Discovery and SAR of Novel 2,3-Dihydroimidazo[1,2-c]quinazoline PI3K Inhibitors: Identification of Copanlisib (BAY 80-6946)

Article abstract of DOI:10.1002/cmdc.201600148

The phosphoinositide 3-kinase (PI3K) pathway is aberrantly activated in many disease states, including tumor cells, either by growth factor receptor tyrosine kinases or by the genetic mutation and amplification of key pathway components. A variety of PI3K isoforms play differential roles in cancers. As such, the development of PI3K inhibitors from novel compound classes should lead to differential pharmacological and pharmacokinetic profiles and allow exploration in various indications, combinations, and dosing regimens. A screening effort aimed at the identification of PI3Kγ inhibitors for the treatment of inflammatory diseases led to the discovery of the novel 2,3-dihydroimidazo[1,2-c]quinazoline class of PI3K inhibitors. A subsequent lead optimization program targeting cancer therapy focused on inhibition of PI3Kα and PI3Kβ. Herein, initial structure–activity relationship findings for this class and the optimization that led to the identification of copanlisib (BAY 80-6946) as a clinical candidate for the treatment of solid and hematological tumors are described.

Full text of DOI:10.1002/cmdc.201600148

DOI: 10.1002/cmdc.201600148
Full Papers
Discovery and SAR of Novel 2,3-Dihydroimidazo[1,2-c]-
quinazoline PI3K Inhibitors: Identification of Copanlisib
(BAY 80-6946)
William J. Scott,*[a] Martin F. Hentemann,[c] R. Bruce Rowley,[c] Cathy O. Bull,[c] Susan Jenkins,[c]
Ann M. Bullion,[c] Jeffrey Johnson,[c] Anikꢀ Redman,[c] Arthur H. Robbins,[c] William Esler,[c]
R. Paul Fracasso,[c] Timothy Garrison,[c] Mark Hamilton,[c] Martin Michels,[d] Jill E. Wood,[c]
Dean P. Wilkie,[c] Hong Xiao,[c] Joan Levy,[c] Enrico Stasik,[e] Ningshu Liu,[e] Martina Schaefer,[f]
Michael Brands,[b] and Julien Lefranc*[b]
Dedicated to the memory of Bruce Rowley, a friend and colleague.
The phosphoinositide 3-kinase (PI3K) pathway is aberrantly ac-
tivated in many disease states, including tumor cells, either by
growth factor receptor tyrosine kinases or by the genetic mu-
tation and amplification of key pathway components. A variety
of PI3K isoforms play differential roles in cancers. As such, the
development of PI3K inhibitors from novel compound classes
should lead to differential pharmacological and pharmacoki-
netic profiles and allow exploration in various indications, com-
binations, and dosing regimens. A screening effort aimed at
the identification of PI3Kg inhibitors for the treatment of in-
flammatory diseases led to the discovery of the novel 2,3-dihy-
droimidazo[1,2-c]quinazoline class of PI3K inhibitors. A subse-
quent lead optimization program targeting cancer therapy fo-
cused on inhibition of PI3Ka and PI3Kb. Herein, initial struc-
ture–activity relationship findings for this class and the optimi-
zation that led to the identification of copanlisib (BAY 80-6946)
as a clinical candidate for the treatment of solid and hemato-
logical tumors are described.
Introduction
The phosphoinositide 3-kinase (PI3K) family of lipid kinases
generates 3’-phosphoinositides that bind to and activate a vari-
ety of cellular targets, initiating a wide range of signal trans-
duction cascades.[1–3] These cascades ultimately induce changes
in a range of cellular processes including proliferation, survival,
differentiation, migration, vesicle trafficking, and chemotaxis.
Class I PI3Ks transmit signals downstream of transmembrane
receptors, signaling from receptor tyrosine kinases via p110a,
p110b and p110d isoforms, and from G-protein-coupled recep-
tors via p110b and p110g isoforms. Class I PI3Ks are divided
into two distinct subclasses based on differences in protein
subunit composition. The class IA PI3Ks are comprised of a cata-
lytic p110 subunit (p110a, p110b or p110d) heterodimerized
with a member of the p85 regulatory subunit family. The clas-
s IA PI3K isoforms associate with activated receptor tyrosine
kinases (RTKs) (including PDGFR, EGFR, VEGFR, IGF1-R, c-KIT,
CSF-R and Met), or with tyrosine phosphorylated adapter pro-
teins, via their p85 regulatory subunits, resulting in stimulation
of the lipid kinase activity.
[a] Dr. W. J. Scott
Global Development, Global Program Management, Bayer HealthCare
Pharmaceuticals Inc., Whippany, NJ 07981 (USA)
E-mail: william.scott@bayer.com
[b] Prof. Dr. M. Brands, Dr. J. Lefranc
Global Drug Discovery, Medicinal Chemistry Berlin,
Bayer Pharma AG, 13353 Berlin (Germany)
E-mail: julien.lefranc@bayer.com
In contrast, the class IB PI3Ks are comprised of a catalytic
p110 subunit (p110g) heterodimerized with a distinct p101 reg-
ulatory subunit.[4,5] Activation of class I PI3K-mediated signaling
has been directly linked to cancer. Somatic mutations or am-
plification of the gene encoding human p110a (PIK3CA) is
known to be causative in many cancers.[5–8] Overexpression or
mutational activation of RTKs leading to increased PI3K signal-
ing occurs in multiple tumor types.[5,9] Loss of function or inac-
tivation of the tumor suppressor PTEN (phosphatase and
tensin homologue) also leads to increased PI3K signaling and
tumor growth.[10–12] This appears to be mediated via the p110b
isoform.[13–15] In addition, aberrant activation of class I PI3Ks has
been associated with both intrinsic and acquired resistance to
targeted therapeutic agents, such as lapatinib or vemurafe-
[c] Dr. M. F. Hentemann, Dr. R. B. Rowley, C. O. Bull, Dr. S. Jenkins, A. M. Bullion,
J. Johnson, A. Redman, Dr. A. H. Robbins, W. Esler, Dr. R. P. Fracasso,
T. Garrison, Dr. M. Hamilton, Dr. J. E. Wood, D. P. Wilkie, H. Xiao, Dr. J. Levy
Former Bayer Research Center, West Haven, CT 16516 (USA)
[d] Dr. M. Michels
Global Drug Discovery, Project Management Drug Discovery,
Bayer Pharma AG, 13353 Berlin (Germany)
[e] E. Stasik, Dr. N. Liu
Global Drug Discovery, TRG Oncology,
Bayer Pharma AG, 13353 Berlin (Germany)
[f] Dr. M. Schaefer
Global Drug Discovery, Structural Biology,
Bayer Pharma AG, 13353 Berlin (Germany)
Supporting information for this article can be found under http://
dx.doi.org/10.1002/cmdc.201600148.
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nib,[16–18] as well as resistance to traditional cytotoxic chemo-
therapy and radiation therapy.[19–21]
teraction with the protein hinge region is obligatory, and activ-
ity can be enhanced via affinity pocket interactions.[30–32] This
led to the development of two major classes of PI3K inhibitors
(Figure 1). There is a family of morpholine-based inhibitors,
typified by pictilisib, buparlisib and apitolisib, having hinge
binding with the relatively weak hydrogen-bond-accepting
morpholine oxygen, and having strong affinity pocket interac-
tions, typically involving both a hydrogen-bond acceptor and
a hydrogen-bond donor.[33] In contrast, PI3Kd-selective inhibi-
tors, such as idelalisib, PIK-39 and AMG-319, possess strong
hinge interactions via a purine moiety combined with induc-
tion of a selectivity pocket.[34–36] A novel approach might in-
volve discovery of a compound class that allows both strong
binding at the hinge and strong affinity pocket interactions.
Herein, we describe the optimization of the 2,3-dihydroimida-
zo[1,2-c]quinazoline class of PI3K inhibitors which strongly
bind at both critical sites, leading to the discovery of copanli-
sib.
A number of PI3K/mTOR, pan-PI3K and selective PI3K inhibi-
tors have entered early clinical development.[3,22–25] Idelalisib
(Figure 1), a PI3Kd-selective inhibitor, has recently been ap-
proved for use in the treatment of two subtypes of indolent
non-Hodgkin’s lymphoma.[26] In contrast, the clinical benefit of
PI3K inhibition in solid tumors has yet to be clearly demon-
strated. This is largely due to the following factors: 1) feedback
activation of physiologic signaling in tumors,[27–29] 2) selectivity
to PI3K and disease relevant PI3K isoforms, 3) both on-target
and off-target toxicity. These factors limited pathway inhibition
and efficacy. Accordingly, there remains a need for the devel-
opment of novel PI3K inhibitors with differing pharmacologic
and pharmacokinetic profiles.
Guided by X-ray crystallographic structures of first-genera-
tion PI3K inhibitors bound to PI3Kg, it has been shown that in-
Results and Discussion
Structure–activity relationships of 2,3-dihydroimidazo[1,2-
c]quinazoline PI3K inhibitors
Initial interest in PI3K inhibitors at Bayer centered around the
development of PI3Kg inhibitors as anti-inflammatory agents
with potential as anti-asthmatic agents.[37] High-throughput
screening for PI3Kg-active leads led to the discovery of the
novel lead 2,3-dihydroimidazo[1,2-c]quinazoline 1 (p110g IC50 =
810 nm, p110b IC50 =4000 nm) (Figure 2). During lead optimiza-
tion efforts, it was established that the enol moiety of structure
1 could be replaced with an amide moiety to minimize poten-
tial for untoward reactivity. In addition, the phenyl substituent
was replaced with a 3-pyridyl moiety, leading to analogue 2. In
addition, it was found that A-ring substitution, such as in com-
pound 3, could affect isoform activity ratios. With this informa-
tion in hand, a program to optimize the p110b and p110a ac-
tivity of the 2,3-dihydroimidazo[1,2-c]quinazoline lead for po-
tential use in cancer therapies was undertaken.
Docking of 2,3-dihydroimidazo[1,2-c]quinazolines with the
published structure for p110g,[31] and making the assumption
that the inhibitors are bound to the ATP binding site, led to
the hypothesis that the dihydroimidazole N1 nitrogen binds at
the protein hinge, while the C-ring sp3 carbons fill a nearby hy-
drophobic pocket. Modeling also suggested that the C5 heter-
Figure 1. PI3K inhibitors.
Figure 2. Optimization of the initial lead.
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ocyclic amide moiety occupies a pocket not used by ATP, and
offers potential hydrogen-bonding opportunities. In addition,
C7 was predicted to point toward the ATP sugar pocket and
C8 toward a channel leading to solvent. Thus, initial synthetic
efforts were directed toward confirmation of the binding hy-
pothesis, as well as toward inhibitor optimization. The binding
model was substantiated after the conclusion of the optimiza-
tion program, via an X-ray structure of copanlisib (39i) bound
to p110g (see below).
