Pyrimidoaminotropanes as Potent, Selective, and Efficacious Small

Subscriber access provided by EXETER UNIV

Article

Pyrimidoaminotropanes as Potent, Selective and Efficacious Small Molecule Kinase Inhibitors of the Mammalian Target of Rapamycin (mTOR) Anthony A Estrada, Daniel G. Shore, Elizabeth Blackwood, Yung-Hsiang Chen, Gauri Deshmukh, Xiao Ding, Antonio G DiPasquale, Jennifer A Epler, Lori S Friedman, Michael Koehler, Lichuan Liu, Shiva Malek, Jim Nonomiya, Daniel Fred Ortwine, Zhonghua Pei, Steve Sideris, Frederic St-Jean, Lan Trinh, Tom Truong, and Joseph P. Lyssikatos J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/jm400194n • Publication Date (Web): 08 Mar 2013 Downloaded from http://pubs.acs.org on March 13, 2013

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Journal of Medicinal Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 34

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

Journal of Medicinal Chemistry

Pyrimidoaminotropanes as Potent, Selective and Efficacious Small Molecule Kinase Inhibitors of the Mammalian Target of Rapamycin (mTOR) Anthony A. Estrada,*,† Daniel G. Shore,† Elizabeth Blackwood,∞ Yung-Hsiang Chen,∆ Gauri Deshmukh,∆ Xiao Ding,∆ Antonio G. DiPasquale,Ω Jennifer A. Epler,∞ Lori S. Friedman,∞ Michael F. T. Koehler,† Lichuan Liu,∆ Shiva Malek,§ Jim Nonomiya,§ Daniel F. Ortwine,† Zhonghua Pei,† Steve Sideris,§ Frederic St-Jean,† Lan Trinh,§ Tom Truong,∞ and Joseph P. Lyssikatos† Departments of †Discovery Chemistry, ∞Translational Oncology, §Biochemical and Cellular Pharmacology, and ∆Drug Metabolism and Pharmacokinetics, Genentech, Inc., 1 DNA Way, South San Francisco, California 94080, United States Ω

X-ray Crystallographic Facility, University of California–Berkeley, 32 Lewis Hall, Berkeley, California 94720, United States

RECEIVED DATE (to be automatically inserted after your manuscript is accepted if required according to the journal that you are submitting your paper to)

Abstract We have recently reported a series of tetrahydroquinazoline (THQ) mTOR inhibitors, which produced a clinical candidate 1 (GDC-0349). Through insightful design, we hoped to discover and synthesize a new series of small molecule inhibitors that could attenuate CYP3A4 time-dependent inhibition commonly observed with the THQ scaffold, maintain or improve aqueous solubility and oral absorption, reduce free drug clearance, and selectively increase mTOR potency. Through key in vitro ACS Paragon Plus Environment

1


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

Page 2 of 34

and in vivo studies, we demonstrate that a pyrimidoaminotropane based core was able to address each of these goals. This effort culminated in the discovery of 20 (GNE-555), a highly potent, selective, metabolically stable and efficacious mTOR inhibitor.

Introduction The mammalian or mechanistic target of rapamycin (mTOR) is a clinically validated target for treatment of human cancers such as renal cell carcinoma, endometrial cancer and mantle cell lymphoma. The PI3K-AKT-mTOR signaling pathway represents a major growth and survival pathway that is dysregulated in many human cancers, is involved in regulating the translation of proteins associated with drug resistance, and controls cell cycle progression and cell survival.1 To date, only the macrolide rapamycin analogs (rapalogs) have been clinically approved as mTOR inhibitors. However, the use of rapalogs as a single-agent therapy in major solid tumors has resulted in only modest objective response rates with the exception of everolimus in combination with exemestane for treatment of hormone receptor positive breast cancer.2,3 The proposed, and recently supported,4,5 rationale for these observations is that rapalogs only inhibit one of two functional multiprotein complexes, mTORC1. The second complex, mTORC2, which has been shown to be an important driver for cancer cell growth and survival, is unaffected by rapalogs. Additionally, there is a postulated feedback loop between mTORC1 and Akt in certain tumor cells whereby mTORC1 inhibition results in upregulation of Akt activity and enhanced cell survival.6,7 Thus, the development of a small molecule that has the potential to inhibit both mTORC1 and mTORC2 has been the focus of several drug discovery efforts,8a-x including our own, and is currently being tested in the clinic. We previously disclosed a variety of saturated-fused pyrimidine mTOR inhibitors including dihydrofuropyrimidines,9 dihydropyrrolopyrimidines,10 and tetrahydroquinazolines (THQs).10,11 The latter culminated in the discovery of a clinical candidate, 1 (GDC-0349, Figure 1).11 During the optimization of the THQ series, we discovered a few potential issues such as CYP3A4 time-dependent

