Identification and Pharmacological Profile of an Indane Based Series

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Identification and pharmacological profile of an indane based series of ghrelin receptor full agonists Cristina Gardelli, Hiroki Wada, Asim Ray, Moya Caffrey, Antonio Llinas, Igor Shamovsky, Joakim Tholander, Joakim Larsson, Ulf Sivars, Leif Hultin, Ulf Andersson, Hitesh J. Sanganee, Kristina Stenvall, Brith Leidvik, Karin Gedda, Lisa Jinton, Marie Ryden Landergren, and Kostas Karabelas J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b00322 • Publication Date (Web): 17 Jun 2018 Downloaded from http://pubs.acs.org on June 18, 2018

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Journal of Medicinal Chemistry

Identification and pharmacological profile of an indane based series of ghrelin receptor full agonists Cristina Gardelli,#,* Hiroki Wada,#,† Asim Ray,# Moya Caffrey,# Antonio Llinas, Igor Shamovsky,# Joakim Tholander,,† Joakim Larsson,#,† Ulf Sivars, Leif Hultin, Ulf Andersson,ǂ Hitesh J. Sanganee,§ Kristina Stenvall,ǁ Brith Leidvik,¤,† Karin Gedda,¤ Lisa Jinton, Marie Rydén Landergren,# Kostas Karabelasǁ,† #

Medicinal Chemistry Department,

DMPK Department,

Bioscience Department, ǁProjects

Department Respiratory, Inflammation and Autoimmunity IMED Biotech Unit, AstraZeneca Gothenburg, 43183 Mölndal, Sweden;

Medicinal Chemistry Department, Cardiovascular and

Metabolic Diseases IMED Biotech Unit, AstraZeneca Gothenburg, 43183 Mölndal, Sweden;

Precision Medicine Laboratories, Precision Medicine and Genomics IMED Biotech Unit,

AstraZeneca Gothenburg, 43183 Mölndal, Sweden; ǂDrug Safety and Metabolism IMED Biotech Unit, AstraZeneca Gothenburg, 43183 Mölndal, Sweden; §Scientific Partnering & Alliances IMED Biotech Unit, AstraZeneca, SK10 4TF Cambridge, United Kingdom; ¤Discovery Sciences IMED Biotech Unit, AstraZeneca Gothenburg, 43183 Mölndal, Sweden. ABSTRACT. Cachexia and muscle wasting are very common among patients suffering from cancer, chronic obstructive pulmonary disease and other chronic diseases. Ghrelin stimulates growth hormone secretion via the ghrelin receptor, which subsequently leads to increase of IGF1 plasma levels. The activation of the GH/IGF-1 axis leads to an increase of muscle mass and

ACS Paragon Plus Environment

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functional capacity. Ghrelin further acts on inflammation, appetite and adipogenesis, and for this reason was considered an important target to address catabolic conditions. We report the synthesis and properties of an indane based series of ghrelin receptor full agonists; they have been shown to generate a sustained increase of IGF-1 levels in dog and have been thoroughly investigated with respect to their functional activity. INTRODUCTION Ghrelin is a 28 amino acid peptidic hormone, characterized by a post-translational noctanoylation on the Serine 3 residue. It is synthesized and secreted by X/A-like cells localized within the oxyntic glands of the mucosa of the gastric fundus.1 It is highly conserved across several species indicating its physiological importance.2 Its pharmacological properties are mediated by the type 1a growth hormone secretagogue receptor (GHS-R1a), a seven transmembrane

G-protein-coupled

receptor

(GPCR)

expressed

predominantly

in

the

hypothalamus and in the pituitary gland. Ghrelin exhibits several biological activities: it is an orexigenic agent, stimulates secretion of growth hormone (GH, an anabolic hormone that stimulates body growth), enhances gastric motility and acts on the cardiovascular system to increase cardiac output. With this wide range of biological activities, the ghrelin receptor represents an interesting target for drug discovery. We were particularly interested in the capability of ghrelin to increase endogenous GH and to elevate the plasma level of a second hormone called insulin-like growth factor-1 (IGF-1); consequently, it could have exciting applications in diseases where skeletal muscles are impaired as in cachexia which is associated with many chronic diseases such as cancer, chronic obstructive pulmonary disease (COPD), chronic heart failure, HIV and many others. The use of ghrelin to reduce cachexia and to increase functional capacity in cachectic patients with COPD has clinical precedence. First, Nagaya et al.3


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demonstrated, in an open-label pilot study, that intravenous administration of human ghrelin for three weeks to cachectic patients with COPD markedly increased growth hormone secretion, food intake, body weight, lean body mass, peripheral and respiratory muscle strength, the Karnofsky performance status score (a marker for functional capacity), as well as distance walked in six minutes. Later SUN11031,4 a synthetic human ghrelin form, was brought into a Phase 2 study where 224 cachectic patients with COPD were treated to determine whether it could enhance body weight, lean body mass and physical performance. Treatment resulted in a rapid and significant increase in body weight and lean body mass although without significant improvement in functional performance measures. However, subgroup analysis revealed a statistically significant increase in functional performance in subjects with more advanced cachexia. Studies in other chronic disease conditions showed reduced muscle wasting and improved exercise capacity after ghrelin treatment in patients with chronic heart failure.5 Employing exogenous ghrelin administration shows that the half-life of total ghrelin is between 10 and 31 minutes in the mammalian bloodstream;6 therefore a number of programs were dedicated to identifying long acting growth hormone secretagogues (GHS) with the potential to increase 24 hours IGF-1 levels.7-8 A number of short acting GHSs have been previously described as full agonists of the ghrelin receptor; a few of them have reached advanced clinical stages for cancer cachexia, frailty in the elderly and gastrointestinal diseases.9-15 A high-throughput screening (HTS) campaign of AstraZeneca´s proprietary collection to identify ghrelin agonists, followed by a hit to lead program, led to the discovery of a series of indane diamides (compounds 1-3) with submicromolar potency (Figure 1, Table 1). This lead series showed partial agonism expressed by the effect of the compounds as a percentage of maximal effect of growth hormone-releasing peptide 6 (GHRP-6). We then embarked on a lead