Synthetic strategy
C-ring SAR was explored during early studies directed toward
development of PI3Kg inhibitors by variation of the diamine
used for imidazoline synthesis.[37] Unsubstituted 2,3-dihydroimi-
dazo[1,2-c]quinazoline, 2, was synthesized starting from 2-cya-
noaniline (4) by treatment with ethylenediamine in the pres-
ence of phosphorus pentasulfide, which afforded aniline 5
(Scheme 1). Cyclization with cyanogen bromide led to tricyclic
amine 7, which was acylated to afford amide 2. The corre-
sponding ring-expanded 3,4-dihydro-2H-pyrimido[1,2-c]quina-
zoline 9 was synthesized in an analogous manner using 1,3-
propylenediamine.
Scheme 2. Synthesis of C-ring analogues 17 and 18.[37] Reagents and condi-
tions: a) H2NCHR1CHR2OH, TEA, THF, quant.; b) POCl3 (crude product used in
the next step); c) aq. NH3, 1508C, 12% (15), 17% (16); d) nicotinic acid,
PyBOP, Hꢂnig’s base, DMF, 808C, 28% (17), 35% (18).
Scheme 3. Synthesis of C-ring analogue 21.[37] Reagents and conditions:
a) MnO2, DMPU, 1508C, 61%; b) BrCN, MeOH, 61%; c) nicotinic acid, PyBOP,
Hꢂnig’s base, DMF, 808C, 20%.
Scheme 1. Synthesis of C-ring analogues 2 and 9.[37] Reagents and conditions:
a) H2N(CH2)n+1NH2, P2S5, 0 to 1008C; b) BrCN, MeOH; c) nicotinic acid, PyBOP,
Hꢂnig’s base, DMF.
afford known aldehyde 23.[38,39] Conversion into benzyl ether
25 followed by ammonium hydroxide/iodine oxidation gave
nitrile 26. Reduction provided aniline 27, and treatment with
ethylenediamine allowed formation of the C-ring. Imidazoline
28 was then cyclized using cyanogen bromide to give tricyclic
amine 29, which was acylated using classical peptide-coupling
conditions to provide amide 30. Treatment of amide 30 with
trifluoroacetic acid afforded deprotected phenol 31, which
could then be alkylated at C8 giving access to the aryl ethers,
32.
To examine the effect of substitution on the C-ring, dichloro-
quinazoline 10 was treated with the appropriate chiral amino-
propanol, and the resulting alcohols 11 and 12 were cyclized
to afford 5-chloro-2,3-dihydroimidazoquinazolines 13 and 14
(Scheme 2). The quinazolines were treated with ammonia, fol-
lowed by nicotinic acid to afford amides 17 and 18.
The effect of the C-ring oxidation state on efficacy was ex-
amined using imidazo[1,2-c]quinazoline 21. Thus, oxidation
(MnO2) of aminodihydroimidazole 5 to aminoimidazole 19 was
followed by cyclization with cyanogen bromide to give 20 and
then by acylation with nicotinic acid to afford amide 21
(Scheme 3).
The C7 methyl ethers of 8-alkoxy-7-methoxy analogues
proved labile to sulfide-based hydrolysis, likely due to the
more hindered methoxy being forced outside of the dihydroi-
midazoquinazoline plane.[40,41] Alkylation of the phenol 33 re-
sulting from hydrolysis of analogue 32g [R8 =3-(morpholin-4-
yl)propyl] led to the desired C7 aryl ethers 34. Alternatively, de-
benzylation of quinazoline 29 at C8 could be achieved using
trifluoroacetic acid. The resulting phenol, 35, was readily con-
verted into amine 36, which upon acylation to 37 could be
used to investigate the amide SAR.
To study variation at the C5, C7, and C8 positions, a more
general approach involving preparation of key intermediate 29
was envisioned (Scheme 4). The synthesis began with treat-
ment of vanillin acetate (22) with fuming nitric acid to readily
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mine, 32e, showed potency against p110a similar to
that of methoxy analogue 32a; however, they were
slightly more potent against p110b. Expanding the
bulk around the amine, such as with piperidine 32 f
or morpholine 32g, slightly improved p110a potency
without modifying the activity against p110b. The
length of the linker between the morpholine moiety
and the core was also investigated. Thus, compound
32h, bearing a two-carbon linker, showed improved
activity however it appeared to be chemically unsta-
ble, while analogue 32i, bearing a four-carbon linker,
showed a drop in potency. In summary, variation of
the alkoxy moiety at C8 had little effect on p110a po-
tency. In contrast, activity against p110b proved
somewhat more sensitive.
Inclusion of an amine base tethered to the core via
an alkyl ether appeared optimal. Compounds with
strong nitrogen bases at C8, such as amine 32d, di-
methylamine 32e or piperidine 32 f, led to high
clearance in PK studies (data not shown), and were
not further pursued.
Scheme 4. General synthetic approach to analogues for SAR studies. Reagents and condi-
tions: a) fuming HNO3, <108C, 41%; b) K2CO3, MeOH, 88%; c) BnBr, K2CO3, DMF, 97%;
d) NH4OH (28%), I2, THF, 95%; e) Fe, AcOH, H2O, 58C to RT, 88%; f) H2N(CH2)2NH2, S8,
1008C, 86%; g) BrCN, TEA, CH2Cl2, 08C to RT, quant.; h) nicotinic acid, PyBOP, Hꢂnig’s
base, DMF, 98%; i) TFA, 608C, 66%; j) R8X, base, DMF; k) Na2S, NMP, 1608C, 51% [R8 =3-
(morpholin-4-yl)propyl, 32g]; l) R7X, base, DMF; m) TFA, 608C, crude starting material
was used; n) 3-(morpholin-4-yl)propyl chloride, Cs2CO3, DMF, 708C, 44%; o) R5CO2H,
PyBOP, Hꢂnig’s base, DMF.
A-ring C7 SAR
The binding hypothesis suggested that modifications
at C7 might allow probing of the ATP sugar pocket.
Variation of the C7 methoxy group of morpholine de-
rivative 32g was initiated by replacement with a pro-
poxy moiety (34a). An increase in potency against
C-ring SAR
p110b was observed in the biochemical assay (Table 3); howev-
Initially, C-ring SAR was probed (Table 1). The binding hypothe-
sis suggested that the pocket filled by ring C left little room for
modification. Consistent with this, expansion to the six-mem-
bered C-ring led to the completely inactive tetrahydropyrimi-
dine, 9. Similarly, activity was lost when a methyl group was in-
troduced at either C2 or C3 (compounds 17 and 18). Oxidation
of the five-membered ring to the corresponding imidazole 21
resulted in decreased activity, by a factor of five relative to di-
hydroimidazole 2. Based on these initial results, there were no
further attempts to optimize SAR at the C-ring. The inability to
accommodate C-ring substitution was later validated with an
X-ray structure of copanlisib (39i) bound to p110g (see below).
er, this effect did not translate to the cellular assays (data not
Table 1. C-ring SAR.
Compd
C-ring analogue
IC50 [nm][a]
p110a
p110b
2
9
n.d.
192
A-ring C8 SAR
The binding hypothesis suggested that substituents at C8
would fit into a channel pointing toward solvent. To probe
this, modifications at the C8 position were investigated
(Table 2). Benzyl ether 30 and phenol 31 were highly potent
against p110a; however, they showed decreased activity
against p110b relative to methoxy analogue 32a. Exchanging
the methyl ether for the ethyl ether (32b) had little influence
on the potency but the bulkier isobutyl ether 32c was seven-
fold less potent against p110b.
n.d.
n.d.
>10000
>10000
17
18
21
>1000
>10000
n.d.
1040
Consistent with the binding model, addition of a polar
moiety attached via an alkoxy linker was tolerated. Thus, ami-
nopropoxy analogue 32d and the corresponding dimethyla-
[a] n.d.=not determined.
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shown). Neither allyl ether 34b nor isobutyl ether 34c offered
any benefit over methyl ether 32g. In contrast, introduction of
a cyclohexyl group (34d) resulted in a significant decrease in
activity against p110b. This could be recovered by addition of
distal polarity, as in tetrahydropyran 34e or pyridine 34 f. Be-
cause no significant improvement was achieved by modifying
the substituents at C7, the initial methoxy moiety was chosen
for additional studies in order to minimize molecular weight.
Table 2. A-ring C8 SAR.
Compd
R8ꢀOꢀ
IC50 [nm][a]
p110a
p110b
B-ring C5 amide SAR
30
6.1
1510
The strongest interactions predicted by the binding hypothesis
involve the dihydroimidazole binding at the hinge, and interac-
tions within the C5 heterocyclic amide pocket. Thus, optimiza-
tion at C5 offered a high potential for improvements in activity,
and a series of amide modification was undertaken multiple
times during the program.
31
8.7
4.5
3.6
210
30.7
76.1
32a
32b
32c
8.0
226
32d
32e
4.7
3.6
22.3
18.0
Amides of A-ring-unsubstituted 5-amino-2,3-dihydroimida-
zo[1,2-c]quinazoline
32 f
2.9
1.8
18.3
20.8
32g
An initial understanding of C5 amide SAR was developed using
the A-ring-unsubstituted 2,3-dihydroimidazo[1,2-c]quinazoline
system (Table 4).[31] Alkyl amides, such as acetamide 38a and
cyclohexyl analogue 38b, were significantly less active than
pyridine 2. The same was observed for aryl amides, for exam-
ple phenyl analogue 38c and thiazole 38d. This decreased ac-
tivity may be due to the inability of these amides to act as hy-
drogen-bond acceptors. Inclusion of a hydrogen-bond accept-
or in the 3’-position, such as in thiazole 38e, led to an activity
equivalent to that of pyridine 2, even in the presence of steric
hindrance. Further addition of a hydrogen-bond donor, for ex-
ample as in aminopyridine 38 f or imidazopyridine 38g, result-
ed in a significant improvement in activity.
32h
0.9
11.2
88.1
32i
n.d.
[a] n.d.=not determined.
Table 3. A-ring C7 SAR.
Amides of 5-amino-7-methoxy-8-[3-(morpholin-4-yl)pro-
poxy]-2,3-dihydroimidazo[1,2-c]quinazoline
C5 Amide SAR for compounds substituted at C8 with an ami-
noalkoxy group revealed similar trends to those observed for
the C8-unsubstituted analogues (Table 5 vs. Table 4). Thus, the
absence of a hydrogen-bond donor, such as in compound
39a, resulted in decreased activity against p110b, while thia-
zole 39b (a direct analogue of 38e) proved slightly more
potent than 32g (Table 5). Moving the pyridine nitrogen from
position 3’ to 4’ (32g vs. 39c) led to reduced activity against
p110b by a factor of three. Addition of a hydrogen-bond
donor, such as an amino group on the pyridine ring as in ana-
logues 39d and 39e, resulted in a slight increase in potency.
