m, 2H), 1.47 (t, J =14 Hz, 3H), 1.02 (t, J =15.2 Hz, 3H). ESI-MS m/z 555.3 [M+H]+ calc. for C31H31FN6O3.


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5-[5-(2-bromoacetyl)-2-ethoxy-phenyl]-1-methyl-3-propyl-6H-pyrazolo[4,3d]pyrimidin-7-one (53) AlCl3 (1.28 g, 9.60 mmol) was added portionwise over 30 minutes to a stirred solution of 14 (1 g, 3.20 mmol) and 2-bromoacetyl bromide (1.29 g, 6.40 mmol) in CH2Cl2 (80 mL) at 0 ºC and the mixture was stirred at 15 ºC for 15.5 hours. Then, the reaction mixture was quenched by addition of water (50 mL) at 0 °C and extracted with CH2Cl2. The combined organic layers were washed with brine, dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue which was purified by column chromatography to obtain pure compound 53 (340 mg, 25%). 1H NMR (CDCl3, 400 MHz): δ 10.81 (br s, 1H), 9.11 (d, J = 2.4 Hz, 1H), 8.14 (dd, J = 2.4, 8.4 Hz, 1H), 7.177.14 (m, 1H), 4.51 (s, 2H), 4.40 (q, J = 6.8 Hz, 2H), 4.29 (s, 3H), 2.97 (t, J =7.4 Hz, 2H), 1.95-1.88 (m, 2H), 1.68-1.58 (m, 3H), 1.06 (t, J = 7.2 Hz, 3H). ESI-MS m/z 433.2 [M+H]+ calc. for C19H21N4O3Br.

5-[2-ethoxy-5-[2-[(4-hydroxy-6-oxo-1H-pyrimidin-2-yl)sulfanyl]acetyl]phenyl]-1methyl-3-propyl-6H-pyrazolo[4,3-d]pyrimidin-7-one (54) To a solution of 4-hydroxy-2-sulfanyl-1H-pyrimidin-6-one (113 mg, 0.78 mmol) and NaOH (63 mg, 1.57 mmol) in H2O (16 mL) was added dropwise a solution of compound 53 (340 mg, 0.78 mmol) in EtOH (8 mL) and then the mixture was stirred at 20 °C for 2 hours. The reaction mixture was quenched by addition of water (20 mL) at 0 °C. Then, the obtained white solid was filtered and purified by prep-HPLC (method 12 described in supporting information) to obtain pure compound 54 (49.8 mg, 13%) as a white solid;



m.p. 232-233 ºC. 1H NMR (DMSO-d6, 400 MHz): δ 12.22 (s, 1H), 8.18 (s, 2H), 7.29 (d, J = 9.0 Hz, 1H), 5.16 (s, 1H), 4.82 (s, 2H), 4.24-4.19 (m, 2H), 4.16 (s, 3H), 2.77 (t, J = 6.8 Hz, 2H), 1.80-1.66 (m, 2H), 1.34 (t, J = 6.2 Hz, 3H), 0.98-0.88 (m, 3H). ESI-MS m/z 497.2 [M+H]+ calc. for C23H24N6O5.

Docking into HDAC1 and HDAC2 Docking simulations into the crystal structure of HDAC2 complexed with N-(4aminobiphenyl-3-yl)benzamide inhibitor 9 (PDB entry 3MAX) [34] and PDE5 complexed with sildenafil (PDB entry 1TBF) [41] were carried out with Gold program [44]. The binding site was defined as a 20-Å sphere around the Zn atom (HDACs). The PLP scoring function was used to rank docking poses, requiring a total of 100 poses per ligand. Optimization parameters were left at the “slow-accurate docking” settings. Previous validation (RMSD < 2 Å) was carried out for docking of the corresponding cocrystallized ligand into HDAC2 and PDE5.

Details about some assays utilized to test these novel molecules had been previously reported by our group: a) HDACs and PDEs enzyme activity assays [12,25,26], b) jumpdilution HDAC1 enzyme activity assay [26], c) acetyl-Histone H3 Lysine 9 (H3K9ac) cellular detection assay (AlphaLisa technology) [26], d) cytotoxicity in THLE-2 cells [12,25,26], e) cytotoxicity in neurons glia cells [12,25,26], f) cytotoxicity in PBMC cells [25], g) PAMPA permeability [12,25,26], h) PDE and HDAC functional response in vitro [25], i) western blot analysis of histones and pCREB [25], j) CYP inhibition [25,26], k) plasma protein binding [25], l) kinetic solubility [25], m) caco-2 permeability [45], n) 52 ACS Paragon Plus Environment

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human and mouse liver microsomal stability [25,26], o) hERG channel test on manual patch-clamp [45] and p) fear conditioning test [46]. Other tests are new or include significant modifications; then, they are explicitly described (below).