ACS Paragon Plus Environment

2

Page 3 of 34

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


inhibition (TDI) that plagued many of the compounds in this series as highlighted in Figure 1. In vitro CYP3A4 TDI raises a potential for clinical drug-drug interactions and idiosyncratic adverse drug reactions.12,13,14 In an attempt to attenuate the commonly observed TDI with the THQ scaffold, as well as address additional potential concerns while maintaining the desirable attributes of the THQ series, we rationalized that a bicyclic system such as the pyrimidoaminotropane (PAT) scaffold could fulfill this role. Through installation of the ethylene bridge at the carbon atoms adjacent to the piperidine nitrogen, we hypothesized that we would observe the following: a) elimination of two potential sites of reactive metabolite formation due to the high strain energy of a resulting iminium bridgehead double bond (Bredt’s rule15,16); b) an increase in sp3 character and a decrease in planarity to maintain or improve aqueous solubility;17,18 c) an increase in metabolic stability; d) a selective increase in mTOR potency through additional lipophilic contacts with a postulated small hydrophobic patch above the plane of the THQ saturated ring system (Figure 2). Additional key ligand-protein interactions that contribute to selective mTOR potency have been previously described and are shown in Figure 2.9,10,11

1 (GDC-0349)!

THQs! •  •  •  • 

PATs!

Attenuate CYP3A4 TDI! Maintain/Increase Aqueous Solubility! Improve Metabolic Stability! Increase Cellular Potency !

•  Avoid Iminium Ion Formation (Bredt’s Rule)! •  Decrease Planarity! •  Introduce Favorable Lipophilic Contacts! with Ehylene Bridge!

Figure 1. Structure of clinical candidate 1, and one of two potential reactive iminium species of the


3


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

Page 4 of 34

THQ series and design rationale for PATs.

Figure 2. Depiction of 7-aza PAT 20 (GNE-555) (yellow) in the active site of the mTOR homology model9 with residues important for binding and selectivity vs. PI3Kγ shown. View is edge-on to the plane of the inhibitor bicyclic ring. Hydrogen bonds to the protein are shown as thin dashed lines. A vdW surface on the protein color-coded by lipophilic potential (tan-brown = lipophilic, cyan-blue = polar, green = between lipophilic and polar) has been added. Note the proximity of the lipophilic portions of the tropane and (S)-3-methylmorpholine ring to the lipophilic portions of the protein. The oxetane ring extends out toward solvent. Image was generated using Benchware3DExplorer (Tripos Inc., St. Louis, MO).

Chemistry


4

Page 5 of 34

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


While the PAT scaffold was unknown in the literature at the time of our investigation, we drew inspiration from a pyrimidine synthetic methodology disclosed by Movassaghi.19,20 We were successful in developing a modification of this protocol to enable flexible and rapid access to N-vinyl tertiary enamide starting materials.21 This methodology provided us with the opportunity to synthesize the desired PAT moiety and quickly test our hypothesis. As depicted in Scheme 1, we decided to initially pursue 6-aza PATs as N-H tropinone is commercially available, and access to the corresponding 7-aza PAT starting material required a multi-step synthesis. Thus, N-Alloc tropinone 2 was condensed with pmethoxybenzylamine in the presence of Ti(OEt)4 and the resulting imine/enamine intermediate is captured with p-nitrobenzoyl chloride to provide tertiary enamide 3. This is followed by pyrimidine formation with the concomitant addition of (S)-3-methylmorpholine, which provides enhanced mTOR potency and selectivity, relative to morpholine, over closely related kinases such as PI3-kinases as previously described.9,10,11 Nitro reduction, ethyl urea formation and N-Alloc deprotection set the stage for various late stage diversifications of the tropane nitrogen. O N AllocN

