Discovery and Optimization of Quinazolinone ... - ACS Publications

Subscriber access provided by UNIV OF NEBRASKA - LINCOLN

Article

Discovery and Optimization of Quinazolinone-pyrrolopyrrolones as Potent and Orally Bioavailable pan-Pim Kinase Inhibitors Liping H. Pettus, Kristin L. Andrews, Shon K. Booker, Jie Chen, Victor J. Cee, Frank Chavez Jr., Yuping Chen, Heather Eastwood, Nadia Guerrero, Bradley James Herberich, Dean Hickman, Brian A Lanman, Jimmy Laszlo III, Matthew Randolph Lee, J. Russell Lipford, Bethany Mattson, Christopher Mohr, Yen Nguyen, Mark H Norman, David Powers, Anthony B. Reed, Karen Rex, Christine Sastri, Nuria A. Tamayo, Paul Wang, Jeffrey T. Winston, Bin Wu, Tian Wu, Ryan P. Wurz, Yang Xu, Yihong Zhou, Andrew S Tasker, and Hui-Ling Wang J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.6b00610 • Publication Date (Web): 10 Jun 2016 Downloaded from http://pubs.acs.org on June 11, 2016

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 88

Journal of Medicinal Chemistry

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 ACS Paragon Plus Environment


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 88

Discovery and Optimization of Quinazolinonepyrrolopyrrolones as Potent and Orally Bioavailable pan-Pim Kinase Inhibitors Liping H. Pettus,*,1 Kristin L. Andrews,2 Shon K. Booker,1 Jie Chen,3 Victor J. Cee,1 Frank Chavez Jr.,1 Yuping Chen,3 Heather Eastwood,2 Nadia Guerrero,4 Bradley Herberich,1 Dean Hickman;3 Brian A. Lanman,1 Jimmy Laszlo III,6 Matthew R. Lee,2 J. Russell Lipford,4 Bethany Mattson,4 Christopher Mohr,2 Yen Nguyen,6 Mark H. Norman,1 David Powers,6 Anthony B. Reed,1 Karen Rex,4 Christine Sastri,4 Nuria Tamayo,1 Paul Wang,6 Jeffrey T. Winston,4 Bin Wu,5 Tian Wu,5 Ryan P. Wurz,1 Yang Xu,3 Yihong Zhou,3 Andrew S. Tasker1, and Hui-Ling Wang1 Department of Therapeutic Discovery 3

1

Medicinal Chemistry;

Pharmacokinetics and Drug Metabolism; 4Oncology Research;

5

2

Molecular Structure;

Pharmaceutics; 6Discovery

Technologies; Amgen Inc., One Amgen Center Drive, Thousand Oaks, CA 91320, USA.

1 ACS Paragon Plus Environment

Page 3 of 88

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


ABSTRACT The high expression of proviral insertion site of Moloney murine leukemia virus kinases (Pim-1, -2, and -3) in cancers, particularly the hematopoietic malignancies, is believed to play a role in promoting cell survival and proliferation while suppressing apoptosis. The three isoforms of Pim protein appear largely redundant in their oncogenic functions. Thus, a pan-Pim kinase inhibitor is highly desirable. However, cell active pan-Pim inhibitors have proven difficult to develop because Pim-2 has a low Km for ATP and therefore, requires a very potent inhibitor to effectively block the kinase activity at cellular ATP concentrations. Herein, we report a series of quinazolinone-pyrrolopyrrolones as potent and selective pan-Pim inhibitors. In particular, compound 17 is orally efficacious in a mouse xenograft model (KMS-12 BM) of multiple myeloma, with 93% tumor growth inhibition at 50 mg/kg QD upon oral dosing.



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

INTRODUCTION The proviral integration site of Moloney murine leukemia virus proteins 1, 2, and 3 (Pim1, Pim-2, and Pim-3) are a family of constitutively active serine/threonine kinases that are involved in the survival and proliferation of hematopoietic cells in response to cytokines, stress, and growth factors signaling downstream of JAK/STAT and NF-κB activation.1,2 The signaling pathway of the Pim kinases parallels that of the PI3K/Akt/mTOR axis and both pathways share overlapping targets, such as Bcl-2-associated death promoter (BAD) and the protein synthesis regulator eIF4E-BP1.3,4 Phosphorylation of BAD increases Bcl-2 activity and thus promotes cell survival, whereas phosphorylation of eIF4E-BP1 releases eIF4E to promote mRNA translation and cellular growth. The Pim-1, -2, and -3 proteins are expressed at low levels in normal tissues but overexpressed in hematologic and solid tumor malignancies including multiple myeloma, acute myeloid leukemia, prostate cancer, and gastric and liver carcinomas.1 Pim triple knockout mice are small but viable, fertile, and show subtle hematologic changes.5,6 Therefore, the inhibition of the Pim kinases presents a promising clinical pathway to treat Pim overexpressing forms of cancers. The kinase domains of the Pim family are structurally unique in the hinge region of the ATP-binding pocket, which contains a tertiary amide (proline) not found in other kinases.7 This unique hinge region feature can be exploited to achieve selectivity against other protein kinases. The three isoforms of Pim kinases share a high level of sequence homology within the family (>61%) and appear largely redundant in their oncogenic functions.8 Thus, a pan-Pim kinase inhibitor is highly desirable. Among the three isoforms, Pim-2 has the lowest Km (~ 4 µM) for ATP, about 100- and 10-fold lower than those of Pim-1 and Pim-3, respectively.9 Hence, cell active pan-Pim inhibitors have been more challenging to identify than Pim-1 or Pim-3 inhibitors. 3 ACS Paragon Plus Environment

Page 4 of 88

Page 5 of 88

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


Many research groups have disclosed ATP-competitive Pim kinase inhibitors of diverse chemical scaffolds with a wide range of cell potency.10,11,12,13 In particular, shown in Figure 1 are the structures of three clinical molecules 27 (PIM447),14 28 (SGI-1776),15 and 29 (AZD1208),16,17 as well as 30, the macrocyclic inhibitor from Amgen.18 While basic Pim inhibitors 27 (Pim-2 Ki = 18 pM) and 29 (Pim-2 Ki = 160 pM) were very potent in enzymatic assays, they displayed >1000-fold enzyme to cell shift. On the other hand, neutral Pim inhibitor 30 had only about 100-fold enzyme to cell shift, but the flat molecule had limited solubility in aqueous medium (40 and 295 µM in PBS (pH 7.4) and FaSIF (pH 6.8), respectively. Herein, we describe the development of a novel series of quinazolinone-pyrrolopyrrolones as potent and selective pan-Pim inhibitors that led to the identification of compound 17, which is orally efficacious in a mouse xenograft model (KMS-12 BM) of multiple myeloma. Figure 1. Structures and potency of publicly disclosed Pim kinases inhibitors.

cpd

Pim-1

Pim-2

Pim-3

KMS-11 luc

Ki (nM) Ki (nM) Ki (nM) EC50 (µM)


KMS-12 BM IC50 (µM)


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

27 (PIM447)*

0.006

0.018

0.009

0.17

28 (SGI-1776)*

16

610

24

5

29 (AZD1208)* 0.017

0.160

0.230

0.67

30#

0.249

0.255

Page 6 of 88

0.25

* Data from Reference 14; # Data from Reference 18. We recently disclosed a highly potent and selective series of 2-aryl-6,7-dihydro-1Hpyrrolo[3,2-c]pyridin-4(5H)-ones, exemplified by 1 (Scheme 1), as pan-Pim kinase inhibitors.19,20 Compound 1 was potent in both biochemical (IC50 = 0.3 nM for both Pim-1 and Pim-2) and cellular (KMS-12 BM, IC50 = 23 nM) assays. In addition, 1 was efficacious in a KMS-12 BM multiple myeloma xenograft model in mice (78% TGI @ 50 mg/kg QD oral dosing). Although 1 possessed desirable pharmacokinetic (PK) properties in rat (CL = 0.4 L/h/kg, VSS = 1.0 L/kg, T1/2 = 3.5 h, %F = 37), it had rapid intravenous (IV) clearance (CL = 3.4 L/h/kg, over liver blood flow) and a short half-life (T1/2 = 0.6 h) in dog. Therefore, this compound was not suitable for further advancement as safety studies in a non-rodent species (such as dog) would have been difficult. We sought to improve the PK profile of this series while maintaining the good potency in vitro and in vivo. The rat and dog plasma protein binding unbound fractions (fu) of 1 were measured (using rapid equilibrium dialysis) to be 1.7% and 6.0%, respectively, which calculated the unbound clearance CLu of 1 in rat and dog to be 23.5 L/h/kg and 56.6 L/h/kg, respectively. The intrinsic clearance (CLint) of 1 in rat liver microsomes (RLM) and dog liver microsomes (DLM) was 189 and 203 µL/min/mg, respectively.21 The CLint of 1 in rat hepatocytes and dog hepatocytes was 39 and 32 µL/min/106 cells, respectively. The in vitro and in vivo pharmacokinetics data of 1 in rat and dog appeared to be poorly correlated. Although metabolic pathways besides hepatic oxidations might be involved, we decided to look 5 ACS Paragon Plus Environment

Page 7 of 88

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


for clues in the microsomal stability first. Metabolite identification studies showed that when 1 was incubated with RLM, compound 2 (derived from hydroxylation of the t-butyl group) was the major metabolite. When 1 was incubated with DLM, compound 3 (formed from dehydrogenation of the 6,7-dihydro-1H-pyrrolo[3,2-c]pyridin-4(5H)-one) was the major metabolite. With this information in hand, we prepared a series of analogs designed to block these oxidations, focusing initially on replacement of the 6,7-dihydro-1H-pyrrolo[3,2-c]pyridin-4(5H)-one with a metabolically more stable bicycle and, subsequently, on the left-hand portion of the molecule, modifying the amino-quinoxaline. Scheme 1. In vitro metabolism of compound 1. F

F

F O

O

O

N

N N HN

HN

RLM

N N

NH

HN

NH

HN

DLM

N

HN

NH

HN

OH 2

1

Pim-1 IC50 0.9 nM Pim-2 IC50 0.3 nM KMS-12 BM IC50 143 nM

Pim-1 IC50 Pim-2 IC50 KMS-12 BM IC50

3 0.3 nM 0.3 nM 23 nM

Pim-1 IC50 0.2 nM Pim-2 IC50 0.1 nM KMS-12 BM IC50 148 nM

RESULTS AND DISCUSSION In Vitro SAR. In biochemical assays, the compounds were evaluated for their ability to inhibit the phosphorylation of a BAD peptide at Serine 112 (S112) by full-length recombinant human Pim-1 and Pim-2 proteins. Our experience across multiple scaffolds in the Pim program had been that Pim-3 activity was in general comparable to that of Pim-1.22,23 Therefore, the Pim3 enzyme assay was not routinely performed. In cell-based assays, the compounds were 6 ACS Paragon Plus Environment


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

measured for their ability to inhibit the phosphorylation of BAD at S112 in KMS-12 BM cells in the presence of 20% fetal bovine serum (FBS) using flow cytometry. KMS-12 BM is a multiple myeloma cell line with endogenous Pim-1, -2, and -3 proteins, and a relatively high level of Pim2 expression.24 For an initial assessment of metabolism, the compounds were evaluated in RLM and HLM, and the data, reported as intrinsic clearance (CLint), were used to prioritize potent compounds for additional PK studies. In the initial phase of this investigation, we focused our SAR work on the right-hand portion of compound 1, searching for a more metabolically stable replacement for the 6,7dihydro-1H-pyrrolo[3,2-c]pyridin-4(5H)-one motif.

As observed previously in our SAR

development for compound 1,20 the C7-F in 1 offered about 3-fold improvement in enzyme and cellular potency over a C7-H in 4 (structure and data are shown in Table 1), whereas in vitro and in vivo metabolic stability in rats was comparable. For this part of SAR investigation, to facilitate syntheses, we elected to keep the left-hand portion of the analogs constant as 3-(tertbutylamino)-2-methylquinoxalin-5-yl and compare their data to that of compound 4, then make a C7-F analog on an optimized right hand piece.

