Discovery of (R)-8-(6-Methyl-4-oxo-1,4,5,6-tetrahydropyrrolo[3,4- b]pyrrol-2-yl)-3-(1-methylcyclopropyl)-2-((1-methylcyclopropyl)amino)quinazolin-4(3 H)-one, a Potent and Selective Pim-1/2 Kinase Inhibitor for Hematological Malignancies

Article abstract of DOI:10.1021/acs.jmedchem.8b01733

Pim kinases are a family of constitutively active serine/threonine kinases that are partially redundant and regulate multiple pathways important for cell growth and survival. In human disease, high expression of the three Pim isoforms has been implicated in the progression of hematopoietic and solid tumor cancers, which suggests that Pim kinase inhibitors could provide patients with therapeutic benefit. Herein, we describe the structure-guided optimization of a series of quinazolinone-pyrrolodihydropyrrolone analogs leading to the identification of potent pan-Pim inhibitor 28 with improved potency, solubility, and drug-like properties. Compound 28 demonstrated on-target Pim activity in an in vivo pharmacodynamic assay with significant inhibition of BAD phosphorylation in KMS-12-BM multiple myeloma tumors for 16 h postdose. In a 2-week mouse xenograft model, daily dosing of compound 28 resulted in 33% tumor regression at 100 mg/kg.

Full text of DOI:10.1021/acs.jmedchem.8b01733

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Article
Discovery of (R)-8-(6-Methyl-4-oxo-1,4,5,6-tetrahydropyrrolo[3,4-
b]pyrrol-2-yl)-3-(1-methylcyclopropyl)-2-((1-
methylcyclopropyl)amino)quinazolin-4(3H)-one, a Potent and
Selective Pim- 1/2 Kinase Inhibitor for Hematological Malignancies
Hui-Ling Wang, Kristin Andrews, Shon K. Booker, Jude Canon, Victor J. Cee, Frank Chavez Jr., Yuping
Chen, Heather Eastwood, Nadia Guerrero, Bradley Herberich, Dean Hickman, Brian A Lanman, Jimmy Laszlo
III, Matthew R Lee, J. Russell Lipford, Bethany Mattson, Christopher Mohr, Yen Nguyen, Mark H Norman,
Liping H. Pettus, David Powers, Anthony B. Reed, Karen Rex, Christine Sastri, Nuria A. Tamayo, Paul Wang,
Jeffrey T. Winston, Bin Wu, Qiong Wu, Tian Wu, Ryan P. Wurz, Yang Xu, Yihong Zhou, and Andrew S Tasker
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b01733 • Publication Date (Web): 09 Jan 2019
Downloaded from http://pubs.acs.org on January 10, 2019
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Discovery of (R)-8-(6-Methyl-4-oxo-1,4,5,6-
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tetrahydropyrrolo[3,4-b]pyrrol-2-yl)-3-(1-methylcyclopropyl)-
2-((1-methylcyclopropyl)amino)quinazolin-4(3H)-one, a Potent
and Selective Pim-1/2 Kinase Inhibitor for Hematological
Malignancies
Hui-Ling Wang,*† Kristin L. Andrews,‡ Shon K. Booker,† Jude Canon,∥ Victor J. Cee,† Frank Chavez,
Jr.,† Yuping Chen,§ Heather Eastwood,‡ Nadia Guerrero,∥ Brad Herberich,† Dean Hickman,§ Brian
A. Lanman,† Jimmy Laszlo, III,# Matthew R. Lee,‡ J. Russell Lipford,∥ Bethany Mattson,∥
Christopher Mohr,‡ Yen Nguyen,# Mark H. Norman,† Liping H. Pettus ,† David Powers,# Anthony
B. Reed,† Karen Rex,∥ Christine Sastri,∥ Nuria Tamayo,† Paul Wang,# Jeffrey T. Winston,∥ Bin
Wu,† Qiong Wu,⊥ Tian Wu,⊥ Ryan P. Wurz,† Yang Xu,§ Yihong Zhou,§ and Andrew S. Tasker†
†
Department of Therapeutic Discovery-Medicinal Chemistry, ‡ Molecular Structure, § Pharmacokinetics and Drug
Metabolism, ∥ Oncology Research, ⊥ Pharmaceutics, # Discovery Technologies, Amgen Research, One Amgen Center
Drive, Thousand Oaks, California 91320, United States
KEYWORDS Pim kinase inhibitor, multiple myeloma, KMS-12BM, oncogene, hematologic cancers
ABSTRACT: Pim kinases are a family of constitutively active serine/threonine kinases which are partially redundant and
regulate multiple pathways important for cell growth and survival. In human disease, high expression of the three Pim
isoforms has been implicated in the progression of hematopoietic and solid tumor cancers, which suggests that Pim kinase
inhibitors could provide patients with therapeutic benefit. Herein, we describe the structure-guided optimization of a series
of quinazolinone-pyrrolodihydropyrrolone analogs leading to the identification of potent pan-Pim inhibitor 28 with
improved potency, solubility and drug-like properties. Compound 28 demonstrated on-target Pim activity in an in vivo
pharmacodynamic assay with significant inhibition of BAD phosphorylation in KMS-12-BM multiple myeloma tumors for 16
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hours post dose. In a 2-week mouse xenograft model, daily dosing of compound 28 resulted in 33% tumor regression at 100
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mg/kg.
INTRODUCTION
Pim proteins were first identified as a preferred proviral integration site of Moloney (Pim) murine leukemia virus in mouse
1
models of virus-induced lymphomas. They are a family of serine/threonine kinases composed of three members (Pim-1, Pim-
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, and Pim-3) that regulate oncogenesis, survival pathways, drug resistance, and migration. The three Pim isoforms are
highly homologous, functionally redundant and often over expressed in subsets of B-cell malignancies4,5 including
6
lymphomas, leukemias, and multiple myeloma, as well as some solid tumors. Pan-Pim kinase inhibitors have been shown to
inhibit proliferation of multiple myeloma cell lines in vitro and in xenograft models, and are expected to provide the most
clinical benefit in patients with this disease.7,8
The research for Pim kinase inhibitors has been very active in recent years,9-18 resulting in three small molecules, SGI-
1
776,19,20 AZD-120821 and PIM447,8,22 which have reached clinical trials. In recent years, efforts at Amgen have also
culminated in the identification of small molecule Pim kinase inhibitors. The structure−activity relationships (SAR) of
numerous diverse scaffolds (Figure 1) have been disclosed including aminothiadiazole 1,23 aminooxadiazole 2,24 3-(pyrazin-
2
-yl)-1H-indazole 3,25 and imidazopyridazine 4.26 Although imidazopyridazine 4 exhibited superior enzymatic and cellular
potency over previously identified compounds (KMS-12 p-BAD IC50 = 28 nM), the moderate to high clearance of this class of
compounds precluded their further advancement.
H
H
N
N
N
N
N
N
S
N
O
N
N
N
NH2
NH
NH
1
2
Pim-1 IC50 = 2.0 nM
Pim-2 IC50 = 10 nM
KMS-12 pBAD IC50 = ND
Pim-1 IC50 = 1.4 nM
Pim-2 IC50 = 2.4 nM
KMS-12 pBAD IC50 = 607 nM
H
N
N
N
N
F
N
N
N
N
H
N
N
F
F
O
F
NH2
3
N
4
H
Pim-1 IC50 = 0.1 nM
Pim-2 IC50 = 1.1 nM
Pim-1 IC50 = 0.024 nM
Pim-2 IC50 = 0.094 nM
KMS-12 pBAD IC50 = 1400 nM
KMS-12 pBAD IC50 = 28 nM
Figure 1. Pim kinase inhibitors previously reported by Amgen.
The naphthyridine pyrrolodihydropiperidinone 5 was another chemotype that was identified from our prior kinase
programs (Figure 2).27 Although this screening hit was a potent Pim-1/2 kinase inhibitor with modest cell potency, it
exhibited poor kinase selectivity. The nitrogen at the 5-position of naphthyridine 5 has favorable H-bonding interactions with
the backbone hydrogen bond donor presented by most kinase hinge residues. The replacement of the naphthyridine with a
methylquinoxaline (6) resulted in not only improved Pim-1/2 potency, but also significantly improved kinase selectivity. Pim-
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/2 kinases are able to accommodate both the naphthyridine and quinoxaline cores because the hinge region of the Pim
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kinases contains a proline residue and therefore lacks the canonical hydrogen-bond donor of all other kinases. In an effort to
diversify the aniline substituent of 5, molecules containing N-alkyl groups (such as compound 6) were found to be similarly
potent. Consideration of the U-shape of these N-alkyl analogs led to the discovery of macrocyclic quinoxaline-
pyrrolodihydropiperidinone 7 as potent Pim-1/2 inhibitors (Figure 2).27
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Pim-1 IC50
Pim-2 IC50
KMS-12 pBAD IC50 225 nM
0.4 nM
1.2 nM
0.8 nM
0.6 nM
112 nM
0.3 nM
0.5 nM
25 nM
O
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Me
8
9
Pim-1 IC50
Pim-2 IC50
0.3 nM
0.2 nM
KMS-12 pBAD IC50 59 nM
Figure 2. Hit-to-lead progression and optimization strategy toward macrocyclic quinoxaline 7 and non-macrocyclic quinazolinone 9.
In parallel with the optimization efforts on the macrocyclic inhibitors, lead optimization efforts on non-macrocyclic analogs
2
8
resulted in the discovery of quinazolinone-pyrrolodihydropyrrolone 8. This compound demonstrated improved metabolic
stability over quinoxaline-pyrrolodihydropiperidinones. Although both 7 and 8 are potent and selective pan-Pim inhibitors
and orally efficacious in mouse xenograft models (KMS-12-BM) of multiple myeloma, the limited solubility (compound 7,
PBS/FaSSIF, 40/295 M; compound 8, 23/104 M respectively) became a potential barrier for further development. Thus,
the challenge was to improve the solubility while maintaining the potency, selectivity and metabolic stability achieved with
compound 8.
The Pim hinge region is unusual in both sequence and conformation; a two-residue insertion gives rise to a wider ATP
binding pocket, and a proline residue in the hinge removes a conserved hydrogen bond donor that can participate in inhibitor
binding. Without this hydrogen bond, van der Waals interactions with the hinge serve to position the ligand. Based on the X-
ray structure of 8 bound to Pim-1 protein (PDB ID: 5IPJ)28 (Figure 3) the dihedral angle between the pyrrole and
quinazolinone is 0° due to the intramolecular H-bond from the pyrrole-NH to the quinazolinone-N. This coplanarity between
the two rings is required for potency but is likely a contributor to the low solubility of compound 8. Because of the wider ATP
binding pocket of Pim, replacement of the methyl group at the 3-position of quinazolinone with various larger substitutions
2
R is feasible to keep the potency and to prevent the molecule tight stacking in the crystalline lattice. Therefore, our strategy
focused on modifying the substituent in structure 9 (Figure 2) to further improve drug-like properties suitable for
development.
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Figure 3. X-ray crystal structure of 8 bound to Pim-1 (PDB ID: 5IPJ).
CHEMISTRY
2
The general synthetic route used to prepare Pim inhibitors is described in Scheme 1. R was introduced early in the
sequence via amide coupling of various amines with commercially available o-aminobenzoic acid 9a in the presence of T3P
or HATU. The amino-quinazolinone scaffold could be accessed through three different routes. Method A: cyclization of amide
9b using triphosgene afforded 2-hydroxyquinazolinone, which was then elaborated to 2-chloroquinazolinone by reacting
with POCl . Displacement of the resulting chloride with amines furnished 2-amino-substituted-quinazolinones 9c. Method B:
3
following the cyclization of amide 9b using triphosgene, 2-hydroxyquinazolinone was treated with BOP and DBU in the
presence of amines to obtain the desired 2-amino-substitued-quinazolinone 9c. Method C: amines 9b were converted to
azides under mild conditions with tert-butyl nitrite and azidotrimethylsilane.29 The resulting aromatic azides were not
isolated, but elaborated to iminophosphoranes by addition of trimethylphosphine. Subsequent heating of the
iminophosphoranes with isocyanates resulted in formation of carbodiimide intermediate, which underwent intramolecular
30
cyclization to furnish the desired 2-amino-substituted-quinazolinones 9c. Method A and B are good for the scale-up. Method
C is a one-pot synthesis through 4 sequential steps and useful to quickly generate SAR. Installation of the pyrrolopyrrolone
was accomplished with a Suzuki coupling between quinazolinones 9c and chiral boronic ester 9d, whose synthesis has been
previously described.28
O
O
O
a
O
b
Br
c
O
Br
Br
N
N
N
N
HN
9
R2
O
R2
NH
OH NH2
9a
R2
NH NH2
9b
O
HN
9c
B
NH
HN
R1
R1
Me
O
N
H
9d
1
1
Scheme 1. Synthesis of compounds 9. Reagents and conditions: (a) R -NH
2
, T3P, DIPEA, EtOAc, rt or R -NH
2
, HATU, Et
3
N, dichloromethane, DMF, rt;
2
2
(b) Method A: (1) triphosgene, (2) POCl
3
, DIPEA, reflux, (3) R -NH
2
, heat; Method B: (1) triphosgene, (2) R -NH
2
, BOP, DBU; Method C: (1) t-BuONO,
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TMSN
5 °C.
3 3 2 3 3 4 2 3 4 2
, MeCN, 0 °C, (2) Me P, (3) R NCO, 60 °C, (4) DBU, 60 °C; (c) Pd dba , XPhos, K PO , dioxane/H O, 80 °C; or XPhos-Pd-G2, K PO , dioxane/H O,
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RESULTS AND DISCUSSION
All analogs were evaluated in Pim biochemical and cellular assays. In the biochemical assays, compounds were assessed 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. According to our experience across multiple scaffolds in the Pim program, the activity of Pim-3 was in
general comparable to Pim-1. Therefore, the Pim-3 enzyme assay was not routinely performed. In cell-based assays, the
compounds were assessed 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 BM31 is a multiple myeloma cell line with expression of Pim-
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, -2, and -3 proteins, Pim-2 is expressed at a high level. 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.
Initially, we systematically increased the size of the alkyl substituent at the 3-position of 2-tert-butylamino quinazolinone
(
Table 1). Replacement of the methyl group (8) with isopropyl (10), cyclopropyl (11), and 1-methylcyclopropyl (12) led to
a slight improvement in Pim activity (enzymatic and cellular), but only the cyclopropyl derivative 11 maintained rat and
human microsomal stability relative to the parent 8. Introduction of a tert-butyl moiety (13) resulted in a 3-fold loss in cellular
potency. Interestingly, installation of a cyclopropylmethyl group (14) restored enzymatic and cellular potency. However,
none of these analogs demonstrated improved solubility relative to 8. Attempting to increase solubility by introducing polar
groups onto the cyclopropylmethyl moiety, both (1-hydroxycyclopropyl)methyl (15) and (1-aminocyclopropyl)methyl (16)
exhibited comparable enzymatic and cellular potencies and compound 16 indeed had good solubility in acidic media. Opening
the cyclopropyl ring of compound 16 to 2-amino-2-methylpropyl derivative 17 led to a greater than 10-fold loss in KMS-12
cell potency.
Next, we investigated heterocyclic substituents at the 3-position of the 2-tert-butylaminoquinazolinone. Replacement of
the methyl group (8) with oxetan-3-ylmethyl (18), (tetrahydrofuran-2-yl)methyl (19 and 20) and (4-methylmorpholin-2-
yl)methyl (21 and 22) were tolerated, especially 19 and 22 demonstrating significant improvements in KMS-12 cell potency.
Unfortunately, their microsomal stability was unacceptable. Analogs equipped with (1-methyl-1H-imidazol-4-yl)methyl (23)
and 1-(1-methyl-1H-imidazol-4-yl)ethyl (24, a racemic mixture) delivered comparable enzymatic and cellular potencies to
the parent 8 with moderate microsomal stability. Compound 24 additionally exhibited improved solubility in all three media.
We had discovered previously that introduction of a (1-methylcyclopropyl)amino group (25) at the 2-position of
quinazolinone improved Pim potency (KMS-12 IC50 = 11 nM) as well as solubility.28 However, the rat PK profile of compound
25 was not ideal with high clearance (2.7 L/h/kg) and low bioavailability (%F = 5). We then turned our attention to
optimization of the 2-(1-methylcyclopropyl)aminoquinazolinone scaffold with the SAR learned from 2-tert-
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butylaminoquinazolinones (Table 1). Replacement of the methyl group (25) with cyclopropyl (26), cyclobutyl (27) and
cyclopropylmethyl group (30) led to a slight improvement in Pim enzymatic activity but a 2- to 5-fold loss in cellular potency
along with an increase in microsomal turnover. Introduction of the 1-methylcyclopropyl moiety (28) retained the excellent
cellular potency with moderate microsomal stability. Installation of t-butyl group (29), as with compound 13, resulted a 100-
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fold loss in cellular potency. Remarkably, the solubility of all these R cyclopropylmethyl and R C1-C4 alkyl analogs (25-30)
were better than their corresponding 2-tert-butylamino quinazolinone analogs (8-14) (Figure 4). Appending a polar group
to cyclopropylmethyl, both (1-hydroxycyclopropyl)methyl (31) and (1-aminocyclopropyl)methyl (32) in this scaffold
resulted a large loss in cellular potency. A racemic mixture of 1-(1-methyl-1H-imidazol-4-yl)ethyl derivative 33 afforded a
similar potency and solubility profile to compound 28. After SFC chiral separation of 33, we obtained two pure single isomers
34 and 35. Compound 35 displayed a better profile than 34. The isomer 35 demonstrated excellent enzymatic and cellular
potency (KMS-12 IC50 = 8 nM) as well as good solubility and moderate microsomal stability.
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Through these SAR efforts, we recognized that 26, 28 and 35 exhibited the best cellular potency with reasonable
microsomal stability and improved solubility. These three most promising analogues were selected for pharmacokinetic
profiling (data presented in Table 2; Parent compounds 8 and 25 28 are also included for comparison). In general, these
molecules demonstrated moderate plasma protein binding, and good permeability. The in vivo PK in male Sprague-Dawley
rats revealed significant differences in oral bioavailability (19-85%), but not in clearance (0.8-1.0 L/h/kg). Compound 28
possessed the most favorable profile (lowest clearance and highest oral bioavailability, even when dosed in a standard
formulation) and was advanced to further evaluation.
m m
A solubility comparison study between the most stable known crystalline form of compound 8 (T = 339 °C) and 28 (T =
2
94 °C) was established at 37 °C using water and three biologically relevant gastrointestinal media, FaSSGF, FaSSIF and
FeSSIF.32 Data for compound 8 are: 6, 62, 14, 118 µM and for compound 28 are: 12, 486, 30, 226 µM respectively. Compound
28 demonstrated a higher solubility than compound 8 in water and biological relevant media. This may be due in part to its
lower melting point (294 °C) versus compound 8 (339 °C).33 Comparison of eight matched pairs in Figure 4 shows that
1
cyclopropylmethyl at R is uniformly more soluble than tert-butyl. A dog PK study of compound 28 revealed low IV clearance
(
0.14 L/h/kg) with Vss of 2.1 L/kg, t1/2 of 11.2 h, and good oral bioavailability (56%). The plasma protein binding of 28 was
also measured and its unbound fractions (f ) were similar across the different species (rat/dog/mouse/human,
.040/0.045/0.033/ 0.039, respectively). In a hERG binding assay, 28 had IC50 of >30 M.
u
0
O
O
N
N
HN
1
R = A:
or B:
R2
NH
HN
R1
Me
Table 1. SAR for substitutions at 2 & 3-position of quinazolinones
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Pim-1
IC50
Pim-2
IC50
(nM) a
KMS-12
pBAD IC50
(nM) a
HLM/RLM
Solubility
(µM)
PBS (pH 7.4) c
Solubility (µM)
FaSSIF (pH 6.8) c
Solubility (µM)
0.01 N HCl (pH 2.0) c
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
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3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Cmpd
8
R1
R2
(µL/min/mg)
nM) a
b
(
A
A