AllocN

a

O

b

N PMB

O 2

AllocN

NO2

R

4: R=NO2

O

c

5: R=NH2

O

N N

d

N

3

R

N

N R

N

f

N

6: R=Alloc 7: R=H

N

N

O N H e

N N H

See Table 1

O N H

N H

Scheme 1. Synthesis of 6-aza PATs via Pyrimidine Synthetic Methodologya


5


Page 6 of 34

a

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

Reagents and conditions: (a) PMBNH2, Ti(OEt)4, CH2Cl2, 40 °C, 16 h, then Et3N, p-NO2BzCl, 0–25 °C, 84%; (b) (S)-3methylmorpholine-4-carbonitrile, Tf2O, 2-ClPyr, CH2Cl2, -78–25 °C, 40%; (c) SnCl2⋅2H2O, EtOH, ∆; (d) triphosgene, Et3N, 1,2-DCE, 0–70 °C, then EtNH2⋅HCl, 25 °C; (e) Pd(PPh3)4, morpholine, THF, 0 °C; (f) see experimental section.

Results and Discussion Table 1. 6-Aza PAT SARa O N

R

N

N N

O N H

potency/selectivity

N H

target modulationb

proliferation

PI3K α/δ (fold)d 875/1090

pAKT IC50 (nM) 3.9

pp70S6K IC50 (nM) 8.2

NCI-PC3 EC50 (nM) 103

MCF7neo/Her2 EC50 (nM) 97

CYP3A4 TDI IC50 (µM)e cleanf

compd 8

R Me

mTOR Ki (nM) 4.2

9c

Me

4.2

147/800

6.7

15

152

158

cleanf

10

Et

6.5

1550/825

3.2

7.4

145

184

cleanf

14

304/548

29

7.7

180

268

>10

21

14/8

45

82

579

971

–

2.6

3770/3770

18

25

260

806

5

7.5

231/325

30

15

180

460

7.3

11 12c

O O

O

13

S

O

O

14 a

Assays described in ref’s 9,22; all biochemical and cellular assay results represent the geometric mean of a minimum of two determinations, and these assays generally produced results within 3-fold of the reported mean. bAssayed in NCI-PC3 cell line. c(5S,8R) tropane isomer. dRatio of Ki values of PI3Kα and δ over mTOR. eTDI IC50 is the concentration of inhibitor that supports 50% inhibition of activity following a 30 min pre-incubation in the presence of NADPH.23 fIC50 could not be measured.

The more active and selective (5R,8S) 6-aza PAT diastereomers shown in Table 1 generally demonstrated good in vitro biochemical (mTOR Ki < 15 nM), cellular (≤ 30 nM) and anti-proliferative (< 500 nM) activities. While simple N-alkyl substitution provided mTOR inhibitors with good activity and selectivity profiles (8–10, Table 1), oxetanyl, sulfonyl and acetyl derivatives (11–14, Table 1) were


6

Page 7 of 34

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


synthesized to attenuate the basicity of the tropane nitrogen and potential risk for hERG off-target activity. The assigned 5R,8S isomers always displayed a superior PI3K/mTOR selectivity index (>100 fold over mTOR) compared to the alternate 5S,8R diastereomer. The observed selectivity, along with docking analyses, was used to assign the stereochemistry of the 6-aza PAT stereoisomers. Docking showed that both stereoisomers could be accommodated by mTOR, whereas one isomer showed a close contact with a Trp residue (W812) in PI3Kγ at the position analogous to K2171 of the mTOR active site (Figure 3). The less bulky K2171 residue in mTOR results in a larger binding cavity that can better accommodate the bridged bicyclic ring and oxetane substituent. Consistent with this observation, 6-aza PAT diastereomers containing smaller N-substituents displayed reduced selectivity differences (e.g. see N-Me isomers 8 and 9, Table 1).