Moving the pyrrole-nitrogen in 4 to the

bridgehead position in 5 (KMS-12 BM, IC50 = 4.56 µM, RLM CLint = 205 µL/min/mg), or homologating the [5,6]-fused bicycle in 4 to the [5,7]-fused bicycle in 6 (KMS-12 BM, IC50 = 1.63 µM, RLM CLint = 169 µL/min/mg) resulted in much weaker cellular potency and higher rat microsomal turnover when compared to 4 (KMS-12 BM, IC50 = 110 nM, RLM CLint = 86 µL/min/mg). Contraction of the [5,6]-fused bicycle in 4 to the [5,5]-fused bicycle gave rise to compound 7, and through substitutions at C6’ (R1 and R2), a set of 5,6-dihydropyrrolo[3,4b]pyrrol-4(1H)-ones, 8, 9, 10, 11, and 12. Compared to 4, the 5,6-dihydropyrrolo[3,4-b]pyrrol4(1H)-ones 7, 8, 9, 10, and 11 have improved cellular potency, with pBAD IC50 values of 44, 65, 7 ACS Paragon Plus Environment

Page 8 of 88

Page 9 of 88

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


30, 22 and 78 nM, respectively. Interestingly, between the set of enantiomers 8 and 9, the (R)6’-Me enantiomer (9) was not only more potent in both enzymatic and cell-based assays, but also had lower microsomal turnover. The [5,5]-fused bicycles, 7, 9, 10, and 11, were further profiled in rat IV/PO PK. Among these four compounds, the C6’-unsubstituted analog 7 had the highest RLM turnover (CLint = 96 µL/min/mg) and the highest IV clearance (1.6 L/h/kg). The (R)-6’-(2aminoethyl) analog 10 had poor rat PK profile, with moderate clearance (CL = 1.4 L/h/kg), high volume of distribution (Vss = 21.2 L/kg) and low bioavailability (%F 99% ee) as a yellow crystalline solid was obtained. 1H NMR (400 MHz, DMSO-d6) δ ppm 12.00 (1H, br.), 7.83 (1H, dd, J = 7.4, 1.2 Hz), 7.64 (2H, m), 7.37 (1H, t, J = 7.7 Hz), 6.85 (1H, d, J = 1.4 Hz), 6.02 (1H, s), 4.57 (1H, q, J = 6.6 Hz), 2.58 (3H, s), 1.56 (9H, s), 1.40 (3H, d, J = 6.7 Hz). m/z (ESI, +ve) 350.0 (M+H)+. Compound 8: (6S)-2-(3-(tert-butylamino)-2-methyl-5-quinoxalinyl)-6-methyl5,6-dihydropyrrolo[3,4-b]pyrrol-4(1H)-one (85 mg, 2.6% yield, >99% ee) as a yellow crystalline solid was obtained. m/z (ESI, +ve) 350.0 (M+H)+. (R)-6-(2-aminoethyl)-2-(3-(tert-butylamino)-2-methylquinoxalin-5-yl)-5,6dihydropyrrolo[3,4-b]pyrrol-4(1H)-one (10). A solution of 6-(2-(benzyloxy)ethyl)-2-(3-(tert-butylamino)-2-methylquinoxalin-5-yl)5,6-dihydropyrrolo[3,4-b]pyrrol-4(1H)-one (10d, 840 mg, 1.79 mmol) in DCM (20 mL) at 0 °C under nitrogen was treated with boron trichloride (1.0 M solution in DCM, 3.94 mL, 3.94 mmol) dropwise. The reaction mixture (rusty color) was stirred at 0 °C for 5 min then RT for 1 h. Additional 1.5 mL of BCl3 (1.0 M in DCM) was added and the mixture stirred at RT for 20 min. It was treated with 10 mL of MeOH dropwise followed by sodium bicarbonate (1.50 g, 17.89 mmol). The solvents were removed under reduced pressure. The residue was purified by silica gel chromatography (5% MeOH in DCM followed by 5% 2 M NH3 in MeOH in DCM) to provide two compounds. The 1st eluent was 2-(3-(tert-butylamino)-2-methylquinoxalin-5-yl)-6(2-chloroethyl)-5,6-dihydropyrrolo[3,4-b]pyrrol-4(1H)-one (100 mg, 0.25 mmol, 14% yield) as an orange color solid. m/z (ESI, +ve) 398.0 (M+H)+. The 2nd eluent was 2-(3-(tert-butylamino)2-methylquinoxalin-5-yl)-6-(2-hydroxyethyl)-5,6-dihydropyrrolo[3,4-b]pyrrol-4(1H)-one mg, 0.40 mmol, 22% yield) as an orange solid. m/z (ESI, +ve) 380.0 (M+H)+.


(150

Page 51 of 88

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


To a suspension of 2-(3-(tert-butylamino)-2-methylquinoxalin-5-yl)-6-(2-hydroxyethyl)5,6-dihydropyrrolo[3,4-b]pyrrol-4(1H)-one (120 mg, 0.316 mmol) in 5 mL of DCM at 0 °C was added triethylamine (220 µL, 1.58 mmol) followed by methanesulfonyl chloride (31 µL, 0.39 mmol). It was stirred at 0 °C for 30 min, and then diluted with 30 mL of DCM, washed sequentially with 5 mL of water, 5 mL of sat’d NaHCO3 and 5 mL of brine. The DCM solution was dried over sodium sulfate and concentrated. The brown residue was dissolved in 1 mL of DMF and treated with sodium azide (62 mg, 0.95 mmol). The mixture was heated in an oil bath at 55 °C for 2 h. It was cooled to RT and diluted with EtOAc (50 mL) and filtered. The filtrate was washed with 2 x 5 mL of water. The EtOAc solution was concentrated and the residue was purified on a silica gel column (2-8% MeOH in DCM) to give 6-(2-azidoethyl)-2-(3-(tertbutylamino)-2-methylquinoxalin-5-yl)-5,6-dihydropyrrolo[3,4-b]pyrrol-4(1H)-one (97 mg, 0.24 mmol, 76% yield) as a brown solid. m/z (ESI, +ve) 405 (M+H)+. To a solution of 6-(2-azidoethyl)-2-(3-(tert-butylamino)-2-methylquinoxalin-5-yl)-5,6dihydropyrrolo[3,4-b]pyrrol-4(1H)-one (92 mg, 0.23 mmol) in 3 mL of THF and water (0.3 mL) at RT was added trimethylphosphine (0.34 mL of 1 M in THF, 0.34 mmol). It was stirred at RT for 45 min, then diluted with 50 mL of EtOAc, and washed with 5 mL of water. The organic layer was concentrated to give an orange amorphous solid containing 6-(2-aminoethyl)-2-(3(tert-butylamino)-2-methylquinoxalin-5-yl)-5,6-dihydropyrrolo[3,4-b]pyrrol-4(1H)-one

(10e)

(about 90% ee). m/z (ESI, +ve) 379.1 (M+H)+. The crude 10e was subjected to chiral SFC [Column: Chiralpak ASH (250 x 30 mm, 5 µ); Mobile Phase: 80:20 (A:B); A: Liquid CO2; B: MeOH containing 20 mM ammonia; Flow Rate: 70 mL/min; Oven Temp: 40 °C; Inlet Pressure: 100 bar; Wavelength: 278 nm]. The 1st eluent was the minor enantiomer: m/z (ESI, +ve) 379.1 (M+H)+. The 2nd eluent was the major enantiomer: (R)-6-(2-aminoethyl)-2-(3-(tert-butylamino)-



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 52 of 88

2-methylquinoxalin-5-yl)-5,6-dihydropyrrolo[3,4-b]pyrrol-4(1H)-one (10) (38 mg, 0.10 mmol, 43% yield, 99% ee) as a yellow amorphous solid. 1H NMR (400 MHz, DMSO-d6) δ ppm 7.81 (1H, dd, J=7.4, 1.4 Hz), 7.71 (1H, s), 7.57 - 7.67 (1H, m), 7.35 (1H, t, J=7.7 Hz), 6.84 (1H, s), 5.99 (1H, s), 4.57 (1H, dd, J=8.4, 4.1 Hz), 3.41 (3H, br.), 2.64 - 2.77 (2H, m), 2.56 (3H, s), 1.92 (1H, dd, J=13.3, 4.3 Hz), 1.57 - 1.65 (1H, m), 1.55 (9H, s). m/z (ESI, +ve) 379.1 (M+H)+. 2'-(3-(tert-Butylamino)-2-methylquinoxalin-5-yl)-1'H-spiro[cyclopropane-1,6'-pyrrolo[3,4b]pyrrol]-4'(5'H)-one (11). This compound was prepared according to the synthetic protocols described for compound 7, starting from β-keto ester 11a. 1H NMR (400 MHz, DMSO-d6) δ ppm 11.64 (1H, br.), 7.78 (1H, d, J = 6.5 Hz), 7.67 (1H, s), 7.60 (1H, m), 7.35 (1H, t, J = 7.8 Hz), 6.90 (1H, m), 6.00 (1H, s), 2.56 (3H, s), 1.52 (9H, s), 1.42 (2H, m), 1.33 (2H, m). m/z (ESI, +ve) 362.1 (M+H)+. 2'-(3-(tert-Butylamino)-2-methylquinoxalin-5-yl)-2,3,5,6-tetrahydro-1'H-spiro[pyran-4,6'pyrrolo[3,4-b]pyrrol]-4'(5'H)-one (12). 2-(4-Aminotetrahydro-2H-pyran-4-yl)-5-(3-(tert-butylamino)-2-methylquinoxalin-5-yl)1H-pyrrole-3-carboxylic acid (12c) was prepared according to the synthetic protocols described for intermediate 7c, starting from β-keto ester 12a. A solution of 2-(4-aminotetrahydro-2Hpyran-4-yl)-5-(3-(tert-butylamino)-2-methylquinoxalin-5-yl)-1H-pyrrole-3-carboxylic acid (12c, 54.7 mg, 0.13 mmol) and diisopropylethylamine (0.13 mL, 0.77 mmol) in DCM (0.65 mL)/DMF (0.65

mL)

was

stirred

at

0

°C

under

nitrogen,

and

(benzotriazol-1-

yloxy)tripyrrolidinophosphonium hexafluorophosphate (HATU) (81 mg, 0.15 mmol) was added. The reaction mixture was stirred at 0 oC for 45 min, then RT for 3 h. It was concentrated under reduced pressure and purified by reverse-phase preparative HPLC [using a Phenomenex Gemini


Page 53 of 88

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


column, 10 micron, C18, 100 Å, 150 x 30 mm, 0.1% TFA in CH3CN/H2O, gradient 10% to 95% over 8 min]. The product fractions were concentrated in a Genevac EZ-2 Evaporator at 55 °C. They were reconstituted in MeOH and applied to a MeOH-washed SiliaPrep Si-carbonate cartridge to generate the free base. The yellow eluent was concentrated under reduced pressure to an orange-yellow solid. LCMS did not give the product (ESI:electrospray positive). A significant yellow coloring remained on the basic column. It was flushed with 2 M NH3/MeOH to give a second yellow solution. The solution was concentrated under reduced pressure to give 2'-(3-(tert-butylamino)-2-methylquinoxalin-5-yl)-1'H-spiro[cyclopropane-1,6'-pyrrolo[3,4b]pyrrol]-4'(5'H)-one (12) (7.4 mg, 14% yield) as a yellow solid. 1H NMR (CDCl3) δ: 12.02 (br., 1H), 7.96 (d, J = 6.3 Hz, 1H), 7.70 (d, J = 6.8 Hz, 1H), 7.39 (t, J = 7.8 Hz, 1H), 7.22 (d, J = 2.5 Hz, 1H), 5.96 (s, 1H), 4.88 (s, 1H), 4.36 (d, J = 2.5 Hz, 2H), 3.97 (t, J = 5.4 Hz, 2H), 2.63 (br., 2H), 2.57 (s, 3H), 1.62 (s, 9H), 1.26 (s, 2H). m/z (ESI, +ve) 407.0 (M+H)+. (R)-2-(3-(tert-butylamino)-2-methylquinoxalin-5-yl)-3-chloro-6-methyl-5,6dihydropyrrolo[3,4-b]pyrrol-4(1H)-one (13). To a suspension of (R)-2-(3-(tert-butylamino)-2-methylquinoxalin-5-yl)-6-methyl-5,6dihydropyrrolo[3,4-b]pyrrol-4(1H)-one (9) (524 mg, 1.50 mmol) in 15 mL of CHCl3 at RT was added N-chlorosuccinimide (260 mg, 1.95 mmol) and the mixture was stirred at RT for 18 h, LCMS showed about 45% conversion. Additional N-chlorosuccinimide (52 mg, 0.38 mmol) was added and the mixture was heated at 50 oC in an oil bath for 90 min. It was cooled to RT, treated with 15 mL of ice cold 0.2 N NaOH, extracted with 50 mL of DCM followed by 50 mL of EtOAc. The combined organic extracts were concentrated and the residue was purified on a silica gel column (50% EtOAc in DCM followed by 5% MeOH in DCM) to give (R)-2-(3-(tert-



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 54 of 88

butylamino)-2-methylquinoxalin-5-yl)-3-chloro-6-methyl-5,6-dihydropyrrolo[3,4-b]pyrrol4(1H)-one (13) (400 mg, 1.04 mmol, 70% yield) as a yellow crystalline solid.

1

H NMR (400

MHz, DMSO-d6) δ ppm 11.77 (1H, br.), 7.78 (2H, m), 7.63 (1H, d, J = 7.2 Hz), 7.41 (1H, t, J = 7.7 Hz), 5.97 (1H, br.), 4.46 (1H, d, J = 6.7 Hz), 2.55 (3H, s), 1.40 (12 H, m). m/z (ESI, +ve) 383.9 (M+H)+. 2-(3-(tert-butylamino)-2-methylquinoxalin-5-yl)-5,6-dihydro-4H-thieno[2,3-c]pyrrol-4-one (14). Step 1. At 0 °C, to a solution of N-(tert-butyl)-2-formylthiophene-3-carboxamide (14a)31 (2.37 g, 11.22 mmol) in 10 mL of MeOH and 10 mL of THF was added sodium borohydride (0.42 g, 11.22 mmol) in small portions. It was stirred at 0 °C for 15 min, then quenched with sat'd NH4Cl (20 mL), and extracted with EtOAc (2 x 50 mL). The combined organic solution was washed with 15 mL of brine and dried, concentrated to give N-(tert-butyl)-2(hydroxymethyl)thiophene-3-carboxamide as a viscous colorless oil. 1H NMR (400 MHz, CHLOROFORM-d) δ ppm 7.13 (2H, q, J = 5.3 Hz), 6.05 (1H, br.), 4.90 (1H, m), 4.78 (2H, d, J = 6.7 Hz), 1.47 (9H, s).

m/z (ESI, +ve) 214.1 (M+H)+.

