Discovery of SP-96, the first non-ATP-competitive Aurora Kinase B inhibitor, for reduced myelosuppression

Article abstract of DOI:10.1016/j.ejmech.2020.112589

Aurora Kinase B is a serine-threonine kinase known to be overexpressed in several cancers, with no inhibitors approved for clinical use. Herein, we present the discovery and optimization of a series of novel quinazoline-based Aurora Kinase B inhibitors. The lead inhibitor SP-96 shows sub-nanomolar potency in Aurora B enzymatic assays (IC₅₀ = 0.316 ± 0.031 nM). We identified the important pharmacophore features resulting in selectivity against receptor tyrosine kinases. Particularly, SP-96 shows >2000 fold selectivity against FLT3 and KIT which is important for normal hematopoiesis. This could diminish the adverse effect of neutropenia reported in the clinical trials of the Aurora B inhibitor Barasertib, which inhibits FLT3 and KIT in addition to Aurora B. Enzyme kinetics of SP-96 shows non-ATP-competitive inhibition which makes it a first-in-class inhibitor. Further, SP-96 shows selective growth inhibition in NCI60 screening, including inhibition of MDA-MD-468, a Triple Negative Breast Cancer cell line.

Full text of DOI:10.1016/j.ejmech.2020.112589

Journal Pre-proof
Discovery of SP-146, the first non-ATP-competitive Aurora Kinase B inhibitor, for
reduced myelosuppression
Naga Rajiv Lakkaniga, Lingtian Zhang, Binyam Belachew, Naresh Gunaganti,
Brendan Frett, Hong-yu Li
PII:
S0223-5234(20)30561-4
DOI:
https://doi.org/10.1016/j.ejmech.2020.112589
EJMECH 112589
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 14 April 2020
Revised Date: 1 June 2020
Accepted Date: 8 June 2020
Please cite this article as: N.R. Lakkaniga, L. Zhang, B. Belachew, N. Gunaganti, B. Frett,
H.-y. Li, Discovery of SP-146, the first non-ATP-competitive Aurora Kinase B inhibitor, for
reduced myelosuppression, European Journal of Medicinal Chemistry, https://doi.org/10.1016/
j.ejmech.2020.112589.
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Urea linker is preferred
over amides
N
N
N
N
Bulkier groups less tolerated.
Halogens are more preferred.
meta position is preferred over
ortho and para.
H
N
H
N
HN
F
Polar groups are more preferred.
Position 6 on quinazoline is highly
unfavorable.
O
Important for selectivity against FLT3/KIT.
1,4- substitution is active on AurB, FLT and KIT.
1,3- substitution is active on AurB only.

Discovery of SP-146, the first non-ATP-competitive Aurora Kinase B
inhibitor, for reduced myelosuppression
Naga Rajiv Lakkaniga1, Lingtian Zhang1, Binyam Belachew 2, Naresh Gunaganti1, Brendan
Frett1, Hong-yu Li 1*
1Department of Pharmaceutical Sciences, College of Pharmacy, University of Arkansas for
Medical Sciences; Little Rock, Arkansas, USA.
2Department of Biochemistry and Molecular Biology, College of Medicine, University of
Arkansas for Medical Sciences; Little Rock, Arkansas, USA.
*Email: HLi2@uams.edu

Abstract
Aurora Kinase B is a serine-threonine kinase known to be overexpressed in several cancers, with
no inhibitors approved for clinical use. Herein, we present the discovery and optimization of a
series of novel quinazoline based Aurora Kinase B inhibitors. The lead inhibitor SP-146 shows
sub-nanomolar potency in Aurora B enzymatic assays (IC50 = 0.316 ± 0.031 nM). We identified
the important pharmacophore features resulting in selectivity against receptor tyrosine kinases.
Particularly, SP-146 shows >2000 fold selectivity against FLT3 and KIT which is important for
normal hematopoiesis. This could eliminate the adverse effect of neutropenia reported in the
clinical trials of the Aurora B inhibitor Barasertib, which inhibits FLT3 and KIT in addition to
Aurora B. Enzyme kinetics of SP-146 shows non-ATP competitive inhibition which makes it a
first-in-class inhibitor. Further, SP-146 shows selective growth inhibition in NCI60 screening,
including inhibition of MDA-MD-468, a Triple Negative Breast Cancer cell line.
Keywords:
Aurora Kinase B; kinase inhibitors; anti-cancer drugs; non-ATP competitive inhibition; structure
activity relationship (SAR); synthetic lethal toxicity