0.34
0.38
59
42
19 / 22
23
104
68
60
26
Me

10
11
12
0.07*
0.17
0.29
0.13*
74 / 121
11
13
<5


A
A
0.12
0.16
35
36
27 / 32
175
36
95
<5
109 / 96

13
14
15
A
A
A
0.21
0.09*
0.13*
0.83
0.08*
0.23*
196
31
195 / 187
71 / 125
58 / 70
<5
<5
<5
58
12
<5
<5
<5
<5

0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0

55
HO

16
A
0.11*
0.24*
48
67 / 77
<5
42
500
H2N
17
18
19
A
A
A

0.56*
0.16*
0.36*
2.2*
0.60*
0.13*
707
126
24
32 / 21
17 / 18
26
6
131
32
347
14
H2N
O



>399 / 319
19
60
<5
O
O
20
A
A
1.8*
1.2*
1.1*
1.6*
341
224
175 / 361
50 / 80
12
<5
15
<5
28
N
O
2
2
1d
256




N
O
2d
A
A
0.55*
0.35*
0.41*
0.16*
38
46
193 / 341
96 / 23
122
22
283
131
500
94
N
N
23
N
N
24
A
0.12
0.7
83
79 / 63
86
489
423
25
B
B
Me

0.20
0.10
0.38
0.09
11
24
20 / 49
42 / 68
56
78
344
144
500
458

2
6
7

2
B
B
0.14*
0.05
0.18*
0.05
57
12
61 / 133
54 / 81
<5
31
379
185
441
490

28

29
30
31
B
B
B
20.3
0.06*
0.08*
3.50
0.12*
0.18*
1,239
21
107 / 177
52 / 87
<5
13
<5
326
179
15
495
214
43


1,805
47 / 65
HO

32
B
B
0.02*
0.03*
0.16*
0.11*
99
19
46 / 72
54 / 80
134
52
280
500
454
500
H2N
N
N
33



N
N
3
4d
B
B
0.65*
0.03
3.2*
0.15
472
8
47 / 107
35 / 92
53
60
435
469
485
464
N
N
3
5d
a
Average of at least two determinations unless indicated by *(n = 1); for full statistical information, see the supporting
information. Single experimental value; estimated clearance from percent parent compound (1 M) remaining following a
0 min incubation in liver microsomes (0.25 mg/mL) and NADPH. n = 1 for solubility data, PBS = phosphate-buffered saline,
FaSSIF = fasted state simulated intestinal fluid. The absolute structure was arbitrarily assigned.
b
c
3
d
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9
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0
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6
7
8
9
0
Figure 4. Solubility comparison for the selected compounds from Table 1
Table 2. Pharmacokinetic properties of most potent analogs
LLC-PK1
Rat
CL
V
ss
t
1/2
(h)
Cmpd
8
P
app
PPB
(L/h/kg)
%Fc
c
c
(
L/kg)
a
b
c
(
µcm/s)
f
u
46
31
23
32
35
0.061
0.061
0.047
0.040
0.032
0.3 d
2.7 d
1.2 e
0.8 e
1.0 e
1.5
1.9
1.2
1.7
1.0
4.6
0.6
1.5
2.2
2.5
39 f
5 f
68 g
85 g
19 g
25
26
28
35
a
b
Average of A–B and B–A diffusion rates across a single layer of LLC-PK1 cells determined at 5 μM compound. Plasma protein binding determined
c
by rapid equilibrium dialysis with 5 μM compound. Pharmacokinetic parameters following administration to male Sprague Dawley rats; mean
values from 3 animals per dosing route. Dosed iv at 2 mg/kg as a solution in DMSO. Dosed iv at 1 mg/kg as a solution in DMSO. Dosed po at 5
mg/kg in 40.0% PEG 400, 60.0% water, adjusted to pH 2.2 with methanesulfonic acid. Dosed po at 5 mg/kg in 1% Tween 80, 2% HPMC, 97% water
d
e
f
g
adjusted to pH 2.2 with methanesulfonic acid.
The selectivity of 28 against a panel of 100 protein and lipid kinases was examined at 1 M in the ScanMAX KINOMEscan
panel offered by DiscoveRx. The S(35), S(10), S(1) selectivity scores, representing the fraction of tested kinases with percent-
of-control (POC) values < 35, 10 and 1% were determined to be 0.04, 0.01 and 0, respectively. Four kinases exhibited POC <
3
5%: CLK4 (34%), MEK3 (31%), JNK3 (11%) and Pim-1 (2%).34 Compound 28 demonstrated better kinase selectivity than
, which against the same panel exhibited S(35), S(10), S(1) selectivity scores of 0.07, 0.06 and 0.03, respectively.28 This
8
selectivity improvement is consistent with the additional hinge residues (two-residue insertion) of Pim-1 kinase resulting in
a wider ATP binding pocket than other kinases. Therefore, the lager methylcyclopropyl group at the 3-position of the
quinazolinone was accommodated by Pim-1 kinase, but not accommodated by other kinases. With the high kinase selectivity,
we believe the in vivo efficacy of 28 is primarily driven by Pim kinase inhibition.
The X-ray crystal structure of 28 bound to Pim-1 protein (PDB ID: 6MT0) in Figure 5A revealed a similar binding mode to
compound 8. The carbonyl oxygen of the pyrrolone engages in a direct H-bonding (N to O distance 2.6 Å) interaction with the
conserved catalytic Lys67. A water-mediated bridge between the carbonyl oxygen of Asn172 and Asp186, and the NH of
pyrrolone was resolved. The pyrrolone is closely packed against the Asp186 side chain (N to O distance 2.8 Å). Another two
water-mediated bridges between the carbonyl oxygen (C4=O) in quinazolinone and the carbonyl oxygen of Pro123, which are
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5
.0 Å apart was also observed. The presence of Pro123 in the ATP-binding hinge of the Pim kinases results in the absence of
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5
6
the canonical backbone NH found in other kinases, and this uniquely accommodates the C5-H of the quinazolinone. In
addition, the favorable hydrophobic contacts between the two methylcyclopropyl groups well filled small lipophilic pockets
under the glycine rich loop and lower hinge region of Pim-1 (shown in Figure 5B) which likely contribute to the excellent
potency and kinase selectivity of 28.
The antiproliferative activity of 8 and 28 across hematologic malignancies was assessed in the multiple cell lines listed in
Table 3, including multiple myeloma and AML cell lines. In general, multiple myeloma cell lines exhibit higher Pim-2
expression, whereas AML cell lines show higher Pim-1 expression. Compound 28 with low double digit picomolar enzymatic
potency in Pim-1/2 resulted in improved IC50 values in the proliferation assay across the panel relative to compound 8.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Table 3. Proliferation activities of compounds 8 and 28 across hematologic malignancies
Proliferati
on
Proliferati
on
Basal Pim-1
levels (counts
per 15 μg)d
Basal Pim-2
levels (counts
per 15 μg)d
Cell Line
IC50 (M)
Cmpd 8 c
IC50 (M)
c
Cmpd 28
KMS-12-BM a
RPMI-8226 a
0.26
0.83
0.72
0.049
0.104
0.667
3547 ± 68.6
1188 ± 4.2
264 ± 16.3
19,067 ± 1074.1
23,228 ± 413.7
10,289 ± 310.4
OPM-2 (PTEN null) a
EOL-1 b
0.02
1.5
0.002
0.093
16,376 ± 243.2
22,004 ± 124.5
1136 ± 59.4
4008 ± 5.7
OCI-M1 b
(erythroleukemia)
a
Multiple myeloma cell line. Acute myeloid leukemia cell line. c IC50 value corresponds to 72 h incubation of compounds in the CellTiter-Glo®
b
d
proliferation assay.
electrochemiluminescence (ECL) per 15 μg protein
Basal Pim-1 and Pim-2 protein levels as measured by MSD assays (n = 2 separate determinations) using
2
6
(A)
(B)
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Figure 5. X-ray crystal structure of 28 bound to Pim-1 (PDB ID: 6MT0). (A) Inhibitor 28 occupies the ATP-binding site; hydrogen bond contacts are
shown with dashed lines; the red dots represent water molecules; the top of the ATP-binding pocket is omitted for clarity. (B) CPK (Corey–Pauling–
Koltun) rendering of 28 illustrating that the two methylcyclopropyl groups form a complementary hydrophobic surface under the P-loop and floor
residues of Pim-1. The figures were generated with the PyMOL Molecular Graphics System, version 1.7.0.1 Schrödinger, LLC.
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The in vivo activity of 28 on Pim-dependent phosphorylation of BAD (Ser 112) was further evaluated in the KMS-12-BM
human multiple myeloma xenograft model in female SCID-beige mice (Figure 6). In this pharmacodynamics (PD) model, we
typically observe a maximum inhibition of 60-70% BAD phosphorylation at Ser-112 for selective Pim kinase inhibitors; the
remainder of BAD phosphorylation is attributed to other kinases, possibly PKB (AKT). 27, 28 Compound 28 dosed orally at 25,
0
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0
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0
5
7
2
0, or 100 mg/kg provided a dose-dependent increase in exposure and nearly all doses achieved a maximum inhibition of 60-
0% p-BAD levels at 6 and 16 hours (Figure 6). We next tested the efficacy of compound 28, choosing a dosing regimen of 10,
5, 50 or 100 mg/kg orally once per day for 14 days. A dose-dependent inhibition of tumor growth was observed with
complete stasis achieved at 50 mg/kg QD and tumor regression observed at 100 mg/kg QD (Figure 7A). All doses were well
tolerated, with minimal impact on body weight over the course of the study (Figure 7B). These data suggest that in
vivo exposures above the in vitro IC50 for at least 16 h with greater than 50% p-BAD inhibition results in significant tumor
growth inhibition (Figure 7C).
Figure 6. The in vivo activity of 28 to impact Pim-dependent phosphorylation of BAD (Ser 112) was evaluated in KMS-12-BM tumors in female
SCID-beige mice. Bars represent %pBAD. Terminal total plasma concentration of 28 is indicated by red circles. Data represent the mean (n = 3) ±
standard deviation, Statistical significance (* p < 0.0001) was evaluated by analysis of variance (ANOVA), followed by Dunnet’s post hoc.
CONCLUSION
In summary, modifications at the 2- and 3-positions of quinazolinone 8 resulted in potent Pim-1/2 kinase inhibitors with
improved properties. Compound 28, bearing two methylcyclopropyl groups at the 2- and 3-positions of the quinazolinone,
demonstrated not only improved Pim potency and kinase selectivity by leveraging more lipophilic interactions, but exhibited
improved solubility over compound 8 in water and biologically relevant media. With the combination of cellular potency,
solubility, good PK profile across species, and potent single agent antitumor activity in a KMS-12-BM mouse xenograft model
(
ED50 = 12 mg/kg), compound 28 emerged as a best in-series candidate. This compound has advanced into additional mouse
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Page 12 of 40
xenograft models of DLBCL and AML, combination studies with standard of care agents and toxicology studies, and the results
will be reported in due course.
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0
Figure 7. Pim inhibition suppresses MM growth in the KMS-12-BM mouse xenograft model. (A) Compound 28 (10, 25, 50, or 100 mg/kg p.o.) is associated
with a dose-dependent inhibition of KMS-12-BM tumor growth. (B) All doses were well tolerated, with minimal impact on body weight over the course of
the study. Data represent the mean (n = 10) ± SEM. (C) Unbound plasma concentration of 28. Data represent the mean (n = 2 / timepoint) Statistical
significance (*p < 0.0001) was determined by repeated measures analysis of variance (RMANOVA) followed by Dunnet’s post hoc.
EXPERIMENTAL SECTION
Pim-1 and Pim-2 Enzyme Assays
The assay for the determination of Pim activity was based on the formation of phosphorylated biotinylated-BAD peptide at
the serine 112 residue (S112) and employed homogeneous time-resolved fluorescence (HTRF) technology to detect the
product in a 384-well plate format. The phosphorylation of biotinylated-BAD (S112) peptide by full length recombinant Pim-1