To the crude N-(tert-butyl)-3-

(hydroxymethyl)thiophene-2-carboxamide in 50 mL of DCM at 0 °C was treated with diisopropylethylamine (3.90 mL, 22.43 mmol) followed by methanesulfonyl chloride (1.04 mL, 13.46 mmol) dropwise. It was stirred at 0 °C for 15 min then RT for 15 min. LC-MS indicated formation of predominantly the chloride (m/z (ESI, +ve) 232.1 (M+H)+). It was diluted with 100 mL of DCM, washed sequentially with 10 mL of ice cold water, 10 mL sat’d NaHCO3 and 10 mL of brine. The DCM extracts were dried over sodium sulfate and concentrated affording a viscous orange oil. At -78 °C, a solution of the obtained viscous orange oil in THF (40 mL) was treated with lithium bis(trimethylsilyl)amide (1 M solution in THF, 16.83 mL, 16.83 mmol). 53 ACS Paragon Plus Environment

Page 55 of 88

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


The resulting dark brown solution was stirred at -78 °C for 10 min, then 0 °C for 10 min. It was quenched with water, and extracted with EtOAc (100 mL). The organic layer was dried over anhydrous sodium sulfate, filtered and concentrated. The residue was purified on a silica gel column (20-50% EtOAc in hexanes) affording 5-(tert-butyl)-5,6-dihydro-4H-thieno[2,3-c]pyrrol4-one (14b, 1.69 g, 8.65 mmol, 77% yield, in about 70% purity) as a yellow crystalline solid. 1H NMR (400 MHz, CHLOROFORM-d) δ ppm 7.30 (1H, d, J = 4.9 Hz), 7.18 (1H, d, J = 4.9 Hz), 4.49 (2H, s), 1.54 (9H, s). m/z (ESI, +ve) 196.1 (M+H)+. Step 2. 5-(tert-Butyl)-5,6-dihydro-4H-thieno[2,3-c]pyrrol-4-one (14b, 370 mg, 1.89 mmol) in DMF (5 mL) was treated with NBS (337 mg, 1.89 mmol) and allowed to stir at RT for 18 h. LC-MS indicated clean conversion to the desired product (m/z (ESI, +ve) 274.0/276.0 (M+H)+). The reaction mixture was treated with water (5 mL), extracted with EtOAc (50 mL). The organic layer was washed with brine (3 x 5 mL) and dried over MgSO4, filtered and concentrated. The crude residue was recrystallized from Et2O and hexanes affording 2-bromo5-(tert-butyl)-5,6-dihydro-4H-thieno[2,3-c]pyrrol-4-one (14c, 176 mg, 0.64 mmol, 34% yield) as a white amorphous solid. 1H NMR (400 MHz, CHLOROFORM-d) δ ppm 7.18 (1H, s), 4.44 (2H, s), 1.53 (9H, s). Step 3. 2-Bromo-5-(tert-butyl)-5,6-dihydro-4H-thieno[2,3-c]pyrrol-4-one (14c, 347 mg, 1.27 mmol) in dioxane (1 mL) and 20% sulfuric acid (1 mL) was heated in a microwave at 135 °C for 1.5 h. It was diluted with 50 mL of EtOAc, neutralized with 5 N NaOH. The organic layer was separated and washed with 5 mL of brine, dried and concentrated. The residue was purified on a silica gel column (35-100% EtOAc in hexanes) affording 2-bromo-5,6-dihydro-4Hthieno[2,3-c]pyrrol-4-one (14d, 70.5 mg, 0.3 mmol, 25% yield) as an off-white crystalline solid.



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

1

Page 56 of 88

H NMR (400 MHz, MeOH) δ ppm 7.29 (1H, s), 4.60 (1H, br.), 4.51 (2H, m). m/z (ESI, +ve)

218.0/220.0 (M+H)+. Step 4. In a 5 mL glass microwave tube, 2-bromo-5,6-dihydro-4H-thieno[2,3-c]pyrrol-4one (14d, 35 mg, 0.16 mmol), dicyclohexyl(2',4',6'-triisopropyl-[1,1'-biphenyl]-2-yl)phosphine (4.59 mg, 9.63 µmol), Pd2(dba)3 (4.41 mg, 4.81 µmol), potassium phosphate (102 mg, 0.48 mmol) and N-(tert-butyl)-3-methyl-8-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)quinoxalin-2amine (AQ2, 65.70 mg, 0.19 mmol) were purged with argon and treated with dioxane (2.0 mL) and water (0.6 mL) and heated in a microwave at 140 °C for 20 min. The reaction mixture was treated with water, extracted with EtOAc, washed with brine and concentrated. Purification on a Gilson reverse phase HPLC (20-95% 0.1%TFA/CH3CN in 0.1%TFA/water)) afforded 2-(3(tert-butylamino)-2-methylquinoxalin-5-yl)-5,6-dihydro-4H-thieno[2,3-c]pyrrol-4-one

2,2,2-

trifluoroacetate (14) (20 mg, 0.04 mmol, 26% yield) as a bright yellow amorphous powder. 1H NMR (400 MHz, DMSO-d6) δ ppm 8.31 (1H, s), 7.86 (1H, dd, J = 7.4, 1.2 Hz), 7.71 (1H, m), 7.69 (1H, s), 7.37 (1 H, t, J = 7.8 Hz), 5.99 (1H, s), 4.52 (2H, s), 2.55 (3 H, m), 1.54 (9H, s). 19F NMR (377 MHz, DMSO-d6) δ ppm -74.50 (s). m/z (ESI, +ve) 353.1 (M+H)+. 2-(3-(tert-Butylamino)-2-methylquinoxalin-5-yl)-4H-thieno[2,3-c]pyrrol-6(5H)-one (15). Step 1. At 0 °C, to a solution of N-(tert-butyl)-3-formylthiophene-2-carboxamide (15a)31 (2.79 g, 13.21 mmol) in 10 mL of MeOH and 10 mL of THF was added sodium borohydride (0.50 g, 13.21 mmol) in small portions. After the reaction was stirred at 0 °C for 15 min, it was quenched with 15 mL of sat’d NH4Cl, and extracted with 2 x 75 mL of EtOAc. The combined organic solution was washed with 15 mL of brine and dried, concentrated to give a sticky oil containing N-(tert-butyl)-3-(hydroxymethyl)thiophene-2-carboxamide that was used as crude. m/z (ESI, +ve) 214.1 (M+H)+.

The crude N-(tert-butyl)-3-(hydroxymethyl)thiophene-255

ACS Paragon Plus Environment

Page 57 of 88

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


carboxamide in 50 mL of DCM at 0 °C was treated with diisopropylethylamine (4.59 mL, 26.4 mmol) followed by methanesulfonyl chloride (1.23 mL, 15.85 mmol) dropwise. It was stirred at 0 oC for 15 min, then RT for 10 min. It was diluted with 100 mL of DCM, washed sequentially with 10 mL of ice cold water, 10 mL sat’d NaHCO3 and 10 mL of brine. The DCM extracts were dried over sodium sulfate and concentrated to give a brown amorphous solid (m/z (ESI, +ve) 232 (M+H)+). The brown solid was dissolved in 15 mL of THF and cooled to -78 °C. Lithium bis(trimethylsilyl)amide (19.8 mL of 1 M solution in THF, 19.8 mmol) was added in dropwise and the resulting dark brown solution was stirred at -78 °C for 10 min, then 0 °C for 10 min. It was quenched with 15 mL of sat’d NH4Cl, and extracted with 2 x 75 mL of EtOAc. The combined organic solution was concentrated. The residue was purified on a silica gel column (25-45% EtOAc in hexanes) to give 5-(tert-butyl)-4H-thieno[2,3-c]pyrrol-6(5H)-one (15b, 1.55 g, 7.94 mmol, 60% yield) as a yellow crystalline solid. 1H NMR (400 MHz, DMSO-D6) δ ppm 7.89 (1 H, d, J=4.7 Hz), 7.17 (1 H, d, J=4.7 Hz), 4.49 (2 H, s), 1.46 (9 H, s). m/z (ESI, +ve) 196.1 (M+H)+. Step 2. At 0 oC, to a suspension of 5-(tert-butyl)-4H-thieno[2,3-c]pyrrol-6(5H)-one (15b, 235 mg, 1.20 mmol) in 0.6 ml of HOAc and 1.2 mL of water was added a solution of bromine (0.06 mL, 1.26 mmol) in 0.6 mL of HOAc. After 30 min at 0 oC, LCMS indicated only 25% conversion. Additional bromine (0.04 mL) was added and the resulting heterogeneous mixture was stirred at RT for 15 min. LCMS indicated 50% conversion. Additional bromine (0.13 mL) in 0.4 mL of HOAc was added and the resulting heterogeneous mixture was stirred at RT for 1 h, then 45 oC for 15 min. LCMS indicated about 80% conversion. It was cooled to RT, diluted with 50 mL of EtOAc, and neutralized with 10 N NaOH to pH of 7. The aqueous layer was discarded; the organic layer was concentrated to give 2-bromo-5-(tert-butyl)-4H-thieno[2,3-



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 58 of 88

c]pyrrol-6(5H)-one (15c, m/z (ESI, +ve) 274/276 (M+H)+) which was used in the next step without purification. Step 3. Crude 15c in 1 mL of dioxane and 1 mL of 15% sulfuric acid was heated in an oil bath at 100 oC for 30 min. LCMS indicated no removal of tert-butyl at all. It was heated in a microwave at 130 oC for 80 min. It was diluted with 50 mL of EtOAc, neutralized to pH of 7 with 5 N NaOH. The organic layer was separated and washed with 5 mL of brine, dried and concentrated. The residue was purified on a silica gel column (35-85% EtOAc in hexanes) to provide 2-bromo-4H-thieno[2,3-c]pyrrol-6(5H)-one (15d, 179 mg, 0.82 mmol, 68% yield, in about 85% pure) as an off white crystalline solid. m/z (ESI, +ve) 218/220 (M+H)+. Step 4. In a 5 mL glass microwave tube, a mixture of N-(tert-butyl)-3-methyl-8-(4,4,5,5tetramethyl-1,3,2-dioxaborolan-2-yl)quinoxalin-2-amine

(AQ2,

dicyclohexyl(2',4',6'-triisopropyl-[1,1'-biphenyl]-2-yl)phosphine

139

mg,

0.41

mmol),

(10.62

mg,

0.02

mmol),

Pd2(dba)3 (10.20 mg, 0.01 mmol), 2-bromo-4H-thieno[2,3-c]pyrrol-6(5H)-one (15d, 81 mg, 0.37 mmol), and potassium phosphate (237 mg, 1.11 mmol) was purged with argon and treated with dioxane (1.5 mL) and water (0.5 mL) and heated in a microwave at 125 °C for 20 min.

The

reaction mixture was treated with 2 mL of 1 N NaOH, extracted with EtOAc (2 x 5 mL). The organic extracts were concentrated. Purification of the brown residue on a silica gel column (eluted with 100% EtOAc)) afforded 2-(3-(tert-butylamino)-2-methylquinoxalin-5-yl)-4Hthieno[2,3-c]pyrrol-6(5H)-one (15) (69 mg, 0.2 mmol, 53% yield) as a yellow crystalline solid. 1

H NMR (400 MHz, DMSO-d6) δ ppm 8.37 (1H, s), 7.92 (1H, dd, J = 7.5, 1.3Hz), 7.74 (1H, m),

7.70 (1H, s), 7.40 (1H, t, J = 7.8 Hz), 6.04 (1H, s), 4.37 (2H, s), 2.58 (3H, s), 1.56 (9H, s). m/z (ESI, +ve) 353.1 (M+H)+.


Page 59 of 88

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


(R)-2-(tert-butylamino)-3-methyl-9-(6-methyl-4-oxo-1,4,5,6-tetrahydropyrrolo[3,4-b]pyrrol2-yl)-4H-pyrido[1,2-a]pyrimidin-4-one (16). Step 1. 2-Amino-3-bromopyridine (3.30 g, 19.07 mmol) and diethyl methylmalonate (7.95 mL, 46.7 mmol) in a 100 mL RBF was fitted with a water-cooled reflux condenser open to air and placed in a 170 °C oil bath. After 2 h, LCMS detected m/z (ESI, +ve ion) 301/303 (M+H)+. The reaction was heated to reflux at 220 °C in a heating block for 18 h. The reaction was cooled to RT and purified by silica gel chromatography (eluted with 0-100% EtOAc/DCM) to afford 9-bromo-2-hydroxy-3-methyl-4H-pyrido[1,2-a]pyrimidin-4-one (16a, 0.61 g, 2.38 mmol, 13% yield) as an orange solid. 1H NMR (400 MHz, DMSO-d6) δ ppm 11.76 (1H, s), 8.88 (1H, dd, J = 7.0, 1.4 Hz), 8.27 (1H, dd, J = 7.2, 1.4 Hz), 7.09 (1H, t, J = 7.2 Hz), 1.98 (3H, s). m/z (ESI, +ve ion) 254.9/257.0 (M+H)+. Step 2. 9-Bromo-2-hydroxy-3-methyl-4H-pyrido[1,2-a]pyrimidin-4-one (16a, 0.42 g, 1.65 mmol) was treated with 3 mL DMF and 1,8-diazabicyclo-[5.4.0]undec-7-ene (0.49 mL, 3.29 mmol) and tert-butylamine (0.86 mL, 8.23 mmol) was added, followed by (benzotriazol-1yloxy)tris(dimethylamino)phosphonium hexafluorophosphate (BOP) (1.09 g, 2.47 mmol). The reaction was sealed and placed in a 70 °C oil bath for 16 h. The reaction was partitioned between sat’d NaHCO3 and EtOAc. The organic layer was washed with sat’d NaHCO3 twice, brine once, and the organics were dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo. The material was purified by silica gel chromatography (40 g column, using 0-50% EtOAc/hexanes). The product-containing fractions were concentrated to afford a yellow foam (0.11 g, 0.35 mmol, 21% yield), as a mixture of two compounds: 75% of 9-bromo-2-(tertbutylamino)-3-methyl-4H-pyrido[1,2-a]pyrimidin-4-one [16b, m/z (ESI, +ve ion) 310/312 (M+H)+], and 25% of 9-bromo-2-(dimethylamino)-3-methyl-4H-pyrido[1,2-a]pyrimidin-4-one 58 ACS Paragon Plus Environment


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 60 of 88

[m/z (ESI, +ve ion) 282/284 (M+H)+]. The mixture was used without further purification in step 3. Step 3. Argon was bubbled into a mixture of potassium phosphate tribasic (0.30 g, 1.41 mmol), XPhos Pd G2 (14 mg, 18 µmol), (R)-6-methyl-2-(4,4,5,5-tetramethyl-1,3,2dioxaborolan-2-yl)-5,6-dihydropyrrolo[3,4-b]pyrrol-4(1H)-one (BE1, 0.19 g, 0.71 mmol), 9bromo-2-(tert-butylamino)-3-methyl-4H-pyrido[1,2-a]pyrimidin-4-one (16b, 0.11 g, 0.35 mmol) in 3 mL dioxane and 0.5 mL water for 1 min. The reaction was placed in a 60 °C oil bath for 30 min. The reaction was cooled and 1.5 g silica gel was added. It was concentrated in vacuo. The residue was purified by silica gel chromatography (eluted with 0-100% of 90/10 DCM/MeOH in DCM). The product-containing fractions were concentrated and the residue was washed with (2 x 3 mL) of MeOH. The solid was collected and dried in vacuo to give (R)-2-(tert-butylamino)-3methyl-9-(6-methyl-4-oxo-1,4,5,6-tetrahydropyrrolo[3,4-b]pyrrol-2-yl)-4H-pyrido[1,2a]pyrimidin-4-one (16) (52 mg, 0.14 mmol, 40% yield) as a white solid.