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optimization project to switch from partial to full agonism by structural alterations as previously reported for other GPCRs.16,17 Furthermore, we optimized against a poor selectivity of the chemical series towards the hERG channel. Herein, we report our efforts and SAR analysis leading to the identification of compound 39, showing the required pharmacological profile for in vivo studies to predict GH/IGF-1 activity in humans.

1

2

3

39

Figure 1. Initial leads in the indane series 1-3 and optimized compound 39.

CHEMISTRY

Indane 1,2 diaminoamides 4, 6, 8-21, 34 and 51 were synthesized in seven steps in overall yield of around 50% from the commercially available (1S, 2R) and (1R, 2R)-aminoindanols following Scheme 1.18 1-Amino-2-hydroxyindane 44 and 52 were Boc protected and an azide group was inserted through mesylation and displacement with NaN3 to give intermediates 47 and 54. Hydrogenation with Pd/C provided amines 48 and 55 which were coupled with the diverse carboxylic acids to provide intermediates 49 and 56. Boc deprotection with tbutyldimethylsilyltriflate and a second amide coupling provided the desired analogs 4, 6, 8-21, 34 and 51. These overall sequences were also applied to provide enantiomers 5 and 7 starting from the opposite enantiomers of 1-amino-2-hydroxyindanes. Scheme 1.aSynthesis of indane diamino derivatives 4, 6, 8-21, 34 and 51.


4

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a (1S,2R)

c

b

44

45

d 47

46

e

g

f

(1S,2S)

48

49

50

a

(1R, 2R)

g

f

56

55

54

53

e

d

b,c

52

4, 8-21, 34, 51

57

6

a

Reagents and conditions: (a) Boc2O, Et3N, DCM, r.t., o/n; (b) MsCl, Et3N, DCM, 0º to r.t., 3h; (c) NaN3, dry DMF, 90ºC, o/n. (d) H2, 10% Pd/C, o/n; (e) RCOOH or 5-methyl-1H-indole-2carboxylic acid, DIPEA, HOBT, DCM, EDCI, r.t.; (f) TBDMSOTf, DCM, 6 h, r.t.; (g) R1COOH, HATU, Et3N, DMF. Compounds reported in Table 3 were obtained from compound 51 or analogs. As an example, 51 was submitted to Boc deprotection of the corresponding morpholine to give compound 32 and methylated through reductive amination to give 23, Scheme 2. Homomorpholines, piperidine, homopiperidine and pyrrolidine derivatives were treated similarly to yield compounds 22-33 and 35-38. Compound 34 was obtained by acetylation of 32 with acetic anhydride. Scheme 2. aSynthesis of compound 23 (as representative of the synthesis of compounds 22-33

and 35-38) and 34.


5


b or c

a

51

Page 6 of 44

32

R = Me, 23 R = Ac, 34

a

Reagents and conditions: (a) TBDMSOTf, DCM, 6 h, r.t.; (b) HCHO, MeOH, NaBH3CN; (c) Ac2O. The reverse amide 39-43 were synthesized from the commercially available ketoester 58,

Scheme 3. Condensation of compound 58 with NH4OAc gave enamine 59 that was reduced with NaBH4 and TFA in THF (caution, exothermic reaction). Boc protection yielded beta amino protected ester 60, in a cis:trans ratio of 1:1. The mixture, was submitted to hydrolysis to obtain acid 61 as a cis:trans 1:6.3 ratio. After HPLC, the pure trans acid 62 was submitted to amide coupling with diverse amines and Boc deprotected. HPLC separation gave the pure enantiomers 63a and 63b. The desired enantiomer 63b was then coupled with a variety of carboxylic acids to give final compounds 39-43, Table 4. Scheme 3. aSynthesis of β-amino acids derivatives 39-43.

b,c

a 58

60, 1:1 trans:cis

59

e 61, 6.3:1 trans:cis

d

h,i

f,g 62, pure trans

39-43 63a and 63b

a

Reagents and conditions: (a) NH4OAc, MeOH, 50°; (b) TFA, NaBH4, THF; (c) Boc2O; (d) MeONa, H2O, MeOH; (e) HPLC; (f) N1,N1-dimethylethane-1,2-diamine or NH2R, HATU, Et3N, DMF-DCM; (g) HCl, dioxane; (h) chiral HPLC; (i) R1COOH, HATU, Et3N, DMF-DCM.