However, dimethylation of the aminopyridine (39 f) led to a de-
crease in activity. Introduction of a second nitrogen in the ring,
such as in pyrazine 39g and pyrimidine 39h, led to a signifi-
cant drop in activity. Surprisingly, the activity could be recov-
ered by introduction of a free amino group in the 4’-position
of the pyrimidine (39i and 39j); in the case of 39j, an addi-
tional methyl substituent was tolerated. Thus, activity was opti-
Compd
R7ꢀOꢀ
IC50 [nm][a]
p110a
p110b
32g
33
1.8
20.8
55.6
n.d.
34a
34b
n.d.
n.d.
7.8
22.6
34c
34d
34e
10.2
n.d.
2.1
29.1
88.5
18.2
19.3
34 f
n.d.
[a] n.d.=not determined.
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Table 4. SAR of amides of A-ring-unsubstituted 5-amino-2,3-dihydroimi-
dazo[1,2-c]quinazoline.
Table 5. SAR of amides of 5-amino-7-methoxy-8-[3-(morpholin-4-yl)pro-
poxy]-2,3-dihydroimidazo[1,2-c]quinazoline.
Compd
ꢀNHꢀ(C=O)ꢀR5
IC50 [nm][a]
Compd
32g
ꢀNHꢀ(C=O)ꢀR5
IC50 [nm][a]
p110a
p110b
p110a
p110b
2
n.d.
192
1.8
20.9
1.6
20.8
38a
38b
n.d.
n.d.
1650
39a
668
7.8
>10000
39b
38c
38d
n.d.
n.d.
71260
532
39c
n.d.
63.7
39d
39e
6.2
0.6
16.6
7.9
38e
38 f
n.d.
176
12.0
49.3
39 f
39g
39h
2.9
n.d.
132
0.5
58.7
351
286
3.7
38g
n.d.
31.1
[a] n.d.=not determined.
mized by the use of both a hydrogen-bond acceptor and a hy-
drogen-bond donor.
39i
(copanlisib,
BAY 80-6946)
Cellular screening
Selected compounds were tested in mechanistic and function-
al cellular assays, allowing differentiation of suitable candidates
(Table 6). Cellular mechanistic activity of compounds was
probed by investigating inhibition of AKT phosphorylation by
PI3K.[42] KPL4, a breast cancer cell line that carries a PIK3CA-acti-
vating mutation, was selected for profiling of anti-proliferative
effects.[42]
39j
0.4
1.0
[a] n.d.=not determined.
Relative activity in cellular assays generally paralleled that
observed in the biochemical assays. Thus, C7-extended ana-
logue 34d proved approximately equivalent to methyl ether
32a, while 4’-aminopyridine 39e was significantly more active.
Surprisingly, 3’-aminopyridine 39d displayed significantly less
activity than expected in the mechanistic assay. Consistent
with the biochemical data, aminopyrimidine 39i and 2’-amino-
4’-methylpyrimidine 39j proved optimal in cellular assays.
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Table 6. Mechanistic and functional cellular activity of selected compounds.
32a[a]
34d[a]
39b
39d[a]
39e[a]
39i
39j
p110a IC50 [nm]
p110b IC50 [nm]
IGF-1-stimulated A549 pAKT(S473) IC50 [nm]
KPL4 proliferation EC50 [nm]
4.5
30.7
30
n.d.
88.5
55
1.6
7.8
60
6.2
16.6
170
n.d.
0.6
7.9
11
0.5
3.7
3.4
3.7
0.4
1.0
0.6
2.3
n.d.
n.d.
86
n.d.
[a] n.d.=not determined.
SAR summary
In summary (see Figure 3), modification of the imidazoline C-
ring, either by substitution, oxidation or ring expansion, result-
ed in a loss of activity, supporting the hypothesis that the N1
nitrogen is involved in hinge binding. The aromatic C5 amide
proved to be the key driver of potency. The amide should pref-
erably have a five- or six-membered monocyclic, or bicyclic, ni-
trogen-containing heterocycle. Inclusion of a hydrogen-bond
donor significantly increased potency, but at the expense of
decreased water solubility and oral bioavailability. The optimal
amide contained an aminopyrimidine moiety.
Figure 4. Influence of A-ring C7/C8/C9 substitution.
Binding mode
After the conclusion of the optimization program, an X-ray
structure of copanlisib (39i) bound to p110g was determined.
The structure is in general agreement with that previously re-
ported for dimethoxy analogue 32a bound to p110g.[32] The
imidazoline N1 nitrogen forms a critical hydrogen bond to
Val882 in the adenine pocket (Figure 5). The majority of known
PI3Ka inhibitors use a morpholine oxygen for this interac-
tion.[32,33]
The C5 aminopyrimidine group fills the affinity pocket, form-
ing hydrogen bonds with Asp836 and Asp841 through the
amino group, and with Lys833 through a pyrimidine nitrogen.
In addition, Asp964 appears to contribute a pi-stacking interac-
tion. Finally, the morpholine group of copanlisib lies over
Trp812, apparently adding a hydrophilic shield to the hydro-
phobic heterocycle. The binding hypothesis, as validated by
the X-ray structure, provides a structural rationale for and vali-
dation of the conclusions generated from the SAR studies.
Figure 3. SAR summary.
Substitution at C7 affords SAR consistent with occupation of
a pocket amenable to polar interactions. Unfortunately, while
tolerated, substitution did not lead to increased potency or
beneficial properties. Thus, the C7 methoxy moiety was consid-
ered optimal. The SAR indicated that inclusion of polar groups
at C8 allowed the tuning of water solubility and PK properties.
Aminoalkoxy substituents were optimal, and balancing of ac-
tivity with PK properties led to the selection of a 3-morpholi-
nylpropoxy moiety at C8 (Figure 3). Thus, overall optimization
led to the selection of analogue 39i, copanlisib (BAY 80-6946),
for further development. In addition, relative to 7,8-dimethoxy
compound 32a, 8,9-dimethoxy analogue 3 was significantly
less active (Figure 4). Removal of either the C7 methoxy moiety
(analogue 40) or the C8 methoxy moiety (analogue 41) from
32a led to decreased activity. This led to the conclusion that
substitution at both C7 and C8 was optimal.
Pharmacology
The pharmacology of copanlisib (39i) has been described in
detail elsewhere;[42] the results are summarized in Table 7. Co-
panlisib is a potent class I PI3K inhibitor with preferential activi-
ty against p110a and p110d as compared with p110b and
p110g. Copanlisib has potent cellular mechanistic activity, in-
hibiting both IGF-1-stimulated AKT phosphorylation in A549
cells, and basal AKT phosphorylation in KPL4 cells. This trans-
lated to the potent inhibition of proliferation in many cell
lines,[42] as exemplified by KPL4.
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rats, 27.6% in male CD1 mice, 42.4% in female
Beagle dogs and 15.8% in male humans). Further-
more, copanlisib has low permeability in the Caco2
assay, with a Papp value for the apical (A) to basal (B)
direction of 56.9ꢁ2.91 nmsꢀ1 at 10 mm in the ab-
sence of a P-glycoprotein (P-gp) inhibitor. Copanlisib
was characterized as a substrate of the efflux trans-
porters P-gp and BCRP. In in vitro studies with differ-
ent cell lines, P-gp (IC50: 7 mm for digoxin and 7.6 mm
for dipyridamole) and BCRP (IC50: 11.5 mm for topote-
can) mediated transport was inhibited.
The pharmacokinetic profile of copanlisib was eval-
uated following administration of single or multiple
intravenous doses to nude rats. The volume of distri-
bution (Vss) was very high in all species investigated
including mouse, rat, dog and monkey (Vss =7–
32 Lkgꢀ1). In rats and dogs, the whole-blood clear-
ance (CL) was 2.8 and 0.74 L(hkg)ꢀ1, respectively, and
thus was 66 and 35%, respectively, relative to hepatic
blood flow. Pharmacokinetics was not dependent on
sex and no relevant drug accumulation was observed
after repeated administration (every second day ꢃ5),
suggesting that there is no change in metabolic pro-
cesses. Thus, the pharmacokinetic profile of copanli-
sib was supportive of initiating clinical studies.
Figure 5. X-ray crystal structure of copanlisib bound to PI3Kg.
Conclusions
Table 7. Pharmacology profile of copanlisib (39i).
The discovery of the novel 2,3-dihydroimidazo[1,2-c]quinazo-
line scaffold has led to the development of potent and selec-
tive PI3K inhibitors which strongly bind at both at the hinge
and the affinity pocket. During the course of optimization,
compound 39i (copanlisib, BAY 80-6946)[42] was identified as
having met program criteria, and as having excellent efficacy
in in vitro and in vivo studies.[43] Copanlisib was selected for
clinical development, and is currently in phase III trials for the
treatment of non-Hodgkin lymphoma (NHL), both as mono-
therapy (CHRONOS-2: NCT02369016) and in combination with
rituximab (CHRONOS-3: NCT02367040) or rituximab-based che-
motherapy (CHRONOS-4: NCT02626455).
Profile
Value
Biochemical profile
p110a IC50 [nm]
p110b IC50 [nm]
p110g IC50 [nm]
p110d IC50 [nm]
0.5
3.7
6.4
0.7
45
mTOR kinase IC50 [nm]
Panel of 220 kinases (10 mm)
<50% inhibition
Cellular mechanistic profile (pAKT-S473)
PI3Ka (KPL4) IC50 [nm]
PI3Kb (LPA-stimulated PC3) IC50 [nm]
PI3Kg (C5a-stimulated Raw 264.7) IC50 [nm]
PI3Kd (IgM-stimulated Raji) IC50 [nm]
mTOR (p-4E-BP1-S65 in ELT3) IC50 [nm]
0.4
10
94
7.4
>500
In vitro antiproliferative activity IC50 [nm]
KPL4 (PIK3CAmut) IC50 [nm]
Experimental Section
3.7
24
2.3
ZR-75-1 (PTENloss) IC50 [nm]
Biochemical IC50 assays:[42] p110a, p100b and p110g activities
were measured by the inhibition of 33P incorporation into phos-
phatidylinositol (PI) in 384-well MaxiSorpꢄ plates coated with PI
(2 mg per well) and phosphatidylserine (PS) (1:1 molar ratio). Each
PI3K isoform assay contained: reaction buffer (9 mL of MOPSO
(50 mm, pH 7.0), NaCl (100 mm), MgCl2 (4 mm), BSA (0.1%)) con-
taining His-tagged N-terminal truncated (DN 1–108) p110a or
p110b protein (7.5 ng), or purified human p110g protein (25 ng)
(Alexis Biochemicals). The reaction was started by adding ATP solu-
tion containing 20 mCimLꢀ1 [33P]ATP (5 mL, 40 mm). After incubation
for 2 h at RT, the reaction was terminated by addition of EDTA solu-
tion (5 mL, 25 mm). The plates were washed and Ultima Goldꢅ
scintillation cocktail (25 mL) was then added. The radioactivity in-
corporated into the immobilized PI substrate was determined with
TMD-8 (CD79mut) IC50 [nm]
In vivo activity [% TGI][a]
KPL4 (14 mgkgꢀ1, Q2D)
TMD-8 (14 mgkgꢀ1, Q2D)
>100%[b]
75%
[a] In mice; TGI=tumor growth inhibition. [b] Tumor regression.