1

H NMR (400 MHz,

DMSO-d6) δ ppm 11.77 (1H, s), 8.83 (1H, dd, J = 7.0, 1.6 Hz), 7.93 (1H, dd, J = 7.0, 1.6 Hz), 7.66 (1H, s), 7.12 (1H, t, J = 7.1 Hz), 6.76 (1H, s), 5.43 (1H, s), 4.52 (1H, q, J = 6.6 Hz), 2.00 (3H, s), 1.44 (9H, s), 1.36 (3H, d, J = 6.7 Hz). m/z (ESI, +ve ion) 366.0 (M+H)+. (R)-2-(tert-butylamino)-3-methyl-8-(6-methyl-4-oxo-1,4,5,6-tetrahydropyrrolo[3,4-b]pyrrol2-yl)quinazolin-4(3H)-one (17). Step 1. A solution of 2-amino-3-bromobenzoic acid (17a, 3.00 g, 13.9 mmol) and methylamine (2.0 M solution in THF, 27.8 mL, 55.5 mmol) in EtOAc (18 mL) at RT was treated with propylphosphonic anhydride (T3P) (50% wt. in EtOAc, 13.2 mL, 20.8 mmol) and stirred at RT for 18 h. It was then quenched with sat’d NaHCO3 (20 mL) and extracted with 2 x 30 mL of EtOAc. The organic layers were combined and washed with brine, dried over anhydrous sodium


Page 61 of 88

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


sulfate, filtered, and concentrated in vacuo to give 2-amino-3-bromo-N-methylbenzamide (17b, 2.9 g, 12.8, 92% yield) as a white solid. 1H NMR (400 MHz, DMSO-d6) δ ppm 8.36 (br. s., 1H), 7.50 (t, J = 8.5 Hz, 2H), 6.52 (t, J = 7.8 Hz, 1H), 6.47 (br. s., 2H), 2.74 (d, J = 1.8 Hz, 3H). MS (ESI, pos. ion) m/z: 229/231 (M+1). Step 2. A solution of 2-amino-3-bromo-N-methylbenzamide (17b, 1.09 g, 4.76 mmol), DIPEA (0.83 mL, 4.76 mmol), and triphosgene (0.49 g, 1.66 mmol) in DCM (48 mL) was stirred at reflux for 18 h when LCMS showed that starting material was still present. More triphosgene (0.21 g, 0.71 mmol) was added, and the reaction mixture was refluxed for 1 h. It was concentrated under reduced pressure and the residue was purified via silica gel flash chromatography (0-100% EtOAc in hexanes) to give 8-bromo-3-methylquinazoline-2,4(1H,3H)dione (17c, 1.19 g, 4.67 mmol, 98% yield) as a white solid.

1

H NMR (400 MHz,

CHLOROFORM-d) δ ppm 3.46 (s, 3H), 7.13 (t, J = 7.92 Hz, 1H), 7.81 (dd, J = 7.92, 1.27 Hz, 1H), 8.12 (dd, J = 8.02, 0.59 Hz, 1H), 8.20 (br., 1H). MS (ESI, pos. ion) m/z: 254.9/257 (M+1). Step 3. A slurry of 8-bromo-3-methylquinazoline-2,4(1H,3H)-dione (17c, 1.14 g, 4.47 mmol), phosphoryl trichloride (4.09 mL, 44.70 mmol), and DIPEA (3.11 mL, 17.88 mmol) was stirred at 100 oC for in an oil bath for 19 h. The reaction mixture was concentrated, diluted with ice, basified with 10 N NaOH to ~pH 10. The mixture was filtered, and washed with water to give a brown solid. The solid was transferred to an Erlenmeyer flask and dissolved in DCM when a small aqueous layer formed; this was partitioned in a separatory funnel. The organic layer was partially concentrated, loaded onto a silica gel column and eluted with 0-40% EtOAc in hexanes to give 8-bromo-2-chloro-3-methylquinazolin-4(3H)-one (17d, 1.03 g, 3.77 mmol, 84% yield) as a light yellow solid. 1H NMR (400 MHz, chloroform-d) δ ppm 3.77 (s, 3H), 7.35



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 62 of 88

(t, J = 7.92 Hz, 1H), 8.02 (dd, J = 7.82, 1.37 Hz, 1H), 8.21 (dd, J = 8.02, 1.37 Hz, 1H). MS (ESI, pos. ion) m/z: 273.9/275.9 (M+1). Step 4. A mixture of 8-bromo-2-chloro-3-methylquinazolin-4(3H)-one (17d, 0.66 g, 2.41 mmol) and tert-butylamine (3.80 mL, 36.20 mmol) in a sealed tube was heated at 80 °C in an oil bath overnight. The mixture was allowed to cool to RT and water was added. The mixture was extracted with EtOAc (3 x). The combined organic layers were dried over anhydrous sodium sulfate, filtered and concentrated to give 8-bromo-2-(tert-butylamino)-3-methylquinazolin4(3H)-one (17e, 0.71 g, 2.30 mmol, 96% yield) as a pale yellow solid.

1

H NMR (400 MHz,

DMSO-d6) δ 7.90 (m, 2H), 7.03 (t, J = 7.73 Hz, 1H), 6.07 (s, 1H), 3.43 (s, 3H), 1.57 (s, 9H). m/z (ESI, +ve ion) 310.0 /312.0 (M+H)+. Step 5. A mixture of (R)-6-methyl-2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-5,6dihydropyrrolo[3,4-b]pyrrol-4(1H)-one

(BE1,

263

mg,

0.90

mmol),

8-bromo-2-(tert-

butylamino)-3-methylquinazolin-4(3H)-one (17e, 200 mg, 0.64 mmol), potassium phosphate tribasic (411 mg, 1.93 mmol) and XPhos Pd G2 (12 mg, 0.016 mmol) in 3 mL of 1,4dioxane/water (3/1) was heated in an oil bath at 45 °C for 30 min. The reaction mixture was allowed to cool to RT, treated with 3 mL of water and stirred for 30 min. The resulting precipitate was collected by filtration, washed with water (2 x 3 mL) and dried to give a light brown solid. The light brown solid was stirred in 3 mL of MeOH for 5 min at RT. The resulting solid was collected by filtration, washed with MeOH (3 mL) and dried to give (R)-2-(tertbutylamino)-3-methyl-8-(6-methyl-4-oxo-1,4,5,6-tetrahydropyrrolo[3,4-b]pyrrol-2yl)quinazolin-4(3H)-one (17) (211 mg, 0.58 mmol, 90% yield) as a tan solid. 1H NMR (400 MHz, DMSO-d6) δ ppm 11.94 (s, 1H), 7.85 - 7.94 (m, 2H), 7.61 (s, 1H), 7.18 (t, J = 7.7 Hz, 1H),


Page 63 of 88

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


6.73 (d, J = 1.0 Hz, 1H), 5.95 (s, 1H), 4.53 (q, J = 6.5 Hz, 1H), 3.48 (s, 3H), 1.51 (s, 9H), 1.37 (d, J = 6.7 Hz, 3H). MS (ESI, pos. ion) m/z: 366 (M+1). (R)-2-(tert-butylamino)-7-fluoro-3-methyl-8-(6-methyl-4-oxo-1,4,5,6-tetrahydropyrrolo[3,4b]pyrrol-2-yl)quinazolin-4(3H)-one (18). 8-Bromo-2-chloro-7-fluoro-3-methylquinazolin-4(3H)-one (18d) was prepared according to the procedures described for intermediate 17d, starting from 18a. 1H NMR (400 MHz, CDCl3) δ ppm 8.22 (1H, m), 7.27 (1H, m), 3.77 (3H, s). m/z (ESI, +ve ion) 290.9/292.9 (M+H)+. A slurry of 8-bromo-2-chloro-7-fluoro-3-methylquinazolin-4(3H)-one (18d, 0.45 g, 1.54 mmol) in tert-butylamine (2.43 mL, 23.16 mmol) was sealed and placed in an 80 °C oil bath. The reaction became a solid mass after 15-30 min. After 1 h, the mass was analyzed by LC/MS and judged complete. The reaction was concentrated under reduced pressure and the residue was purified by silica gel chromatography (eluted with 0-50% EtOAc/hexanes) to afford 8-bromo-2(tert-butylamino)-7-fluoro-3-methylquinazolin-4(3H)-one (18e, 0.47 g, 1.44 mmol, 94% yield) as a white solid.

1

H NMR (400 MHz, CDCl3) δ ppm 8.06 (1H, m), 6.90 (1H, m), 4.55 (1H, br.),

3.46 (3H, s), 1.63 (9H, s). m/z (ESI, +ve ion) 328/330 (M+H)+. Argon was bubbled into a mixture of tris (dibenzylideneacetone) dipalladium (0) (14 mg, 15 µmol), 2-(dicyclohexylphosphino)-2',4',6',-tri-isopropyl-1,1'-biphenyl (15 mg, 30 µmol), (R)6-methyl-2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-5,6-dihydropyrrolo[3,4-b]pyrrol4(1H)-one (BE1, 0.16 g, 0.61 mmol), 8-bromo-2-(tert-butylamino)-7-fluoro-3-methylquinazolin4(3H)-one (18e, 0.10 g, 0.30 mmol), potassium phosphate (0.19 g, 0.91 mmol) in 2 mL dioxane and 0.4 mL water for 1 min. The reaction was sealed and heated to 80 °C for 2 h. The reaction was cooled and transferred to a RBF with some MeOH and adsorbed onto 1.5 g silica gel and dried in vacuo. The material was purified by silica gel chromatography (eluted with 0-100% of 62 ACS Paragon Plus Environment


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 64 of 88

90/10 DCM/MeOH in DCM) to afford (R)-2-(tert-butylamino)-7-fluoro-3-methyl-8-(6-methyl-4oxo-1,4,5,6-tetrahydropyrrolo[3,4-b]pyrrol-2-yl)quinazolin-4(3H)-one (18) (0.06 g, 0.15 mmol, 51% yield) as an off-white solid.

1

H NMR (400 MHz, DMSO-d6) δ ppm 11.76 (1H, s), 7.96

(1H, dd, J = 8.8, 6.3 Hz), 7.58 (1H, s), 7.07 (1H, dd, J = 10.0, 8.8 Hz), 6.35 (1H, t, J = 1.8 Hz), 6.01 (1H, s), 4.50 (1H, q, J = 6.7 Hz), 3.45 (3H, s), 1.38 (9H, s), 1.35 (3H, d, J = 6.7 Hz).

19

F

NMR (377 MHz, DMSO-d6) δ ppm -105.07 (s). m/z (ESI, +ve ion) 384.1 (M+H)+. (R)-2-(tert-Butylamino)-3-methyl-8-(6-methyl-4-oxo-1,4,5,6-tetrahydropyrrolo[3,4b]pyrrol-2-yl)pyrido[3,4-d]pyrimidin-4(3H)-one (19). Step 1. A solution of 3-amino-2-chloroisonicotinic acid (600 mg, 3.48 mmol) and N,Ndiisopropylethylamine (0.60 mL, 3.48 mmol) in 1,4-dioxane (5 mL) in a glass tube was treated with triphosgene (0.51 mL, 3.48 mmol). The tube was sealed and heated in a microwave reactor (Personal Chemistry) at 80 °C for 20 min. It was cooled with an ice bath and treated with methylamine (33% wt. solution in absolute ethanol, 4.67 mL, 34.8 mmol) dropwise (exothermic) and stirred for 20 min. Additional triphosgene (0.25 mL) was added and the reaction was heated at 50 °C in an oil bath for 15 h. The mixture was cooled to RT and quenched with water. The suspension was filtered and the solid was dried to give 8-chloro-3-methylpyrido[3,4d]pyrimidine-2,4(1H,3H)-dione (19a, 324 mg, 1.53 mmol, 44% yield) as a white solid. 1H NMR (400 MHz, DMSO-d6) δ ppm 11.32 (1 H, s), 8.21 (1H, d, J = 5.1 Hz), 7.84 (1H, d, J = 5.1 Hz), 3.27 (3 H, s). m/z (ESI, +ve ion) 212.0 (M+H)+. Step 2. To a 50-mL RBF was added 8-chloro-3-methylpyrido[3,4-d]pyrimidine2,4(1H,3H)-dione

(19a,

320

mg,

1.51

mmol)

and

(benzotriazol-1-

yloxy)tris(dimethylamino)phosphonium hexafluorophosphate (BOP) (803 mg, 1.81 mmol) in DMF (5 mL) followed by t-butylamine (2.38 mL, 22.68 mmol). The suspension turned clear and


Page 65 of 88

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


was heated at 40 °C for 8 h. It was quenched with water (30 mL) and the suspension was filtered and the solid was dried to give 2-(tert-butylamino)-8-chloro-3-methylpyrido[3,4-d]pyrimidin4(3H)-one (19b, 255 mg, 0.95 mmol, 63% yield) as a white solid. 1H NMR (400 MHz, CHLOROFORM-d) δ ppm 8.09 (1H, d, J = 5.1 Hz), 7.82 (1H, d, J = 5.1 Hz), 4.62 (1H, br. s.), 3.49 (3H, s), 1.62 (9H, s). m/z (ESI, +ve ion) 267.0 (M+H)+. Step 3. To a 50-mL RBF was added 2-(tert-butylamino)-8-chloro-3-methylpyrido[3,4d]pyrimidin-4(3H)-one (19b, 250 mg, 0.93 mmol) and (R)-6-methyl-2-(4,4,5,5-tetramethyl1,3,2-dioxaborolan-2-yl)-5,6-dihydropyrrolo[3,4-b]pyrrol-4(1H)-one (BE1, 295 mg, 1.12 mmol) in 1,4-dioxane (4 mL) and water (1 mL) followed by potassium phosphate (0.233 mL, 2.81 mmol) and XPhos Pd G2 (36.9 mg, 0.047 mmol). The reaction mixture was stirred at 40 °C for 3 h, then diluted with water (30 mL). The mixture was extracted with CHCl3/iPrOH (4:1, 3 x 30 mL) and the combined organic layers were dried, filtered and concentrated. The residue was triturated with DCM and the suspension was filtered. The filtrate was concentrated and purified with RP-HPLC (10-70% MeCN in water with 0.1% TFA in 30 min) to give (R)-2-(tertbutylamino)-3-methyl-8-(6-methyl-4-oxo-1,4,5,6-tetrahydropyrrolo[3,4-b]pyrrol-2yl)pyrido[3,4-d]pyrimidin-4(3H)-one (19) (72 mg, 0.20 mmol, 21% yield) as an orange solid as a TFA salt. 1H NMR (400 MHz, DMSO-d6) δ ppm 12.01 (1H, s), 8.27 (1H, d, J = 5.1 Hz), 7.51 7.72 (3H, m), 6.10 (1H, s), 4.49 (1H, q, J=6.6 Hz), 3.50 (3H, s), 1.61 (9H, s), 1.39 (3H, d, J=6.7 Hz). m/z (ESI, +ve ion) 367.0 (M+H)+.