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Absolute configuration of key compound 39 and its enantiomer 40 was assigned by vibrational circular dichroism (VCD) analyses in an indirect way. Direct VCD analysis of the final compounds 39 and 40 gave inconclusive results and therefore smaller analog intermediates 65a and 65b were prepared for VCD analyses, Scheme 4. The pure trans racemic ester 62 was submitted to chiral separation to obtain the two enantiomers 62a and 62b. Further manipulation of these two enantiomers led to N,N dimethyl amides 65a and 65b whose absolute configuration was assigned by VCD. Once the absolute configuration of the analogs 65a and 65b was assigned, the absolute configuration of the Boc protected acids 62a and 62b was revealed as well. Compounds 39 and 40 were resynthesized starting from these intermediates and their respective 1S,2S and 1R,2R absolute configuration was assigned. For VCD spectra see Supplementary Material. Scheme 4. Synthesis of the intermediates 65a and 65b for the elucidation of the absolute configuration of the indane compounds 39-40 through VCD.

a 62 pure racemic trans

62a

c

b 62a

62b

65a

64a 40

b 62b

c 64b

65b

39


7


Page 8 of 44

a

Reagents and conditions: (a) chiral HPLC; (b) NHMe2, DIPEA, HATU, PyBOP, dry DMF; (c) HCl 4M in dioxane. RESULTS AND DISCUSSION The binding activity of the compounds to the GHS-R1a receptor was measured in a competition experiment with [125I]-ghrelin in membranes prepared from HEK293 cell line. The agonistic activity of the compounds was assessed by measuring intracellular myo-Inositol 1 phosphate (IP1). D-myo-Inositol 1-phosphate as a surrogate of D-myo-inositol 1,4,5-triphosphate serves to monitor G protein-coupled receptor activation. As a comparison, ghrelin, Capromorelin and Ibutamoren data are reported as well. The indane lead series showed pEC50 between 6.8 and 7.5 in the functional assay and a moderate metabolic stability in human liver microsomes. The compounds were characterized by a low agonism and a lack of human ERG selectivity. The initial medicinal chemistry work focused on extending the chemical diversity of the series. It confirmed the required stereochemistry in positions 1 and 2 of the indane core and assessed the characteristics of the pharmacophoric features for further optimization. O

N

N

NH

O

N

NH H N N H

NH H N

N H

O

4

O

N

NH H N

O

O

5

O

H N N H

N H

O

6

7

Figure 2. Compounds 4-7 made to assess the desired stereochemistry. Table 1. Potency in binding and functional assay, agonism and potency in hERG assay of compounds 1-7, ghrelin, Capromorelin and Ibutamoren. Binding[a]

Functional assay[b]

Entry 1

pIC50

IC50 (µM)

6.4

0.43

pEC50 and agonism

[c]

(%)

6.8 (75)

EC50 (µM) 0.160

hERG pIC50[d] 5.8


8

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2

7.0

0.098

7.5 (52)

0.035

5.4

3

6.7

0.189

7.3 (61)

0.047

< 5.0

4

7.8

0.016

7.7 (54)

0.021

5.1

5

6.0

0.93

6.6 (40)

0.226

5.6

6

7.2

0.069

7.0 (84)

0.10

4.6

7

5.9

1.26

5.8 (101)

1.5

5.2

ghrelin3

8.05

0.009

7.7 (82%)

0.021

-

Capromorelin9

7.7

0.022

9.04 (87%)

0.0009

-

Ibutamoren11

8.7

0.002

9.4 (90%)

0.0004

-

n= 40 n= 9 n= 42

[a]

In vitro assay. Binding assay to detect compounds binding to the GHS-R1a expressed in HEK293 cells, by competing with [125I]-ghrelin. Values are the mean of at least 2 independent determinations; for reference compounds n ≥ 8. [b,c]In vitro assay. Agonist activation of the ghrelin receptor results in a production of IP1. IP1 is accumulated in the cell in presence of LiCl and measured by time resolved fluorescence. Values are the mean of at least 2 independent determinations. The assay validation resulted in a CIR value of +/- 0.07 and the MDR value of 0.1 (on a log scale) when tested at 2 separate occasions. For reference compounds n ≥ 9. [d]Effect of the compounds on the voltage-dependent potassium channel encoded by the human ether-ago-go-related gene (hERG). The preferred stereochemistry was assessed through the synthesis of the four diastereoisomers 47, Figure 2. The (1S,2S) trans enantiomer 4 proved to have higher affinity than its enantiomer 5 with a pIC50 of 7.8 vs 6.0 in the binding assay. The cis diastereoisomers 6 and 7 showed higher agonism but lower potency. The preferred conformations of nine ghrelin full agonists reported in the literature including SM130686,19 MK-677 (Ibutamoren),20 ONO-7643 (Anamorelin),21 CP-424391 (Capromorelin),22 CP-464709,7 SB791016 and GSK894490A,23 L-163540,24 and aryl sulphonamide25 were superimposed with those of our leads, using the software Phase (Schrödinger LLC).26 The calculations indicate three pharmacophoric features, a basic (A), an aromatic residue (B) and a H-bond acceptor (C) which are likely relevant for the interaction with the ghrelin receptor,


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Page 10 of 44

Figure 3. These features are consistent with the putative binding site proposed by a published homology model of the ghrelin receptor that was built based on (1) the X-ray structure of the inactive state of rhodopsin and (2) results of site-directed mutagenesis studies.27 To understand the origin for the observed structure-activity relationships, the identified pharmacophore features and established mutational hits28 have to be linked to a putative binding mode of the compounds in the binding site of the receptor. The basic center A likely forms a salt-bridge to the acidic residue Glu-124 of the receptor, a very important mutational hit, which is used as a major anchoring site for agonists, antagonists and inverse agonists in many published attempts to derive potency and efficacy from binding mode.28,29,30 The aromatic moiety B forms π−π and/or hydrophobic interactions with the Phe-309 residue and the H-bond acceptor C forms a chargereinforced H-bond donor with the basic residue, Arg-283, two other important mutational hits.