Preclinical pharmacokinetics
The preclinical pharmacokinetics of copanlisib (39i) has been
presented in detail elsewhere.[42] Copanlisib has a high plasma-
free fraction across all species tested (42.5% in male Wistar
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a BetaPlate Liquid Scintillation Counter. Each IC50 value reported is
the mean of a minimum of two experiments.
4-(Benzyloxy)-3-methoxy-2-nitrobenzonitrile (26): Iodine (272 g,
1.1 mol) was added to a mixture of aldehyde 25 (220 g, 766 mmol)
and ammonium hydroxide (28% solution, 3 L) dissolved in THF
(5 L). After 16 h the reaction mixture was treated with sodium sul-
fite (49 g, 383 mmol), then concentrated under reduced pressure
to afford a thick slurry. The slurry was filtered, washed with water
(250 mL) and air-dried to give nitrile 26 (206 g, 95%): 1H NMR
([D6]DMSO): d=3.91 (s, 3H), 5.35 (s, 2H), 7.40 (m, 3H), 7.49 (m,
2H), 7.59 (d, 1H), 7.89 ppm (d, 1H).
General procedures: Air- and moisture-sensitive liquids and solu-
tions were transferred via syringe or cannula, and introduced into
reaction vessels through rubber septa. Commercial grade reagents
and solvents were used without further purification. The term ‘con-
centrated under reduced pressure’ refers to use of a Bꢂchi rotary
evaporator at approximately 15 mmHg. Thin-layer chromatography
was performed on precoated glass-backed silica gel 60A F254
250 mm plates.
2-Amino-4-(benzyloxy)-3-methoxybenzonitrile (27): A degassed
solution of nitrobenzonitrile 26 (185 g, 651 mmol) in glacial acetic
acid (3500 mL) and water (10 mL) was cooled to 58C and treated
with iron powder (182 g, 3.25 mol). After 3 d at RT the reaction
mixture was filtered through Celiteꢄ, and the filtrate was concen-
trated under reduced pressure. The oil, thus obtained, was treated
with saturated aqueous NaCl solution, neutralized with sodium bi-
carbonate solution and extracted with CH2Cl2. The resulting emul-
sion was filtered through Celiteꢄ, after which the organic layer was
separated, washed with saturated aqueous NaCl solution, dried
(Na2SO4) and concentrated under reduced pressure to give 27
NMR: Routine 1D-NMR spectroscopy was performed on either
a 300 or 400 MHz Varian Mercury Plus spectrometer, except where
otherwise indicated. Samples were dissolved in deuterated sol-
vents. Chemical shifts, recorded on the ppm scale, are referenced
to the appropriate solvent signals (e.g., for 1H NMR spectra:
[D6]DMSO, 2.49 ppm; CDCl3, 7.26 ppm).
GC–MS: Electron-impact mass spectra (EI-MS) were obtained using
a Hewlett Packard 5973 mass spectrometer equipped with a Hew-
lett Packard 6890 gas chromatograph with a J&W HP-5 column
(30 mꢃ0.32 mm, 0.25 mm). The ion source was maintained at
2508C and spectra were scanned from 50–550 amu at 0.34 s per
scan.
1
(145 g, 88%): H NMR ([D6]DMSO): d=3.68 (s, 3H), 5.15 (s, 2H), 5.69
(s, 2H), 6.47 (d, 1H), 7.15 (d, 1H), 7.32–7.44 ppm (m, 5H).
3-(Benzyloxy)-6-(4,5-dihydro-1H-imidazol-2-yl)-2-methoxyaniline
(28): A mixture of nitrile 27 (144 g, 566 mmol) and sulfur (55 g,
1.7 mol) in ethylenediamine (800 mL) was degassed for 30 min,
then heated at 1008C. After 16 h the reaction mixture was cooled
to RT and then filtered. The filtrate was concentrated under re-
duced pressure, diluted with saturated sodium bicarbonate solu-
tion and extracted with EtOAc. The organic layer was washed with
saturated aqueous NaCl solution, dried (Na2SO4), filtered and con-
centrated under reduced pressure. The resulting solids were recrys-
tallized from EtOAc/hexane giving 28 (145 g, 86%): 1H NMR
([D6]DMSO): d=3.33 (s, 2H), 3.67 (s, 3H), 3.76 (brs, 2H), 5.11 (s,
2H), 6.32 (d, 1H), 6.64 (m, 1H), 6.92 (m, 2H), 7.14 (d, 1H), 7.27–
7.48 ppm (m, 5H).
Synthesis: Compounds 5–21, 38a–38g, 40 and 41 were synthe-
sized as previously reported.[37]
4-Formyl-2-methoxy-3-nitrophenyl acetate (23): Fuming nitric
acid (2.2 L) under nitrogen was cooled to 08C at which time vanil-
lin acetate (22; 528 g, 2.7 mol) was added portionwise, keeping
the internal temperature below 108C. After 2 h the resulting mix-
ture was poured into ice with stirring. The slurry was filtered and
the resulting solids were washed with water (3ꢃ100 mL) and air-
dried. After 2 d the solids were heated in CH2Cl2 (3000 mL) until
complete dissolution. The solution was allowed to cool to RT while
hexane (3000 mL) was added dropwise. The solids were collected
by filtration, washed with hexane (500 mL) and air-dried to give 23
1
8-(Benzyloxy)-7-methoxy-2,3-dihydroimidazo[1,2-c]quinazolin-5-
amine (29): A mixture of amine 28 (100 g, 336 mmol) and triethyla-
mine (TEA) (188 mL) in CH2Cl2 (3 L) was cooled to 08C and treated
with cyanogen bromide (78.4 g, 740 mmol). The reaction mixture
was stirred and allowed to warm to RT gradually. After 16 h it was
diluted with saturated sodium bicarbonate solution and extracted
with CH2Cl2. The organic layer was washed with saturated sodium
bicarbonate solution followed by multiple washes with saturated
aqueous NaCl solution. The organic layer was dried (Na2SO4) and
concentrated under reduced pressure to give 29 (130 g, quant.):
1H NMR ([D6]DMSO): d=3.81 (s, 3H), 4.13 (m, 2H), 4.32 (m, 2H),
5.31 (s, 2H), 7.30–7.48 ppm (m, 7H); MS (ESI+) m/z: 323 [M+H]+.
(269 g, 41%): H NMR ([D6]DMSO): d=2.40 (s, 3H), 3.87 (s, 3H), 7.75
(d, 1H), 7.94 (d, 1H), 9.90 ppm (s, 1H).
4-Hydroxy-3-methoxy-2-nitrobenzaldehyde (24): A mixture of
acetate 23 (438 g, 1.8 mol) and K2CO3 (506 g, 3.7 mol) in MeOH
(4 L) was stirred at RT for 16 h. Then, the reaction mixture was con-
centrated under reduced pressure to afford a viscous oil. The resi-
due was dissolved in water, and the solution was acidified using
HCl (2n) and extracted with EtOAc. The organic layer was washed
with saturated aqueous NaCl solution, dried (MgSO4) and filtered.
The solution was concentrated under reduced pressure to 1/3
volume and the resulting solid was collected by filtration to give
1
24 (317 g, 88%): H NMR ([D6]DMSO): d=3.82 (s, 3H), 7.19 (d, 1H),
7.68 (d, 1H), 9.69 ppm (s, 1H); MS (ESI+) m/z: 198 [M+H]+.
N-[8-(Benzyloxy)-7-methoxy-2,3-dihydroimidazo[1,2-c]quinazo-
lin-5-yl]nicotinamide (30): Amine 29 (21 g, 65 mmol) and nicotinic
acid (12 g, 97.7 mmol) were suspended in DMF (240 mL). N,N-Diiso-
propylethylamine (DIPEA) (33.7 g, 260.4 mmol) and then (benzotria-
4-(Benzyloxy)-3-methoxy-2-nitrobenzaldehyde (25): Phenol 24
(155 g, 786 mmol) was dissolved in DMF (1500 mL) and the stirred
solution was treated with K2CO3 (217 g, 1.57 mol) followed by
benzyl bromide (161 g, 0.94 mol). The reaction mixture was stirred
for 16 h, then concentrated under reduced pressure and separated
between water (2 L) and EtOAc (2 L). The organic layer was washed
with saturated aqueous NaCl solution (3ꢃ2 L), dried (Na2SO4) and
concentrated under reduced pressure. The resulting solids were tri-
turated with Et2O (1 L) to give 25 (220 g, 97%): 1H NMR
([D6]DMSO): d=3.05 (s, 3H), 5.36 (s, 2H), 7.39 (m, 3H), 7.49 (m,
1H), 7.51 (m, 1H), 7.58 (d, 1H), 7.87 (d, 1H), 9.77 ppm (s, 1H); MS
(ESI+) m/z: 288 [M+H]+.
zol-1-yloxy)tripyrrolidinophosphonium
hexafluorophosphate
(PyBOP; 51 g, 97.7 mmol) were added and the resulting mixture
was stirred with overhead stirring at RT for 3 d. The resultant pre-
cipitate was isolated by vacuum filtration. After repeated washing
with EtOAc, the material was dried under reduced pressure with
slight heating to give 30 (27.3 g, 98%): 1H NMR ([D6]DMSO+
2 drops [D]TFA): d=3.78 (s, 3H), 4.04 (t, 2H), 4.36 (t, 2H), 5.18 (s,
2H), 7.16 (m, 6H), 7.27 (d, 1H), 7.37 (d, 1H), 7.82 (d, 1H), 7.89
(brm, 1H), 8.84 (d, 1H), 8.89 (brm, 1H), 9.32 ppm (s, 1H); MS
(ESI+) m/z: 428 [M+H]+.