or Pim-2 protein was detected with streptavidin:allophycocyanin conjugate and an europium (Eu) labeled antibody directed
against phosphorylated-BAD (S112). Excitation of Eu by a high energy laser light (337 nm) led to a transfer of energy to the
APC molecule and resulted in an emission at 665 nm. The fluorescence was directly proportional to the amount of
phosphorylated BAD peptide present in the reaction. Compounds were prepared in DMSO by conducting 3-fold serial
dilutions to give a 22-point dosing curve having a high dose of 1 μM. A reference compound was included on each assay plate
[Costar 3658] in order to validate that plate; on one plate of every assay run, two additional reference compounds were
included. The reaction buffer consisted of 45 mM Hepes, pH 7.0, 15 mM NaCl, and 1 mM MgCl . The quench/detection buffer
2
consisted of 50 mM Tris, 100 mM NaCl, 0.05% BSA, 0.1% Tween, and 3 mM EDTA, biotinylated BAD peptide (Biopeptide), 10
mM ATP (Sigma), labeled pBAD (S112) mAb (Cell Signaling and PerkinElmer) [with 0.05% BSA and 2 mM DTT added], and
streptavidin:allophycocyanin [PerkinElmer], and final concentrations, either Pim-1 enzyme [5 pM] or Pim-2 enzyme [0.5 pM],
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DMSO [1%], BAD-LANCE-conjugated BAD (S112) [0.5 μM], ATP [1.5 μM], streptavidin:allophycocyanin [0.002 mg/mL], and
biotinylated-BAD (S112) mAb [100 pM]. Initial incubations were carried out at RT (22 °C) for 30 min for both Pim-1 and Pim-
2. Pim enzyme was added to compound in buffer, and plates were incubated for 30 min. Biotinylated BAD and ATP were
added, and plates were incubated for 1 h. A mixture of labeled pBAD (S112) mAb and quench/detection buffer were added
and incubated for 2 h. Fluorescence was measured by an HTRF Envision microplate reader. For each plate, percent of control
1
2
3
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8
9
1
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4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
(
POC) values were calculated for each well. Values for the IC50 were estimated using a standard three- or four-parameter
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
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0
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9
0
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0
logistic model.
KMS-12 BM pBAD Assay
The flow cytometry assay for determination of the Pim activity in the KMS-12 BM cell line (DSMZ catalogue no. ACC 551)
measured levels of phospho-BAD normalized against total BAD protein levels. Protocols: compounds were initially diluted in
DMSO by conducting 2-fold serial dilutions to give a 22-point dosing curve having a high dose of 30 μM. Exponentially growing
6
KMS-12 BM cells (50 μL, between 0.5 and 1.5 × 10 /mL, DSMZ) in assay media (RPMI/20% heat inactivated FBS/1×
NaPyruvate/1× NEAA/1× PSG (pen/strep glutamine)) were added to a 384-well plate containing 200 nL of compound. The
2
cell plates were then incubated with compound for 110 min at 37 °C, 5% CO . BD Phosflow Lyse/Fix (BD Biosciences) was
diluted to 2× with assay media. Then 50 μL of the diluted BD Phosflow Lyse/Fix was added to each well. The cell plates were
incubated for 15 min at RT. The plates were spun for 15 s at 2 K RPM then aspirated. Staining Media (1× PBS with 0.5% FBS)
was added (80 μL). The plates again were spun for 15 s at 2 K RPM then aspirated. BD Perm/Wash Buffer (1×, 50 μL, BD
Biosciences) was added. The cell plates were then incubated for >30 min at RT in the dark. The plates were spun for 15 s at 2
K RPM and then aspirated. Staining media (1× PBS with 0.5% FBS) was added (80 μL). The plates were spun for 15 s at 2 K
RPM and then aspirated and additional staining media (1× PBS with 0.5% FBS) was added (80 μL). The plates were spun for
1
5 s at 2 K RPM and then aspirated. Rabbit antihuman pBAD Ser112 Ab (Cell Signaling) was diluted in staining media (1:120).
The diluted pBAD Ab (10 μL) was added to each well. The cell plates were incubated for >1h at RT. The plates were spun for
15 s at 2 K RPM then aspirated. Staining media (1× PBS with 0.5% FBS) was added (80 μL). Goat anti-rabbit Alexa-647
(Invitrogen) was diluted in staining media (1:4000). The diluted goat anti-rabbit Alexa-647 (70 μL) was added to each well.
The cell plates were then incubated for >30 min at RT in the dark. The plates were spun for 15 s at 2 K RPM and then aspirated.
Staining media (1× PBS with 0.5% FBS) was added (80 μL). The plates were spun for 15 s at 2 K RPM and then aspirated and
additional staining media (1× PBS with 0.5% FBS) was added (80 μL). The plates were spun for 15 s at 2 K RPM and then
aspirated. Staining Media (1× PBS with 0.5% FBS) was added (30 μL). The plates were read on a BD LSRII, and results were
calculated according to the assay protocols ((%Phosphoprotein = ((2 × Phospho signal)/(Phospho signal + Total signal)) ×
100)).
KMS-12 BM Cell Viability Assay
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KMS-12 BM cells were seeded into 96-well tissue culture plates at 5000 cells/well in full growth medium, RPMI1640/20%
heat-inactivated FBS and were allowed to acclimate overnight in an incubator (37 °C; 5% CO ) prior to treatment. The
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2
following day, cells were treated with compounds that had been serially diluted first in DMSO and then into full growth
medium, which was then added onto the cells to achieve a dose–response curve with a final high dose of 10 μM. DMSO alone
was used as a control in all viability assays. Cellular viability was assessed after 72 h of treatment using the CellTiter-Glo
luminescent viability assay reagent (Promega Inc.). Plates were read on the EnVision Multilabel plate reader (PerkinElmer),
and IC50 values were calculated using the statistical curve-fitting program XLFit (IDBS software).
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KMS-12 BM Xenograft Efficacy
Animals were cared for in accordance to the Guide for the Care and Use of Laboratory Animals (eighth edition, National
Research Council (US)). All research protocols were approved by the Institutional Animal Care and Use Committee. The effect
of compound 28 on tumor growth inhibition (TGI) in vivo was evaluated in the KMS-12 BM human multiple myeloma
6
xenograft model. Female SCID-beige mice were injected with 10 KMS-12 BM cells at a ratio of 1:1 cell culture media to
3
Matrigel. Animals were randomized into treatment groups when tumors reached approximately 200 mm . Vehicle (25% PEG
4
1
00, 10% Kollidon VA 64, 0.1% ascorbic acid, pH 1.5 with MSA) or compound 28 was administered orally at 10, 25, 50, or
00 mg/kg once daily. Body weights and tumor volumes were measured twice per week. The tumor volume was calculated
3
as L × W × H and expressed in mm . Tumor volume data represent the mean volume (n = 10) ± standard error of the mean
(SEM). Statistical significance was determined by repeated measures followed by Dunnett’s post hoc analysis. Statistical
calculations were made through the use of JMP software v8.0.2 interfaced with SAS v9.1 (SAS Institute, Inc., Cary, NC).
KMS-12 BM PD Studies
The ability of compound 28 to impact Pim-dependent phosphorylation of BAD (Ser-112) was evaluated in vivo. When
average tumor size reached ~300 mm3, mice were randomized into treatment groups (n=3). Mice were treated with a single
dose orally with vehicle or compound 28 at 25, 50, or 100 mg/kg. Tumors were harvested at 6 and 16 h post dose. Tumor
lysates were generated and analyzed by MSD as per manufacturer’s recommendation for levels of phosphorylated BAD. Bars
represent levels of phosphorylated BAD and are expressed as percent of vehicle; circles represent total plasma concentration
(
μM).
Solubility Assays
Compounds as 10 mM DMSO stock solutions were dispensed into 96-deep-well plates at a volume of 10 μL 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 drying down was complete, a Tecan Evo liquid handler was used to transfer 200 μL of each of the buffers/solvent to the
corresponding plate copy. The buffers used were 1× PBS (pH 7.4), fasted state simulated intestinal fluid (FaSIF, pH 6.8) and
0
.01 N HCl (pH 2.0), and the solvent DMSO was used to create the standard plate for comparison. The plates were sealed and
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Page 15 of 40
Journal of Medicinal Chemistry
centrifuged at 1000 rpm for 1 min to push all liquid from walls to the bottom of the wells. The plates were shaken at 1500
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
4000 rpm for 30 min. The supernatant was analyzed by LC/MS (at 215 nm, 2 μL injection volume). Peak area in PBS, fasted
state simulated intestinal fluid and 0.01 N HCl aqueous were compared to DMSO standard to determine solubility, accurate
within the range of 5−500 μM.
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Chemistry
Unless otherwise noted, all reagents were commercially available and used as received. All final compounds possessed
purity ≥95% as determined by high performance liquid chromatography (HPLC). The HPLC methods used the following
conditions: Zorbax SB-C18 column (50 mm × 3.0 mm, 3.5 μm) at 40 °C with a 1.5 mL/min flow rate; solvent A of 0.1% TFA in
water, solvent B of 0.1% TFA in MeCN; 0.0–3.0 min, 5–95% B in A; 3.0–3.5 min, 95% B in A; 3.5–3.51 min, 5% B in A. Flow
from the UV detector was split (50:50) to the MS detector, which was configured with APIES as ionizable source. The UV
1
detection wavelengths were 210 and 254 nm. H NMR spectra were recorded on a 300 or 400 MHz Bruker NMR spectrometer
at ambient temperature. Data are reported as follows: chemical shift (ppm, δ units) from an internal standard, multiplicity (s
=
singlet, d = doublet, t = triplet, q = quartet, m = multiplet, and br = broad), coupling constant (Hz), and integration. All
microwave-assisted reactions were performed in sealed reaction vials using a Personal Chemistry Emrys Optimizer
microwave synthesizer. Analytical thin-layer chromatography (TLC) was performed using JT Baker silica gel plates precoated
with a fluorescent indicator. Silica gel chromatography was performed using either an ISCO Companion or Biotage medium
pressure liquid chromatography system.
(R)-2-(tert-Butylamino)-3-isopropyl-8-(6-methyl-4-oxo-1,4,5,6-tetrahydropyrrolo[3,4-b]pyrrol-2-yl)quinazolin-
4(3H)-one (10). (Method A) Step-1: 2-Amino-3-bromo-N-isopropylbenzamide. To a slurry of 2-amino-3-bromobenzoic
acid (1.00 g, 4.63 mmol) in 10 mL of EtOAc in an ice bath was added T3P (1-propanephosphonic acid cyclic anhydride, 50
wt% in EtOAc, 3.00 mL, 5.09 mmol) followed by isopropylamine (1.19 mL, 13.89 mmol). The ice bath was removed, and the
3
solution stirred rapidly at ambient temperature for 30 min. The reaction was partitioned between saturated NaHCO and