Figure 3. Conformation of 2 mapped into the pharmacophore model. Color spheres illustrate the optimal locations of the three points pharmacophore features: A (basic, red), B (aromatic, amber) and C (H-bond acceptor, green). Only polar hydrogens are illustrated.

In the course of the optimization we then identified compound 8, reasonably potent and, for the first time, showing a 93% maximal response associated with pEC50 of 7.8, Table 2. This


10

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encouraging result led to further docking studies in a homology model now based on the newly available X-ray crystallographic structure of A2a agonist bound to the human adenosine A2a receptor (Brookhaven PDB access code 3QAK),31 guiding the design of further molecules. Compound 8 was studied in detail in respect of the bicyclic heteroaryl moiety; indole showed to be one of the best groups and its substitution pattern was quickly investigated, Table 2. The presence of the aminopyridine usually led to compounds with a high percentage of agonism as in compounds 14-17 and 19-21; only compound 18 being an outlier.

R=

8

9

10

11

12

13

all pIC50< 6

Figure 4. SAR around the aminopyridine moiety of compound 8. The SAR around the 4-amino pyridine moiety was very narrow. Figure 4 shows several analogs with replacements of the amino pyridine ring (9-13), all with pIC50 < 6. The interactions of the amino pyridine moiety with residues Gln-120, Phe-121 and Glu-124 seem to be crucial for full agonism at the ghrelin receptor and minor structural alterations lead to interconversion of full agonists to partial agonists or antagonists as has been previously observed with other GPCRs,16 Figure 5. Consistent with previous observations, the agonists under study form a superficial binding mode with no interactions with a more deeply located mutational hit Asp-99, which are thought to be an interaction site for more profoundly bound antagonists and inverse agonists.28,29,30 Residue Phe-286, which has been shown to be a mutational hit for more bulky peptidic agonists and inverse agonists are located far from the putative binding mode of


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the compact indane-based agonists, consistent with suggested binding modes for other smallmolecule agonists.27

N NH 2 NH

O

H N O

N

8

Figure 5. Interactions of compound 8 with the homology model of ghrelin receptor as structural drivers for full agonism. The binding mode of 8 was obtained by energy minimization of the complex using the OPLS3 force field.32 Table 2. Potency in binding, functional assay, agonism and logD of compounds 8, 14-21. N N H2 O

NH

H N

R O

Binding[a] Entry

Functional assay[b] LogD[d]

R pIC50 IC50 (µM) pEC50 and agonism

8

6.6

0.246

7.8 (93) (n = 80)

[c]

(%) EC50 (µM) 0.015

4.1


12

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14

7.1

0.089

7.6 (74)

0.023

2.4

15

7.9

0.014

8.6 (88)

0.003

3.1

16

7.9

0.013

8.0 (82)

0.003

2.9

17

7.5

0.035

7.9 (73)

0.014

3.0

6.7

0.187

7.4 (65)

0.044

2.1

19

7.3

0.049

7.6 (93)

0.025

2.4

20

-

-

6.9 (84)

0.135

3.1

21

-

-

8.1 (83)

0.007

4.0

CN

18

N H

[a,b,c]

See footnotes in Table 1. [d]Distribution coefficient expressing partitioning of compounds between 1-octanol and aqueous buffer at pH 7.4. However the aminopyridine based derivatives, although full agonists, were quite lipophilic and during the work to explore the influence of individual residues on ghrelin receptor agonism, we designed the morpholine compound 23. As shown in Figure 6, the interaction with Gln-120 is now engaged by the morpholine oxygen atom. Both epimers at the morpholine ring stereocenter were investigated (compounds 23 and 24), Table 3. Compound 23 with S configuration at the morpholine ring maintained a potency with pEC50 of 7.7 in the functional assay and showed the desired full agonism (91), while the epimer 24 showed a drop both in pEC50 (6.6) and in agonism


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(65%). To challenge the role of the morpholine oxygen, piperidine analogs were prepared (compounds 25 and 26) and they showed lower agonism, 70 and 69 % respectively. The more potent analog 22 with higher lipophilicity and metabolic instability was not further elaborated. Q-120

F-121 E-124

F-312

8

F-279

Q-120

F-312 F-309 F-279

F-121 E-124

23 F-309

R-283

R-283

Figure 6. Residues of human ghrelin receptor within the homology model that interact with compounds 8 and 23. Ionic, polar, charge-reinforced H-bonding and hydrophobic interactions are shown in red, amber, purple and green, respectively. Table 3. Potency in binding, functional assay, agonism and logD of compounds 22-38.