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N-(8-Hydroxy-7-methoxy-2,3-dihydroimidazo[1,2-c]quinazolin-5-
yl)nicotinamide (31): Benzyl ether 30 (20 g, 45.1 mmol) was added
portionwise over 1 h to a round-bottom flask containing trifluoro-
acetic acid (TFA) (400 mL) precooled with an ice bath. The reaction
mixture was heated at 608C and allowed to stir at this temperature
for 17 h, at which time it was cooled to RT. Then, it was concentrat-
ed under reduced pressure. The resulting residue was taken up in
CH2Cl2 and hexane, and the solution was concentrated under re-
duced pressure. The material thus obtained was dissolved in
MeOH/CH2Cl2 (1:1, 250 mL) and concentrated under reduced pres-
sure. The resulting solids were dried overnight under reduced pres-
tert-Butyl [3-({7-methoxy-5-[(pyridin-3-ylcarbonyl)amino]-2,3-di-
hydroimidazo[1,2-c]quinazolin-8-yl}oxy)propyl]carbamate (32d’):
Phenol 31 (1.5 g, 4.4 mmol) was dissolved in DMF (50 mL) contain-
ing a few drops of water. Cs2CO3 (7.24 g, 22.2 mmol) and NaI
(0.80 g, 5.3 mmol) were added, followed by tert-butyl (3-bromopro-
pyl)carbamate (3.18 g, 13.3 mmol). The mixture was stirred at
1008C overnight. After the mixture was cooled to RT, the volatiles
were removed under reduced pressure. The residue was diluted
with MeOH/CH2Cl2 (1:9), and solids were removed by filtration. The
filtrate was concentrated and purified by silica gel flash column
chromatography (0–10% MeOH/CH2Cl2) to afford 32d’ (1.13 g,
51%): 1H NMR ([D6]DMSO+2 drops [D]TFA): d=1.37 (s, 9H), 1.94
(m, 2H), 3.15 (t, 2H), 4.03 (s, 3H), 4.26 (m, 4H), 4.57 (m, 2H), 6.96
(brt, 1H), 7.47 (d, 1H), 7.82 (m, 1H), 8.03 (d, 1H), 8.79 (m, 1H), 8.91
(m, 1H), 9.46 ppm (m, 1H).
1
sure with low heat to give 31 (bis-TFA salt; 17.3 g, 66%): H NMR
([D6]DMSO+2 drops [D]TFA): d=3.98 (s, 3H), 4.21 (m, 2H), 4.54 (m,
2H), 7.17 (d, 1H), 7.59 (m, 1H), 7.85 (d, 1H), 8.53 (d, 1H), 8.78 (d,
1H), 9.38 (s, 1H), 12.21 (brs, 1H), 13.41 ppm (s, 1H); MS (ESI+) m/
z: 338 [M+H]+; HRMS-ESI m/z [M+H]+ calcd for C17H16N5O3:
338.1253, found: 338.1252.
N-[8-(3-Aminopropoxy)-7-methoxy-2,3-dihydroimidazo[1,2-c]qui-
nazolin-5-yl]nicotinamide hydrotrifluoroacetate (32d): Carba-
mate 32d’ (1.2 g, 2.3 mmol) was dissolved in a mixture of TFA
(6 mL) and CH2Cl2 (24 mL). The mixture was stirred at RT overnight,
then concentrated under reduced pressure to afford a viscous
yellow oil. Acetonitrile was added to the mixture, and the desired
product 32d was isolated by vacuum filtration as a white solid
(TFA salt; 0.55 g, 47%): 1H NMR ([D6]DMSO+2 drops [D]TFA): d=
2.13 (m, 2H), 3.02 (m, 2H), 4.01 (s, 3H), 4.25 (m, 2H), 4.35 (t, 2H),
4.57 (m, 2H), 7.45 (d, 1H), 7.73 (m, 1H), 7.92 (brm, 2H), 8.06 (d,
1H), 8.69 (m, 1H), 8.87 (m, 1H), 9.43 ppm (m, 1H).
N-(7,8-Dimethoxy-2,3-dihydroimidazo[1,2-c]quinazolin-5-yl)nico-
tinamide (32a):[32] Potassium tert-butylate (69.9 mg, 623 mmol) was
added to a slurry of phenol 31 (210 mg, 623 mmol) in DMF (5 mL),
followed by dimethyl sulfate (12 mL, 120 mmol). The reaction mix-
ture was stirred at RT for 2 h. Dimethyl sulfate (12 mL, 120 mmol)
was again added and the reaction mixture was stirred at RT for
a further 2 h, then concentrated under reduced pressure and dis-
solved in CH2Cl2/MeOH (1:1, 5 mL). Potassium tert-butylate
(69.9 mg, 623 mmol) and dimethyl sulfate (12 mL, 120 mmol) were
again added. The reaction mixture was stirred at RT for 1 h, then
concentrated under reduced pressure and purified by flash chro-
matography (MeOH/EtOAc) to give 32a (140 mg, 65%): 1H NMR
([D6]DMSO): d=4.00 (s, 4H), 4.06 (s, 3H), 4.25–4.33 (m, 2H), 4.56–
4.64 (m, 2H), 7.53 (d, 1H), 8.03–8.13 (m, 2H), 9.05 (d, 2H),
9.56 ppm (s, 1H); MS (ESI+) m/z: 352 [M+H]+; HRMS-ESI m/z [M+
H]+ calcd for C18H18N5O3: 352.1410, found: 352.1408.
N-{8-[3-(Dimethylamino)propoxy]-7-methoxy-2,3-dihydroimida-
zo[1,2-c]quinazolin-5-yl}nicotinamide (32e):
A suspension of
phenol 31 (bis-TFA salt; 2.00 g, 3.54 mmol), 3-chloro-N,N-dimethyl-
propan-1-amine hydrochloride (839 mg, 5.31 mmol) and Cs2CO3
(5.76 g, 17.7 mmol) in DMF (40 mL) was heated in a sealed tube at
1208C for 3 h. The reaction mixture was then cooled to RT and
concentrated under reduced pressure. Ice was added to the mix-
ture and the precipitate was collected by filtration and dried under
reduced pressure to give 32e, without further purification (1 g,
N-(8-Ethoxy-7-methoxy-2,3-dihydroimidazo[1,2-c]quinazolin-5-
yl)nicotinamide (32b): Ethyl methanesulfonate (68 mL, 0.66 mmol)
was added to a suspension of phenol 31 (TFA salt; 150 mg,
0.33 mmol) and Cs2CO3 (433 mg, 1.33 mmol) in DMF (7 mL). The re-
action mixture was stirred at RT for 48 h, then concentrated under
reduced pressure. The crude mixture was purified by flash column
chromatography (0–2% MeOH/CH2Cl2) to give 32b (97 mg, 80%):
1H NMR (500 MHz, [D6]DMSO+3 drops [D]TFA): d=1.47 (t, 3H),
4.04 (s, 3H), 4.28–4.33 (m, 2H), 4.37 (q, 2H), 4.59–4.64 (m, 2H), 7.52
(d, 1H), 8.07 (dd, 1H), 8.09 (d, 1H), 9.02–9.09 (m, 2H), 9.58 ppm (s,
1H); HRMS-ESI m/z [M+H]+ calcd for C19H20N5O3: 366.1566, found:
366.1569.
1
67%): H NMR (500 MHz, [D6]DMSO+3 drops [D]TFA): d=2.26 (dd,
1H), 2.87 (s, 3H), 3.27–3.32 (m, 2H), 4.05 (s, 3H), 4.27–4.32 (m, 2H),
4.37 (t, 2H), 4.61 (dd, 2H), 7.50 (d, 1H), 7.86–7.92 (m, 1H), 8.11 (dd,
1H), 8.83–8.89 (m, 1H), 8.96–9.00 (m, 1H), 9.52 ppm (s, 1H); MS
(ESI+) m/z: 423 [M+H]+; HRMS-ESI m/z [M+H]+ calcd for
C22H27N6O3: 423.2145, found: 423.2145.
N-{7-Methoxy-8-[3-(piperidin-1-yl)propoxy]-2,3-dihydroimida-
zo[1,2-c]quinazolin-5-yl}nicotinamide (32 f): Phenol 31 (bis-TFA
salt; 150 mg, 265 mmol) and 1-(3-chloropropyl)piperidine hydro-
chloride (158 mg, 796 mmol) were added to a suspension of NaH
(41.4 mg, 1.72 mmol) in DMF (4 mL). The reaction mixture was
stirred at 508C for 2 h, then NaH (41.4 mg, 1.72 mmol) was added
again. After 2 h further NaH (41.4 mg, 1.72 mmol) was added,
along with 1-(3-chloropropyl)piperidine hydrochloride (158 mg,
796 mmol). The reaction mixture was stirred at 508C overnight,
then cooled to RT and carefully quenched by the addition of water.
The organic phase was extracted with CH2Cl2 and the extract was
concentrated under reduced pressure; trituration with EtOAc gave
N-(8-Isobutoxy-7-methoxy-2,3-dihydroimidazo[1,2-c]quinazolin-
5-yl)nicotinamide (32c): A suspension of phenol 31 (bis-TFA salt;
150 mg, 265 mmol) and Cs2CO3 (346 mg, 1.06 mmol) in DMF (5 mL)
was stirred at RT for 1.5 h. 2-Methylpropan-1-ol (51 mL, 560 mmol)
and TEA (78 mL, 560 mmol) were solubilized in anhydrous DMF
(2 mL), and this solution was slowly added to the previous suspen-
sion. The reaction mixture was stirred at RT for 48 h. Cs2CO3
(346 mg, 1.06 mmol) was again added to the mixture which was
stirred for an additional 48 h at 508C. The reaction mixture was
cooled to RT, then concentrated under reduced pressure. The
crude mixture was purified by flash column chromatography (0–
2% MeOH/CH2Cl2) to give 32c (90 mg, 86%): 1H NMR (500 MHz,
[D6]DMSO+3 drops [D]TFA): d=1.06 (d, 6H), 2.16 (dt, 1H), 4.05 (s,
3H), 4.08 (d, 2H), 4.25–4.32 (m, 2H), 4.56–4.63 (m, 2H), 7.51 (d,
1H), 7.83 (dd, 1H), 8.07 (d, 1H), 8.80 (d, 1H), 8.94 (dd, 1H),
9.49 ppm (d, 1H); HRMS-ESI m/z [M+H]+ calcd for C21H24N5O3:
394.1879, found: 394.1880.
1
32 f (45 mg, 36%): H NMR (500 MHz, [D6]DMSO+3 drops [D]TFA):
d=1.42 (d, 1H), 1.64–1.76 (m, 3H), 1.85 (d, 2H), 2.28 (dd, 2H),
2.92–3.00 (m, 2H), 3.23–3.29 (m, 2H), 3.53 (d, 2H), 4.04 (s, 3H),
4.25–4.31 (m, 2H), 4.37 (t, 2H), 4.57–4.63 (m, 2H), 7.49 (d, 1H), 7.77
(dd, 1H), 8.11 (d, 1H), 8.73 (d, 1H), 8.91 (dd, 1H), 9.47 ppm (s, 1H);
HRMS-ESI m/z [M+H]+ calcd for C25H31N6O3: 463.2458, found:
463.2455.