EtOAc. The organic layer was washed with saturated NaCl, and the organics were dried over anhydrous sodium sulfate,
filtered, and concentrated in vacuo to give 2-amino-3-bromo-N-isopropylbenzamide (1.00 g, 3.89 mmol, 84 % yield) as a white
1
solid. H NMR (400 MHz, CDCl
3
) δ ppm 7.51 (dd, J = 7.9, 1.5 Hz, 1H), 7.25 (d, J = 1.4 Hz, 1H), 6.53 (t, J = 7.8 Hz, 1H), 5.72 - 6.15
m, 3H), 4.15 - 4.33 (m, 1H), 1.27 (d, J = 6.7 Hz, 6H), m/z (ESI, +ve) 257.0/259.0 (1:1) (M+H)+.
(
Step-2: 8-Bromo-3-isopropylquinazoline-2,4(1H,3H)-dione. A slurry of 2-amino-3-bromo-N-isopropylbenzamide
(1.00 g, 3.89 mmol) and tri-phosgene (0.46 g, 1.56 mmol) in 50 mL of dichloromethane was heated to reflux overnight. The
reaction mixture was cooled to room temperature and concentrated in vacuo to give 8-bromo-3-isopropylquinazoline-
1
2
,4(1H,3H)-dione (1.10 g, 3.89 mmol, 100%) as a white solid. H NMR (400 MHz, CDCl
3
) δ ppm 8.02 - 8.25 (m, 1H), 7.98 (br.
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Journal of Medicinal Chemistry
Page 16 of 40
s., 1H), 7.78 (d, J = 7.8, 1.4 H z, 1H), 7.11 (t, J = 7.9 Hz, 1H), 5.27 (dt, J = 13.9, 6.9 Hz, 1H), 1.56 (s, 6H). m/z (ESI, +ve) 282.9/284.9
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(1:1) (M+H)+
Step-3: 8-Bromo-2-chloro-3-isopropylquinazolin-4(3H)-one.
,4(1H,3H)-dione (1.11 g, 3.89 mmol), POCl (1.79 mL, 19.60 mmol) and DIPEA (1.36 mL, 7.84 mmol) was heated to reflux for
h. The reaction reached 50% conversion. Additional DIPEA (1.36 mL, 7.84 mmol) was added and the reaction stirred
A
mixture of 8-bromo-3-isopropylquinazoline-
2
2
3
overnight. The reaction was cooled, treated with ice, and agitated. The slurry was poured onto 20 mL of 10N NaOH aqueous
solution in ice bath and stirred rapidly for 20 min. The fine precipitate was collected by filtration and rinsed with water (3 x
0
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8
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9
0
2
0 mL). The solid was dried in vacuo to give 8-bromo-2-chloro-3-isopropylquinazolin-4(3H)-one (0.978 g, 3.24 mmol, 83%)
1
as a tan solid. H NMR (400 MHz, CDCl
3
) δ ppm 8.18 (dd, J = 8.0, 1.4 Hz, 1H), 8.00 (dd, J = 7.8, 1.4 Hz, 1H), 7.33 (t, J = 7.9 Hz,
1H), 5.31 (br s, 1H), 1.67 (d, J = 7.0 Hz, 6H). m/z (ESI, +ve) 301.0/302.9 (4:5) (M+H)+.
Step-4: 8-Bromo-2-(tert-butylamino)-3-isopropyl-quinazolin-4(3H)-one.
A
slurry of 8-bromo-2-chloro-3-
isopropylquinazolin-4(3H)-one (0.40 g, 1.33 mmol) and tert-butylamine (4.18 mL, 39.8 mmol) was heated in a sealed tube at
0 °C overnight. The reaction was treated with ice water, and dichloromethane. The cloudy aqueous layer was extracted with
dichloromethane and EtOAc. The combined organics were dried over anhydrous Na SO , filtered, and concentrated in vacuo.
8
2
4
The crude material was purified by silica gel chromatography (eluted with 0-40% EtOAc/hexanes) to afford 8-bromo-2-(tert-
1
butylamino)-3-isopropylquinazolin-4(3H)-one (0.39 g, 1.15 mmol, 87%) as an oil which slowly solidified. H NMR (400 MHz,
3
CDCl ) δ ppm 8.05 (dd, J = 7.9, 1.5 Hz, 1H), 7.83 (dd, J = 7.6, 1.4 Hz, 1H), 6.97 (t, J = 7.7 Hz, 1H), 5.55 (br s, 1H), 4.68 (br s, 1H),
1
.63 (s, 9H), 1.55 (d, J = 7.2 Hz, 6H). m/z (ESI, +ve) 338.0/340.0 (1:1) (M+H)+.
Step-5:
(R)-2-(tert-Butylamino)-3-isopropy-8-(6-methy4-oxo-1,4,5,6-tetrahydropyrrolo[3,4-b]pyrro2-
yl)quinazolin-4(3H)-one. Argon was bubbled into a mixture of Pd
2
dba
3
(7.45 mg, 0.008 mmol), XPhos (7.75 mg, 0.016
28
mmol), (R)-6-methy2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-5,6-dihydropyrrolo[3,4-b]pyrro4(1H)-one (9d ; 85.0
mg, 0.33 mmol), 8-bromo-2-(tert-butylamino)-3-isopropylquinazolin-4(3H)-one (55.0 mg, 0.16 mmol), potassium phosphate
(104 mg, 0.49 mmol) in 1 mL of dioxane and 0.2 mL of water for 1 min. The reaction was sealed and heated to 80 °C for 2 h.
The reaction mixture was cooled and concentrated onto silica gel in vacuo. The material was purified by silica gel
chromatography (using 0-100% 90/10 dichloromethane /MeOH in dichloromethane) to afford (R)-2-(tert-butylamino)-3-
isopropy-8-(6-methy4-oxo-1,4,5,6-tetrahydropyrrolo[3,4-b]pyrro2-yl)quinazolin-4(3H)-one (44.0 mg, 0.112 mmol, 69 %) as
1
a light yellow solid. H NMR (400 MHz, DMSO-d
6
) δ ppm 11.78 (s, 1H), 7.84 (ddd, J = 14.9, 7.6, 1.6 Hz, 2H), 7.59 (s, 1H), 7.15
(
t, J = 6.7 Hz, 1H), 6.63 (d, J = 1.6 Hz, 1H), 5.72 (s, 1H), 4.94 (quin, J = 6.7 Hz, 1H), 4.51 (q, J = 6.8 Hz, 1H), 1.52 (d, J = 6.8 Hz,
= 2.09 min (>95%); m/z (ESI, +ve ion) 394.1 (M+H)+.
H), 1.48 (s, 9H), 1.36 (d, J = 6.7 Hz, 3H), LC−MS t
R
6
(R)-2-(tert-Butylamino)-3-cyclopropyl-8-(6-methyl-4-oxo-1,4,5,6-tetrahydropyrrolo[3,4-b]pyrrol-2-
yl)quinazolin-4(3H)-one (11). (Method A) Step-1: 2-Amino-3-bromo-N-cyclopropylbenzamide. To a heterogenous
mixture of 2-amino-3-bromobenzoic acid (8.00 g, 37.0 mmol) in EtOAc (80 mL) at 0 °C was added 1-propanephosphonic acid
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Page 17 of 40
cyclic anhydride (T3P) (50 wt.% solution in ethyl acetate, 24.0 mL, 40.7 mmol) followed by cyclopropylamine (2.57 mL, 37.0
mmol). The ice bath was removed, and the reaction was allowed to stir at room temperature for 1 h. Saturated NaHCO (aq.)
was added and the mixture was stirred for 5 min. The layers were separated, and the organic layer was washed with saturated
aqueous NaHCO and brine. The aqueous layer was extracted with EtOAc (3 x 80 mL). The combined organic layers were
dried over anhydrous Na
Journal of Medicinal Chemistry
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3
3
2
SO
4
, filtered and concentrated to afford 2-amino-3-bromo-N-cyclopropylbenzamide (6.00 g, 23.5
1
mmol, 64% yield) as a white solid. H NMR (400 MHz, CDCl
3
) δ ppm 7.50 (dd, J = 7.8, 1.37 Hz, 1H), 7.20 (dd, J = 7.9, 1.27 Hz,
0
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2
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H), 6.50 (t, J = 7.8 Hz, 1H), 6.13 (br. s., 2H), 2.85 (tq, J = 7.0, 3.5 Hz, 1H), 0.83 - 0.91 (m, 2H), 0.57 - 0.64 (m, 2H). The -NH peak
was not observed. m/z (ES, +ve) 255.1/257.0 (1:1) (M+H)+.
Step-2: 8-Bromo-3-cyclopropylquinazoline-2,4(1H,3H)-dione. Triphosgene (2.79 g, 9.41 mmol) was added to a
solution of 2-amino-3-bromo-N-cyclopropylbenzamide (6.00 g, 23.5 mmol) in dichloromethane (235 mL) at room
temperature. The mixture was heated to reflux and stirred for 17 h. The resulting cloudy reaction mixture was cooled and
concentrated to afford 8-bromo-3-cyclopropylquinazoline-2,4(1H,3H)-dione (6.61 g, 23.5 mmol, 100% yield) as a pale yellow
1
solid. H NMR (400 MHz, CDCl
3
) δ ppm 8.09 (d, J = 8.0 Hz, 1H), 8.06 (br. s., 1H), 7.78 (dd, J = 7.8, 1.2 Hz, 1H), 7.10 (t, J = 7.9 Hz,
H), 2.74 - 2.84 (m, 1H), 1.21 (q, J = 7.0 Hz, 2H), 0.83 - 0.91 (m, 2H). m/z (ES, +ve) 281.0/283.0 (1:1) (M+H)+.
1
Step-3: 8-Bromo-2-chloro-3-cyclopropylquinazolin-4(3H)-one. To A mixture of 8-bromo-3-cyclopropylquinazoline-
2,4(1H,3H)-dione (5.00 g, 17.8 mmol), POCl (8.3 mL, 89.0 mmol) and DIPEA (12.4 mL, 71.1 mmol) was stirred at reflux
3
overnight. The reaction was then cooled and concentrated. The brown syrup was cooled in an ice bath and then ice (100 mL)
was added to the reaction mixture. The brown sludge was added to 100 mL of 10M aqueous NaOH and stirred for 20 min.
The mixture was filtered, and the brown solid was washed with water, and then dissolved in CH
was dried over Na SO , filtered, and concentrated to afford 8-bromo-2-chloro-3-cyclopropylquinazolin-4(3H)-one (4.54 g,
5.16 mmol, 85% yield) as a brown solid. H NMR (400 MHz, CDCl
2 2
Cl and filtered. The filtrate
2
4
1
1
3
) δ ppm 8.17 (dd, J = 8.0, 1.6 Hz, 1H), 7.99 (dd, J = 7.8, 1.6
Hz, 1H), 7.32 (t, J = 7.9 Hz, 1H), 3.02 (tt, J = 7.1, 4.0 Hz, 1H), 1.33 - 1.43 (m, 2H), 0.94 - 1.04 (m, 2H). m/z (ES, +ve) 298.9/301.0
(4:5) (M+H)+.
Step-4:
8-Bromo-2-(tert-butylamino)-3-cyclopropylquinazolin-4(3H)-one.
8-bromo-2-chloro-3-cyclopropyl-
quinazolin-4(3H)-one (213 mg, 0.71 mmol) was mixed with tert-butylamine (0.90 mL, 8.56 mmol) and heated at 90 °C in a
sealed tube for 24 h. The reaction mixture was concentrated to remove the excess tert-butylamine and the crude residue was
purified by silica gel chromatography (using a gradient of 0-5% MeOH in dichloromethane) to afford 8-bromo-2-(tert-
butylamino)-3-cyclopropylquinazolin-4(3H)-one (224 mg, 0.66 mmol, 94% yield) as an off-white crystalline solid. 1H NMR
(
400 MHz, CDCl
3
) δ ppm 8.02 (dd, J = 8.0, 1.4 Hz, 1H), 7.82 (dd, J = 7.7, 1.5 Hz, 1H), 6.94 (t, J = 7.7 Hz, 1H), 5.42 (s, 1H), 2.62 -
2.71 (m, 1H), 1.61 (s, 9H), 1.28 - 1.35 (m, 2H), 0.87 - 0.95 (m, 2H). m/z (ESI, +ve) 335.9/337.9 (1:1) (M+H)+.
Step-5:
(R)-2-(tert-Butylamino)-3-cyclopropyl-8-(6-methyl-4-oxo-1,4,5,6-tetrahydropyrrolo[3,4-b]pyrrol-2-
yl)quinazolin-4(3H)-one. To
a
mixture of (R)-6-methyl-2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-5,6-
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Page 18 of 40
dihydropyrrolo[3,4-b]pyrrol-4(1H)-one (9d; 1.69 g, 6.44 mmol) and 8-bromo-2-(tert-butylamino)-3-cyclopropylquinazolin-
4(3H)-one (1.48 g, 4.39 mmol) in 1,4-dioxane (15 mL) and water (2.5 mL) was added potassium phosphate (2.80 g, 13.2
mmol). The reaction mixture was bubbled with Argon for 3 min, and then XPhos-Pd-G2 (0.17 g, 0.22 mmol) was added and
the mixture was stirred at 40 °C for 1.5 h. The reaction 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
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6
4
over MgSO , filtered and the filtrate was treated with Si-thiol (ca. 20 g) and stirred 48 h. The mixture was filtered and
0
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3
4
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6
7
8
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2
3
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concentrated. The residue was purified by silica gel chromatography (using a gradient of 0-15% MeOH in dichloromethane)
1
6
to afford the title compound (1.09 g, 2.78 mmol, 63.4%) as a light yellow solid. H NMR (400 MHz, DMSO-d ) δ ppm 11.97 (s,
1H), 7.87 (dd, J = 7.5, 1.5 Hz, 1H), 7.83 (dd, J = 7.8, 1.6 Hz, 1H), 7.61 (s, 1H), 7.14 (t, J = 7.7 Hz, 1H), 6.72 (d, J = 1.4 Hz, 1H), 6.12
(s, 1H), 4.49 - 4.57 (m, 1H), 2.82 - 2.90 (m, 1H), 1.52 (s, 9H), 1.36 (d, J = 6.7 Hz, 3H), 1.25 (d, J = 5.7 Hz, 2H), 0.73 - 0.82 (m, 2H).
LC−MS t
= 1.90 min (>95%); m/z (ESI, +ve) 392.0 (M+H)+.
R
(R)-2-(tert-Butylamino)-8-(6-methyl-4-oxo-1,4,5,6-tetrahydropyrrolo[3,4-b]pyrrol-2-yl)-3-(1-
methylcyclopropyl)quinazolin-4(3H)-one
methylcyclopropyl)benzamide.
(12).
(Method
C)
Step-1:
2-Amino-3-bromo-N-(1-
2-(3H-[1,2,3]-Triazolo[4,5-b]pyridin-3-yl)-1,1,3,3-tetramethylisouronium
hexafluorophosphate (HATU; 1.94 g, 5.09 mmol) was added to a suspension of 2-amino-3-bromobenzoic acid (1.00 g, 4.63
mmol), 1-methylcyclopropanamine hydrochloride (0.55 g, 5.09 mmol), and triethylamine (1.6 mL, 12.04 mmol) in a mixture