Binding[a] Entry

R

R1

Functional assay[b] LogD[d]

pIC50

IC50 (µM)

pEC50 and agonism[c] (%)

EC50 (µM)

22

7.9

0.014

8.9 (81)

0.0014

3.7

23

6.6

0.241

7.7 (91)

0.021

2.6

24

2.9

6.6 (65)

0.274

2.4

(n = 5)

(n = 80)


14

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25

6.3

0.528

6.8 (70)

0.170

1.9

26

6.3

0.556

7.6 (69)

0.028

2.1

7.0

0.107

7.4 (97)

0.036

2.1

6.7

0.189

7.1 (79)

0.076

1.6

29

7.2

0.071

8.0 (91)

0.01

2.3

30

1.0

6.6 (83)

0.262

2.3

31

-

-

7.7 (72)

0.021

3.7

32

-

-

7.3 (92)

0.052

1.8

33

-

-

6.9 (55)

0.137

2.6

34

-

-

< 5.0 (-)

>7.7

2.2

35

6.5

0.288

6.9 (61)

0.129

1.4

36

6.5

0.343

6.6 (65)

0.25

1.4

37

6.7

0.184

< 6.1 (84)

>0.74

1.8

27

28

a

a


15


38

7.5

0.029

Page 16 of 44

7.7 (57)

0.019

1.7

[a,b,c,d]

See footnotes in Table 2. Increase of lipophilicity, in order to fill the hydrophobic pocket, with a homopiperidine ring as in compound 27 gave the desired agonism of 97%. The (S)-configured homomorpholine derivative 29 showed a pEC50 of 8 and 91% agonism while the epimer 30 was lower in potency and agonism. The position of the oxygen in this ring is relevant as shown by the loss in agonism of the regioisomeric compound 31 and the corresponding demethylated compound 33. Acetylation of the basic nitrogen led to the inactive acetyl derivative 34. Pyrrolidine derivatives 35-38 did not offer any advantage in terms of potency or agonism. Unfortunately, the morpholine derivative 23, although being a potent full agonist, was not a candidate to progress. The compound showed a significant amount of reactive metabolites formed in human microsomes and rat hepatocytes. Metabolite identification using KCN and MeONH2 as trapping agents confirmed the formation of iminium ions and aldehyde adducts on the morpholine ring in both the parent and the demethylated morpholine 32 (main metabolite), (hydroxylation of the morpholine ring and dehydration result in the formation of iminium ions that are unstable, but can be trapped by forming cyanide stable adducts which can be detected by LC-MS/MS). The mechanism of formation of iminium intermediates and aldehydes by oxidative dealkylation on morpholine groups has been previously described,33-35 more recently in the case of the bioactivation of Foretinib.36 Moreover, all the morpholine derivatives showed quite significant brain penetration, which according to our hypothesis at that point of time, was unnecessary to achieve the desired effect (see below), but could cause undesired side effects. Brain penetration was measured as Kpu,u, which is the ratio between the compound free concentration in brain and its free concentration in plasma.37


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Looking for a morpholine bioisoster, we designed the reverse amide 39 where the carbonyl directly attached to the indane might engage in the same interaction with Gln-120 as the morpholine oxygen to maintain full agonism (Figure 7). At the same time, we hoped that reducing permeability and increasing efflux, would keep these compounds out of the brain. Q-120

F-121

E-124

F-121

E-124

Q-120

39

23

Figure 7. Morpholine diamide 23 and reverse amide 39. Table 4 shows a series of reverse amides. The stereochemistry was confirmed with the enantiomer 40 and the isomer cis (1R,2S) 42 being less potent. By increasing lipophilicity (as with compound 41), a very high potency could be obtained, pIC50 of 10 but at the cost of metabolic instability. Methylation of the NH of the reverse amide as in compound 43 was possible, but the resulting compound was less metabolically stable. Table 4. Potency in binding, functional assay, agonism, logD and metabolic stability of compounds 39-43.

Entry 39 (1S, 2S)

40 (1R, 2R)

41 (1S, 2S)

R

Binding[a] IC50 pIC50 (µM)

Functional assay[b] EC50 pEC50 and [c] Agonism (%) (µM) 8.8 (89) 0.002

LogD[d]

Clint HLM[e]

hERG[f] pIC50

2.4

9

4.5

8.4

0.004

6.3

0.56

6.2 (85)

0.63

2.3

3

4.9

-

-

10 (96)

0.0001

3.0

132

30%inh. @ 11µM

(n = 9)


17


42

Page 18 of 44

42%inh. @ 11µM 50%inh. 43 7.9 0.014 8.3 (84) 0.005 2.5 26 (1S, 2S) @ 11µM [a,b,c,d] See footnotes in Table 2. [e]In vitro clearance in human liver microsomes (µL/min/mg). [f] See footnote [d] in Table 1. cis (1R, 2S)

-

-

6.6 (85)

0.236

2.3

-

Compound 39 (MW 404, LogD 2.4, PSA 81, solubility 100 µM) without significant inhibition of the hERG channel, (pEC50 4.5) showed a promising profile. Its plasma protein binding is 4, 8 and 12% in rat, dog and human respectively. The intrinsic metabolic clearance is variable depending on the species. Compound 39 is quite stable in vitro with low hepatic intrinsic clearance in human ( 5%) was measured to be far above (~10 fold higher) the filtration clearance. This extrahepatic clearance mechanism, together with a significant bile clearance explained the underprediction of clearance in vivo (rather than a misleading low rat hepatic clint in hepatocytes due to the low permeability and high efflux in rat hepatocytes). Metabolite identification in vivo in urine, bile and plasma showed that the main