Figure 3. Superposition of low energy docked models of tropanes 11 (pink) and 12 (green). PI3Kγ


7


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

Page 8 of 34

residues are in cyan and the corresponding mTOR residues are in grey. mTOR numbering is shown. Note the closer contact of the oxetane ring to the Trp ring in PI3Kγ (W812 in PI3Kγ; K2171 in mTOR) for 11, consistent with its increased mTOR selectivity relative to 12. Both 11 and 12 are well accommodated by mTOR due to the less bulky K2171 residue providing a larger binding cavity. In this view, W2239 in mTOR occurs behind the inhibitor oxetane rings of 11 and 12, and does not sterically clash. Image was generated using Benchware3DExplorer (Tripos Inc., St. Louis, MO).

In addition to the promising activity and selectivity profiles, the 6-aza PATs shown in Table 1 also exhibited little to no CYP3A4 TDI risk and good aqueous solubility (all kinetic solubilities > 75 µM). Furthermore, we were encouraged by the collective profiles of the 6-aza PATs when compared to the corresponding 6-aza THQ analogs. This is exemplified in Figure 4 with 11 and 6-aza THQ, 15. The CYP3A4 TDI was attenuated, the aqueous solubility improved, and a 4-5 fold increase in antiproliferative activity was noted.24 These encouraging results provided confidence that the PAT scaffold could address the inherent issues associated with the lead THQ series. Additionally, we already knew that the 7-aza THQ analogs were superior to the 6-aza THQ variants.10 Before testing this relationship in the 7-aza PAT series, which required a multi-step synthesis to access the tropinone starting material, we wanted to obtain in vivo validation for the 6-aza PAT series.


8

Page 9 of 34

Journal of Medicinal Chemistry O

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

N

O N

N O

N N H

N H

15: 6-aza THQ!

11: 6-aza PAT!

mTOR Ki (µM)!

0.018!

0.014!

NCI-PC3 EC50 (µM)!

0.797!

0.180!

MCF7 EC50 (µM)!

1.2!

0.268!

Kinetic Solubility (µM)!

40!

186!

CYP 3A4 TDI IC50 (µM)!

2.5!

>10!

Figure 4. Matched pair comparison of 6-aza THQ (15) and 6-aza PAT (11).

Pharmacokinetic profiles of 8 and 9 were assessed in mouse PK studies. Both analogs demonstrated low unbound clearance values (207 and 53 mLmin-1kg-1, Table 2) and exhibited a good in vitro–in vivo correlation with mouse microsomal stability data. Additionally, these two compounds had excellent bioavailability (114 and 82%, Table 2) and high free fraction in human plasma. Inhibitor 8 was chosen for in vivo target modulation studies of the mTOR signaling pathway using NCR nude mice inoculated subcutaneously with NCI-PC3 prostate cancer xenografts. The PI3K/Akt/mTOR pathway is activated in this PTEN-null cell line and mTOR inhibitors have previously been reported to be effective in this model.25 Dose dependent on-target reduction of pS6RP (mTORC1 readout) and pAkt (mTORC2 readout) were demonstrated from 25 to 50 mg/kg (Figure 5), with 50 and 100 mg/kg drug level cohorts demonstrating reduced pAkt and pS6RP recovery at the 6 and 10 hour time points. This inhibition correlated with the plasma concentration, where total plasma levels of >1 µM (unbound plasma levels >0.32 µM) translate to >50% knockdown of both markers. With these positive results, the focus now shifted to the regioisomeric 7-aza PAT series that we hoped would follow the same pattern as the THQ


9


series and yield improved overall profiles compared to the 6-aza PATs. Table 2. Mouse PK Profiles of 6-aza PATs.

mouse PKa iv (1 mg/kg) microsomes Clhepb -1 -1 (mLmin kg ) h/r/mc 12/18/42

compd 8

Cl (Clu)d -1 -1 (mLmin kg ) 67 (207)

po (5 mg/kg)

Vss (Lkg-1) 4.5

t1/2 (h) 0.80

F (%) 114

PPB (%) h/r/mc 38/27/68

9 15/28/58 34 (53) 2.6 1.07 82 19/20/46 Compound dosed po as a suspension in MCT and iv as a 60% PEG solution. bClhep predicted from incubation with microsomes. ch/r/m = human/rat/mouse. dClu = unbound clearance = total clearance/[(100 – % mouse PPB)/100]. a

#!!$

7$4819$ 7$4:(;

Pyrimidoaminotropanes as Potent, Selective, and Efficacious Small

Recommend Documents