of DMF (3.5 mL) and dichloromethane (3.5 mL) and the resulting mixture was stirred at 23 °C for 2 h. The reaction mixture
was then diluted with dichloromethane (50 mL), and the resulting solution was sequentially washed with water (2 × 40 mL),
0
.5N aqueous NaOH (40 mL), and brine (40 mL), then concentrated in vacuo and purified by silica gel chromatography (using
a gradient 0–80% EtOAc in hexanes) to yield 2-amino-3-bromo-N-(1-methylcyclopropyl)benzamide (960 mg, 3.57 mmol,
1
7
1
7% yield) as a white solid. H NMR (400 MHz, CDCl
3
)  ppm 7.49 (d, J = 8.0 Hz, 1H), 7.19 (d, J = 7.2 Hz, 1H), 6.50 (t, J = 7.3 Hz,
H), 6.31 (br. s., 1H), 6.10 (br. s., 2H), 1.47 (br. s., 3H), 0.84 (br. s., 2H), 0.74 (br. s., 2H). m/z (ESI, +ve) 268.9/271.0 (1:1)
(M+H)+.
Step-2: 8-Bromo-2-(tert-butylamino)-3-(1-methylcyclopropyl)quinazolin-4(3H)-one. 2-(tert-Butyl nitrite (0.35 mL,
.65 mmol) and azidotrimethylsilane (0.28 mL, 2.12 mmol) were sequentially added (dropwise) to a suspension of 2-amino-
-bromo-N-(1-methylcyclopropyl)benzamide (476 mg, 1.77 mmol) in MeCN (10 mL) at 0 °C. The resulting mixture was
2
3
stirred at 0 °C for 5 min, and then allowed to warm to 23 °C and stir for 15 min. Trimethylphosphine (1.0M in THF; 2.12 mL,
2
2
.12 mmol) was added dropwise, and the resulting solution was stirred at 23 °C for 5 min. tert-Butyl isocyanate (0.23 mL,
.03 mmol) was added, and the resulting mixture was stirred at 23 °C for 10 min, and then at 60 °C for 45 min. 1,8-
Diazabicyclo-[5.4.0]undec-7-ene (DBU; 0.32 mL, 2.12 mmol) was added, and the resulting brown suspension was heated at
60 °C for 20 min. The reaction mixture was subsequently cooled to ambient temperature, half-saturated aqueous sodium
bicarbonate (50 mL) was added, and the resulting mixture was extracted with dichloromethane (3 × 50 mL). The combined
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Journal of Medicinal Chemistry
organic extracts were dried over anhydrous sodium sulfate, filtered, and concentrated onto silica gel. The crude material was
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
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4
5
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5
5
5
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5
5
5
6
purified by silica gel chromatography (using a gradient 0–60% Et
2
O/hexanes) to obtain 8-bromo-2-(tert-butylamino)-3-(1-
1
methylcyclopropyl)-quinazolin-4(3H)-one (291 mg, 0.83 mmol, 47% yield) as a white solid: H NMR (400 MHz, CDCl
3
)  ppm
8
.01 (dd, J = 7.9, 1.3 Hz, 1H), 7.81 (dd, J = 7.7, 1.3 Hz, 1H), 6.94 (t, J = 7.8 Hz, 1H), 5.43 (br. s., 1H), 1.62 (s, 9H), 1.41 (s, 3H), 1.21
1.37 (m, 2H), 0.88 - 1.02 (m, 2H). m/z (ESI, +ve) 349.9/352.0 (1:1) (M+H)+.
-
Step-3:
(R)-2-(tert-Butylamino)-8-(6-methyl-4-oxo-1,4,5,6-tetrahydropyrrolo[3,4-b]pyrrol-2-yl)-3-(1-
solution of 8-bromo-2-(tert-butylamino)-3-(1-
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
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4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
methylcyclopropyl)quinazolin-4(3H)-one.
A
methylcyclopropyl)quinazolin-4(3H)-one (129 mg, 0.37 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 (9d; 135 mg, 0.51 mmol), potassium phosphate (234 mg, 1.10 mmol), and
chloro(2-dicyclohexylphosphino-2',4',6'-triisopropyl-1,1'-biphenyl)[2-(2'-amino-1,1'-biphenyl)]palladium(II) (XPhos-Pd-
G2; 14.5 mg, 0.02 mmol) in a mixture of 1,4-dioxane (1.3 mL) and water (0.3 mL) was stirred under argon at 45 °C for 1.5 h.
Additional potassium phosphate (234 mg, 1.10 mmol), XPhos-Pd-G2 (14.5 mg, 0.02 mmol), and (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 (135 mg, 0.51 mmol) were added, and the
resulting solution was stirred under argon at 45 °C for 1 h. The reaction mixture was then concentrated onto silica gel and
purified by silica gel chromatography (using a gradient 50–100% EtOAc/hexanes). The collected product was triturated with
MeOH (3 mL), and the resulting suspension was vacuum filtered. The collected solid was washed with ethyl ether (8 mL) and
dried in vacuo to provide (R)-2-(tert-butylamino)-8-(6-methyl-4-oxo-1,4,5,6-tetrahydropyrrolo[3,4-b]pyrrol-2-yl)-3-(1-
1
methylcyclopropyl)quinazolin-4(3H)-one (12) (65.2 mg, 0.16 mmol, 44% yield) as a light-yellow solid. H NMR (400 MHz,
DMSO-d )  ppm 11.89 (br. s., 1H), 7.81 - 7.90 (m, 2H), 7.60 (s, 1H), 7.16 (t, J = 7.6 Hz, 1H), 6.70 (s, 1H), 5.68 (d, J = 3.9 Hz, 1H),
6
4
.52 (q, J = 6.7 Hz, 1H), 1.53 (s, 9H), 1.47 (s, 3H), 1.36 (d, J = 6.7 Hz, 3H), 1.14 - 1.27 (m, 2H), 1.11 - 1.14 (m, 1H), 0.91 - 1.01
(
m, 1H) LC−MS t
. R
= 2.48 min (>95%); m/z (ESI, +ve) 406.0 (M+H)+.
(R)-3-(tert-Butyl)-2-(tert-butylamino)-8-(6-methyl-4-oxo-1,4,5,6-tetrahydropyrrolo[3,4-b]pyrrol-2-
yl)quinazolin-4(3H)-one (13). (Method C) The title compound was prepared analogous to compound 12 (Method C). Step-
1: 2-Amino-3-bromo-N-(tert-butyl)benzamide. The title compound (1.29 g, 4.76 mmol, 48.9 % yield) was prepared using 2-
amino-3-bromobenzoic acid (2.10 g, 9.72 mmol), 2-methylpropan-2-amine (1.2 mL, 11.66 mmol), HATU (4.80 g, 12.64 mmol),
1
DIPEA (2.5 mL, 14.58 mmol) and DMF (9.7 mL): H NMR (400 MHz, CDCl
3
)  ppm 7.48 (dd, J = 8.0, 1.4 Hz, 1H), 7.22 (dd, J =
7
.8, 1.2 Hz, 1H), 6.51 (t, J = 7.8 Hz, 1H), 5.95 (br. s., 2H), 5.82 (br. s., 1H), 1.46 (s, 9H). m/z (ESI, +ve) 271.0/273.0 (1:1) (M+H)+.
Step-2: 8-Bromo-3-(tert-butyl)-2-(tert-butylamino)quinazolin-4(3H)-one. The title compound (1.17 g, 3.32 mmol,
9.8 % yield) was prepared using 2-amino-3-bromo-N-(tert-butyl)benzamide (1.29 g, 4.76 mmol), tert-butyl nitrite (0.95 mL,
.14 mmol), azidotrimethylsilane (0.76 mL, 5.71 mmol), trimethylphosphine (5.71 mL, 5.71 mmol), 2-isocyanato-2-
6
7
1
methylpropane (0.63 mL, 5.47 mmol) and DBU (0.78 mL, 5.23 mmol): H NMR (400 MHz, CDCl )  ppm 7.89 (dd, J = 7.8, 1.4
3
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Journal of Medicinal Chemistry
Page 20 of 40
Hz, 1H), 7.76 (dd, J = 7.7, 1.4 Hz, 1H), 6.89 (t, J = 7.7 Hz, 1H), 4.42 (s, H), 1.75 (s, 9H), 1.59 (s, 9H). m/z (ESI, +ve) 352.1/354.0
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
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3
3
3
3
3
3
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4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
(1:1) (M+H)+.
Step-3:
yl)quinazolin-4(3H)-one. The title compound (74 mg, 0,18 mmol, 12.2 % yield) was prepared using 8-bromo-3-(tert-butyl)-
-(tert-butylamino)quinazolin-4(3H)-one (525 mg, 1.49 mmol), (R)-6-methyl-2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-
(R)-3-(tert-Butyl)-2-(tert-butylamino)-8-(6-methyl-4-oxo-1,4,5,6-tetrahydropyrrolo[3,4-b]pyrrol-2-
2
yl)-5,6-dihydropyrrolo[3,4-b]pyrrol-4(1H)-one (9d, 781 mg, 2.98 mmol), XPhos-Pd-G2 (59 mg, 0.08 mmol), and potassium
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
phosphate (949 mg, 4.47 mmol) in 1,4-dioxane (12.0 mL)/water (3.0 mL): H NMR (400 MHz, CDCl
3
)  ppm 12.34 (br. s., 1H),
7
.92 (dd, J = 7.6, 1.6 Hz, 1H), 7.86 (dd, J = 7.8, 1.6 Hz, 1H), 7.13 (t, J = 7.7 Hz, 1H), 6.80 (d, J = 1.4 Hz, 1H), 6.32 (s, 1 H), 4.66 (q,
R
J = 6.5 Hz, 1H), 4.59 (s, 1H), 1.77 (s, 9H), 1.64 (s, 9H), 1.52 (d, J = 6.7 Hz, 3H). LC−MS t = 2.35 min (>95%); m/z (ESI, +ve)
408.0 (M+H)+.
(
R)-2-(tert-Butylamino)-3-(cyclopropylmethyl)-8-(6-methyl-4-oxo-1,4,5,6-tetrahydropyrrolo[3,4-b]pyrrol-2-
yl)quinazolin-4(3H)-one (14). Step-1: 8-Bromoquinazoline-2,4(1H,3H)-dione. Urea (3.78 g, 63.0 mmol) and 2-amino-3-
bromo-benzoic acid (4.00 g, 18.52 mmol) were combined and heated at 170 °C, open to air, in an oil bath. After 3 h, additional
urea (3.78 g, 63.0 mmol) was added and stirring continued. After 5 h, the reaction was judged complete and cooled, treated
with water, filtered, and the solid dried overnight under vacuum to give 4.7 g. The material was treated with water and stirred
rapidly for 3 h to give a fine suspension. The solid was collected by filtration and dried in vacuo overnight, to give 8-
1
bromoquinazoline-2,4(1H,3H)-dione (4.30 g, 17.8 mmol, 96% yield) as a light yellow solid. H NMR (400 MHz, DMSO-d
6
) δ
ppm 11.50 (br. s., 1H), 10.25 (br. s., 1H), 7.93 (dq, J = 7.8, 1.4 Hz, 2H), 7.13 (t, J = 7.8 Hz, 1H), m/z (ESI, +ve ion) 240.9/242.9
(
1:1) (M+H)+.
3
Step-2: 8-Bromo-2,4-dichloroquinazoline. To a slurry of 8-bromoquinazoline-2,4-diol (4.30 g, 17.8 mmol) in POCl (8.17
mL, 89.0 mmol) was added DIPEA (15.5 mL, 89.0 mmol) in small ~ 2 mL portions. The reaction became hot and was cooled
in an ice bath for the rest of DIPEA addition. The slurry was fitted with a water-cooled reflux condenser and drying tube and
heated to reflux. After 4 h, the reaction was nearly complete. The reaction was heated 1 h additional, cooled, and poured onto
ice. While still cold the mixture was treated with 10N NaOH until pH >10. The resulting very fine orange solid was collected
by filtration. The solid was rinsed water and dried in vacuo to give 8-bromo-2,4-dichloroquinazoline (4.30 g, 15.5 mmol, 87%
1
yield) as an orange solid. H NMR (400 MHz, CDCl
3
) δ ppm 8.25-8.31 (m, 2H), 7.61 (dd, J=8.2, 7.6 Hz, 1H), m/z (ESI, +ve ion)
2
76.9/278.9/280.9 (M+H)+.
Step-3: 8-Bromo-2-chloroquinazolin-4(3H)-one. To a biphasic mixture of NaOH 1 N aq. (10.8 mL, 10.8 mmol) and THF
11 mL) was added 8-bromo-2,4-dichloroquinazoline (1.50 g, 5.40 mmol) in one portion. The reaction became dark red. After
(
20 min the reaction mixture was cooled in an ice/water bath and acidified with AcOH until pH ~7. The resulting mixture was
concentrated to 1/2 volume under vacuum (precipitate observed) and cooled in an ice/water bath. The slurry was filtered
and the solid rinsed with water. The orange solid was collected and dried in vacuo to give 8-bromo-2-chloroquinazolin-4(3H)-
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Journal of Medicinal Chemistry
) δ ppm 13.49 (br. s., 1H), 8.15 (dd, J = 7.8, 1.4 Hz, 1H), 8.09
1
one (1.10 g, 4.24 mmol, 79% yield). H NMR (400 MHz, DMSO-d
6
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
(dd, J = 7.9, 1.5 Hz, 1H), 7.45 (t, J = 7.8 Hz, 1H). m/z (ESI, +ve ion) 258.9/260.9 (1:1) (M+H)+.
Step-4: 8-Bromo-2-chloro-3-(cyclopropylmethyl)quinazolin-4(3H)-one. In a sealed tube, sodium hydride (60 %
dispersion in mineral oil, 58 mg, 1.44 mmol) was added to a solution of 8-bromo-2-chloroquinazolin-4(3H)-one (0.34 g, 1.31
mmol) in DME (5.2 mL)/DMF (1.3 mL) at 0 °C. After stirring for 5 min, lithium bromide in THF (1.75 mL, 2.62 mmol) was
added, and the reaction mixture was allowed to warm to room temperature.
After stirring for 5 min,
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(
bromomethyl)cyclopropane (0.38 mL, 3.93 mmol) was added, and the reaction vessel was sealed and heated to 60 °C for 16
h. The reaction mixture was diluted with dichloromethane (100 mL) and washed with saturated aqueous sodium bicarbonate