(R)-3-Methyl-8-(6-methyl-4-oxo-1,4,5,6-tetrahydropyrrolo[3,4-b]pyrrol-2-yl)-2neopentylquinazolin-4(3H)-one (20).



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 66 of 88

Step 1. To a solution of 2-amino-3-bromobenzoic acid (2.00 g, 9.26 mmol) and methylamine (2.0 M solution in THF, 18.52 mL, 37.04 mmol) in 10 mL EtOAc at 0 °C was added T3P (6.48 mL, 10.18 mmol). The reaction was sealed and stirred at RT for 18 h. The reaction was partitioned between sat’d NaHCO3 and EtOAc. The organic layer was washed with brine, dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo to give 2-amino3-bromo-N-methylbenzamide (20a, 1.32 g, 5.76 mmol, 62% yield) as a white solid. 1H NMR (400 MHz, DMSO-d6) δ ppm 8.36 (1H, d, J = 3.5 Hz), 7.41-7.57 (2H, m), 6.40-6.57 (3H, m), 2.74 (3H, d, J = 4.5 Hz). m/z (ESI, +ve ion) 228.9/230.9 (M+H)+. Step 2. To a solution of 2-amino-3-bromo-N-methylbenzamide (20a, 0.20 g, 0.87 mmol) in 2 mL dioxane at RT was added t-butylacetyl chloride (0.13 mL, 0.96 mmol). The reaction became cloudy over 5 min. After 1 h additional t-butylacetyl chloride (0.13 mL) was added. After 1 h, the reaction was sealed and placed in a 100 °C oil bath. After 30 min, the reaction was homogeneous. The reaction was heated an additional 1.5 h, cooled, and allowed to stir overnight. The reaction was partitioned between sat’d aqueous sodium bicarbonate and EtOAc.

The

organic layer was washed with sat’d aqueous sodium bicarbonate once, brine once, and the organics were dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo to give 8bromo-3-methyl-2-neopentylquinazolin-4(3H)-one (20b, 0.28 g, 0.90 mmol, 100% yield) as an off-white solid. 1H NMR (400 MHz, chloroform-d) δ ppm 8.23 (1H, dd, J = 7.9, 1.5 Hz) 8.00 (1H, dd, J = 7.6, 1.4 Hz) 7.27-7.31 (1H, m) 3.65 (3H, s) 2.81 (2H, s) 1.19 (9H, s). m/z (ESI, +ve ion) 309.0/311.0 (M+H)+. Step 3. Argon was bubbled into a mixture of X-Phos Pd G2 (0.014 g, 0.018 mmol), potassium

phosphate

(0.23

g,

1.07

mmol),

(R)-6-methyl-2-(4,4,5,5-tetramethyl-1,3,2-

dioxaborolan-2-yl)-5,6-dihydropyrrolo[3,4-b]pyrrol-4(1H)-one (BE1, 0.14 g, 0.53 mmol), 865 ACS Paragon Plus Environment

Page 67 of 88

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


bromo-3-methyl-2-neopentylquinazolin-4(3H)-one (20b, 0.11 g, 0.36 mmol) in 2 mL dioxane and 0.4 mL water for 1 min. The reaction was placed in a 45 °C oil bath. After 1 h, the reaction was cooled and adsorbed onto 1 g silica gel. The material was purified by silica gel chromatography (24 g column using 0-10% MeOH in DCM) to afford (R)-3-methyl-8-(6methyl-4-oxo-1,4,5,6-tetrahydropyrrolo[3,4-b]pyrrol-2-yl)-2-neopentylquinazolin-4(3H)-one (20) (0.08 g, 0.22 mmol, 62% yield) as an off-white solid. 1H NMR (400 MHz, DMSO-d6) δ ppm 12.21 (1H, s), 8.19 (1H, dd, J = 7.6, 1.4 Hz), 7.97 (1H, dd, J = 7.9, 1.3 Hz), 7.65 (1H, s), 7.48 (1H, t, J = 7.8 Hz), 6.97 (1H, d, J = 1.4 Hz), 4.56 (1H, q, J = 6.7 Hz), 3.63 (3H, s), 3.04 (2H, s), 1.38 (3H, d, J = 6.7 Hz), 1.09 (9H, s). m/z (ESI, +ve ion) 365.1 (M+H)+. (R)-2-(3-(tert-Butylamino)-2-methyl-1,1-dioxido-2H-benzo[e][1,2,4]thiadiazin-5-yl)-6methyl-5,6-dihydropyrrolo[3,4-b]pyrrol-4(1H)-one (21). Step 1. 3-Bromo-2-nitro aniline (Combi-Blocks, 4.32 g, 19.91 mmol) was stirred in concentrated HCl (20 mL, 237 mmol) at 0 °C and treated with sodium nitrite (1.44 g, 20.87 mmol) in water (3 mL) dropwise and allowed to stir at ca. 5-10 °C for 30 min. The suspension was filtered and the diazonium solution was simultaneously added with sodium sulfite (2.51 g, 19.91 mmol) in water (10 mL) to a solution of concentrated HCl (45 mL), anhydrous copper (II) sulfate (0.32 g, 1.99 mmol), sodium sulfite (2.51 g, 19.91 mmol) and water (10 mL). The addition was done in ca. 10-15 min. The reaction mixture was stirred for another 30 min at 0 °C and then filtered through a sintered glass frit washing with cold water. The resulting light yellow solid was collected and dried under high vacuum overnight to give 3-bromo-2-nitrobenzene-1sulfonyl chloride (21a) which was used as crude. Step 2. The crude 21a in 5 mL of THF was treated with methanamine (2.0 M in THF) (12.98 mL, 25.96 mmol) at 0 °C and stirred at RT for 48 h. LC-MS indicated a small amount of 66 ACS Paragon Plus Environment


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 68 of 88

the desired product [m/z (ESI, +ve) 316.8/318.8 (M+H)+] was formed. The reaction mixture was treated with Hunig's base (1.28 mL, 7.32 mmol) and heated to 50 °C for 18 h. The reaction mixture was concentrated and the crude residue was purified on the ISCO Combiflash RF (40 g Thomson SingleStep column, using a gradient of 20-100% EtOAc in hexanes) affording 3bromo-N-methyl-2-nitrobenzenesulfonamide (21b, 556 mg, 1.88 mmol, 26% yield) as a light yellow crystalline solid. 1H NMR (400 MHz, CHLOROFORM-d) δ ppm 8.00 (1H, dd, J = 8.0, 1.2 Hz), 7.92 (1H, dd, J = 8.1, 1.1 Hz), 7.53 (1H, t, J = 8.0 Hz), 4.63-4.79 (1H, m), 2.75 (3H, d, J = 5.1 Hz). m/z (ESI, +ve) 316.8/318.8 (M+H)+. Step 3. 3-Bromo-N-methyl-2-nitrobenzenesulfonamide (21b, 556 mg, 1.88 mmol) was treated with platinum (10% wt. on activated carbon, 184 mg, 0.09 mmol) and MeOH (20 mL) and fitted with a balloon filled with hydrogen.

The flask was purged (5 x) with

vacuum/hydrogen backfill cycles and allowed to stir overnight at RT. The reaction mixture was filtered through a 0.45 um acrodisc to remove the Pt/C washing with MeOH and concentrated to dryness under high vacuum affording 2-amino-3-bromo-N-methylbenzenesulfonamide (21c, 477 mg, 1.80 mmol, 95% yield) as a yellow crystalline solid. This material was used as crude. m/z (ESI, +ve) 266.1/268.1 (M+H)+. Step 4. To a suspension of crude 2-amino-3-bromo-N-methylbenzenesulfonamide (21c, 477 mg, 1.799 mmol) in MeCN (6.60 mL) at 0 °C was added tert-butyl nitrite (90% pure grade, 0.35 mL, 2.70 mmol) followed by azidotrimethylsilane (0.28 mL, 2.16 mmol) dropwise. Gas was evolution was observed. The solution was allowed to warm up to RT and stirred for 1 h. The reaction mixture was treated with tert-butyl nitrite (0.35 mL) and azidotrimethylsilane (0.28 mL) again and allowed to stir for 3 h. The reaction mixture was concentrated. The orange residue was purified by chromatography on an ISCO Combiflash RF (25 g Thomson SingleStep 67 ACS Paragon Plus Environment

Page 69 of 88

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


column, using a gradient of 20-80% EtOAc in hexanes) affording 2-azido-3-bromo-Nmethylbenzenesulfonamide (21d, 106 mg, 0.37 mmol, 20% yield) as a light yellow crystalline solid. 1H NMR (400 MHz, CHLOROFORM-d) δ ppm 7.97 (1H, dd, J = 7.8, 1.4 Hz), 7.80 (1H, m), 7.25 (1H, m), 5.02 (1 H, d, J=4.9 Hz), 2.64 (3 H, m). m/z (ESI, +ve) 291.1/293.1 (M+H)+. Note: A byproduct was isolated whose mass was consistent with 5-bromo-2-methyl-2Hbenzo[e][1,2,3,4]thiatriazine 1,1-dioxide (70 mg, 0.25 mmol, 14% yield) as a light yellow crystalline solid.

1

H NMR (400 MHz, CHLOROFORM-d) δ ppm 8.08 - 8.15 (1H, m), 7.94 -

8.01 (1H, m), 7.62 (1H, t, J = 7.9 Hz), 3.93 (3H, s). m/z (ESI, +ve) 275.9/277.9 (M+H)+. Step 5. 2-azido-3-bromo-N-methylbenzenesulfonamide (21d, 106 mg, 0.364 mmol) in DCM (4.0 mL) was treated with triphenylphosphine (115 mg, 0.437 mmol) resulting in a bright yellow solution and the mixture was allowed to stir at RT for 3 h. The DCM was removed under reduced pressure and the crude reaction mixture was treated with THF (4 mL) and 2-isocyanato2-methylpropane (0.051 mL, 0.437 mmol) and heated to 80 °C in a heating block for 2 h. The reaction mixture was treated with DBU (0.066 mL, 0.437 mmol) and stirred overnight at 80 °C. It was heated to 120 °C for 4 h. LC-MS indicated ca. 35% conversion to the desired product [m/z (ESI, +ve) 345.9/347.9 (M+H)+]. The reaction mixture was concentrated and the crude residue was purified on a reverse phase HPLC (Gemini Phenomenex; 30 x 150 mm, 5 u, 30-95% 0.1%TFA/CH3CN in 0.1% TFA/water) to give 5-bromo-3-(tert-butylamino)-2-methyl-2Hbenzo[e][1,2,4]thiadiazine 1,1-dioxide (21e, 56 mg, 0.16 mmol, 45% yield) as a white solid. 1H NMR (400 MHz, DMSO-d6) δ ppm 7.92 (1H, dd, J = 7.8, 1.4 Hz), 7.70 (1H, dd, J = 7.8, 1.4 Hz), 7.11 (1H, t, J = 7.8 Hz), 7.07 (1H, s), 3.31 (3H, s), 1.52 (9H, s). m/z (ESI, +ve) 345.8/347.8 (M+H)+.