PDE and HDAC functional response in vivo To confirm the ability of 29a to inhibit HDAC and PDE in the brain, the compound (20 mg/kg) was administered to WT mice (n=3). 15 min, 30 min or one hour later, mice were sacrificed and their hippocampus was quickly dissected from the brains. Total tissue homogenates were obtained by homogenizing the hippocampus in a lysis buffer containing Tris HCl 10 mM, NaF 1 mM, NaVO4 0.1 mM, sodium dodecyl sulfate (SDS) 2% and protease inhibitors. Western blot was carried out to analyze pCREB-Ser133 and AcH3K9.

Pharmacokinetic study of compound 29a in plasma samples Compound 29a was measured in plasma samples using an Acquity UPLC system (Waters, Manchester, UK) coupled to a Xevo-TQ MS triple quadrupole mass spectrometer with electrospray ionization (ESI) source. Solutions of compound 29a were prepared by dissolving the solid in dimethyl sulfoxide (DMSO) and this solution was made up to a final volume by addition of a mixture of Tween 20 and 0.9% NaCl (1/1/8, v:v:v, DMSO/Tween 20/saline). A drug dosage of 20 mg/kg was administered as a single intraperitoneal injection. Blood was collected at predetermined times post injection (0.25, 0.75, 2, 5, 8 and 24 h) into EDTA-containing 53 ACS Paragon Plus Environment


tubes and plasma was obtained via centrifugation (4˚ C, 1200 rpm, 10 min). Samples were analyzed the same day right after extraction. Details about the chromatographic separation and compound quantification are described in the Supporting Information. The pharmacokinetic parameters were obtained by fitting the plasma concentration-time data to a non-compartmental model with the WinNonlin software (v6.3, Pharsight, Mountain View, CA). The estimated parameters are the Area Under the Curve computed to the last observation (AUClast), half-life of the product (t½), clearance (Cl) and volume of distribution (V).

Determination of brain to plasma concentration ratio of 29a at Tmax Pharmacokinetic study of compound 29a showed that the maximum time to reach the peak plasma concentration (Tmax) was reached at 15 minutes. To obtain the brain to plasma (B/P) ratio at Tmax, compound 29a was injected (20 mg/kg, i.p.) to mice (n=5). Then, 15 minutes after injection, plasma was collected and the brain was removed.

Brains were homogenized using a Branson 250 ultrasonic probe sonicator (Branson, Danbury, Connecticut, USA) after the addition of 670 µL PBS/g tissue. 50 µL of the brain homogenate were subjected to the sample preparation procedure described in the pharmacokinetics section. Chromatographic separation, compound detection and quantification of both plasma and brain samples was carried out as described previously in the pharmacokinetics section.


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ASSOCIATED CONTENT Supporting Information Details about purification methods, synthesis of intermediates, purities and HPLC traces for final compounds, PK parameters and Figure S1.

AUTHOR INFORMATION Corresponding author *For

J. O.: phone, +34 948 19 47 00, ext 2044. E-mail, [email protected]

Notes These authors declare no competing financial interest.

ABBREVIATIONS 9-BBN, 9-borabicyclo[3.3.1]nonane; ACN, acetonitrile; AD, Alzheimer’s disease; ADME, absorption, distribution, metabolism and excretion; ATP, adenosine 5'triphosphate; BBB, blood-brain barrier; BOC, tert-butoxycarbonyl; BPO, benzoyl peroxide; BSA, bovine serum albumin; cAMP, 3',5'-cyclic adenosine monophosphate; cGMP, 3',5'-cyclic guanosine monophosphate; CNS, central nervous system; CREB, cAMP response element-binding protein; DIC, N,N′-diisopropylcarbodiimide; DIEA, diethanolamine, DMAP, 4-(N,N-dimethylamino)pyridine; DMF, dimethylformamide; DMSO, dimethylsulfoxide; EDC·HCl, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride; EDTA, ethylenediaminetetraacetic acid; EGTA, ethylene glycol-bis(2-