(2 x 100 mL); the organic layer was separated, dried over sodium sulfate, and concentrated to give 8-bromo-2-chloro-3-
(cyclopropylmethyl)quinazolin-4(3H)-one (0.35 g, 1.12 mmol, 85 % yield) as a crude red oil, which was used for next step
without purification. m/z (ESI, +ve) 312.9/314.9 (4:5) (M+H)+.
Step-5: 8-Bromo-2-(tert-butylamino)-3-(cyclopropylmethyl)quinazolin-4(3H)-one. A solution of 8-bromo-2-chloro-
3
-(cyclopropylmethyl)quinazolin-4(3H)-one (0.35 g, 1.12 mmol) and 2-methylpropan-2-amine (0.59 mL, 5.58 mmol) in
DMSO (2.79 mL) was stirred at 90 °C for 16 h. The reaction mixture was diluted with dichloromethane (100 mL) and washed
with saturated aqueous brine (2 x 100 mL). The organic layer was separated, dried over sodium sulfate, and concentrated.
The crude product was adsorbed onto silica and was purified by silica gel chromatography (using a gradient 0–30%
EtOAc/hexanes) to give 8-bromo-2-(tert-butylamino)-3-(cyclopropylmethyl)quinazolin-4(3H)-one (264 mg, 0.75 mmol, 67.5
1
%
yield) as a brown solid. H NMR (400 MHz, CDCl
3
)  ppm 8.05 (dd, J = 7.9, 1.5 Hz, 1H), 7.84 (dd, J = 7.6, 1.37 Hz, 1H), 6.97
(
t, J=7.8 Hz, 1H), 4.80 (s, 1H), 3.97 (d, J = 6.5 Hz, 2H), 1.62 (s, 9H), 1.00 - 1.12 (m, 1H), 0.62 - 0.69 (m, 2H), 0.44 - 0.52 (m, 2H).
m/z (ESI, +ve) 350.0/352.0 (1:1) (M+H)+.
Step-6:
(R)-2-(tert-Butylamino)-3-(cyclopropylmethyl)-8-(6-methyl-4-oxo-1,4,5,6-tetrahydropyrrolo[3,4-
b]pyrrol-2-yl)quinazolin-4(3H)-one. A mixture of 8-bromo-2-(tert-butylamino)-3-(cyclopropylmethyl)quinazolin-4(3H)-
one (129 mg, 0.37 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 (9d, 212 mg, 0.81 mmol), XPhos-Pd-G2 (14.5 mg, 0.02 mmol), and potassium phosphate (235 mg, 1.11 mmol) in
1
,4-dioxane (2.9 mL)/water (0.7 mL) was stirred at 90 °C for 1 h. The reaction mixture was diluted with dichloromethane
75 mL) and washed with saturated aqueous brine (2 x 50 mL). The organic layer was separated, dried over sodium sulfate,
and concentrated. The crude product was adsorbed onto silica and purified by silica gel chromatography (using a gradient
(
0
1
–6% 2 M ammonia in MeOH/dichloromethane) to give (R)-2-(tert-butylamino)-3-(cyclopropylmethyl)-8-(6-methyl-4-oxo-
,4,5,6-tetrahydropyrrolo[3,4-b]pyrrol-2-yl)quinazolin-4(3H)-one (84 mg, 0.21 mmol, 56.2 % yield) as an yellow solid. 1
H
NMR (400 MHz, CDCl )  ppm 12.43 (br. s., 1H), 8.04 (dd, J = 7.8, 1.6 Hz, 1H), 8.01 (dd, J = 7.7, 1.5 Hz, 1H), 7.21 (t, J = 7.8 Hz,
3
1H), 6.82 (d, J = 1.4 Hz, 1H), 5.91 (s, 1H), 5.01 (s, 1H), 4.66 (q, J = 6.7 Hz, 1H), 4.06 (dd, J = 6.4, 2.5 Hz, 2H), 1.67 (s, 9H), 1.53 (d,
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Journal of Medicinal Chemistry
Page 22 of 40
R
= 2.23 min (>95%); m/z (ESI, +ve) 406.1
J = 6.7 Hz, 3H), 1.04 - 1.14 (m, 1H), 0.66 - 0.75 (m, 2H), 0.49 - 0.56 (m, 2H). LC−MS t
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
(M+H)+.
(R)-2-(tert-Butylamino)-3-((1-hydroxycyclopropyl)methyl)-8-(6-methyl-4-oxo-1,4,5,6-tetrahydropyrrolo[3,4-
b]pyrrol-2-yl)quinazolin-4(3H)-one (15). (Method C) Step-1: (1-((tert-
Butyldimethylsilyl)oxy)cyclopropyl)methanamine. A mixture of 1-(aminomethyl)-cyclopropanol (1.00 g, 11.5 mmol) and
DIPEA (6.0 mL, 34.4 mmol) in dichloromethane (25 mL) at 0 °C under nitrogen was treated with (t-butyldimethylsilyl
trifluoromethanesulfonate (3.2 mL, 13.8 mmol). The reaction mixture was allowed to warm to ambient temperature over 1
h. The reaction was partitioned between saturated aqueous sodium bicarbonate and dichloromethane. The aqueous layer
was extracted with dichloromethane, and the combined organics were dried over anhydrous sodium sulfate, filtered, and
concentrated in vacuo. The crude material was purified by silica gel chromatography (using a gradient 0–10% 2M ammonia
in MeOH/dichloromethane). The product-containing fractions (stained blue in CAM) were concentrated to afford 2.65 g of
an orange solid. The material was treated with saturated aqueous sodium bicarbonate and dichloromethane. The aqueous
layer was extracted with dichloromethane (2X), and the combined organics were dried over anhydrous sodium sulfate,
filtered, and concentrated under vacuum to give (1-((tert-butyldimethylsilyl)oxy)cyclopropyl)methanamine (1.14 g, 5.66
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
mmol, 49.3 % yield) as an orange oil: H NMR (400 MHz, CDCl
3
)  ppm 2.70 (s, 2H), 1.95 - 2.49 (br. s, 2H), 0.88 (s, 9H), 0.73 -
0.79 (m, 2H), 0.50 - 0.55 (m, 2H), 0.12 (s, 6H).
Step-2:
2-Amino-3-bromo-N-((1-((tert-butyldimethylsilyl)-oxy)cyclopropyl)methyl)benzamide.
The
title
compound (1.16 g, 2.90 mmol, 59.8 % yield, yellow solid) was prepared analogous to compound 10, Step-1 using T3P (50
wt.% solution in ethyl acetate, 3.4 mL, 5.35 mmol), DIPEA (1.3 mL, 7.29 mmol), 2-amino-3-bromobenzoic acid (1.05 g, 4.86
1
mmol), (1-((tert-butyldimethylsilyl)oxy)cyclopropyl)methanamine (1.18 g, 5.83 mmol), and EtOAc (20 mL): H NMR (400
MHz, CDC
3
)  ppm 7.49 - 7.56 (m, 1H), 7.33 (dd, J = 7.9, 1.3 Hz, 1H), 6.56 (t, J = 7.8 Hz, 1H), 6.44 (br. s., 1H), 6.01 (br. s., 2H),
3
.48 (d, J = 5.1 Hz, 2H), 0.88 (s, 9H), 0.80 - 0.86 (m, 2H), 0.63 - 0.68 (m, 2H), 0.14 (s, 6H). m/z (ESI, +ve ion) 399.0/401.0 (1:1)
M+H)+.
(
Step-3: 8-Bromo-2-(tert-butylamino)-3-((1-((tert-butyldimethylsilyl)oxy)cyclopropyl)methyl)quinazolin-4(3H)-
one. The title compound (300 mg, 0.62 mmol, 45.3 % yield, white solid) was prepared analogous to compound 12, Step-2
using 2-amino-3-bromo-N-((1-((tert-butyldimethylsilyl)oxy)cyclopropyl)methyl) benzamide (550 mg, 1.38 mmol), MeCN
(
10 mL), tert-butyl nitrite, 90% (0.25 mL, 2.07 mmol), trimethylsilyl azide (0.22 mL, 1.65 mmol), trimethylphosphine (1.0 M
1
in THF, 1.65 mL, 1.65 mmol), tert-butyl isocyanate (0.18 mL, 1.58 mmol), DBU (0.25 mL, 1.65 mmol): H NMR (400 MHz,
CDCl )  ppm 8.01 (dd, J=7.8, 1.4 Hz, 1H), 7.85 (dd, J=7.6, 1.4 Hz, 1H), 6.96 (t, J=7.8 Hz, 1H), 5.61 (s, 1H), 4.36 (s, 2H), 1.64 (s,
3
H), 0.90 - 0.95 (m, 2H), 0.88 (s, 9H), 0.70 - 0.75 (m, 2H), 0.20 (s, 6H). m/z (ESI, +ve ion) 480.0/482.0 (1:1) (M+H)+.
9
Step-4:
(R)-2-(tert-Butylamino)-3-((1-((tert-butyldimethylsilyl)oxy)cyclopropyl)methyl)-8-(6-methyl-4-oxo-
1,4,5,6-tetrahydropyrrolo[3,4-b]pyrrol-2-yl)quinazolin-4(3H)-one. The title compound (139 mg, 0.26 mmol, 83 % yield,
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Journal of Medicinal Chemistry
white foam) was prepared analogous to compound 12, Step-3 using potassium phosphate tribasic (265 mg, 1.25 mmol),
XPhos-Pd-G2 (12 mg, 0.02 mmol), (R)-6-methyl-2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-5,6-dihydropyrrolo[3,4-
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
b]pyrrol-4(1H)-one
(9d;
164
mg,
0.62
mmol),
8-bromo-2-(tert-butylamino)-3-((1-((tert-
butyldimethylsilyl)oxy)cyclopropyl)methyl)quinazolin-4(3H)-one (150 mg, 0.31 mmol) and dioxane (3 mL) / water (0.5 mL):
1H NMR (400 MHz, CDCl
3
) δ ppm 12.54 (br s, 1 H), 8.01 (br s, 2 H), 7.15 - 7.24 (m, 2 H), 6.83 (br s, 1 H), 5.75 (br s, 1 H), 4.67 (br
d, J=6.7 Hz, 1 H), 4.40 (q, J=15.2 Hz, 2 H), 1.53 (br d, J=5.3 Hz, 3 H), 1.24 (br s, 9 H), 0.97 (br s, 2 H), 0.87 (br s, 9 H), 0.78 (br d,
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
J=5.3 Hz, 2 H), 0.19 (br s, 6 H). m/z (ESI, +ve ion) 536.2 (M+H)+.
Step-5:
(R)-2-(tert-Butylamino)-3-((1-hydroxycyclopropyl)methyl)-8-(6-methyl-4-oxo-1,4,5,6-
tetrahydropyrrolo[3,4-b]pyrrol-2-yl)quinazolin-4(3H)-one. To a solution of (R)-2-(tert-butylamino)-3-((1-((tert-
butyldimethylsilyl)oxy)cyclopropyl)methyl)-8-(6-methyl-4-oxo-1,4,5,6-tetrahydropyrrolo-[3,4-b]pyrrol-2-yl)quinazolin-
4
(3H)-one (139 mg, 0.26 mmol) in THF (3 mL) under nitrogen was added TBAF(1.0 M in THF, 0.39 mL, 0.39 mmol) at room
temperature and stirred for 30 min. The resulting mixture was concentrated in vacuo and purified by silica gel
chromatography (using a gradient 0–10% 2M ammonia in MeOH/dichloromethane) to afford the title compound (76 mg, 0.18
1
mmol, 69.5 % yield) as an off-white solid. H NMR (400 MHz, DMSO-d
6
)  ppm 12.00 (s, 1H), 7.76 - 8.04 (m, 2H), 7.62 (s, 1H),
7
.36 (s, 1H), 7.17 (t, J = 7.7 Hz, 1H), 6.75 (s, 1H), 6.65 (s, 1H), 4.54 (q, J = 6.7 Hz, 1H), 4.20 (s, 2H), 1.50 (s, 9H), 1.37 (d, J = 6.7
Hz, 3H), 0.79 - 0.88 (m, 2H), 0.73 - 0.79 (m, 2H). LC−MS t
R
= 1.87 min (>95%); m/z (ESI, +ve ion) 422.1 (M+H)+.
(R)-3-((1-Aminocyclopropyl)methyl)-2-(tert-butylamino)-8-(6-methyl-4-oxo-1,4,5,6-tetrahydropyrrolo[3,4-
b]pyrrol-2-yl)quinazolin-4(3H)-one (16). (Method C) Step-1: tert-Butyl (1-((2-amino-3-
bromobenzamido)methyl)cyclopropyl)carbamate. The title compound (392 mg, 1.02 mmol, 76 % yield, white solid) was
prepared analogous to compound 12, Step-1 using 2-amino-3-bromobenzoic acid (290 mg, 1.34 mmol), tert-butyl 1-
(
aminomethyl)cyclopropylcarbamate (0.30 g, 1.61 mmol), DIPEA (0.7 mL, 4.03 mmol), HATU (561 mg, 1.48 mmol), DMF (1.3
1
mL) and dichloromethane (1.3 mL): H NMR (400 MHz, CDCl
3
)  ppm 8.00 (br s, 1 H), 7.41 - 7.55 (m, 2 H), 6.53 (t, J = 7.9 Hz,
1
H), 6.26 (br s, 2 H), 5.11 (br s, 1 H), 3.42 (d, J = 3.9 Hz, 2 H), 1.43 (s, 9 H), 0.90 (br d, J = 8.2 Hz, 4 H). m/z (ESI, +ve ion)
384.0/386.0 (1 :1) (M+H)+.
Step-2: tert-Butyl (1-((8-bromo-2-(tert-butylamino)-4-oxoquinazolin-3(4H)-yl)methyl)cyclopropyl)carbamate.
The title compound (70 mg, 0.15 mmol, 19.6 % yield, white solid) was prepared analogous to compound 12, Step-2 using tert-
butyl (1-((2-amino-3-bromobenzamido)methyl)cyclopropyl)carbamate (295 mg, 0.77 mmol), MeCN (5.1 mL), tert-butyl
nitrite, 90% (0.15 mL, 1.15 mmol), azidotrimethylsilane (0.12 mL, 0.92 mmol), trimethylphosphine (1.0 M in THF, 0.92 mL,
1
0
.92 mmol), tert-butyl isocyanate (0.01 mL, 0.84 mmol), and 1,8-diazabicyclo[5.4.0]undec-7-ene (0.13 mL, 0.84 mmol): H
NMR (400 MHz, CDCl )  ppm 7.98 (d, J = 7.8 Hz, 1H), 7.82 (d, J = 7.2 Hz, 1H), 6.91 (t, J = 7.8 Hz, 1H), 6.86 (br. s., 1H), 5.05 (br.
3
s., 1H), 4.40 (br. s., 2H), 1.66 (s, 9H), 1.46 (s, 9H), 0.65 - 0.82 (m, 4H). m/z (ESI, +ve ion) 465.0/467.0 (1 :1) (M+H)+.
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Step-3: (R)-tert-Butyl (1-((2-(tert-butylamino)-8-(6-methyl-4-oxo-1,4,5,6-tetrahydropyrrolo[3,4-b]pyrrol-2-yl)-4-
oxoquinazolin-3(4H)-yl)methyl)cyclopropyl)carbamate. The title compound (55 mg, 0.11 mmol, 78 % yield, pale green
solid) was prepared analogous to compound 12, Step-3 using tert-butyl (1-((8-bromo-2-(tert-butylamino)-4-oxoquinazolin-
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
3
5
(4H)-yl)methyl)cyclopropyl)carbamate (63 mg, 0.14 mmol), (R)-6-methyl-2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-
,6-dihydropyrrolo[3,4-b]pyrrol-4(1H)-one (71 mg, 0.27 mmol), X-Phos precatalyst generation II (5.33 mg, 6.77 µmol) and
1
potassium phosphate (86 mg, 0.41 mmol) and 1,4-dioxane (3.2 mL)/water (0.8 mL): H NMR (400 MHz, CDCl
3
) δ ppm 12.50
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(br s, 1 H), 7.99 (ddd, J = 7.8, 6.5, 1.6 Hz, 2 H), 7.17 (t, J = 7.8 Hz, 1 H), 6.96 (br s, 1 H), 6.81 (d, J = 1.2 Hz, 1 H), 5.58 (s, 1 H),