1.0 Introduction
Aurora Kinase B is a serine-threonine kinase known to play an important role in mitosis[1]. It is
a part of Chromosomal Passenger Complex (CPC) along with three other proteins named Inner
Centromere Protein (INCENP), Survivin and Borealin, and functions as the enzymatic module of
the protein complex[2]. It acts mainly in the M Phase and is crucial for ensuring proper
microtubule-chromosome attachments and cytokinesis[3]. Overexpression of Aurora B is
reported in several cancers such as breast cancer[4], colorectal cancer[5], prostate cancer[6],
lung cancer[7], and leukemia[8]. There are several studies which relate genomic instability
with increased Aurora B activity, establishing it as a therapeutic target in cancer therapy[9–
11]. Over the last two decades, several drug discovery projects focused on the inhibition of
Aurora kinase B[12–14] and resulted in a few promising molecules (Figure 1). These studies also
gave important insights into the use of Aurora B inhibitors in combination therapy. Studies show
that the Aurora B inhibitors have synergistic effect when used in combination with other
therapies including chemotherapeutic drugs [15,16], and radiotherapy[17]. However, there are
no Aurora B inhibitors approved for clinical use.
‘Synthetic lethality’ is an emerging concept in cancer therapy where simultaneous loss of two
proteins leads to non-viable phenotypes whereas a loss of only one of them is tolerated.
However, if both the targets are crucial for normal cellular functions, it can lead to ‘synthetic
lethal toxicity’[18]. One example of this scenario is Barasertib (AZD1152), a potent Aurora B
inhibitor[19] currently in clinical trials. Although it shows sub-nanomolar potency against
Aurora B, it also inhibits FLT3 and KIT.[20,21] Research suggests that these two receptor
tyrosine kinases are important for normal hematopoiesis and are ‘anti-targets’ to each other.
Thus, simultaneous inhibition of FLT3 and KIT causes ‘synthetic lethal’ myelosuppression[22].
Because cancer patients are already prescribed chemotherapy that results in myelosuppression, it
is imperative that Aurora B precision medicine is designed to limit this potential dose-limiting
side effect. Further, results from the clinical trials with barasertib show that 70% of patients
reported febrile neutropenia,[23,24] which is a form of myelosuppression. This emphasizes the
need to investigate and develop Aurora B inhibitors with better selectivity against FLT3 and KIT
to reduce the incidence of myelosuppression with Aurora B therapy. Further, barasertib and other
known Aurora B inhibitors are ATP-competitive. Since ATP is present in high cellular

concentrations, the ATP-competitive inhibitors need to be dosed at higher concentrations. This
challenge can be overcome by the discovery of inhibitors which do not compete with ATP.
These can be dosed at lower concentrations and are thus beneficial for improved side-effects
profile.
Figure 1: Representative Aurora B inhibitors
As a part of the continued effort to discover kinase inhibitors with enhanced therapeutic
profiles[25–31], a kinase-directed fragment (KDF) library, as previously described[27], was
screened against Aurora B. Frag-1 was identified from the screening efforts with an IC50 of
116.33 ± 10.67 nM against Aurora B. Preliminary follow-on screening, supplemented with
computer aided drug discovery (CADD), suggested Frag-1 to be a good starting point.
Visualization of the docking model revealed an unoccupied pocket which can be exploited to
improve the activity of the scaffold by extending the terminal aniline group towards the pocket
(Figure 2). An iterative cycle of synthesis and following enzymatic assays were performed to
understand the structure-activity relationship (SAR) and identify a lead molecule. Herein, we
discuss the discovery of a highly selective Aurora B inhibitor, SP-146, based on the KDF hit