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4-(3-Chloropropyl)morpholine hydrochloride (32g’): To a solution
of 1-bromo-3-chloropropane (45 g, 0.29 mol) in toluene (100 mL),
morpholine (38 g, 0.44 mol) was added. The solution was stirred at
848C for 3 h, during which time a precipitate formed. After the so-
lution was cooled to RT, the precipitate was isolated by vacuum fil-
tration and washed with Et2O, and the solid was discarded. The
mother liquor was acidified with HCl (4n) in dioxane (72 mL,
0.29 mol), which resulted in the desired product precipitating as
the HCl salt. The volatiles were removed under reduced pressure,
and the resultant solid was dried to give 32g’ (HCl salt; 53 g,
to RT and concentrated under reduced pressure. The resulting
slurry was diluted with water (100 mL) and the mixture was adjust-
ed to pH 7 by the slow addition of aqueous HCl (1n); the mixture
was stirred for 2 h. The solid was collected via vacuum filtration
and washed with water (50 mL), and finally triturated with CH2Cl2/
heptane (1:1, 10 mL). High-vacuum drying overnight gave 33
(0.49 g, 51%): 1H NMR ([D6]DMSO+2 drops [D]TFA): d=2.14–2.26
(m, 2H), 3.03–3.17 (m, 2H), 3.32–3.54 (m, 4H), 3.60–3.72 (m, 2H),
3.95–4.05 (m, 2H), 4.18–4.35 (m, 4H), 4.51–4.64 (m, 2H), 7.38 (d,
1H), 7.68 (dd, 1H), 7.82 (d, 1H), 8.63 (s, 1H), 8.84 (dd, 1H), 9.42 (s,
1H), 13.39 ppm (s, 1H); MS (ESI+) m/z: 451 [M+H]+.
1
90%): H NMR ([D6]DMSO): d=2.21 (m, 2H), 3.03 (m, 2H), 3.15 (m,
2H), 3.39 (m, 2H), 3.74 (t, 2H), 3.77–3.94 (m, 4H), 11.45 ppm (brs,
1H).
N-{8-[3-(Morpholin-4-yl)propoxy]-7-propoxy-2,3-dihydroimida-
zo[1,2-c]quinazolin-5-yl}nicotinamide (34a): Phenol 33 (TFA salt;
370 mg, 655 mmol) and K2CO3 (452 mg, 3.28 mmol) were suspend-
ed in DMF (3 mL), and 1-iodopropane (130 mL, 1.3 mmol) was
added. The reaction mixture was stirred at RT for 5 h, then the vol-
atiles were removed under reduced pressure. The residue was puri-
fied by flash chromatography (0–7% MeOH/CH2Cl2) to give 34a as
an oil that solidified on standing. Trituration with cold acetone/
MeOH (9:1) gave a white solid (65 mg, 20%): MS (ESI+) m/z: 493.1
[M+H]+; HRMS-ESI m/z [M+H]+ calcd for C26H33N6O4: 493.2563,
found: 493.2559.
N-{7-Methoxy-8-[3-(morpholin-4-yl)propoxy]-2,3-dihydroimida-
zo[1,2-c]quinazolin-5-yl}nicotinamide (32g): Phenol 31 (bis-TFA
salt; 5.00 g, 8.84 mmol) was solubilized in DMF (81 mL) and Cs2CO3
(14.4 g, 44.2 mmol) was added. The mixture was stirred at RT for
1.5 h, then 4-(3-chloropropyl)morpholine hydrochloride (32g’;
2.65 g, 13.3 mmol) was added. The reaction mixture was stirred at
608C for 20 h, then the crude mixture was concentrated under re-
duced pressure. The crude material was stirred in water (200 mL)
for 1 h, and the suspension was filtered and dried under reduced
pressure to give 32g (3.26 g, 79%): 1H NMR ([D6]DMSO+2 drops
[D]TFA): d=2.25 (m, 2H), 3.18 (m, 2H), 3.31 (m, 2H), 3.52 (m, 2H),
3.65 (brt, 2H), 4.00 (s, 3H), 4.04 (m, 2H), 4.23 (m, 2H), 4.34 (brt,
2H), 4.54 (m, 2H), 7.43 (d, 1H), 8.04 (d, 1H), 9.01 ppm (s, 2H); MS
(ESI+) m/z: 465 [M+H]+; HRMS-ESI m/z [M+H]+ calcd for
C24H29N6O4: 465.2250, found: 465.2249.
N-{7-(Allyloxy)-8-[3-(morpholin-4-yl)propoxy]-2,3-dihydroimida-
zo[1,2-c]quinazolin-5-yl}nicotinamide (34b): To an orange-brown
solution of phenol 33 (50.0 mg, 111 mmol) in DMF (2.0 mL) K2CO3
(5m; 62 mL, 330 mmol) was added, followed by allyl bromide (14 mL,
170 mmol). The resulting pale-yellow mixture was stirred at RT for
1 d. Then, the reaction mixture was concentrated under reduced
pressure. The residue was taken up in CH2Cl2 and a small volume
of MeOH, and the slurry was filtered. The filtrate was purified by
flash chromatography (5–10% MeOH/CH2Cl2) to give 34b as
a white solid (10 mg, 18%): MS (ESI+) m/z: 491 [M+H]+.
N-{7-Methoxy-8-[2-(morpholin-4-yl)ethoxy]-2,3-dihydroimida-
zo[1,2-c]quinazolin-5-yl}nicotinamide (32h): Phenol 31 (1.34 g,
3.97 mmol) was solubilized in DMF (50 mL) and NaH (60% in min-
eral oil; 794 mg, 19.9 mmol) was added. The reaction mixture was
stirred at RT for 20 min, then 4-(2-chloroethyl)morpholine hydro-
chloride (1.48 g, 7.94 mmol) was added in one portion. The reac-
tion mixture was stirred at 1008C for 16 h, then was cooled to RT
and concentrated under reduced pressure. The crude material was
solubilized in chloroform and the solution was washed with water.
The organic phase was dried (Na2SO4), filtered and concentrated
under reduced pressure. The crude material was purified by flash
column chromatography to give 32h (400 mg, 22%): HRMS-ESI m/
z [M+H]+ calcd for C23H27N6O4: 451.2094, found: 451.2097.
N-{7-Isobutoxy-8-[3-(morpholin-4-yl)propoxy]-2,3-dihydroimida-
zo[1,2-c]quinazolin-5-yl}nicotinamide (34c): To a slurry of phenol
33 (100 mg, 222 mmol) in DMF (4.0 mL) was added NaH (16 mg,
670 mmol); gas evolution was observed upon the dissolution of 33.
After 30 min, 1-bromo-2-methylpropane (38 mg, 277 mmol) was
added. The resulting orange solution was stirred at RT for 17 h.
Then, further 1-bromo-2-methylpropane (40 mg, 292 mmol) was
added and the mixture was stirred for 3 h. Water was added, and
the mixture was adjusted to pH 7 with a few drops of aqueous HCl
(1n). The mixture was extracted with CH2Cl2 and the combined ex-
tracts were dried (MgSO4) and concentrated under reduced pres-
sure. Purification by flash chromatography (5–10% MeOH/CH2Cl2)
gave 34c as a white solid (30 mg, 26%): MS (ESI+) m/z: 507 [M+
H]+.
N-[8-(4-Chlorobutoxy)-7-methoxy-2,3-dihydroimidazo[1,2-c]qui-
nazolin-5-yl]nicotinamide (32i’): A mixture of 1-bromo-4-chloro-
butane (57.0 mg, 333 mmol), phenol 31 (102 mg, 302 mmol) and
Cs2CO3 (296 mg, 907 mmol) in DMF (10 mL) was stirred at RT for
2 d. Then, the reaction mixture was filtered and the filtrate was
concentrated under reduced pressure. The crude material was puri-
fied by flash column chromatography to give 32i’ (60 mg, 48%).
N-{7-(Cyclohexylmethoxy)-8-[3-(morpholin-4-yl)propoxy]-2,3-di-
hydroimidazo[1,2-c]quinazolin-5-yl}nicotinamide (34d): A slurry
of phenol 33 (74.0 mg, 164 mmol) in N,N-dimethylacetamide (2 mL)
was treated with NaH (60% in mineral oil; 13.1 mg, 329 mmol) for
30 min while being stirred vigorously. To the resulting solution was
added (bromomethyl)cyclohexane (37 mL, 250 mmol) and Ag2CO3
(67.9 mg, 246 mmol); the mixture was stirred overnight and then
heated at 1008C for 2 h. Dilution with water (20 mL) resulted in
a deep purple mixture which was filtered through a pad of Celiteꢄ.
The pad was washed with water and CH2Cl2. The filtrate was sepa-
rated and the aqueous layer was extracted with CH2Cl2. The com-
bined organic layers were concentrated to dryness under reduced
pressure. The residue was dissolved in CH2Cl2 and purified by flash
chromatography (MeOH/EtOAc 2:8 to MeOH/CH2Cl2 5:95) to give
34d as a white solid (10 mg, 11%): MS (ESI+) m/z: 547 [M+H]+;
N-{7-Methoxy-8-[4-(morpholin-4-yl)butoxy]-2,3-dihydroimida-
zo[1,2-c]quinazolin-5-yl}nicotinamide (32i): A mixture of chloride
32i’ (50.0 mg, 117 mmol) and morpholine (1.0 mL, 11.4 mmol) in di-
oxane (4 mL) was held at reflux for 18 h. The reaction mixture was
then cooled to RT and concentrated under reduced pressure.
Water was added to the mixture, and the precipitate was collected
by filtration and washed with a mixture of MeOH/Et2O to give 32i
(14 mg, 25%), without further purification.
N-{7-Hydroxy-8-[3-(morpholin-4-yl)propoxy]-2,3-dihydroimida-
zo[1,2-c]quinazolin-5-yl}nicotinamide (33): Methyl ether 32g
(1.0 g, 2.15 mmol) in N-methyl-2-pyrrolidinone (20 mL) was heated
at 1008C and Na2S (0.84 g, 10.76 mmol) was added portionwise.
The reaction mixture was heated at 1608C for 10 min, then cooled
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HRMS-ESI m/z [M+H]+ calcd for C30H39N6O4: 547.3033, found:
547.3033.
pressure, dry-loaded onto silica gel and purified by chromatogra-
phy (CH2Cl2/MeOH 95:5!CH2Cl2/MeOH!9:1!CH2Cl2/2m NH3 in
MeOH 95:5 ! CH2Cl2/2m NH3 in MeOH 85:15). The resultant oil
was triturated with hexanes/EtOAc (1:1, 15 mL) to afford 36 as
N-{8-[3-(Morpholin-4-yl)propoxy]-7-[(tetrahydro-2H-pyran-4-yl)-
methoxy]-2,3-dihydroimidazo[1,2-c]quinazolin-5-yl}nicotinamide
(34e): To a slurry of phenol 33 (63.0 mg, 140 mmol) in DMF
(2.0 mL) was added NaH (60% in mineral oil; 8 mg, 210 mmol).