18

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metabolite was not the N-demethylation of the tertiary amine as expected, but the hydroxylation of the 5-methyl of the indole ring, first to the aldehyde and finally to the carboxylic acid. Cyp Reaction Phenotyping showed that hydroxylation of the methyl group of the indole was catalysed mainly by CYP 2C19. Based on its properties, 39 was considered an appropriate tool compound to be studied in a dog pharmacokinetic/pharmacodynamic model to understand the relationship between the plasma concentration of the drug and its effect on GH and IGF-1 after a single dose of compound. We further were interested in probing the importance of CNS penetration for the efficacy of a ghrelin agonist. Literature has discordant hypotheses about the relevance of CNS penetration for efficacy. Compound 39 was designed for low CNS penetration. It presented a low intrinsic permeability (0.64 10-6 cm/s) and a high efflux (67.2), sufficient to keep the compound effectively out of the brain (Kpu,u < 0.020) while still getting an adequate absorption in dog, Table 5. As a comparator, compound 22 with higher CNS penetration was added to the study. CNS penetration is expressed as Brainunbound / Plasmaunbound ratio, which was 0.292 for compound 22 and < 0.020 for compound 39. Compound 22 with a short half life (1.2 h in dog) and high CNS penetration and compound 39 with an oral half life of 13.2 h and low CNS penetration were profiled against ghrelin, Figure 8. Table 5. Dog pharmacokinetics data and CNS penetration value of compounds 39 and 22.

Entry

Dog PK[a] Cl, Vd, t1/2, F

Caco-2 Caco-2 PappBtA[b] PappAtB[b] pH=7.4 pH=7.4 (1E-6 cm/s) (1E-6 cm/s)

Caco-2 Efflux Ratio[b]

Caco-2 Perm.AtB[e] pH=6.5 (1E-6 cm/s)

CNS[g]

22

28, 2.1, 1.2, 39

15.3[c]

7.5[c]

0.5[c]

44.4

0.292

39

26, 5.8, 13.2, 9

0.6[d]

17.7[d]

67.2[d]

85%. Protocol for ghrelin IP1 agonist assay. In HEK293s cells stably expressing human GHS receptor,39 agonist activity was assessed by measuring intracellular myo-Inositol 1 phosphate (IP1) using the IP-One Tb (HTRF) assay kit (Cisbio International).40,41 Solutions were prepared as described by the supplier. Test compounds or DMSO (200 nl per well, final DMSO concentration 0.2%) were plated in 384-well assay plates (Greiner Lumitrac 200). Cryopreserved cells were thawed, washed in Dulbecco´s modified Eagle medium (DMEM; Invitrogen) containing 10% foetal calf serum (FCS; Hyclone) and resuspended in stimulation buffer (10 mM HEPES, 1 mM CaCl2, 0.5 mM MgCl2, 4.2 mM KCl, 146 mM NaCl, 5.5 mM glucose, 50 mM


31


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LiCl, pH7.4; supplied in the assay kit) supplemented with 0.02% bovine serum albumin (BSA; Sigma) to a density of 0.75E6 cells/ml. Cell suspension (20 µl/well) was added to the compound plate and incubated for 90 minutes at 37°C in 5% CO2/95% air. Following lysis of cells using lysis buffer (supplied in the assay kit) in the presence of IP1-d2 (5 µl/well) and anti-IP1 cryptate (5µl/well) for 60 minutes at room temperature, fluorescence was measured on the Pherastar (BMG Labtech) at 612 and 665 nm, respectively. The effect of the compounds was expressed as a percentage of maximal effect of GHRP-6 (5µM; Bachem). The concentration and percent effect of the test compound was fitted using a Sigmoidal Dose-Response Model (Genedata Screener) and potency (EC50) and efficacy (% agonism) were determined. The results obtained in this assay are shown in Tables 1-4. Pharmacokinetics in male Han Wister rats. Male Han Wister rats (7–8 weeks old, body weight 200–300 g) were kept in plastic cages with free access to standard rat diet and water. The animals were maintained at a temperature of 20–25°C with a 12-hour light/dark cycle and relative humidity of 40%-70% before the experiment. All studies were approved by the corresponding local ethical committees. IV formulation: Appropriate weight of compound was weighed and dissolved into the required volume of vehicle with vortex and sonication applied to reach the target concentration. The vehicle was composed of 5% DMSO, 95% SBE-B-CD (30% w/v in water). This solution was pH adjusted with 1M HCl and 1M NaOH, to the final pH of 7.5. This IV bolus was filtered before administration with formulations stirred at room temperature for at least 2 minutes before dosing. The final measured dose was typically ca. 1 mg/Kg. Two animals were administered the IV bolus via tail vein. The cannulation of the jugular vein was pre-treated to the rats 2-3 days prior