Frag-1. To the best of our knowledge, SP-146 is the first inhibitor of Aurora B which shows non-
ATP-competitive inhibition.
2.0 Chemistry
Scheme 1: Synthesis of target compounds 16-35 and 36-38
The synthetic route for target molecules 16-35 and 36-38 is depicted is Scheme 1. Briefly,
cyclization of commercially available 2-amino-5-chlorobenzoic acid with formamidine acetate
resulted in intermediate 3a. Chlorination of 3a with thionyl chloride provided the substituted
quinazoline 4a, which upon nucleophilic substation with tert-butyl (3-aminophenyl)carbamate
provided intermediate 5a in high yields. Using Suzuki-Miyaura coupling[32], compounds 6-10
were synthesized from various boronic acids and subsequently deprotected using a solution of
10% trifluoroacetic acid in dichloroethane. Isocyanates were prepared from commercially
available amines using triphosgene and were reacted with intermediates 11-15. The final
coupling reaction provided compounds 16-33 in good to excellent yields. For compounds 34-35,
the same procedure was followed except 2-amino-4-chlorobenzoic acid was used in place of 2-

amino-5-chlorobenzoic acid. For compounds 36-38, intermediate 11 was coupled to various
substituted benzoic acids via EDC coupling.
Scheme 2: Synthesis of target compounds 42-44
Final compounds 42-44 were synthesized as depicted in Scheme 2. Intermediate 3a was coupled
with 1-methylpyrazole-4-boronic acid pinacol ester via Suzuki-Miyaura coupling to form
intermediate 39, which was chlorinated with thionyl chloride to obtain intermediate 40.
Nucleophilic substitution of 40 with 3-amino benzoic acid provided compound 41. The final
compounds 42-44 were synthesized by coupling 41 with various substituted anilines.
3.0 Results and Discussion
3.1 Inhibitors Design

Figure 2: Molecular docking of Frag-1 in Aurora B reveals unoccupied pocket.
Quinazoline moiety is frequently used in kinase inhibitors to mimic adenosine of ATP[33].
Through the in-house KDF library screening, we discovered Frag-1 with moderate activity on
Aurora B. Molecular docking of Frag-1 in Aurora B crystal structure reveals that nitrogen of the
quinazoline engages Ala173 in a strong hydrogen bond. The N-methyl pyrazole orients towards
the solvent front and is not involved in any interactions. However, the kinase pocket is not fully
occupied, with some room next to the terminal phenyl groups. We designed compounds by
extending the terminal -NH2 group of Frag-1 towards the unoccupied region of Aurora B pocket.
3.2 In-vitro evaluation
We initially constructed derivatives 16-20 with various substitutions on the terminal aromatic
ring, which was predicted to extend into the lipophilic pocket. Compounds 17 and 18, with IC50
values of 939.1 ± 45.85 nM and 197.033 ± 5.55 nM respectively, suggested that bulky groups are
less tolerated. Compounds 19 and 20, with meta substituted -CF3 and -Br groups respectively,
showed IC50 values of 29.893 ± 2.71 nM and 90.623 ± 7.45 nM respectively. This hinted that
the halogen substitution is preferred. To further determine preferred aromatic substitution, we
synthesized compounds 21 and 22, with chlorine at the meta- and para- positions. The results
indicate that substitution on the meta position is preferred. Also, comparisons between 20 and 22
demonstrate that smaller halogens are preferred. We then synthesized compounds 23-25, with
fluorine at the ortho, meta and para positions, and discovered that the meta position had highest
Aurora B inhibition. The IC50 values improved when chlorine was replaced by fluorine,
supporting our initial hypothesis that smaller substitutions are preferred. Also, the p-fluoro and

o-fluoro derivatives exhibited decreased activity compared to m-fluoro confirming that the meta-
substitution on the terminal phenyl ring is preferred for Aurora B inhibition.
#
R
Aurora B IC50
(nM)
#
R
Aurora B IC50
(nM)
222.1 ± 19.41
22.836 ± 1.49
16
22
939.1 ± 45.85
197.03 ± 5.55
29.893 ± 2.71
2.981 ± 0.32
0.316 ± 0.031
15.083 ± 0.99
17
18
19
23
SP-146
(24)
25
90.623 ± 7.45
209.53 ± 14.88
35.3 ± 1.62
20
21
26
27
29.823 ± 2.25
Barasertib
0.326 ± 0.018
Table 1: Aurora B inhibition of compounds 16-27.
Molecular docking of SP-146 in Aurora B (Figure 4) showed that the N-methyl pyrazole moiety
orients towards the solvent front. To determine structure activity relationships (SAR) at this
region, we replaced N-methyl pyrazole of compound SP-146 with a thiophene. However, this
resulted in a drastic increase in IC50. Since groups that interact with water are most appropriate at
the solvent front, we investigated 4-methoxy phenol and 4-methyl sulfonyl moieties. Compounds
30 and 32 showed IC50 values of 185.3 ± 9.26 nM and 9.968 ± 0.905 nM, respectively. Likely
from a more favourable interaction at the solvent front, the more polar 4-methyl sulfonyl
exhibited stronger Aurora B inhibition. However, neither substitution improved activity
compared to SP-146, although 32 exhibited low nanomolar inhibition. To explore substitution