1
a solid (171 mg, 44%): H NMR ([D6]DMSO): d=1.87 (m, 2H), 2.35
(m, 4H), 2.42 (t, 2H), 3.55 (m, 4H), 3.69 (s, 3H), 3.88 (m, 4H), 4.03
(t, 2H), 6.73 (m, 3H), 7.43 ppm (d, 1H); MS (ESI+) m/z: 360.3 [M+
H]+.
After
30 min
4-(bromomethyl)tetrahydro-2H-pyran
(27 mL,
210 mmol) was added. The resulting orange solution was stirred at
RT for 17 h. Then, further 4-(bromomethyl)tetrahydro-2H-pyran
(27 mL, 210 mmol) was added and the mixture was stirred for 3 h.
Water was added and the mixture was adjusted to pH 7 with a few
drops of aqueous HCl (1n). The mixture was extracted with CH2Cl2
and the combined extracts were dried, filtered and concentrated
under reduced pressure. Purification by flash chromatography (5–
10% MeOH/CH2Cl2) gave 34e as a white solid (20 mg, 26%): MS
(ESI+) m/z: 549 [M+H]+.
N-{7-Methoxy-8-[3-(morpholin-4-yl)propoxy]-2,3-dihydroimida-
zo[1,2-c]quinazolin-5-yl}-1,3-benzodioxole-5-carboxamide (39a):
To a vigorously stirred solution of amine 36 (50% purity; 154 mg,
214 mmol) in DMF (2.0 mL) containing piperonylic acid (1,3-benzo-
dioxole-5-carboxylic acid; 53.4 mg, 321 mmol) and PyBOP (167 mg,
321 mmol) was added DIPEA (150 mL, 860 mmol). After 20 min
a thick slurry formed, and stirring was continued for 4 h. The slurry
was filtered and the solid was washed with EtOAc to give 39a as
1
a white solid (60 mg, 55%): H NMR (500 MHz, [D6]DMSO+3 drops
N-{8-[3-(Morpholin-4-yl)propoxy]-7-[(pyridin-4-yl)methoxy]-2,3-
[D]TFA): d=2.29 (dd, 2H), 3.14–3.21 (m, 2H), 3.33–3.37 (m, 2H),
3.54 (d, 2H), 3.70 (t, 2H), 4.01–4.06 (m, 2H), 4.03–4.04 (m, 3H),
4.22–4.27 (m, 2H), 4.36 (t, 2H), 4.52–4.57 (m, 2H), 6.15 (s, 2H), 7.03
(d, 1H), 7.44 (d, 1H), 7.67 (d, 1H), 7.88–7.93 (m, 1H), 8.05 ppm (d,
1H); MS (ESI+) m/z: 508 [M+H]+; HRMS-ESI m/z [M+H]+ calcd
for C26H30N5O6: 508.2196, found: 508.2198.
dihydroimidazo[1,2-c]quinazolin-5-yl}nicotinamide
(34 f):
To
a slurry of phenol 33 (100 mg, 222 mmol) in DMF (2.0 mL), NaH
(60% in mineral oil; 27 mg, 670 mmol) was added; gas evolution
was observed upon the dissolution of 33. After 20 min, 4-(chloro-
methyl)pyridine hydrochloride (1:1; 54.6 mg, 333 mmol) was added.
The resulting orange mixture was stirred at RT; after 2 h a thick
slurry formed. Stirring was continued overnight. Then, water was
added and the mixture was adjusted to pH 7 with a few drops of
aqueous HCl (1n). The slurry was filtered to give 34 f as a white
N-{7-Methoxy-8-[3-(morpholin-4-yl)propoxy]-2,3-dihydroimida-
zo[1,2-c]quinazolin-5-yl}-2,4-dimethyl-1,3-thiazole-5-carboxa-
mide (39b): Amine 36 (80% purity; 5.00 g, 11.1 mmol) was dis-
solved in DMF (1.2 mL), and 2,4-dimethyl-1,3-thiazole-5-carboxylic
acid (2.62 g, 16.7 mmol), PyBOP (8.69 g, 16.7 mmol) and DIPEA
(7.8 mL, 45 mmol) were sequentially added. The reaction mixture
was stirred at RT overnight, cooled, then filtered. The white solid
was triturated with EtOAc, filtered and dried to give 39b (450 mg,
8%).
1
solid (40 mg, 35%): H NMR (500 MHz, [D6]DMSO+3 drops [D]TFA):
d=2.22–2.29 (m, 2H), 3.08–3.16 (m, 2H), 3.28–3.33 (m, 2H), 3.50
(d, 2H), 3.70 (t, 2H), 4.01 (d, 2H), 4.30 (t, 2H), 4.41 (t, 2H), 4.61 (t,
2H), 5.64 (brs, 2H), 7.56 (d, 1H), 7.81–7.92 (m, 1H), 8.17 (d, 1H),
8.28 (brs, 2H), 8.75–8.89 (m, 1H), 8.93–9.02 (m, 1H), 9.11 (brs, 2H),
9.49 ppm (brs, 1H); MS (ESI+) m/z: 542 [M+H]+.
N-{7-Methoxy-8-[3-(morpholin-4-yl)propoxy]-2,3-dihydroimida-
zo[1,2-c]quinazolin-5-yl}isonicotinamide (39c): To a vigorously
stirred solution of amine 36 (50% purity; 100 mg, 139 mmol) in
DMF (2.0 mL) was added isonicotinic acid (25.7 mg, 209 mmol), fol-
lowed by DIPEA (97 mL, 560 mmol) and PyBOP (109 mg, 209 mmol).
After a few seconds a clear brown solution was obtained, then
a thick slurry immediately formed. Stirring was continued over-
night. The slurry was filtered and the solid was washed with EtOAc
to give 39c as a white solid (30 mg, 46%): MS (ESI+) m/z (%): 465
(100) [M+H]+; HRMS-ESI m/z [M+H]+ calcd for C24H29N6O4:
465.2250, found: 465.2249.
5-Amino-7-methoxy-2,3-dihydroimidazo[1,2-c]quinazolin-8-ol
bis(trifluoroacetate) (35): Amine 29 (30 g, 93 mmol) was added
portionwise over 1 h to a round-bottom flask containing TFA
(400 mL) precooled with an ice bath. The reaction mixture was
heated at 608C and allowed to stir at this temperature for 17 h, at
which time it was cooled to RT and then concentrated under re-
duced pressure. The resulting residue was taken up in CH2Cl2 and
hexanes, and concentrated under reduced pressure. The material
thus obtained was dissolved in MeOH/CH2Cl2 (1:1, 250 mL) and
concentrated under reduced pressure. The resulting solid was
dried overnight under reduced pressure with low heat to give 35
1
(44.7 g, quant.): H NMR ([D6]DMSO): d=3.64 (s, 3H), 4.00 (m, 2H),
4.15 (brt, 2H), 6.87 (m, 1H), 7.61 ppm (m, 1H); MS (ESI+) m/z: 233
[M+H]+.
2-Amino-N-{7-methoxy-8-[3-(morpholin-4-yl)propoxy]-2,3-dihy-
droimidazo[1,2-c]quinazolin-5-yl}isonicotinamide (39d): Amine
36 (100 mg, 0.28 mmol) was dissolved in DMF (3 mL), and 2-amino-
isonicotinic acid (38 mg, 0.28 mmol) was added. PyBOP (217 mg,
0.42 mmol) and DIPEA (0.15 mL, 0.83 mmol) were sequentially
added, and the mixture was stirred at RT overnight and then con-
centrated under reduced pressure. Purification by HPLC gave 39d
7-Methoxy-8-[3-(morpholin-4-yl)propoxy]-2,3-dihydroimida-
zo[1,2-c]quinazolin-5-amine (36): Phenol bis(trifluoroacetate) 35
(500 mg, 1.1 mmol) was dissolved in CH2Cl2 (10 mL), and TEA
(0.75 mL, 5.4 mmol) was added. The suspension was stirred at RT
for 1.5 h, after which time 5-amino-7-methoxy-2,3-dihydroimida-
zo[1,2-c]quinazolin-8-ol hydrotrifluoroacetate was isolated. Thus
prepared, this compound was dissolved in DMF (10 mL). Cs2CO3
(1.41 g, 4.3 mmol) and chloride 32g’ (218 mg, 1.1 mmol) were
added, and the mixture was stirred at 708C for 30 min. Additional
chloride 32g’ (109 mg, 0.55 mmol) and Cs2CO3 (350 mg, 1.1 mmol)
were added, and stirring was continued for 1 h. Another portion of
chloride 32g’ (109 mg, 0.55 mmol) was added, and the tempera-
ture was increased to 758C. After 3 h the reaction mixture was
cooled to RT and filtered through a pad of Celiteꢄ, washing with
MeOH and CH2Cl2. The filtrate was concentrated under reduced
1
(50 mg, 37%): H NMR ([D6]DMSO): d=2.25 (m, 2H), 3.13 (m, 2H),
3.31 (brt, 2H), 3.50 (d, 2H), 3.66 (t, 2H), 3.99 (brs, 2H), 4.01 (s, 3H),
4.27 (dd, 2H), 4.35 (brt, 2H), 4.50 (dd, 2H), 7.38 (dd, 1H), 7.50 (d,
1H), 7.75 (s, 1H), 8.06 (d, 1H), 8.08 (s, 1H), 8.42 (brs, 1H), 10.15
(brs, 1H), 13.25 ppm (brs, 1H); MS (ESI+) m/z: 480.3 [M+H]+.