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aminoethylether)-N,N,N′,N′-tetraacetic acid; ESI-MS, electrospray ionisation mass spectrometry, Et3N, triethylamine; EtOAc, ethyl acetate; EtOH, ethanol; FBS, fetal bovine serum; FDA, Food and Drug Administration; HDAC, histone deacetylase; HEPES,

4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic

acid;

HOBt,

hydroxybenzotriazole; HPLC, high-performance liquid chromatography; LCMS, liquid chromatography–mass spectrometry; MeOH, methanol; MOA, mechanism of action; Mw, molecular weight; MW, microwave; NADPH, nicotinamide adenine dinucleotide phosphate hydrogen; NIS, N-iodosuccinimide; NMM, N-methylmorpholine; NMR, nuclear magnetic resonance; PAMPA, parallel artificial membrane permeability assay; PBMCs, peripheral blood mononuclear cells; PBS, phosphate buffered saline; Pd2(dba)3, tris(dibenzylideneacetone)dipalladium(0),

Pd(dppf)Cl2,

[1,1′-

bis(diphenylphosphino)ferrocene]dichloropalladium(II); PDE, phosphodiesterase; PFP, pentafluorophenyl; PK, pharmacokinetic; PSA, polar surface area; pTau, phosphorylated Tau; PTFE, polytetrafluoroethylene; PVDF, polyvinylidene difluoride; rt, room temperature; Rt, retention time; SAR, structure-activity relationship; TBS, tris-buffered saline; TEA, triethanolamine; TFA, trifluoroacetic acid; THF, tetrahydrofuran; THP, tetrahydropyranyl; THPONH2, N-(tetrahydro-2H-pyran-2-yloxy)amine; TLC, thin-layer chromatography; TMS, tetramethylsilane; TPSA, total polar surface area; UPLC, ultra performance liquid chromatography; UV, ultraviolet; WT, wild type; xantphos, 4,5bis(diphenylphosphino)-9,9-dimethylxanthene,

X-Phos,

2′,4′,6′-triisopropylbiphenyl; ZBG, zinc binding group.

ACKNOWLEDGEMENTS


2-dicyclohexylphosphino-

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We thank the Foundation for Applied Medical Research, University of Navarra (Pamplona, Spain) as well as to Fundación Fuentes Dutor for financial support. This work has been partially supported through Ministerio de Economía y Competitividad (FIS PI12/00710), and FSE (Inncorpora-Torres Quevedo grant), PTQ-12-05641 (A.U.) and PTQ-14-07320 (I. dM.). This work was supported by grants from FIS projects (11/02861 and 14/01244).

Authors Contributions:

Conception and design: O.R., J.S.-A., M.C.-T., A.G.-O. and J.O. Development of methodology: O. R., J.S.-A., M.C.-T., I.DM., A.U., H.T., W.W., M.X., A.G.-O. and J.O. Acquisition of data: O.R., J.S.-A., M.P.-

G., C.G.-B., A.U., A.E., E.S., M.E., S.U.,

H.T. and W.W. Analysis and interpretation of data: O. R., J.S.-A., M.C.-T., I.DM., A.U., A.E., H.T., W.W., A.G.-O. and J.O. Writing and/or revision of the manuscript:

O.R.,

I.DM., M.C.-T., A.G.-O. and J.O. Study supervision: M.C.-T., A.G.-O. and J.O.

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For Table of Contents Use Only

Discovery of in-vivo chemical probes for treating Alzheimer´s disease: Dual phosphodiesterase 5 (PDE5) and class I histone deacetylasesselective inhibitors.

Obdulia Rabal,1,5 Juan A. Sánchez-Arias,1,5 Mar Cuadrado-Tejedor,2,3,5 Irene de Miguel,1 Marta Pérez-González,2 Carolina García-Barroso,2 Ana Ugarte,1 Ander Estella-Hermoso de Mendoza,1 Elena Sáez,1 Maria Espelosin,2 Susana Ursua,2 Tan Haizhong,4 Wu Wei,4 Xu Musheng,4 Ana Garcia-Osta,2,5 and Julen Oyarzabal.1,5,*


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Discovery of in Vivo Chemical Probes for Treating Alzheimer's

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