5
.11 (s, 1 H), 4.66 (q, J = 6.9 Hz, 1 H), 4.49 (br s, 2 H), 1.70 (s, 9 H), 1.53 (d, J = 6.7 Hz, 3 H), 1.46 (s, 9 H), 0.79 (br s, 2 H), 0.74
(br d, J=3.9 Hz, 2 H). m/z (ESI, +ve ion) 521.1 (M+H)+.
Step-4: (R)-3-((1-Aminocyclopropyl)methyl)-2-(tert-butylamino)-8-(6-methyl-4-oxo-1,4,5,6-tetrahydropyrrolo
[
3,4-b]pyrrol-2-yl)quinazolin-4(3H)-one. Trifluoroacetic acid (0.2 mL, 2.88 mmol) was added to a heterogenous solution
of (R)-tert-butyl (1-((2-(tert-butylamino)-8-(6-methyl-4-oxo-1,4,5,6-tetrahydropyrrolo[3,4-b]pyrrol-2-yl)-4-oxoquinazolin-
(4H)-yl)methyl)cyclopropyl)carbamate (50 mg, 0.10 mmol) in dichloromethane (0.5 mL) at room temperature. The solution
turned clear and yellow upon addition of TFA. The reaction stirred for 2 h and then saturated NaHCO (aq.) was added. The
mixture was extracted with CH Cl (3X). The combined organic layers were washed with brine and dried over anhydrous
3
3
2
2
sodium sulfate, filtered and concentrated to give (R)-3-((1-aminocyclopropyl)methyl)-2-(tert-butylamino)-8-(6-methyl-4-
1
oxo-1,4,5,6-tetrahydropyrrolo[3,4-b]pyrrol-2-yl)quinazolin-4(3H)-one (35 mg, 0.08 mmol, 87 % yield) as a yellow solid. H
NMR (400 MHz, DMSO-d
J = 7.7 Hz, 1H), 6.75 (d, J = 1.4 Hz, 1H), 4.55 (q, J = 6.5 Hz, 1H), 4.05 (s, 2H), 1.52 (s, 9H), 1.38 (d, J = 6.5 Hz, 3H), 0.83 - 0.89 (m,
H), 0.61 - 0.69 (m, 2H). The peaks of -NH and -NH2 were not observed. LC−MS t = 1.32 min (>95%); m/z (ESI, +ve ion) 421.1
6
)  ppm 12.12 (s, 1H), 7.92 (dd, J = 7.4, 1.4 Hz, 1H), 7.84 (dd, J = 7.8, 1.4 Hz, 1H), 7.62 (s, 1H), 7.13 (t,
2
R
M+H)+.
(
(R)-3-(2-Amino-2-methylpropyl)-2-(tert-butylamino)-8-(6-methyl-4-oxo-1,4,5,6-tetrahydropyrrolo[3,4-b]pyrrol-
2-yl)quinazolin-4(3H)-one (17). (Method C) Step-1: tert-Butyl(1-(2-amino-3-bromobenzamido)-2-methylpropan-2-
yl)carbamate. The title compound (1.57 g, 4.06 mmol, 88% yield, white solid) was prepared analogous to compound 16,
Step-1 using 2-amino-3-bromobenzoic acid (1.00 g, 4.63 mmol), 2-N-boc-2-methylpropane-1,2-diamine hydrochloride salt
(
1.25 g, 5.55 mmol), DIPEA (3.2 mL, 18.50 mmol), HATU (1.94 g, 5.09 mmol), DMF (4.5 mL) and dichloromethane (4.5 mL):
1H NMR (400 MHz, CDCl
3
) δ ppm 7.79 (br s, 1 H), 7.50 (dd, J = 7.8, 1.2 Hz, 1 H), 7.43 (d, J = 7.8 Hz, 1 H), 6.55 (t, J=7.9 Hz, 1 H),
.17 (br s, 2 H), 4.71 (br s, 1 H), 3.57 (d, J = 5.7 Hz, 2 H), 1.44 (s, 9 H), 1.34 (s, 6 H). m/z (ESI, +ve ion) 386.0/388.0 (1:1) (M+H)+.
6
Step-2: tert-Butyl(1-(8-bromo-2-(tert-butylamino)-4-oxoquinazolin-3(4H)-yl)-2-methylpropan-2-yl)carbamate.
The title compound (133 mg, 0.28 mmol, 35.3% yield, white solid) was prepared analogous to compound 16, Step-2 using
tert-butyl(1-(2-amino-3-bromobenzamido)-2-methylpropan-2-yl)carbamate (311 mg, 0.80 mmol), MeCN (5.2 mL), tert-butyl
nitrite, 90% (0.16 mL, 1.21 mmol), azidotrimethylsilane (0.13 mL, 0.97 mmol), trimethylphosphine (1M in THF, 0.97 mL, 0.97
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mmol), tert-butyl isocyanate (0.01 mL, 0.89 mmol), and 1,8-diazabicyclo[5.4.0]undec-7-ene (0.13 mL, 0.89 mmol): H NMR
(400 MHz, CDCl )  ppm 8.01 (dd, J = 8.0, 1.4 Hz, 1H), 7.83 (dd, J = 7.6, 1.4 Hz, 1H), 6.93 (t, J = 7.7 Hz, 1H), 6.77 (br. s., 1H), 4.69
Journal of Medicinal Chemistry
1
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
3
(s, 1H), 3.79 - 4.63 (m, 2H), 1.64 (s, 9H), 1.47 (s, 9H), 1.35 (br. s., 6H). m/z (ESI, +ve ion) 467.0/469.1 (1:1) (M+H)+.
Step-3: (R)-tert-Butyl (1-(2-(tert-butylamino)-8-(6-methyl-4-oxo-1,4,5,6-tetrahydropyrrolo[3,4-b]pyrrol-2-yl)-4-
oxoquinazolin-3(4H)-yl)-2-methylpropan-2-yl)carbamate. The title compound (125 mg, 0.24 mmol, 89 % yield, pale
yellow solid) was prepared analogous to compound 16, Step-3 using tert-butyl(1-(8-bromo-2-(tert-butylamino)-4-
oxoquinazolin-3(4H)-yl)-2-methylpropan-2-yl)carbamate (125 mg, 0.27 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 (175 mg, 0.67 mmol), X-Phos precatalyst generation II (10.5
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
mg, 0.01 mmol) and potassium phosphate (170 mg, 0.80 mmol) and 1,4-dioxane (2.2 mL)/water (0.6 mL): H NMR (400 MHz,
DMSO-d ) 11.86 (s, 1 H), 7.89 (q, J=1.6 Hz, 1 H), 7.87 (q, J=1.6 Hz, 1 H), 7.63 (s, 1 H), 7.17 (t, J=7.6 Hz, 1 H), 7.11 (s, 1 H), 6.75
s, 1 H), 6.69 (d, J=1.4 Hz, 1 H), 4.53 (q, J=6.4 Hz, 1 H), 3.97 (br. s., 2H), 1.51 (s, 9 H), 1.42 (s, 9 H), 1.37 (d, J=6.7 Hz, 3 H), 1.15
6
(
-
1.32 (m, 6 H). m/z (ESI, +ve ion) 523.1 (M+H)+.
Step-4: (R)-3-(2-Amino-2-methylpropyl)-2-(tert-butylamino)-8-(6-methyl-4-oxo-1,4,5,6-tetrahydropyrrolo[3,4-
b]pyrrol-2-yl)quinazolin-4(3H)-one. The title compound (55 mg, 0.13 mmol, 61.8 % yield, off-white solid) was prepared
analogous to compound 16, Step-4 using trifluoroacetic acid (0.47 mL, 6.31 mmol), (R)-tert-butyl (1-(2-(tert-butylamino)-8-
(6-methyl-4-oxo-1,4,5,6-tetrahydropyrrolo[3,4-b]pyrrol-2-yl)-4-oxoquinazolin-3(4H)-yl)-2-methylpropan-2-yl)carbamate
1
(110 g, 0.21 mmol) and dichloromethane (2.0 mL). H NMR (400 MHz, DMSO-d
6
)  ppm 12.14 (br. s., 1H), 7.91 (dd, J = 7.5, 1.5
Hz, 1H), 7.85 (dd, J = 7.8, 1.4 Hz, 1H), 7.61 (s, 1H), 7.13 (t, J = 7.7 Hz, 1H), 6.74 (d, J = 1.4 Hz, 1H), 4.54 (q, J = 6.3 Hz, 1H), 3.97
(
br. s., 2H), 1.50 (s, 9H), 1.37 (d, J = 6.7 Hz, 3H), 1.15 (br. s., 6H). The peaks of -NH and -NH2 were not observed. LC−MS t
R
=
1
.32 min (>95%); m/z (ESI, +ve ion) 423.0 (M+H)+.
(
R)-2-(tert-Butylamino)-8-(6-methyl-4-oxo-1,4,5,6-tetrahydropyrrolo[3,4-b]pyrrol-2-yl)-3-(oxetan-3-ylmethyl)-
quinazolin-4(3H)-one (18). Step-1: 8-Bromo-2-chloro-3-(oxetan-3-ylmethyl)quinazolin-4(3H)-one. The title
compound (132 mg, 0.40 mmol, 52 % yield, orange solid) was prepared analogous to compound 14, Step-4 using sodium
hydride (60% dispersion in mineral oil, 34 mg, 0.85 mmol), 8-bromo-2-chloroquinazolin-4(3H)-one (200 mg, 0.77 mmol),
lithium bromide (1.5M in THF, 1.0 mL, 1.54 mmol), 3-(iodomethyl)oxetane (168 mg, 0.85 mmol) and DME (0.5 mL)/ DMF
1
(
0.5 mL): H NMR (400 MHz, DMSO-d
6
)  ppm 8.17 (dd, J = 7.8, 1.4 Hz, 1H), 8.11 (dd, J = 7.9, 1.3 Hz, 1H), 7.47 (t, J = 7.9 Hz, 1H),
4
.63 (dd, J = 7.9, 6.2 Hz, 2H), 4.42 - 4.53 (m, 4H), 3.47 (dt, J = 14.2, 7.3 Hz, 1H). m/z (ESI, +ve) 329.0/331.0 (1:1) (M+H)+.
Step-2: 8-Bromo-2-(tert-butylamino)-3-(oxetan-3-ylmethyl)-quinazolin-4(3H)-one. The title compound (86 mg, 0.24
mmol, 81 % yield, white solid) was prepared analogous to compound 14, Step-5 using 8-bromo-2-chloro-3-(oxetan-3-
1
ylmethyl)quinazolin-4(3H)-one (95 mg, 0.29 mmol), tert-butylamine (0.45 mL, 4.32 mmol), and NMP (0.2 mL): H NMR (400
MHz, DMSO-d )  ppm 7.91 (d, J = 7.6 Hz, 2H), 7.02 (t, J = 7.8 Hz, 1H), 6.15 (s, 1H), 4.59 (dd, J = 8.0, 6.1 Hz, 2H), 4.48 (d, J = 7.0
6
Hz, 2H), 4.42 (t, J = 6.3 Hz, 2H), 3.29 - 3.37 (m, 1H), 1.55 (s, 9H). m/z (ESI, +ve) 366.0/368.0 (1:1) (M+H)+.
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Step-3:
(R)-2-(tert-Butylamino)-8-(6-methyl-4-oxo-1,4,5,6-tetrahydropyrrolo[3,4-b]pyrrol-2-yl)-3-(oxetan-3-
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
ylmethyl)-quinazolin-4(3H)-one. The title compound (45 mg, 0.11 mmol, 45% yield, off-white solid) was prepared
analogous to compound 14, Step-6 using 8-bromo-2-(tert-butylamino)-3-(oxetan-3-ylmethyl)quinazolin-4(3H)-one (86 mg,
0
.24 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 (62
mg, 0.24 mmol), XPhos-Pd-G2 (9.3 mg, 0.01 mmol), potassium phosphate (151 mg, 0.71 mmol), and 1,4-dioxane (0.8
1
mL)/water (0.2 mL): H NMR (400 MHz, DMSO-d
6
)  ppm 11.79 (s, 1H), 7.82 - 7.92 (m, 2H), 7.59 (s, 1H), 7.18 (t, J = 7.7 Hz,
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
5
H), 6.65 (d, J = 1.6 Hz, 1H), 6.00 (s, 1H), 4.60 (dd, J = 7.9, 6.0 Hz, 2H), 4.48 - 4.57 (m, 3H), 4.43 (t, J = 6.3 Hz, 2H), 4.08 (q, J =
= 2.17 min (>95%); m/z (ESI, +ve ion) 422.5 (M+H)+.
R
.3 Hz, 1H), 1.47 (s, 9H), 1.36 (d, J = 6.7 Hz, 3H). LC−MS t
2
-(tert-Butylamino)-8-((R)-6-methyl-4-oxo-1,4,5,6-tetrahydropyrrolo[3,4-b]pyrrol-2-yl)-3-(((R)-
tetrahydrofuran-2-yl)methyl)quinazolin-4(3H)-one (19). (Method B) Step-1: (R)-2-Amino-3-bromo-N-
(tetrahydrofuran-2-yl)methyl)benzamide. The title compound (1.35 g, 4.50 mmol, 88 % yield, light yellow solid) was
(
prepared analogous to compound 10, Step-1 using 2-amino-3-bromobenzoic acid (1.10 g, 5.09 mmol) and (R)-(-)-
tetrahydrofurfurylamine (0.57 g, 5.60 mmol), DIPEA (2.7 mL, 15.3 mmol) and T3P (50 wt% in EtOAc, 3.9 mL, 6.11 mmol) and
1
EtOAc (20 mL): H NMR (400 MHz, DMSO-d
6
)  ppm 8.46 (t, J = 6.1 Hz, 1H), 7.53 (d, J = 7.8 Hz, 2H), 6.53 (t, J = 7.8 Hz, 1H), 6.42
(
(
s, 2H), 3.97 (quin, J = 6.3 Hz, 1H), 3.73 - 3.82 (m, 1H), 3.59 - 3.68 (m, 1H), 3.21 - 3.31 (m, 2H), 1.75 - 1.96 (m, 3H), 1.53 - 1.64
m, 1H). m/z (ESI, +ve) 299.0/301.0 (1:1) (M+H)+.
Step-2: (R)-8-Bromo-3-((tetrahydrofuran-2-yl)methyl)-quinazoline-2,4(1H,3H)-dione. The title compound (1.45 g,
4.46 mmol, 99 % yield, tan solid) was prepared analogous to compound 10, Step-2 using (R)-2-amino-3-bromo-N-
(
(tetrahydrofuran-2-yl)methyl)benzamide (1.34 g, 4.49 mmol), triphosgene (0.47 g, 1.57 mmol) and dichloromethane (20
1
mL): H NMR (400 MHz, DMSO-d
6
)  ppm 10.57 (s, 1H), 7.95 - 8.03 (m, 2H), 7.17 (t, J = 7.8 Hz, 1H), 4.14 - 4.23 (m, 1H), 4.04 -
4
+
.12 (m, 1H), 3.72 - 3.87 (m, 2H), 3.58 - 3.65 (m, 1H), 1.87 - 1.98 (m, 2H), 1.75 - 1.86 (m, 1H), 1.60 - 1.70 (m, 1H). m/z (ESI,
ve) 325.0/327.0 (1:1) (M+H)+.
Step-3:
(R)-8-Bromo-2-(tert-butylamino)-3-((tetrahydrofuran-2-yl)methyl)quinazolin-4(3H)-one.
A
glass
microwave reaction vessel was charged with (R)-8-bromo-3-((tetrahydrofuran-2-yl)methyl)quinazoline-2,4(1H,3H)-dione
(221 mg, 0.68 mmol) and (benzotriazol-1-yloxy)tris(dimethylamino)phosphonium hexafluorophosphate (451 mg, 1.02
mmol) in DMF (2 mL) followed by 1,8-diazabicyclo-[5.4.0]undec-7-ene (0.20 mL, 1.36 mmol). The reaction mixture was
stirred for 5 min and tert-butylamine (0.36 mL, 3.40 mmol) was added. The reaction was stirred at 40 °C for 30 min and
allowed to cool to room temperature. The mixture was added ice water and stirred for 5 min. The resulting precipitate was
collected by filtration, washed with water and MeOH to give (R)-8-bromo-2-(dimethylamino)-3-((tetrahydrofuran-2-
yl)methyl)quinazolin-4(3H)-one (188 mg, 0.50 mmol, 73.0 % yield) as a light yellow solid. 1H NMR (400 MHz, DMSO-d
) 
6
ppm 7.88 - 7.98 (m, 2H), 7.03 (t, J = 7.8 Hz, 1H), 6.90 (s, 1H), 4.07 - 4.29 (m, 3H), 3.69 - 3.89 (m, 2H), 2.01 (dt, J = 12.4, 6.2 Hz,
1H), 1.79 - 1.93 (m, 2H), 1.55 - 1.66 (m, 1H), 1.54 (s, 9H). m/z (ESI, +ve) 380.0/382.0 (1:1) (M+H)+.
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1
Journal of Medicinal Chemistry
Step-4:
(2-(tert-Butylamino)-8-((R)-6-methyl-4-oxo-1,4,5,6-tetrahydropyrrolo[3,4-b]pyrrol-2-yl)-3-(((R)-
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
tetrahydrofuran-2-yl)methyl)quinazolin-4(3H)-one. The title compound (154 mg, 0.35 mmol, 73.1 % yield, tan solid) was