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 70 of 88

Step 6. A 100-mL RBF was charged with (R)-6-methyl-2-(4,4,5,5-tetramethyl-1,3,2dioxaborolan-2-yl)-5,6-dihydropyrrolo[3,4-b]pyrrol-4(1H)-one (BE1, 70 mg, 0.26 mmol), XPhos Pd G2 (6.25 mg, 7.94 µmol), potassium phosphate (101 mg, 0.47 mmol) and 5-bromo-3-(tertbutylamino)-2-methyl-2H-benzo[e][1,2,4]thiadiazine 1,1-dioxide (21e, 55 mg, 0.16 mmol) in 1,4-dioxane (4 mL)/water (0.67 mL) followed by purging the flask with argon. The mixture was stirred at 40 °C for 1.5 h. The mixture was diluted with EtOAc (50 mL) and water (10 mL). The layers were separated and the aqueous layer was extracted with EtOAc (3 x 50 mL). The combined organic layers were dried (MgSO4), filtered. The crude residue was purified on a silica gel column (0-10% MeOH in DCM) affording a viscous light yellow oil. It was suspended in ether

and

filtered

to

give

(R)-2-(3-(tert-butylamino)-2-methyl-1,1-dioxido-2H-

benzo[e][1,2,4]thiadiazin-5-yl)-6-methyl-5,6-dihydropyrrolo[3,4-b]pyrrol-4(1H)-one

(21)

(45

mg, 0.11 mmol, 71% yield) as a white amorphous powder after drying in the vacuum oven overnight. 1H NMR (400 MHz, DMSO-d6) δ ppm 11.67 (1H, s), 7.77 (1H, dd, J = 7.6, 1.6 Hz), 7.63 (1H, dd, J = 7.8, 1.4 Hz), 7.59 (1H, s), 7.25 (1H, t, J = 7.8 Hz), 6.97 (1H, s), 6.51 (1H, d, J = 1.6 Hz), 4.48 (1H, q, J = 6.7 Hz), 3.24 (3H, s), 1.37 (3H, br.), 1.35 (9H, s). m/z (ESI, +ve) 402.0 (M+H)+. (R)-3-methyl-8-(6-methyl-4-oxo-1,4,5,6-tetrahydropyrrolo[3,4-b]pyrrol-2-yl)-2-((1methylcyclopropyl)amino)quinazolin-4(3H)-one (22). Step 1. A sealable flask was charged with 2-amino-3-iodobenzoic acid (22a, Bosche Scientific, catalog # A12065; 10.0 g, 38.0 mmol) and methyl isothiocyanate (5.6 g, 76 mmol) in EtOH (120 mL) followed by triethylamine (7.9 mL, 57.0 mmol). The flask was sealed and heated in an oil bath at 100 °C for 4 h behind a blast shield. The mixture was cooled to RT and the solvents were removed in vacuo. The residue was triturated with ether (40 mL), filtered, and


Page 71 of 88

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


dried to give 8-iodo-3-methyl-2-thioxo-2,3-dihydroquinazolin-4(1H)-one (11.2 g, 35.3 mmol, 93% yield) as an off-white solid. The material was used without further purification. 1H NMR (400 MHz, DMSO-d6) δ ppm 9.97 (1H, br.), 8.23 (1H, d, J = 8.4 Hz), 8.00 (1H, d, J = 8.0 Hz), 7.15 (1H, t, J = 7.5 Hz), 3.65 (3H, s). m/z (ES, +ve) 318.8 (M+H)+. To a mixture of 8-iodo-3methyl-2-thioxo-2,3-dihydroquinazolin-4(1H)-one (11.2 g, 35.3 mmol) and potassium carbonate (9.7 g, 70.6 mmol) in THF (100 mL) was added iodomethane (6.6 mL, 106.0 mmol). The reaction mixture was heated to reflux for 6 h, and then cooled to RT. The mixture was poured into ice/water (300 mL) and stirred for 10 min. The suspension was filtered and the solid was washed with cold water (20 mL) followed by cold ether (20 mL). The solid was dried to give 8iodo-3-methyl-2-(methylthio)quinazolin-4(3H)-one (22b, 11.4 g, 97% yield) as an off-white solid. The crude material was used in the next step. 1H NMR (400 MHz, DMSO-d6) δ ppm 8.31 (1H, dd, J = 7.6, 1.4 Hz), 8.08 (1H, dd, J = 7.8, 1.4 Hz), 7.19 (1H, t, J = 7.7 Hz), 3.51 (3H, s), 2.73 (3H, s). m/z (ES, +ve) 332.9 (M+H)+. Step 2. To a stirred suspension of 8-iodo-3-methyl-2-(methylthio)quinazolin-4(3H)-one (22b, 15.00 g, 45.20 mmol) in 400 mL of dichloromethane at 0 °C was added 3chlorobenzoperoxoic acid (15.59 g of 77% max. Aldrich, 70 mmol) portion wise over 10 min. The ice bath was removed and the reaction mixture was allowed to stir at RT for 30 min. It was diluted with 200 mL of DCM, washed sequentially with ice cold 2 x 50 mL of sat’d Na2CO3, 2 x 50 mL of sat’d Na2SO3 solution, and 15 mL of brine. The organic solution was dried over sodium sulfate, filtered, and concentrated under reduced pressure to give yellow crystalline solid containing a mixture of 8-iodo-3-methyl-2-(methylsulfinyl)quinazolin-4(3H)-one and 8-iodo-3methyl-2-(methylsulfonyl)quinazolin-4(3H)-one.

The crude material in 40 mL of THF and 40

mL of water at RT was treated with LiOH-H2O (6.60 g, 275.00 mmol). The reaction mixture 70 ACS Paragon Plus Environment


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 72 of 88

was stirred in an oil bath at 75 °C for 2.5 h. It was concentrated to half of its volume under reduced pressure. The precipitated solid was filtered, rinsed with 2 x 5 mL of water followed by 2 x 5 mL of ether. The off-white crystalline solid was dried in a vacuum oven at 45 oC for 48 h to give 2-hydroxy-8-iodo-3-methylquinazolin-4(3H)-one (22c, 12.79 g, 42.30 mmol, 94% yield). The crude material was used in the next step. m/z (ES, +ve) 302.9 (M+H)+. Step 3. At RT, 2-hydroxy-8-iodo-3-methylquinazolin-4(3H)-one (22c, 12.8 g, 42.3 mmol) was mixed with phosphorus oxychloride (46.5 mL, 508.0 mmol). The resulting white suspension was treated with N,N-diisopropylethylamine (14.7 mL, 85.0 mmol) dropwise. The heterogeneous mixture was heated in an oil bath at 105 °C for 26 h. LCMS indicated the reaction was about 60% conversion.

It was cooled to RT, and treated with additional phosphorus

oxychloride (12 mL) and N,N-diisopropylethylamine (15 mL). The resulting dark homogeneous solution was heated at 110 °C in an oil bath for 24 h. LCMS indicated the reaction was about >95% conversion. It was concentrated under reduced pressure. The brown sticky solution was treated with 50 mL of toluene and concentrated under reduced pressure again. The brown residue was cooled with an ice bath, ice was added to the flask, followed by 5 N NaOH solution till pH >9. It was extracted with 3 x 250 mL of EtOAc. The combined organic solution was washed with 2 x 25 mL of brine, dried over sodium sulfate, filtered and concentrated. The brown residue was purified via silica gel chromatography (25-50% EtOAc in hexanes) to afford 2-chloro-8-iodo-3-methylquinazolin-4(3H)-one (22d, 10.5 g, 32.8 mmol, 77% yield) as an offwhite crystalline solid. 1H NMR (400 MHz, chloroform-d) δ ppm 8.27 (m, 2 H), 7.22 (t, J=7.82 Hz, 1 H), 3.77 (s, 3 H). m/z (ES, +ve) 328.8 (M+H)+. Step 4. A heterogeneous mixture of 1-methylcyclopropanamine hydrochloride (0.47 g, 4.38 mmol), 2-chloro-8-iodo-3-methylquinazolin-4(3H)-one (22d, 0.80 g, 2.50 mmol) and 71 ACS Paragon Plus Environment

Page 73 of 88

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


triethylamine (2.10 mL, 15.10 mmol) in 2 mL of THF in a sealed glass tube was heated in an oil bath at 80 °C for 5 h. It was diluted with EtOAc, washed water followed by brine. The organic extract was concentrated and the residue was purified on a silica gel column (25-55% EtOAc in hexanes) to afford 8-iodo-3-methyl-2-((1-methylcyclopropyl)amino)quinazolin-4(3H)-one (22e, 0.71 g, 2.00 mmol, 80% yield) as a yellow crystalline solid. 1H NMR (400 MHz, DMSO-d6) δ ppm 8.14 (1H, dd, J = 7.5, 1.3 Hz), 7.93 (1H, dd, J = 7.9, 1.3 Hz), 7.49 (1H, s), 6.89 (1H, t, J = 7.7 Hz), 3.32 (3H, m), 1.58 (3H, s), 0.84 (2H, m), 0.74 (2H, m). m/z (ES, +ve) 355.9 (M+H)+. Step 5. A mixture of 2-(dicyclohexylphosphino)-2',4',6',-tri-isopropyl1,1'-biphenyl (17 mg, 0.036 mmol), tris (dibenzylideneacetone) dipalladium (0) (16 mg, 0.018 mmol), potassium phosphate tribasic monohydrate (311 mg, 1.35 mmol), (R)-6-methyl-2-(4,4,5,5-tetramethyl1,3,2-dioxaborolan-2-yl)-5,6-dihydropyrrolo[3,4-b]pyrrol-4(1H)-one (BE1, 130 mg, 0.49 mmol) and 8-iodo-3-methyl-2-((1-methylcyclopropyl)amino)quinazolin-4(3H)-one (22e, 160 mg, 0.45 mmol) in dioxane (2.5 mL) and water (0.6 mL) in a sealed glass tube was heated in an Initiator microwave reactor (Personal Chemistry, Biotage AB, Inc., Upssala, Sweden) at 105 °C for 35 min. It was partitioned between 25 mL of EtOAc and 2 mL of 0.5 N of NaOH. The organic layer was separated and concentrated under reduced pressure. The crude material was purified by silica gel (eluted with a gradient of 1-5% MeOH in DCM) to afford (R)-3-methyl-8-(6-methyl-4oxo-1,4,5,6-tetrahydropyrrolo[3,4-b]pyrrol-2-yl)-2-((1-methylcyclopropyl)amino)quinazolin4(3H)-one (22) (85 mg, 0.23 mmol, 52% yield), as a brown crystalline solid. 1H NMR (400 MHz, DMSO-d6) δ ppm 13.02 (1H, br.), 8.13 (1H, dd, J = 7.6, 1.4 Hz), 7.83 (1H, dd, J = 7.8, 1.4 Hz), 7.69 (2H, d, J = 3.1 Hz), 7.18 (1H, t, J = 7.7 Hz), 6.96 (1H, s), 4.64 (1H, q, J = 6.6 Hz), 3.40 (3H, s), 1.56 (3H, s), 1.37 (3H, d, J = 6.7 Hz), 1.01 (2H, m), 0.86 (2H, m). MS (ESI, pos. ion) m/z: 364.0 (M+1). 72 ACS Paragon Plus Environment


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 74 of 88

(R)-2-((1-hydroxy-2-methylpropan-2-yl)amino)-3-methyl-8-(6-methyl-4-oxo-1,4,5,6tetrahydropyrrolo[3,4-b]pyrrol-2-yl)quinazolin-4(3H)-one (23). Step 1. A glass microwave reaction vessel was charged with 2-chloro-8-iodo-3methylquinazolin-4(3H)-one (22d, 160 mg, 0.50 mmol) and 2-amino-2-methyl-1-propanol (0.24 mL, 2.50 mmol) in DMSO (1.0 mL). The reaction mixture was stirred and heated at 80 °C for 22 h. It was cooled to RT, treated with water (10 mL) and extracted with EtOAc (3 x 10 mL). The combined organic layers were dried over anhydrous Na2SO4, filtered and concentrated. The crude material was purified by silica gel chromatography (50-100% EtOAc in hexanes) to provide 2-((1-hydroxy-2-methylpropan-2-yl)amino)-8-iodo-3-methylquinazolin-4(3H)-one (23e, 110 mg, 0.30 mmol, 59% yield) as a light-yellow solid. 1H NMR (400 MHz, chloroform-d) δ ppm 8.11 (ddd, J = 7.58, 5.92, 1.37 Hz, 2H), 6.90 (t, J = 7.82 Hz, 1H), 4.81 (s, 1H), 3.89 (d, J = 5.87 Hz, 2H), 3.62 - 3.69 (m, 1H), 3.50 (s, 3H), 1.62 (s, 6H). m/z (ESI, +ve) 374.0 (M+H)+. Step 2. A glass microwave reaction vessel was charged with 2-((1-hydroxy-2methylpropan-2-yl)amino)-8-iodo-3-methylquinazolin-4(3H)-one (23e, 105 mg, 0.28 mmol), (R)-6-methyl-2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-5,6-dihydropyrrolo[3,4-b]pyrrol4(1H)-one (BE1, 221 mg, 0.84 mmol), XPhos Pd G2 (11 mg, 14 µmol), potassium phosphate (179 mg, 0.84 mmol) in dioxane (2.5 mL) and water (0.6 mL). The reaction mixture was stirred and heated in an oil bath at 45 °C for 90 min. Water (5 mL) and EtOAc (5 mL) were added and the layers were separated. The aqueous layer was extracted with EtOAc (2 x 5 mL). The combined organic layers were dried over anhydrous Na2SO4, filtered and concentrated. The crude material was purified by silica gel chromatography (0-10% MeOH in DCM), to provide (R)-2-((1-hydroxy-2-methylpropan-2-yl)amino)-3-methyl-8-(6-methyl-4-oxo-1,4,5,673 ACS Paragon Plus Environment

Page 75 of 88

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


tetrahydropyrrolo[3,4-b]pyrrol-2-yl)quinazolin-4(3H)-one (23) (55 mg, 0.14 mmol, 51% yield) as a tan solid. 1H NMR (400 MHz, DMSO-d6) δ ppm 12.03 (s, 1 H), 7.90 (ddd, J = 10.32, 7.78, 1.47 Hz, 2 H), 7.61 (s, 1 H), 7.18 (t, J = 7.73 Hz, 1 H), 6.71 (d, J = 1.37 Hz, 1 H), 5.79 (s, 1 H), 5.17 (t, J = 5.67 Hz, 1 H), 4.53 (q, J = 6.59 Hz, 1 H), 3.59 (m, 2 H), 3.48 (s, 3 H), 1.46 (d, J = 9.78 Hz, 6 H), 1.38 (d, J = 6.65 Hz, 3 H). m/z (ESI, +ve) 382.1 (M+H)+. (R)-2-(tert-butyl(methyl)amino)-3-methyl-8-(6-methyl-4-oxo-1,4,5,6-tetrahydropyrrolo[3,4b]pyrrol-2-yl)quinazolin-4(3H)-one (24). Step 1. A mixture of 2-chloro-8-iodo-3-methylquinazolin-4(3H)-one (22d, 2.44 g, 7.61 mmol) and t-butylamine (16.00 mL, 152 mmol) was heated at 80 °C in a sealed tube for 6 h. The reaction mixture was transferred to a RBF and concentrated to remove excess of t-butylamine. The residue was then treated with water and extracted with EtOAc/Et2O 1:1 (2 x 75 mL), dried over

MgSO4,

filtered

and

concentrated

affording

2-(tert-butylamino)-8-iodo-3-

methylquinazolin-4(3H)-one (24e, 2.68 g, 7.50 mmol, 99% yield) as a yellow amorphous solid. 1