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to the study. Blood samples (0.2 mL) were collected from the jugular vein at the following intervals: Pre-dose, 2, 5, 10, 30 min, 1, 2, 4, 8 and 24 hours post dose. PO formulations: Appropriate weight of compound was weighed and dissolved into the required volume of vehicle with vortex and sonication applied to reach the target concentration. The vehicle was composed of 5% DMSO, 95% SBE-B-CD (30% w/v in water). The final measured dose was typically ca. 2 mg/Kg. Blood samples (0.2 mL) were collected from Jugular vein at the following time intervals: Pre-dose, 15, 30 min, 1, 1.5, 2, 3, 4, 8 and 24 hours after oral dosing. Each sample was transferred into plastic micro centrifuge tubes containing K2-EDTA and placed on wet ice prior to centrifugation for plasma. Blood samples were centrifuged at 10000 rpm for 2 min. at 4ºC to obtain plasma. Samples were stored in a freezer at -75±15 ºC prior to analysis. Plasma samples were analysed by an LC-MS/MS method. Pharmacokinetic calculations were performed using WinNonlin (PhoenixTM, version 6.1) Pharmacokinetics in male Beagle Dogs. Group-housed male/female beagle dogs (2-5 years old, body weight 12-15 Kg) were fasted overnight prior to dosing, food was offered ca. 4h postdose following collection of the 4 h blood sample (oral phase) and ca. 3 h post dose following collection of the 3 h blood sample (intravenous phase). Tap water was available ad libitum. All studies were approved by the corresponding local ethical committees. IV formulation: Appropriate weight of compound was weighed and dissolved into the required volume of vehicle with vortex and sonication applied to reach the target concentration. The vehicle was composed of 5% DMSO, 95% SBE-B-CD (30% w/v in water). This solution was pH adjusted with 1M HCl and 1M NaOH, to the final pH of 7.5 and filtered before administration with formulations stirred overnight at room temperature before dosing. The final measured dose was typically ca. 1 mg/Kg. Compound was administered to two animals as a 15 min. intravenous


33


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infusion via the cephalic vein. The cannulation of the jugular vein was pre-treated to the rats 2-3 days prior to the study. Blood samples (ca 1 mL) were collected from the jugular vein at the following intervals: 0.03, 0.08, 0.17, 0.33, 0.67, 1, 2, 4, 6, 12 and 24 hours post dose. PO formulations: Appropriate weight of compound was weighed and dissolved into the required volume of vehicle with vortex and sonication applied to reach the target concentration. The vehicle was composed of 5% DMSO, 95% SBE-B-CD (30% w/v in water) or 0.5% HPMC/0.1% Tween 80 in water (when oral suspension was required). The final measured dose was typically ca. 2 mg/Kg. Compound was administered orally via gastric gavage. Blood samples (ca 1 mL) were collected from Jugular vein at the following time intervals: 0.17, 0.33, 0.67, 1, 2, 3, 4, 6, 12 and 24 hours after oral dosing. Each sample was transferred into plastic micro centrifuge tubes containing K2-EDTA and placed on wet ice prior to centrifugation for plasma. Blood samples were centrifuged at 10000 rpm for 2 min. at 4ºC to obtain plasma. Samples were stored in a freezer at -75±15 ºC prior to analysis. Plasma samples were analysed by an LC-MS/MS method. Pharmacokinetic calculations were performed using WinNonlin (PhoenixTM, version 6.1) CNS penetration (Kp,uu). The amount (as free concentration in brain) of compound 22 and 39 present in the brain at steady state in relation to that in blood was measured according to the method described in literature.37 Animal Study Protocol. The present study was approved by the local Ethical committee in Gothenburg, Sweden. (2010-04-01). Experimental procedures. Female, four to eight years old Beagles from Kennel Rååhöjden were used in these studies. Four dogs were included in each group. All dogs were fed at 14:30 each day, starting at least one week before the first experiment, and after the last sample on study


34

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day. All dogs were weighed and orally dosed at 7:30. Blood samples were obtained before dosing, 15, 30 and 45 minutes, 1, 2, 4, 7, 12 and 24 h after dose. The blood was collected in K2E EDTA coated BD Vacutainer® 2 ml vials and centrifuged at 4°C, 3.0 g for 5 minutes and 125 µl plasma was transferred to 0.5 ml Eppendorf tubes or 96-well plates (compound level) and kept frozen (< -20°C compound level, < -80°C, IGF-1) until analysis. The total blood volume obtained was minimized for each blood sample. The maximally allowed volume was 13 ml per experiment. Dogs were allowed to rest for at least three weeks when 10% of circulating blood volume (80 ml for a 13 kg dog) was obtained. Bioanalytical procedures. IGF-1 was determined with an IGF-1 ELISA kit purchased from Mediagnost®, Germany, catalogue number E20. The assay is performed according to the manufacturer´s instructions. The primary readout is the change in IGF-1 levels from baseline in relation to plasma levels of the compound. Paired student T test is used for statistical check at each time point. Changes with P values below 5% were considered to be significant. hERG Assay. hERG currents are recorded from hERG-expressing CHO cells. AUTHOR INFORMATION Corresponding Author *Phone: +46 31 7761567. Fax +46 31 7763792. Email: [email protected]. Notes The authors declare no competing financial interest. Present Addresses †H.W. now at Sygnature Discovery Limited, BioCity, Pennyfoot Street, Nottingham, UK. †J.T. now at Gothenburg University, Gothenburg, Sweden.