6-Amino-N-{7-methoxy-8-[3-(morpholin-4-yl)propoxy]-2,3-dihy-
droimidazo[1,2-c]quinazolin-5-yl}nicotinamide (39e): To amine
36 (150 mg, 417 mmol) and 6-aminonicotinic acid (69.2 mg,
501 mmol) dissolved in anhydrous DMF (10 mL) was added DIPEA
(218 mL, 1.25 mmol), then PyBOP (261 mg, 501 mmol), and the reac-
tion mixture was stirred at RT for 18 h. Amide 39e was collected
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Full Papers
1
by filtration, washed with water (20 mL) and oven-dried (112 mg,
56%): 1H NMR ([D6]DMSO): d=1.88–2.02 (m, 2H), 2.32–2.42 (m,
4H), 2.43–2.47 (m, 2H), 3.53–3.63 (m, 4H), 3.90 (s, 3H), 4.03 (dd,
4H), 4.15 (t, 2H), 6.43 (d, 1H), 6.63 (s, 2H), 7.01 (d, 1H), 7.58 (d,
1H), 8.04 (dd, 1H), 8.78 (d, 1H), 12.88 ppm (s, 1H); MS (ESI-) m/z:
478 [MꢀH]+.
vacuum filtration to give 39i (42.7 mg, 40%): H NMR ([D6]DMSO+
2 drops [D]TFA): d=2.25 (m, 2H), 3.18 (m, 2H), 3.31 (m, 2H), 3.52
(m, 2H), 3.65 (brt, 2H), 4.00 (s, 3H), 4.04 (m, 2H), 4.23 (m, 2H), 4.34
(brt, 2H), 4.54 (m, 2H), 7.43 (d, 1H), 8.04 (d, 1H), 9.01 (s, 2H);
1H NMR of the bis-HCl salt (500 MHz, [D6]DMSO): d=2.30–2.37 (m,
2H), 3.11 (brs, 2H), 3.25–3.31 (m, 2H), 3.48 (d, J=12.1 Hz, 2H),
3.83–3.90 (m, 2H), 3.95–4.00 (m, 2H), 4.01 (s, 3H), 4.17–4.22 (m,
2H), 4.37 (t, J=6.0 Hz, 2H), 4.47 (t, J=9.7 Hz, 2H), 7.40 (d, J=
9.2 Hz, 1H), 7.54 (s, 2H), 8.32 (d, J=9.2 Hz, 1H), 8.96 (s, 2H), 11.46
(brs, 1H), 12.92 (brs, 1H), 13.41 (brs, 1H); 13C NMR (125 MHz,
[D6]DMSO): d=23.09, 45.22, 46.00, 51.21, 53.38, 61.54, 63.40, 67.09,
101.18, 112.55, 118.51, 123.96, 132.88, 134.35, 148.96, 157.25,
160.56, 164.96, 176.02 ppm; MS (ESI+) m/z: 481 [M+H]+.
6-(Dimethylamino)-N-{7-methoxy-8-[3-(morpholin-4-yl)propoxy]-
2,3-dihydroimidazo[1,2-c]quinazolin-5-yl}nicotinamide
(39 f):
Amine 36 (50% purity; 150 mg, 209 mmol) and 6-(dimethylamino)-
nicotinic acid (69.4 mg, 417 mmol) were suspended in DMF (2.0 mL)
and DIPEA (110 mL, 630 mmol). Then, solid PyBOP (217 mg,
417 mmol) was added all at once. The mixture was stirred at RT for
3 d. The precipitate was collected by filtration, washed with EtOAc
and dried in a vacuum oven at 508C for 1 h to give 39 f (53.7 mg,
51%).
2-Amino-4-methylpyrimidine-5-carboxylic acid (39j’): To a solu-
tion of ethyl 2-amino-4-methylpyrimidine-5-carboxylate (1.00 g,
5.52 mmol) in MeOH (27 mL) and THF (41 mL) was added NaOH
(2n, 14 mL), and the reaction mixture was stirred at RT overnight.
Then, the mixture was neutralized with HCl (1n, 20 mL), concen-
trated to ~30 mL under reduced pressure and filtered to give 39j’
(0.6 g, 71%): MS (ESI+) m/z: 154 [M+H]+.
N-{7-Methoxy-8-[3-(morpholin-4-yl)propoxy]-2,3-dihydroimida-
zo[1,2-c]quinazolin-5-yl}pyrazine-2-carboxamide (39g): Amine 36
(50% purity; 150 mg, 209 mmol) and pyrazine-2-carboxylic acid
(51.8 mg, 417 mmol) were suspended in DMF (1.0 mL) and DIPEA
(110 mL, 630 mmol). Solid PyBOP (217 mg, 417 mmol) was added all
at once. The mixture was stirred at RT for 7 d. The white solids
were collected by filtration, washed copiously with EtOAc and
dried in a vacuum oven at 458C overnight to give 39g (59 mg,
61%).
2-Amino-N-{7-methoxy-8-[3-(morpholin-4-yl)propoxy]-2,3-dihy-
droimidazo[1,2-c]quinazolin-5-yl}-4-methylpyrimidine-5-carboxa-
mide (39j): To a solution of amine 36 (100 mg, 278 mmol) and acid
39j’ (42.6 mg, 278 mmol) in anhydrous DMF (3.0 mL) was added
DIPEA (150 mL, 830 mmol) and PyBOP (217 mg, 417 mmol). The mix-
ture was stirred at RT overnight. The precipitate was collected by
filtration and washed with MeOH to give 39j (93 mg, 68%): MS
(ESI+) m/z: 495 [M+H]+.
N-{7-Methoxy-8-[3-(morpholin-4-yl)propoxy]-2,3-dihydroimida-
zo[1,2-c]quinazolin-5-yl}pyrimidine-5-carboxamide (39h): Amine
36 (80% purity; 100 mg, 0.22 mmol) was dissolved in DMF (5 mL),
and pyrimidine-5-carboxylic acid (41 mg, 0.33 mmol) was added.
PyBOP (173 mg, 0.33 mmol) and DIPEA (0.16 mL, 0.89 mmol) were
sequentially added, and the mixture was stirred at RT overnight.
EtOAc was added, and the precipitate was isolated by vacuum fil-
tration to give 39h (12 mg, 11%): 1H NMR ([D6]DMSO+2 drops
[D]TFA): d=2.27 (m, 2H), 3.16 (m, 2H), 3.33 (m, 2H), 3.52 (m, 2H),
3.67 (brt, 2H), 4.00 (m, 2H), 4.02 (s, 3H), 4.26 (m, 2H), 4.35 (brt,
2H), 4.59 (m, 2H), 7.47 (d, 1H), 8.05 (d, 1H), 9.39 (s, 1H), 9.48 (s,
2H); MS (ESI+) m/z: 466 [M+H]+; HRMS-ESI m/z [M+H]+ calcd
for C23H28N7O4: 466.2203, found: 466.2203.
X-ray structure of copanlisib (BAY 80-6946, 39i) in complex with
PI3Kg: Protein was expressed in insect cells and purified using Ni
affinity chromatography, ion-exchange chromatography (Resour-
ce Q) and size exclusion chromatography (Superdex 200 26/60).
The protein was concentrated to 5 mgmLꢀ1 in Tris (20 mm, pH 7.2),
(NH4)2SO4 (0.5 mm), ethylene glycol (1%), CHAPS (0.02%) and DTT
(5 mm). Prior to crystallization, copanlisib (2 mm) was added to the
protein. Crystals were obtained using the sitting drop method by
mixing an equal volume of protein and reservoir solution (1 mL+
1 mL). Crystals were obtained using PEG 4000 (19%), (NH4)2SO4
(0.15m) and Tris (0.1m, pH 7.5). Data were collected at the syn-
chrotron facility at the SLS in Villigen, Switzerland. The structure
was solved using 2CHX as search model. The structure was refined
using REFMAC within the CCP4 suite. The crystallographic data for
the structure have been deposited with the RCSB Protein Data
Bank (PDB) with access code 5G2N.
2-Aminopyrimidine-5-carboxylic acid (39i’): Sodium (1Z)-2-(dime-
thoxymethyl)-3-methoxy-3-oxoprop-1-en-1-olate (1.37 g, 6.9 mmol),
prepared as reported,[37] was dissolved in DMF (12 mL), and guani-
dine hydrochloride (640 mg, 6.7 mmol) was added. The mixture
was stirred at 1008C for 1 h, then cooled to RT and diluted with
water. Methyl 2-aminopyrimidine-5-carboxylate precipitated as
a light yellow solid, which was isolated by vacuum filtration
1
(510 mg, 50%): H NMR ([D6]DMSO): d=3.79 (s, 3H), 7.56 (brs, 2H),
8.67 (s, 2H). Methyl 2-aminopyrimidine-5-carboxylate (300 mg,
2.0 mmol) was dissolved in MeOH (5 mL) containing a few drops of
water. Lithium hydroxide (122 mg, 5.1 mmol) was added, and the
reaction mixture was stirred at 608C overnight. The mixture was
concentrated under reduced pressure, then diluted with water, and
the solution was adjusted to pH 4 with HCl (1n). 2-Aminopyrimi-
dine-5-carboxylic acid (39i’) precipitated as a white solid, which
was isolated by vacuum filtration (244 mg, 90%): 1H NMR
([D6]DMSO): d=7.44 (brs, 2H), 8.63 (s, 2H), 12.73 (brs, 1H).
Acknowledgements
We thank Dr. S. Gruendemann and Dr. G. Depke for their support
regarding analytical data, and C. Moldenhauer, S. Korthals, and
Dr. K. Greenfield for valuable technical support with the manu-
script. We thank Proteros Biostructures for the X-ray structure de-
termination of copanlisib. We also thank Dr. F. von Nussbaum for
very stimulating discussions and Prof. H. Wild for his support.
2-Amino-N-{7-methoxy-8-[3-(morpholin-4-yl)propoxy]-2,3-dihy-
droimidazo[1,2-c]quinazolin-5-yl}pyrimidine-5-carboxamide (BAY
80-6946, 39i): Amine 36 (80% purity; 100 mg, 0.22 mmol) was dis-
solved in DMF (5 mL), and acid 39i’ (46 mg, 0.33 mmol) was
added. PyBOP (173 mg, 0.33 mmol) and DIPEA (0.16 mL,
0.89 mmol) were sequentially added, and the mixture was stirred
at RT overnight. EtOAc was added, and the solids were isolated by
Keywords: phosphoinositide 3-kinase
kinases · PI3K inhibitors · X-ray crystallography
· copanlisib · lipid
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Published online on && &&, 0000
&
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ÝÝ These are not the final page numbers!

FULL PAPERS
The phosphoinositide 3-kinase (PI3K)
pathway is aberrantly activated in many
tumors. The presence of various PI3K
isoforms and their differential roles in
cancers makes them ideal candidates
for targeted inhibition. A PI3Kg screen-
ing hit led to the discovery of the novel
2,3-dihydroimidazo[1,2-c]quinazoline
class of PI3K inhibitors. Herein we de-
scribe initial structure–activity relation-
ship findings for this class and the opti-
mization that led to the identification of
copanlisib (BAY 80-6946) as a clinical
candidate.
W. J. Scott,* M. F. Hentemann,
R. B. Rowley, C. O. Bull, S. Jenkins,
A. M. Bullion, J. Johnson, A. Redman,
A. H. Robbins, W. Esler, R. P. Fracasso,
T. Garrison, M. Hamilton, M. Michels,
J. E. Wood, D. P. Wilkie, H. Xiao, J. Levy,
E. Stasik, N. Liu, M. Schaefer, M. Brands,
J. Lefranc*
&& – &&
Discovery and SAR of Novel 2,3-
Dihydroimidazo[1,2-c]-
quinazoline PI3K Inhibitors:
Identification of Copanlisib (BAY 80-
6946)
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&
These are not the final page numbers! ÞÞ