H NMR (400 MHz, CHLOROFORM-d) δ ppm 8.10 (2H, m), 6.87 (1H, t, J = 7.7 Hz), 4.45 (1H,

br.), 3.46 (3H, s), 1.65 (9H, s). m/z (ESI, +ve ion) 358.0 (M+H)+. Step 2. To a solution of 2-(tert-butylamino)-8-iodo-3-methylquinazolin-4(3H)-one (24e, 400 mg, 1.12 mmol) in 3 mL of DMF at 0 oC was added sodium hydride (60% wt. in mineral oil) (81 mg, 2.02 mmol). The resulting yellow suspension was stirred at RT for 15 min then cooled at 0 oC, and treated with iodomethane (70 µL, 1.12 mmol). It was stirred at RT for 90 min, then treated with 5 mL of sat’d NH4Cl. The precipitated white solid was filtered, rinsed with 2 x 3 mL of water followed by 2 x 2 mL of ether. The filtrate was discarded. The white solid was dried in a vacuum oven at 40 oC for 2 h to afford 2-(tert-butyl(methyl)amino)-8-iodo-



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 76 of 88

3-methylquinazolin-4(3H)-one (24f, 355 mg, 0.95 mmol, 85% yield). 1H NMR (400 MHz, DMSO-d6) δ ppm 8.23 (1H, dd, J = 7.6, 1.4 Hz), 8.02 (1H, dd, J = 7.8, 1.4 Hz), 7.09 (1H, t, J = 7.6 Hz), 3.48 (3 H, s), 2.74 (3 H, s), 1.49 (9 H, s). m/z (ESI, +ve ion) 372.0 (M+H)+. Step 3. A mixture of (R)-6-methyl-2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-5,6dihydropyrrolo[3,4-b]pyrrol-4(1H)-one (BE1, 275 mg, 1.05 mmol), 2-(tert-butyl(methyl)amino)8-iodo-3-methylquinazolin-4(3H)-one (24f, 300 mg, 0.81 mmol), potassium phosphate tribasic (515 mg, 2.42 mmol) and XPhos Pd G2 (31 mg, 0.04 mmol) in 1,4-dioxane (4 mL)/water (1.2 mL) was heated in an oil bath at 50 °C for 1 h. Additional XPhos Pd G2 (15 mg, 0.02 mmol) and BE1 (50 mg, 0.19 mmol) were added to the reaction mixture and heating was continued at 50 oC for 35 min. It was cooled to RT, treated with 5 mL of water, and extracted with 50 mL of EtOAc. The EtOAc layer was washed with 5 mL of brine, concentrated and the residue was purified on a silica gel column (eluted with 1-5% MeOH in EtOAc) to give (R)-2-(tertbutyl(methyl)amino)-3-methyl-8-(6-methyl-4-oxo-1,4,5,6-tetrahydropyrrolo[3,4-b]pyrrol-2yl)quinazolin-4(3H)-one (24) (160 mg, 0.42 mmol, 52% yield) as a brown crystalline solid. 1H NMR (400 MHz, DMSO-d6) δ ppm 11.85 (1H, br.), 8.00 (2H, m), 7.64 (1H, s), 7.42 (1H, t, J = 7.7 Hz), 6.83 (1H, s), 4.54 (1H, d, J = 6.5 Hz), 3.56 (3H, s), 2.75 (3H, s), 1.37 (3H, d, J = 6.7 Hz), 1.33 (9H, s). m/z (ESI, +ve ion) 380.0 (M+H)+. (R)-2-(2,2-dimethylpyrrolidin-1-yl)-3-methyl-8-(6-methyl-4-oxo-1,4,5,6tetrahydropyrrolo[3,4-b]pyrrol-2-yl)quinazolin-4(3H)-one (25). A mixture of 2,2-dimethylpyrrolidine hydrochloride (280 mg, 2.062 mmol, Enamine Ltd., cat # EN300-62047), 8-bromo-2-chloro-3-methylquinazolin-4(3H)-one (17d, 470 mg, 1.72 mmol) and N,N-diisopropylethylamine (1.20 mL, 6.87 mmol) in 2 mL of DMSO was heated in a microwave at 140 oC for 35 min. It was partitioned between 10 mL of water and 50 mL of


Page 77 of 88

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


EtOAc. The organic layer was separated and concentrated. The residue was purified on a silica gel column (35-55% EtOAc in hexanes) to give 8-bromo-2-(2,2-dimethylpyrrolidin-1-yl)-3methylquinazolin-4(3H)-one (25e, 497 mg, 1.48 mmol, 86% yield) as a brown amorphous solid. 1

H NMR (400 MHz, DMSO-d6) δ ppm 7.93 (2H, d, J = 8.0 Hz), 7.06 (1H, t, J = 7.8 Hz), 3.57

(2H, t, J = 7.0 Hz), 3.48 (3H, s), 1.941 (2H, m), 1.80 (2H, m), 167 (6H, s). m/z (ESI, +ve) 336/338 (M+H)+. A mixture of 8-bromo-2-(2,2-dimethylpyrrolidin-1-yl)-3-methylquinazolin-4(3H)-one (25e, 320 mg, 0.95 mmol), (R)-6-methyl-2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-5,6dihydropyrrolo[3,4-b]pyrrol-4(1H)-one (BE1, 324 mg, 1.23 mmol), potassium phosphate (606 mg, 2.86 mmol) and XPhos Pd G2 (37.4 mg, 0.048 mmol) in 1,4-dioxane (4 mL) / water (1.2 mL) was heated in an oil bath at 50 °C for 1 h. It was cooled to room temperature. The mixture was partitioned between 5 mL of water and 50 mL of EtOAc. The EtOAc layer was washed with 5 mL of brine, concentrated and the residue was purified on a silica gel column (1-5% MeOH in EtOAc) to give (R)-2-(2,2-dimethylpyrrolidin-1-yl)-3-methyl-8-(6-methyl-4-oxo1,4,5,6-tetrahydropyrrolo[3,4-b]pyrrol-2-yl)quinazolin-4(3H)-one (25) (289 mg, 0.73 mmol, 78% yield) as a brown crystalline solid. 1H NMR (400 MHz, DMSO-d6) δ ppm 11.67 (1H, br.), 7.93 (1H, m), 7.85 (1H, m), 7.59 (1H, s), 7.26 (1H, t, J = 7.7 Hz), 6.61 (1H, s), 4.51 (1H, d, J = 6.7 Hz), 3.56 (2H, m), 3.44 (3H, s), 1.97 (2H, m), 1.77 (2H, m), 1.47 (3H, s), 1.43 (3H, s), 1.36 (3H, d, J = 6.7 Hz). m/z (ESI, +ve) 392.0 (M+H)+. (R)-2-(3-(tert-butylamino)-6-fluoro-2-methylquinoxalin-5-yl)-6-methyl-5,6dihydropyrrolo[3,4-b]pyrrol-4(1H)-one (26).



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 78 of 88

Step 1. 2-Bromo-1,3-difluoro-4-nitrobenzene (26a), Combi-Blocks, 27.15 g, 114 mmol) was treated with ammonium carbonate (10.96 g, 114 mmol) and DMF (200 mL) followed by Et3N (47.7 mL, 342 mmol) and stirred at RT for 48 h. The mixture was treated with water and extracted with DCM (300 mL). The DCM layer was washed with water (3 x 200 mL) and brine (3 x 200 mL), dried over MgSO4, filtered and concentrated affording crude 2-bromo-3-fluoro-6nitroaniline (26b, 26 g, 111 mmol, 97% yield) as a bright yellow amorphous solid. 1H NMR (400 MHz, CDCl3) δ ppm 8.22 (1H, dd, J = 9.6, 5.9Hz), 6.81 (2H, br.), 6.56 (2H, dd, J = 9.6, 7.2 Hz). 19

F NMR (376 MHz, CDCl3) δ ppm -90.92 (s). m/z (ESI, +ve ion) 234.9/236.9 (M+H)+. Step 2. A 500 mL RBF containing Pt/C (5% wt., 6.00 g, 1.54 mmol) and 2-bromo-3-

fluoro-6-nitroaniline (26b) (20.85 g, 89 mmol) were treated with EtOH (250 mL) and stirred under an atmosphere of H2 (balloon) for 22 h. LC-MS indicated ca. 35% conversion to the desired material (m/z (ESI, +ve ion) 204.9/206.9 (M+H)+). Another balloon of H2 was added and it was stirred for another 16 h resulting in >90% conversion to the desired product. The suspension was filtered through a plug of Celite washing with EtOH and concentrated affording crude 3-bromo-4-fluorobenzene-1,2-diamine (18 g, 88 mmol, 99% yield) as a black/purple viscous oil. 1H NMR (400 MHz, CDCl3) δ ppm 6.60 (1H, dd, J = 8.5, 5.2 Hz), 6.47 (1H, m). 19F NMR (376 MHz, CDCl3) δ ppm -116.48 (s). m/z (ESI, +ve ion) 204.9/206.9 (M+H)+. Step 3. A 500 mL RBF charged with 3-bromo-4-fluorobenzene-1,2-diamine (26c) (18.00 g, 88.00 mmol), DMF (100 mL), ethyl 2-bromopropionate (11.54 mL, 89.00 mmol) and NaHCO3 (7.60 g, 90.00 mmol) was heated to 90 °C with a reflux condenser for 30 min, then at 120 °C for 15 h. The reaction mixture was cooled to RT, treated with brine and extracted with EtOAc (2 x 200 mL), washed with brine (3 x) and dried over Na2SO4, filtered and concentrated


Page 79 of 88

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


affording crude 8-bromo-7-fluoro-3-methyl-3,4-dihydroquinoxalin-2(1H)-one (26d, 21.75 g, 84.00 mmol, 96% yield) as an orange-brown viscous oil. The material was used in the subsequent step without further purification. 1H NMR (400 MHz, DMSO-d6) δ ppm 9.67 (s, 1H) 6.80 (m, 1H) 6.60 (m, 1H) 6.27 (m, 1H) 1.25 (d, J = 6.46 Hz, 3H). 19F NMR (377 MHz, DMSOd6) δ ppm -120.49 (s). m/z (ESI, +ve ion) 259.0/261.0 (M+H)+. Step 4. A mixture of water (14 mL) and 30% H2O2 (30.0 mL, 294 mmol) was added slowly dropwise to a solution of 8-bromo-7-fluoro-3-methyl-3,4-dihydroquinoxalin-2(1H)-one (26d) (21.7 g, 84.0 mmol) in 1 N NaOH (168 mL) and MeOH (140 mL). The flask was fitted with a reflux condenser and heated at 85 °C for 3 h. LC-MS indicated ca. 87% conversion to the desired product (m/z (ESI, +ve ion) 257.0/259.0 (M+H)+). The reaction mixture was cooled to RT and acidified with 2 N HCl to ca. pH 6 and was diluted with CHCl3/IPA (4:1) (100 mL), added to a separatory funnel. The resulting suspension was filtered through a sintered glass frit, washing with water and dried affording 8-bromo-7-fluoro-3-methylquinoxalin-2(1H)-one (5.8 g, 22.0 mmol, 22% yield) as a light brown solid. 1H NMR (400 MHz, DMSO-d6) δ ppm 11.83 (1H, br.), 7.78 (1H, m), 7.34 (1H, t, J = 8.4 Hz), 2.43 (3H, s).

19

F NMR (377 MHz, DMSO-d6) δ ppm

-104.13 (s). m/z (ESI, +ve ion) 257.0/259.0 (M+H)+. The aqueous solution was extracted with CHCl3:i-PrOH = 9:1 (6 x 100 mL), dried over MgSO4, filtered and concentrated affording additional 8-bromo-7-fluoro-3-methylquinoxalin-2(1H)-one (26e, 12.5 g, 49.0 mmol, 58% yield) as a dark brown amorphous solid (ca. 80% purity). The material was used in the subsequent step without further purification. Step 5. In a 500-mL round-bottomed flask, a mixture of 8-bromo-7-fluoro-3methylquinoxalin-2(1H)-one (26e, 3.57 g, 13.89 mmol) and POCl3 (20.0 mL) was heated at 90



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 80 of 88

°C for 1.5 h with a reflux condenser. The reaction mixture was cooled to RT and most of the excess POCl3 was removed under reduced pressure. The mixture was treated with EtOAc (100 mL), cooled in an ice bath, treated with ice chips and 1 N NaOH slowly. After phase separation, the organic layer was washed with brine (50 mL), dried over MgSO4, filtered and concentrated to afford the crude product as a brown solid. The crude residue was purified by silica gel chromatography

(10-100%

DCM

in

hexanes)

affording

5-bromo-3-chloro-6-fluoro-2-

methylquinoxaline (26f, 1.88 g, 6.82 mmol, 49% yield) as a light orange crystalline solid. 1H NMR (400 MHz, CDCl3) δ ppm 8.01 (1H, dd, J = 9.3, 5.4 Hz), 7.58 (1H, dd, J = 9.1, 8.1 Hz), 2.86 (3H, s).

19

F NMR (376 MHz, CDCl3) δ ppm -99.07 (s). m/z (ESI, +ve ion) 275.0/277.0

(M+H)+. Step 6. A mixture of 5-bromo-3-chloro-6-fluoro-2-methylquinoxaline (26f, 5.00 g, 18.15 mmol) and t-butylamine (9.62 mL, 91.00 mmol) in DMSO (50 mL) in a sealed tube was heated at 100 °C for 2 h. After cooling to RT, the mixture was diluted with EtOAc and sat’d NaHCO3 (aq.). The layers were separated and the aqueous layer was extracted with EtOAc (3 x). The combined organic layers were washed with water and brine and dried over anhydrous Na2SO4, filtered and concentrated. The crude material was absorbed onto a plug of silica gel and purified by silica gel chromatography (0-20% EtOAc in hexanes) to provide 8-bromo-N-(tert-butyl)-7fluoro-3-methylquinoxalin-2-amine (26g) (4.70 g, 15.06 mmol, 83% yield) as an orange solid. 1

H NMR (400 MHz, CHLOROFORM-d) δ ppm 7.71 (1H, dd, J = 9.0, 5.7 Hz), 7.14 (1H, t, J =

8.6 Hz), 4.82 (1H, br.), 2.51 (3H, s), 1.64 (9H, s).

19

F NMR (376 MHz, CHLOROFORM-d) δ

ppm -103.85 (s). m/z (ESI, +ve) 312.0, 314.0 (M+H)+.