position on the quinazoline ring, compounds 34-35 were synthesized, in which the pyrazole was
moved from the position 7 to 6. However, this substantially attenuated the activity, indicating
that the substitution at the position 6 is highly unfavourable.
#
R1
R2
R3
AKB IC50 (nM)
H
3-F
75.87 ± 2.63
28
H
H
4-F
3-F
1008 ± 47.08
185.3 ± 9.26
29
30
H
H
4-F
3-F
2150.6 ± 187.2
9.968 ± 0.905
31
32
H
4-F
63.07 ± 5.76
33
34
35
H
H
3-F
4-F
6580.6 ± 596.12
> 15 µM
Table 2: Aurora B inhibition of compounds 28-35.
To further explore the SAR, the urea linker was changed to amine. Compounds 36-38 were
synthesized by replacing the urea linker with amide. Although the SAR pattern was retained, this
change lead to decrease in activity. As expected, compound 36 with 4-methoxy substitution

showed significant reduction in activity. Compound 37 and 38 with halogen substitutions
showed better activity compared to compound 36. Also, compound 37 showed better activity
compared to 38 confirming that smaller halogens are preferred. Compounds 39-41 were
constructed by flipping the amide group. We again observed similar SAR with no improvement
in activity. Compound 39 with the bulkier isopropyl group showed the highest IC50. Compound
40 with 3-fluoro substitution showed decreased activity compared to its urea counterpart.
However, 3-fluoro substitution showed better activity compared to 4-fluoro confirming that the
meta position is most preferred. These results concluded that urea linker is more preferred for
Aurora B inhibition compared to amide linkers.
Aurora B IC50
#
X
Y
R
(nM)
NH
NH
C=O
C=O
C=O
NH
4-OCH3
3-F
141.8 ± 9.23
61.2 ± 4.2
36
37
38
42
43
44
NH
3-Br
238.45 ± 14.28
944.2 ± 73.45
71.63 ± 6.17
310.6 ± 21.4
C=O
C=O
C=O
4-isopropyl
3-F
NH
NH
4-F
Table 3: Aurora B inhibition of compounds 36-38 and 42-44.
3.3 Selectivity against FLT3 and KIT

The goal of this project was to discover an Aurora B inhibitor which is not active on FLT3 and
KIT. This was achieved by exploring the SAR on the central phenyl group. We observed that
when the substitution pattern on the central phenyl ring is changed from 1,3- to 1,4-, as depicted
in Figure 3A, the activity on Aurora B is retained. However, while SP-146 did not inhibit
FLT3/KIT, compound 45 showed low nanomolar potency against these anti-targets. In-house
screening of SP-146 on other receptor tyrosine kinases (RTKs) namely EGFR, RET and HER2
showed IC50 value ≥ 2µM. These results indicate that the substitution pattern on the central
phenyl ring is crucial not only for selectivity against FLT3/KIT, but also on other RTKs. To
determine the sub-type selectivity, we screened SP-146 against Aurora A and observed IC50
value of 18.975±1.435 nM. This shows that SP-146 is about 60 times selective for Aurora B over
Aurora A.
A
Compound 45
SP-146
N
N
N
N
N
N
N
N
F
H
N
H
N
HN
HN
O
O
N
H
N
H
F
AKB IC50 = 0.336 ± 0.027 nM
FLT3 IC50 = 8.237 ± 0.718 nM
KIT IC50 = 12.04 ± 0.36 nM
AKB IC50 = 0.316 ± 0.031 nM
FLT3 IC50 = 1475.6 ± 60.07 nM
KIT IC50 = 1307.6 ± 56.14 nM
Figure 3: (A) Substitution pattern on the central phenyl ring is crucial for selectivity against
FLT3, KIT and several other tyrosine kinases. (B) Increased DNA content of H460 cells when
treated with SP-146.
3.4
Enzyme kinetics studies