35


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†J.L. now at Red Glead Discovery AB, Lund, Sweden. †B.L. now at Sahlgrenska University Hospital, Gothenburg, Sweden. †K.K. now at Bioglan AB, Malmö, Sweden. Orcid Cristina Gardelli: 0000-0003-1169-2180 Antonio Llinas: 0000-0003-4620-9363 Igor Shamovsky: 0000-0002-2881-9531 Author Contributions The manuscript was written through contributions from CG, AL, IS, LH, UA, LJ. All authors have given approval to the final version of the manuscript. ACKNOWLEDGMENT We thank Wuxi chemists for their work in the synthesis and purification of many of the reported compounds. We thank our colleagues at AstraZeneca R&D whose testing of the compounds in hERG, logD, Caco, metabolic stability, CNS assays and PK studies provided the results which are described and the analytical team for purity and HRMS data. We thank Werngard Czechtizky for her insightful comments in the preparation of the manuscript.

ABBREVIATIONS USED

AcOH, acetic acid; Boc2O, di-tert-butyl dicarbonate; CH3CN, acetonitrile; CNS, central nervous system; COPD, chronic obstructive pulmonary disease; DCM, dichloromethane; DEA, diethylamine; DIEA, diisopropylethylamine; DMF, dimethylformamide; DMSO, dimethyl sulfoxide;

EDCI,

1-ethyl-3-(3-dimethylaminopropyl)carbodiimide;

EDTA,

ethylenediaminetetraacetic acid; Et3N, triethylamine; EtOAc, ethyl acetate; EtOH, ethanol; ESI,


36

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electrospray ionization; GH, growth hormone; GHS-R, growth hormone secretagogue receptor; GPCR, G-protein coupled receptor; HCHO, formaldehyde; HOBt, 1-hydroxybenzotriazole; HPLC, high -performance liquid chromatography; HRMS, high resolution mass spectroscopy; iPrOAc, isopropyl acetate; IGF, insulin growth factor; iv, intravenous; KCl, potassium chloride; KHSO4, potassium hydrogen sulfate; LC-MS, liquid chromatography-mass spectrometry; MeOH, methanol; MgCl2, magnesium chloride; MgSO4, magnesium sulfate; MTBE, methyl tertbutyl ether; NaHCO3, sodium bicarbonate; NaCl, sodium chloride; NaBH3CN, sodium cyanoborohydride;

NaBH4,

pharmacodynamics;

PE,

sodium

petroleum

borohydride; ether;

PK,

Na2SO4,

sodium

pharmacokinetics;

sulfate;

PD,

TBDMSOTf,

tert-

butyldimethylsilyl triflate; THF, tetrahydrofuran; VCD, vibrational circular dichroism. ASSOCIATED CONTENT Supporting

Information.

Vibrational

Circular

Dichroism

assignment

of

absolute

configuration of compounds 65a and 65b; homology model of ghrelin receptor with bound compound 8 in pdb format; analytical data of compounds 1-22, 24-31, 33-38, 41-43; representative curves for a full and a partial agonist. Molecular Formula Strings (provided as a separate file, (.csv)). REFERENCES 1.

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32. Harder, E.; Damm, W.; Maple, J.; Wu, C.; Reboul, M.; Xiang, J.W.; Wang, L.; Lupyan, D.; Dahlgren, M. K.; Knight, J. L.; Kaus, J. W.; Cerutti, D. S.; Krilov, G.; Jorgensen, W. L.; Abel, R.; Friesner, R. A. OPLS3: a force field providing broad coverage of drug-like small molecules and proteins. J. of Chemical Theory and Computation 2016, 12, 281-296. 33. Masic, L. P. Role of cyclic tertiary amine bioactivation to reactive iminium species: structure toxicity relationship. Curr. Drug Metabolism 2011, 12 (1), 35-50. 34. Zhang, Z.; Chen, Q.; Li, Y.; Doss, G. A.; Dean, B. J.; Ngui, J. S.; Silva Elipe, M.; Kim, S.; Wu, J. Y.; Dininno, F.; Hammond, M. L.; Stearns, R. A.; Evans, D. C.; Baillie, T. A. and Tang, W. In vitro bioactivation of dihydrobenzoxathiin selective estrogen receptor modulators by cytochrome P450 3A4 in human liver microsomes: formation of reactive iminium and quinone type metabolites. Chem. Research Toxicol. 2005, 18 (4), 675-685. 35. Park, B. K.; Boobis; A.; Clarke, S.; Goldring, C. E.; Jones, D.; Kenna, J. G.; Lambert, C.; Laverty, H. G.; Naisbitt, D. J.; Nelson, S.; Nicoll-Griffith, D. A.; Obach, R. S.; Routledge P.; Smith, D. A.; Tweedie, D. J.; Vermeulen, N.; Williams, D. P.; Wilson, I. D.; Baillie, T. A. Managing the challenge of chemically reactive metabolites in drug development. Nat. Rev. Drug Discovery 2011, 10, (4), 292-306. 36. Kadi, A.; Amer, S. M.; Darwish, H. W.; Attwa, M. W. LC-MS/MS reveals the formation of aldehydes and iminium reactive intermediates in foretinib metabolism: phase I metabolic profiling. RSC Advances 2017, 7, 36279-36287. 37. Hammarlund-Udenaes, M.; Fridén, M.; Syvänen, S., Gupta, A. On the rate and extent of drug delivery to the brain. Pharm. Res. 2008, 25 (8), 1737-1750.


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Table of Contents graphic

Arg-283

Gln-120 Phe-121 Phe-309

Glu-124

Phe-279

8

Phe-312


44

Identification and Pharmacological Profile of an Indane Based Series

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