Page 81 of 88

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


Step 7. To a 250-mL RBF was added (R)-6-methyl-2-(4,4,5,5-tetramethyl-1,3,2dioxaborolan-2-yl)-5,6-dihydropyrrolo[3,4-b]pyrrol-4(1H)-one (BE1, 5.52 g, 19.40 mmol) and 8-bromo-N-(tert-butyl)-7-fluoro-3-methylquinoxalin-2-amine (26g, 6.06 g, 19.40 mmol) in 1,4dioxane (40 mL)/water (12 mL) followed by potassium phosphate (12.35 g, 58.20 mmol) and XPhos Pd G2 (0.763 g, 0.97 mmol). The reaction mixture was heated in an oil bath at 45 °C for 1 h. The mixture was added 10 mL of ice water. The resulting precipitate was collected by filtration, washed with water and dried under vacuo to give some brown solid that contained the desired product. The filtrate was extracted with EtOAc (3 x 100 mL). The organic extracts were combined and dried with (MgSO4), filtered and concentrated. The residue was combined with the brown solid that contained the desired product, loaded on a silica gel column and eluted with 1-10% MeOH in DCM to give (R)-2-(3-(tert-butylamino)-6-fluoro-2-methylquinoxalin-5-yl)-6methyl-5,6-dihydropyrrolo[3,4-b]pyrrol-4(1H)-one (26) (3.86 g, 10.51 mmol, 54% yield) as a light yellow solid. 1H NMR (400 MHz, DMSO-d6) δ ppm 11.88 (s, 1H), 7.70 (dd, J = 8.9, 6.0 Hz, 1H), 7.61 (s, 1H), 7.29 (dd, J = 10.4, 9.0 Hz, 1H), 6.46 (t, J = 1.8 Hz, 1H), 6.08 (s, 1H), 4.52 (q, J = 6.7 Hz, 1H), 2.53 (s, 3H), 1.44 (s, 9H), 1.37 (d, J = 6.7 Hz, 3H). MS (ESI, pos. ion) m/z: 368 (M+1). C. Solubility Assays in PBS and FaSIF Compounds as 10 mM DMSO stock solutions were dispensed into 96 deep well plates at a volume of 10 uL per well. Four copies of identical plates were created. The DMSO was removed in a Genevac Evaporator for 2.5 h, at 40 °C, under full vacuum. After dry down was complete, a Tecan Evo Liquid Handler was used to transfer 200 uL of each of the buffers/solvent to the corresponding plate copy. The buffers used were 1x PBS (pH 7.4), and fasted state simulated intestinal fluid (FaSIF, pH 6.8), and the solvent DMSO was used to create the standard 80 ACS Paragon Plus Environment


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 82 of 88

plate for comparison. The plates were sealed and centrifuged at 1,000 rpm for 1 min to push all liquid from walls to the bottom of the wells. The plates were shaken at 1,500 rpm on a 3 mm radius orbital shaker for 1 h. The samples were equilibrated at RT for 72 h. The plates were centrifuged at 4,000 rpm for 30 min. The supernatant was analyzed by LC/MS (at 215 nm, 2 µL injection volume). Peak area in PBS, and fasted state simulated intestinal fluid were compared to DMSO standard to determine solubility, accurate within the range of 5-500 µM.

ASSOCIATED CONTENT Supporting Information. Statistical analysis of data presented in Tables 1 and 2. Atomic coordinates and experimental data of 17/Pim-1 (PDB code 5IPJ). Mouse body weight for the KMS-12 BM xenograft study presented in Figure 3. KINOMEscan data of 17. This material is available free of charge via the Internet at http://pubs.acs.org. AUTHOR INFORMATION Corresponding Author *E-mail: [email protected]; 805-447-6964. Author Contributions All authors have given approval to the final version of the manuscript. ACKNOWLEDGMENT We thank Wes Barnhart, Kyung Gahm, and Sam Thomas for chiral SFC separation of intermediates and final compounds; Chris Wilde for NMR studies. ABBREVIATIONS 81 ACS Paragon Plus Environment

Page 83 of 88

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


BAD, Bcl-2-associated death promoter; BM, bone marrow; CL, clearance; CPK, Corey-PaulingKoltun; DMAC, N,N-dimethylacetamide; FBS, fetal bovine serum; HLM, human liver microsome; RLM, rat liver microsome; PBS, phosphate-buffered saline; Pim, proviral insertion site of moloney murine leukemia; PK, pharmacokinetic; POC, percent of control; PPB, plasma protein binding; QD, once a day; SAR, structure-activity relationship; SFC, supercritical fluid chromatography. REFERENCES (1) Brault, L.; Gasser, C.; Bracher, F.; Huber, K.; Knapp, S.; Schwaller, J. PIM serine/threonine kinases in the pathogenesis and therapy of hematologic malignancies and solid cancers. Haematologica 2010, 95, 1004–1015. (2) Nawijn, M. C.; Alendar, A.; Berns, A. For better or for worse: the role of Pim oncogenes in tumorigenesis. Nat. Rev. Cancer 2011, 11, 23–34. (3) Yan, B.; Zemskova, M.; Holder, S.; Chin, V.; Kraft, A.; Koskinen, P. J.; Lilly, M. The PIM-2 kinase phosphorylates BAD on serine 112 and reverses BAD-induced cell death. J. Biol. Chem. 2003, 278, 45358–45367. (4) Fox, C. J.; Hammerman, P. S.; Cinalli, R. M.; Master, S. R.; Chodosh, L. A.; Thompson, C. B. The serine/threonine kinase Pim-2 is a transcriptionally regulated apoptotic inhibitor. Genes & Dev. 2003, 17, 1841–1854. (5) An, N.; Kraft, A. S.; Kang, Y. Abnormal hematopoietic phenotypes in Pim kinase triple knockout mice. J. Hematol. Oncol. 2013, 6, Article # 12. (6) Mikkers, H.; Nawijn, M.; Allen, J.; Brouwers, C,; Verhoeven, E.; Jonkers, J.; Berns, A. Mice Deficient for All PIM Kinases Display Reduced Body Size and Impaired Responses to Hematopoietic Growth Factors. Mol. Cell Biol., 2004, 24, 6104–6115. 82 ACS Paragon Plus Environment


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 84 of 88

(7) Qian, K. C.; Wang, L.; Hickey, E. R.; Studts, J.; Barringer, K.; Peng, C.; Kronkaitis, A.; Li, J.; White, A.; Mische, S.; Farmer, B. Structural Basis of Constitutive Activity and a Unique Nucleotide Binding Mode of Human Pim-1 Kinase. J. Biol. Chem. 2005, 280, 6130–6137. (8) Blanco-Aparicio, C.; Carnero, A. Pim kinases in cancer: diagnostic, prognostic and treatment opportunities. Biochem. Pharmacol. 2013, 85, 629–643. (9) Garcia, P. D.; Langowski, J. L.; Wang, Y.; Chen, M.; Castillo, J.; Fanton, C.; Ison, M.; Zavorotinskaya, T.; Dai, Y.; Lu, J.; Niu, X.; Basham, S.; Chan, J.; Yu, J.; Doyle, M.; Feycht, P.; Warne, R.; Narberes, J.; Tsang, T.; Fritsch, C.; Kauffmann, A.; Pfister, E.; Drueckes, P.; Trappe, J.; Wilson, C.; Han, W.; Lan, J.; Nishiguchi, G.; Lindvall, M.; Bellamacina, C.; Aycinena, A.; Zang, R.; Holash, J.; Burger, M. T. Pan-PIM Kinase Inhibition Provides a Novel Therapy for Treating Hematologic Cancers. Clin. Cancer Res. 2014, 20, 1834–1845. (10) Arunesh, G. M.; Shanthi, E.; Krishna, M. H.; Kumar, J. S.; Viswanadhan, V. N. Small molecule inhibitors of PIM1 kinase: July 2009 to February 2013 patent update. Expert Opin. Ther. Patents 2014, 24, 5–17. (11) Merkel, A. L.; Meggers, E.; Ocker, M. PIM1 kinase as a target for cancer therapy. Expert Opin. Investig. Drugs 2012, 21, 425–436. (12) Drygin, D.; Haddach, M.; Pierre, F.; Ryckman, D. M. Potential Use of Selective and Nonselective Pim Kinase Inhibitors for Cancer Therapy. J. Med. Chem. 2012, 55, 8199–8208. (13) Hu, H; Wang, X.; Chan, G. K. Y.; Chang, J. H.; Do, S.; Drummond, J.; Ebens, A.; Lee, W.; Ly, J.; Lyssikatos, J. P.; Murray, J.; Moffat, J. G.; Chao, Q.; Tsui, V.; Wallweber, H.; Kolesnikov, A.

Discovery of 3,5-substituted 6-azaindazoles as potent pan-Pim inhibitors.

Bioorg. Med. Chem. Lett. 2015, 25, 5258–5264.


Page 85 of 88

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


(14) Burger, M. T.; Nishiguchi, G.; Han, W.; Lan, J.; Simmons, R.; Atallah, G.; Ding, Y.; Tamez; V.; Zhang, Y.; Mathur, M; Muller, K.; Bellamacina, C.; Lindvall, M. K.; Zang, R.; Huh, K.; Feucht, P.; Zavorotinskaya, T.; Dai, Y.; Basham, S.; Chan, J.; Ginn, E.; Aycinena, A.; Holash, J.; Castillo, J.; Langowski, J. L.; Wang, Y.; Chen, M. Y.; Lambert, A.; Fritsch, C.; Kauffmann, A.; Pfister, E.; Vanasse, K. G.; Garcia, P. D. Identification of N-(4-((1R,3S,5S)-3Amino-5-methylcyclohexyl)pyridin-3-yl)-6-(2,6-difluorophenyl)-5-fluoropicolinamide (PIM447), a Potent and Selective Proviral Insertion Site of Moloney Murine Leukemia (PIM) 1, 2, and 3 Kinase Inhibitor in Clinical Trials for Hematological Malignancies. J. Med. Chem. 2015, 58, 8373–8386. (15) Mumenthaler, S. M.; Ng, P. Y. B.; Hodge, A.; Bearss, D.; Berk, G.; Kanekal, S.; Redkar, S.; Taverna, P.; Agus, D. B.; Jain, A. Pharmacologic inhibition of Pim kinases alters prostate cancer cell growth and resensitizes chemoresistant cells to taxanes. Mol. Cancer Ther. 2009, 8, 2882–2893. (16) Keeton, E. K.; McEachern, K.; Dillman, K. S.; Palakurthi, S.; Cao, Y.; Grondine, M. R.; Kaur, S.; Wang, S.; Chen, Y.; Wu, A.; Shen, M.; Gibbons, F. D.; Lamb, M. L.; Zheng, X.; Stone, R. M.; DeAngelo, D. J.; Platanias, L. C.; Dakin, L. A.; Chen, H.; Lyne, P. D.; Huszar, D. AZD1208, a potent and selective pan-Pim kinase inhibitor, demonstrates efficacy in preclinical models of acute myeloid leukemia. Blood 2014, 123, 905–913. (17) Dakin, L. A.; Block, M. H.; Chen, H.; Code, E.; Dowling, J. E.; Feng, X.; Ferguson, A. D.; Green, I.; Hird, A. W.; Howard, T.; Keeton, E. K.; Lamb, M. L.; Lyne, P. D.; Pollard, H.; Read, J.; Wu, A. J.; Zhang, T.; Zheng, X. Discovery of novel benzylidene-1,3-thiazolidine-2,4diones as potent and selective inhibitors of the PIM-1, PIM-2, and PIM-3 protein kinases. Bioorg. Med. Chem. Lett. 2012, 22, 4599–4604.



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 86 of 88

(18) Cee, V. J.; Chavez, F., Jr.; Herberich, B.; Lanman, B. A.; Pettus, L. H.; Reed, A. B.; Wu, B.; Wurz, R. P.; Andrews, K. L.; Chen, J.; Hickman, D.; Laszlo, J., III; Lee, M. R.; Guererro, N.; Mattson, B. K.; Nguyen, Y.; Mohr, C.; Rex, K.; Sastri, C. E.; Wang, P.; Wu, Q.; Wu, T.; Xu, Y.; Zhou, Y.; Winston, J. T.; Lipford, J. R.; Tasker, A. S.; Wang, H-L. Discovery and Optimization of Macrocyclic Quinoxaline-pyrrolo-dihydropiperidinones as Potent Pim-1/2 Kinase Inhibitors. ACS Med. Chem. Lett., 2016, 7, 408–412. (19) Cee, V. J.; Chavez, F., Jr.; Chen, J. J.; Harrington, E. H.; Herberich, B.; Jackson, C. L. M.; Lanman, B. A.; Nguyen, T. T.; Norman, M. H.; Pettus, L. H.; Reed, A. B.; Smith, A. L.; Tamayo, N. A.; Tasker, A. S.; Wang, H-L; Wu, B.; Wurz, R. P. Preparation of fused pyridinones and related compounds as Pim kinase inhibitors and their use in the treatment of cancer. US 20150329538, November 19, 2015. (20) Wang, H-L.; Cee, V. J.; Chavez, F. Jr.; Herberich, B.; Lanman, B. A.; Pettus, L. H.; Reed, A. B.; Wu, B.; Wurz, R. P.; Andrews, K. L.; Chen, J.; Hickman, D.; Huang, X.; Laszlo, J. III; Lee, M. R.; Guerrero, N.; Mattson, B. K.; Nguyen, Y.; Mohr, C.; Rex, K.; Lipford, J. R.; Tasker, A. S. Discovery and Optimization of Quinoxaline-pyrrolodihydropiperidinones as Potent Pim-1/2 Kinase Inhibitors. 35th National Medicinal Chemistry Symposium, Chicago, IL, United States, June 26-29, 2016, Poster Session. (21) Microsome stability studies: the parent compound (1 µM) was incubated in liver microsomes (rat, dog and human, 0.25 mg/mL) in potassium phosphate (66.7 mM, pH 7.4) buffered with NADPH (1 mM) at 37 °C for 30 min. Intrinsic clearance, CLint (µL/min/mg), was estimated from the amount of parent compound remaining. Under these conditions, a cutoff of CLint

Discovery and Optimization of Quinazolinone ... - ACS Publications

Recommend Documents