Enzyme kinetics revealed that inhibition of SP-146 is not ATP-competitive (Figure S1). Kinase
inhibitors are generally classified into two major categories: Type 1 and Type 2 inhibitors. The
Type-1 inhibitors compete with ATP at the binding pocket, while the Type-2 inhibitors do not
compete with ATP. Since ATP is present in high cellular concentrations, a larger dose of type-1
inhibitors is required to show the desired effect. The Aurora B inhibitors reported in literature are
all ATP-competitive. To the best of our knowledge, SP-146 is the first inhibitor which displays
non-ATP competitive inhibition of Aurora B. This facilitates dosing the compound at lower
concentrations which help in improving the selectivity profile.
3.5
Cellular assays
We have dosed H460 cells with SP-146. As illustrated in Figure 3B, we observed that cells
treated with SP-146 showed increased cell volume with enormous nucleus. As mentioned
previously, Aurora B plays a crucial role in mitosis. Upon Aurora B inhibition, the cell division
is arrested, and the cell doesn’t undergo cytokinesis resulting in polyploid cells. This increased
DNA content is a characteristic phenotype of Aurora B inhibition.
Further, SP-146 was submitted to NCI60 screening. The results of the screening are provided in
Supplemental Data. These results indicate that SP-146 is not promiscuous, rather selective for a
few cell lines. While it shows GI50 > 1µM for most of the cell lines, it shows potent inhibition of
the cell lines tabulated in Table 4. In particular, it shows GI50=107 nM on MDA-MB-468 which
is a triple negative breast cancer (TNBC) cell line. Although Hormone Receptor positive
(ER/PR+) and HER2+ breast cancers have better therapies, precision medicine for TNBC is yet
to be developed. The role of Aurora B in breast cancers, including TNBC, is well
established[34,35]. Given that SP-146 is an Aurora B inhibitor and that it shows good activity on
MDA-MB-468, further studies on its role in TNBC is an important topic for future research. We
also determined the growth inhibition of MCF-7 cells when treated with selected compounds of
this series and observed good enzymatic-cellular inhibition corelation. The results are illustrated
in S3.
Cell-line
SP-146 GI50

MDA-MB-468
CCRF-CEM
COLO 205
A498
107 nM
47.4 nM
50.3 nM
53.2 nM
Table 4: GI50 values of SP-146 on cell lines from NCI60 panel
3.6 Molecular modelling
Molecular docking of SP-146 in the Aurora B crystal structure is shown in Figure 4. The
quinazoline moiety forms a crucial hydrogen bond with Ala173 of the hinge region. The two
nitrogens of the urea linker engage Ala233 in strong hydrogen bonding. As expected, the urea
linker and the terminal substituted phenyl ring extend in the region previously unoccupied by
Frag-1. The N-methyl pyrazole moiety is exposed to the solvent front. This pose was subjected
to 1µs unbiased atomistic molecular dynamics simulation. The simulated analysis revealed that
both the hydrogen bonds remained stable through the entire duration.
Figure 4: Binding pose of SP-146 in the crystal structure of Aurora B (PDB ID: 5EYK).
Hydrogen bonds are depicted in blue.
4.0 Conclusion