prepared analogous to compound 10, Step-5 using (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 (9d, 206 mg, 0.63 mmol), (R)-8-bromo-2-(tert-butylamino)-3-((tetrahydrofuran-2-
yl)methyl)quinazolin-4(3H)-one (184 mg, 0.48 mmol), potassium phosphate (308 mg, 1.45 mmol) and XPhos-Pd-G2 (19.0
1
mg, 0.02 mmol) and 1,4-dioxane (4 mL)/water (1.2 mL): H NMR (400 MHz, DMSO-d
6
)  ppm 11.94 (s, 1H), 7.85 - 7.94 (m,
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
3
H), 7.62 (s, 1H), 7.19 (t, J = 7.6 Hz, 1H), 6.98 (s, 1H), 6.74 (d, J = 1.0 Hz, 1H), 4.50 - 4.58 (m, 1H), 4.13 - 4.32 (m, 3H), 3.72 -
.88 (m, 2H), 2.05 (dq, J = 12.3, 6.1 Hz, 1H), 1.81 - 1.93 (m, 2H), 1.55 - 1.67 (m, 1H), 1.49 (s, 9H), 1.37 (d, J = 6.7 Hz, 3H). LC−MS
= 2.43 min (>95%); m/z (ESI, +ve) 436.0 (M+H)+.
R
t
2-(tert-Butylamino)-8-((R)-6-methyl-4-oxo-1,4,5,6-tetrahydropyrrolo[3,4-b]pyrrol-2-yl)-3-(((S)-
tetrahydrofuran-2-yl)methyl)quinazolin-4(3H)-one (20). (Method B) The title compound was prepared analogous to
1
compound 19 using (S)-(+)-tetrahydrofurfurylamine (0.57 g, 5.60 mmol) in the Step-1: H NMR (400 MHz, DMSO-d
6
)  ppm
1
1.95 (s, 1H), 7.90 (ddd, J = 11.3, 7.6, 1.4 Hz, 2H), 7.62 (s, 1H), 7.19 (t, J = 7.6 Hz, 1H), 6.98 (s, 1H), 6.74 (d, J = 1.2 Hz, 1H), 4.54
(
q, J = 6.5 Hz, 1H), 4.11 - 4.34 (m, 3H), 3.70 - 3.90 (m, 2H), 2.00 - 2.13 (m, 1H), 1.81 - 1.94 (m, 2H), 1.55 - 1.68 (m, 1H), 1.49 (s,
= 2.43 min (>95%); m/z (ESI, +ve) 436.0 (M+H)+.
H), 1.38 (d, J = 6.7 Hz, 3H). LC−MS t
R
9
Isomer 1 and isomer 2 of 2-(tert-Butylamino)-8-((R)-6-methyl-4-oxo-1,4,5,6-tetrahydropyrrolo[3,4-b]pyrrol-2-
yl)-3-((4-methylmorpholin-2-yl)methyl)quinazolin-4(3H)-one (21 and 22). (Method B) Step-1: 2-Amino-3-bromo-N-
((4-methylmorpholin-2-yl)methyl)benzamide. The title compound (0.85 g, 2.59 mmol, 33.7 % yield, white solid) was
prepared analogous to compound 12, Step-1 using 2-amino-3-bromobenzoic acid (1.66 g, 7.68 mmol), (4-methylmorpholin-
1
2
-yl)methanamine (1.00 g, 7.68 mmol), HATU (3.21 g, 8.45 mmol), DIPEA (1.6 mL, 9.22 mmol) and DMF (5 mL): H NMR (300
MHz, DMSO-d
6
) δ ppm 8.51 (br t, J = 5.3 Hz, 1H), 7.54 (d, J = 7.7 Hz, 2H), 6.54 (t, J = 7.8 Hz, 1H), 6.46 (br s, 2H), 3.87 (br d, J =
1
1
1.5 Hz, 1H), 3.67 (br d, J = 9.4 Hz, 1H), 3.55 (br t, J = 11.6 Hz, 1H), 3.31 (td, J = 13.5, 6.7 Hz, 2H), 3.17 (s, 2H), 2.97 (br d, J =
1.3 Hz, 1H), 2.84 (br d, J = 11.8 Hz, 1H), 2.40 (s, 3H). m/z (ESI, +ve) 327.9/329.9 (1:1) (M+H)+.
Step-2: 8-Bromo-3-((4-methylmorpholin-2-yl)methyl)-quinazoline-2,4(1H,3H)-dione. The title compound (300 mg,
0.85 mmol, 42.8 % yield, white solid) was prepared analogous to compound 10, Step-2 using 2-amino-3-bromo-N-((4-
methylmorpholin-2-yl)methyl)benzamide (650 mg, 1.98 mmol) and tri-phosgene (206 mg, 0.69 mmol) and dichloromethane
1
(
16 mL): H NMR (300 MHz, DMSO-d
6
) δ ppm 10.78 - 11.01 (m, 1H), 10.69 (br s, 1H), 7.99 (d, J = 7.7 Hz, 2H), 7.18 (t, J=7.8 Hz,
1
2
H), 4.10 (br d, J = 8.9 Hz, 2H), 3.89 - 4.04 (m, 2H), 3.76 (br t, J = 11.6 Hz, 1H), 3.50 (br d, J = 11.1 Hz, 1H), 2.95 - 3.09 (m, 1H),
.86 (br s, 1H), 2.72 (s, 3H). m/z (ESI, +ve) 353.9/355.8 (1:1) (M+H)+.
Step-3: 8-Bromo-2-(tert-butylamino)-3-((4-methylmorpholin-2-yl)methyl)quinazolin-4(3H)-one. The title
compound (254 mg, 0.62 mmol, 73.3 % yield, white solid) was prepared analogous to compound 19, Step-3 using 8-bromo-
3-((4-methylmorpholin-2-yl)methyl)quinazoline-2,4(1H,3H)-dione (300 mg, 0.85 mmol), (benzotriazol-1-
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Journal of Medicinal Chemistry
Page 28 of 40
yloxy)tris(dimethylamino)phosphonium hexafluorophosphate (562 mg, 1.27 mmol), and tert-butylamine (0.45 mL, 4.23
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
1
mmol) and DMF (5.2 mL): H NMR (300 MHz, DMSO-d
6
) δ ppm 7.92 (t, J = 6.6 Hz, 2H), 7.04 (t, J = 7.5 Hz, 1H), 6.58 (s, 1H), 3.97
- 4.16 (m, 2H), 3.77 - 3.95 (m, 2H), 3.56 (br t, J = 11.1 Hz, 1H) 2.74, (br d, J = 11.4 Hz, 1H), 2.54 - 2.66 (m, 2H), 2.18 (s, 3H), 1.81
br t, J = 10.4 Hz, 1H), 1.53 (s, 9H). m/z (ESI, +ve) 408.9/410.9 (1:1) (M+H)+.
(
Step-4:
2-(tert-Butylamino)-8-((R)-6-methyl-4-oxo-1,4,5,6-tetrahydropyrrolo[3,4-b]pyrrol-2-yl)-3-((4-
methylmorpholin-2-yl)methyl)quinazolin-4(3H)-one. The title compound (250 mg, 0.54 mmol, 87 % yield, yellow solid)
was prepared analogous to compound 10, Step-5 using (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 (9d, 325 mg, 1.24 mmol), potassium phosphate (527 mg, 2.48 mmol), XPhos-Pd-G2
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(24 mg, 0.03 mmol), 8-bromo-2-(tert-butylamino)-3-((4-methylmorpholin-2-yl)methyl)quinazolin-4(3H)-one (254 mg, 0.62
1
mmol), and 1,4-dioxane (6 mL)/water (1.5 mL): H NMR (300 MHz, DMSO-d
6
) δ ppm 11.91 (br s, 1H), 7.89 (t, J = 6.7 Hz, 2H),
7
.61 (s, 1H), 7.19 (t, J = 7.7 Hz, 1H), 6.72 (s, 1H), 6.62 (s, 1H), 4.53 (q, J = 6.2 Hz, 1H), 4.16 (br s, 2H), 3.83 - 3.98 (m, 2H), 3.57
(
br t, J = 10.8 Hz, 1H), 2.77 (br d, J = 11.5 Hz, 1H), 2.60 (br d, J = 12.0 Hz, 1H), 2.18 (s, 3H), 1.93 - 2.06 (m, 1H), 1.80 (br t, J =
1
0.8 Hz, 1H), 1.47 (s, 9H), 1.37 (d, J = 6.4 Hz, 3H). m/z (ESI, +ve) 465.2 (M+H)+.
Step-5: Isomer 1 and isomer 2 of 2-(tert-Butylamino)-8-((R)-6-methyl-4-oxo-1,4,5,6-tetrahydropyrrolo[3,4-
b]pyrrol-2-yl)-3-((4-methylmorpholin-2-yl)methyl)quinazolin-4(3H)-one (21 and 22). The above racemic mixture of
2-(tert-butylamino)-8-((R)-6-methyl-4-oxo-1,4,5,6-tetrahydropyrrolo[3,4-b]pyrrol-2-yl)-3-((4-methylmorpholin-2-
yl)methyl)quinazolin-4(3H)-one was purified by SFC for chiral separation (Chiralpak IDH, 250 x 20 mm, 5 μm, 25% MeOH
(
containing 20 mM Ammonia)/CO2, 70 mL/min, 165 bar) to obtain two isomers: Isomer 1 (21, Peak 1, 49.6 mg, >99% ee,
1
white solid) and isomer 2 (22, Peak 2, 44.4 mg, >99% ee, light yellow solid). Isomer 1 (21): H NMR (300 MHz, DMSO-d
6
)
1
4
3
1.91 (br s, 1H), 7.89 (t, J = 6.8 Hz, 2H), 7.61 (s, 1H), 7.19 (t, J = 7.7 Hz, 1H), 6.72 (s, 1H), 6.62 (s, 1H), 4.53 (q, J = 6.7 Hz, 1H),
.16 (br s, 2H), 3.79 - 3.98 (m, 2H), 3.57 (br t, J = 11.1 Hz, 1H), 2.77 (br d, J = 11.5 Hz, 1H), 2.60 (br d, J = 11.7 Hz, 1H), 2.18 (s,
H), 1.99 (br t, J = 10.0 Hz, 1H), 1.80 (br t, J = 10.8 Hz, 1H), 1.47 (s, 9H), 1.37 (d, J = 6.4 Hz, 3H). LC−MS t = 1.48 min (>95%);
) δ ppm 11.90 (s, 1H), 7.89 (t, J = 6.6 Hz, 2H), 7.62
R
+
1
m/z (ESI, +ve) 465.0 (M+H) . Isomer 2 (22): H NMR (300 MHz, DMSO-d
6
(s, 1H), 7.19 (t, J = 7.6 Hz, 1H), 6.72 (s, 1H), 6.61 (s, 1H), 4.43 - 4.60 (m, 1H), 4.08 - 4.27 (m, 2H), 3.80 - 4.01 (m, 2H), 3.58 (br t,
J = 11.1 Hz, 1H), 2.79 (br d, J = 11.4 Hz, 1H), 2.62 (br d, J = 11.4 Hz, 1H), 2.20 (s, 3H), 1.96 - 2.12 (m, 1H), 1.73 - 1.91 (m, 1H),
1
.47 (s, 9H), 1.37 (d, J = 6.6 Hz, 3H). LC−MS t
= 1.48 min (>95%); m/z (ESI, +ve) 465.0 (M+H)+.
R
2-(tert-Butylamino)-3-((1-methyl-1H-imidazol-4-yl)methyl)-8-(6-methyl-4-oxo-1,4,5,6-tetrahydropyrrolo[3,4-
b]pyrrol-2-yl)quinazolin-4(3H)-one (23). (Method B) Step-1: 2-Amino-3-bromo-N-((1-methyl-1H-imidazol-4-
yl)methyl)benzamide. The title compound (752 mg, 2.43 mmol, 52.5% yield, orange solid) was prepared analogous to
compound 10, Step-1 using 2-amino-3-bromobenzoic acid (1.00 g, 4.63 mmol) and (1-methyl-1H-imidazol-4-yl)methylamine
1
(514 mg, 4.63 mmol), DIPEA (2.4 mL, 13.89 mmol), T3P (50 wt% in EtOAc, 4.42 mL, 6.94 mmol) and EtOAc (20 mL): H NMR
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Journal of Medicinal Chemistry
(
400 MHz, DMSO-d6)  ppm 8.87 (t, J = 5.5 Hz, 1 H), 7.54 (td, J = 8.1, 1.3 Hz, 2 H), 7.08 (d, J = 1.0 Hz, 1 H), 6.79 (d, J = 1.2 Hz, 1
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
H), 6.46 - 6.54 (m, 3 H), 4.48 (d, J = 5.5 Hz, 2 H), 3.65 (s, 3 H)m/z (ESI, +ve) 308.9/310.9 (1:1) (M+H)+.
Step-2: 8-Bromo-3-((1-methyl-1H-imidazol-4-yl)methyl)quinazoline-2,4(1H,3H)-dione. The title compound (313
mg, 0.93 mmol, 38.4 % yield, white solid) was prepared analogous to compound 10, Step-2 using 2-amino-3-bromo-N-((1-
methyl-1H-imidazol-4-yl)methyl)benzamide (752 mg, 2.43 mmol) and triphosgene (253 mg, 0.85 mmol) and
1
dichloromethane (20 mL): H NMR (400 MHz, DMSO-d
6
)  ppm 10.85 (s, 1H), 8.95 (s, 1H), 7.96 - 8.05 (m, 2H), 7.63 (s, 1H),
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
7
.20 (t, J=7.8 Hz, 1H), 5.12 (s, 2H), 3.79 (s, 3H). m/z (ESI, +ve) 334.9/336.9 (1:1) (M+H)+.
Step-3: 8-Bromo-2-(tert-butylamino)-3-((1-methyl-1H-imidazol-4-yl)methyl)quinazolin-4(3H)-one. The title
compound (164 mg, 0.42 mmol, 69.4% yield, yellow oil) was prepared analogous to compound 19, Step-3 using 8-bromo-3-
((1-methyl-1H-imidazol-4-yl)methyl)quinazoline-2,4(1H,3H)-dione (203 mg, 0.61 mmol), (benzotriazol-1-
yloxy)tris(dimethylamino)phosphonium hexafluorophosphate (BOP, 402 mg, 0.91 mmol), tert-butylamine (0.32 mL, 3.03
mmol) and DMF (2 mL): m/z (ESI, +ve) 389.9/391.9 (1:1) (M+H)+.
Step-4:
(2-(tert-Butylamino)-8-((R)-6-methyl-4-oxo-1,4,5,6-tetrahydropyrrolo[3,4-b]pyrrol-2-yl)-3-(((R)-
tetrahydrofuran-2-yl)methyl)quinazolin-4(3H)-one. The title compound (111 mg, 0.25 mmol, 59.3 % yield, white solid)
was prepared analogous to compound 10, Step-5 using (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 (9d, 132 mg, 0.50 mmol), (R)-8-bromo-2-(tert-butylamino)-3-((tetrahydrofuran-2-
yl)methyl)quinazolin-4(3H)-one (164 mg, 0.42 mmol), potassium phosphate (268 mg, 1.26 mmol) and XPhos-Pd-G2 (16.5
1
mg, 0.02 mmol) and 1,4-dioxane (3 mL)/water (1 mL): H NMR (400 MHz, DMSO-d
6
)  ppm 11.94 (br. s., 1H), 7.79 - 7.93 (m,
3
H), 7.57 - 7.70 (m, 2H), 7.22 (s, 1H), 7.17 (t, J = 7.7 Hz, 1H), 6.72 (s, 1H), 5.08 (s, 2H), 4.53 (q, J = 6.1 Hz, 1H), 3.64 (s, 3H), 1.52
(
s, 9H), 1.36 (d, J = 6.5 Hz, 3H). LC−MS t
R
= 1.44 min (>95%); m/z (ESI, +ve) 446.0 (M+H)+.
2-(tert-Butylamino)-3-(1-(1-methyl-1H-imidazol-4-yl)ethyl)-8-((R)-6-methyl-4-oxo-1,4,5,6-
tetrahydropyrrolo[3,4-b]pyrrol-2-yl)quinazolin-4(3H)-one (24). (Method B) Step-1: 2-amino-3-bromo-N-(1-(1-
methyl-1H-imidazol-4-yl)ethyl)benzamide. The title compound (511 mg, 1.58 mmol, 56.2% yield, orange solid) was
prepared analogous to compound 10, Step-1 using 2-amino-3-bromobenzoic acid (608 mg, 2.81 mmol) and 1-(1-methyl-1H-
imidazol-4-yl)ethanamine (352 mg, 2.81 mmol), DIPEA (1.5 mL, 8.44 mmol), T3P (50 wt% in EtOAc, 2.7 mL, 4.22 mmol) and
EtOAc (5 mL): m/z (ESI, +ve) 322.9/324.9 (1:1) (M+H)+.
Step-2: 8-Bromo-3-(1-(1-methyl-1H-imidazol-4-yl)ethyl) quinazoline-2,4(1H,3H)-dione. The title compound (561
mg, 1.58 mmol, 100 % yield, white solid) was prepared analogous to compound 10, Step-2 using 2-amino-3-bromo-N-((1-
methyl-1H-imidazol-4-yl)ethyl)benzamide (511 mg, 1.58 mmol) and triphosgene (164 mg, 0.55 mmol) and dichloromethane
1
(
(
16 mL): H NMR (400 MHz, DMSO-d
6
)  ppm 10.71 (br. s., 1H), 9.01 (s, 1H), 7.97 (dd, J = 18.6, 7.8 Hz, 2H), 7.70 (s, 1H), 7.18
t, J = 7.8 Hz, 1H), 6.15 (q, J = 6.5 Hz, 1H), 3.84 (s, 3H), 1.76 (d, J = 6.8 Hz, 3H). m/z (ESI, +ve) 348.8/350.8 (1:1) (M+H)+.
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