In conclusion, based on fragment screening, a novel Aurora Kinase B inhibitor, SP-146, was
discovered with the following characteristics: (1) It shows sub-nanomolar potency in enzymatic
assays. (2) It shows improved selectivity profile compared to barasertib. SP-146 is >2000-fold
selective against FLT3 and KIT, which is important to evade synthetic lethal toxicity. (3) SP-146
displays non-ATP-competitive inhibition. (4) SP-146 showed growth inhibition of selective
NCI60 cell lines, including MDA-MB-468. These results indicate that SP-146 will serve as an
excellent lead compound in the search of precision medicine for both Aurora B inhibition and
Triple Negative Breast Cancer.
5.0 Experimental
5.1 Chemistry: Synthetic procedures
5.1.1 General procedure for the synthesis of isocyanates
To a solution of 1 eq. of triphosgene in dichloromethane, a 0.1 M solution of aniline (3 eq.) and
triethylamine (3 eq.) was added very slowly at room temperature. The cloudy reaction mixture
was stirred at RT until it becomes colorless solution. The solvent was then evaporated under
vacuum to obtain the crude isocyanate which was used without further purification.
5.1.2 General procedure for the synthesis of urea derivatives
The isocyanate obtained through the general procedure 5.1.2 was dissolved in dichloromethane.
To this, a solution of the corresponding amine and triethylamine in DMF was added dropwise at
room temperature. The reaction was stirred for 15 minutes at RT and the solvent was
concentrated under vacuum. The crude was washed with water to remove aqueous impurities.
The precipitate was filtered and then washed with methanol. The precipitate was filtered and
then dried under vacuum to obtain the desired urea derivatives in high purities.
5.1.3 General procedure for the synthesis of amide derivatives
1 equivalent was carboxylic acid derivative, 1.5 equivalents of the corresponding amine and 1.5
equivalents of triethylamine were dissolved in dimethylformamide under nitrogen. To this 1
equivalent of EDC and 2.5 equivalents of HOAt were added. The reaction mixture was stirred at
RT overnight. The solvent was evaporated under vacuum. The crude was washed with water to
remove aqueous impurities. The precipitate was filtered and then washed with methanol. The

precipitate was filtered and then dried under vacuum to obtain the desired urea derivatives in
high purities.
5.1.4 General procedure for the synthesis of compounds 6-9
A solution of 1 eq. of 5a and 1.5 eq. of boronic acid in DMF/H20 (4:1) was purged with nitrogen
gas and 0.03 eq. of Pd2(dba)3, 0.09 eq. of PCy3 and 3 eq. of Na2CO3 were added to it. The
reaction was stirred at 110ᵒC overnight. After the completion of the reaction, the reaction mixture
was added to ice. The precipitate was filtered, dried under vacuum and used without further
purification.
5.1.5 General procedure for the synthesis of compounds 11-15
To a solution of the boc-protected amines dichloromethane, 5 eq. of trifluoroacetic acid was
added and stirred at room temperature. The reaction progressed was monitored with TLC. After
the completion of the reaction, the solvent was evaporated under vacuum. The crude was
dissolved in water and washed with dichloromethane. Saturated NaOH solution was added to
adjust the pH to 9. The precipitate was filtered and dried under vacuum to obtain the desired
compounds.
5.1.6 Synthesis of the quinazoline scaffold
To a solution of 1 eq. of chloro-substituted 2-aminobenzoic acid in 2-methoxyethanol, 3 eq. of
formamidine acetate was added and stirred at 130ᵒC overnight. On cooling the reaction mixture,
a precipitate crashed out which was filtered, washed with water and dried under vacuum to
obtain substituted quinazolin-4(3H)-ones. It was then dissolved in thionyl chloride and stirred at
110ᵒC for 2 hours. The solvent was then evaporated under vacuum, and the crude was dissolved
in ethyl acetate and washed with saturated NaHCO3. The organic layer was dried over anhydrous
Na2SO4 and concentrated to afford the chloro substituted quinazoline scaffold.
5.2 Experimental of all the target compounds
5.2.1 N1-(7-(1-methyl-1H-pyrazol-4-yl)quinazolin-4-yl)benzene-1,3-diamine (Frag-1):
1
Yield: 99%; H NMR (400 MHz, DMSO-d6)
δ
9.45 (s, 1H), 8.48 (d,
J
= 8.0 Hz, 2H), 8.38 (s,
= 7.8 Hz, 1H),
1H), 8.08 (s, 1H), 7.88 (s, 1H), 7.81 (d,
J
= 8.6 Hz, 1H), 7.08 (s, 1H), 6.98 (t, J

6.92 (d,
J
= 7.8 Hz, 1H), 6.33 (d,
158.00, 155.38, 150.84, 149.27, 140.12, 137.48, 137.31, 129.54, 129.09,
J
= 7.6 Hz, 1H), 5.08 (s, 2H), 3.87 (s, 3H). 13C NMR (100
MHz, DMSO-d6)
δ
124.14, 124.10, 122.30, 121.28, 113.63, 110.92, 110.35, 108.65, 39.23; ; LC-MS (ESI): m/z
305.2710 (M+H)+ .
5.2.2
1-(4-methoxyphenyl)-3-(3-((7-(1-methyl-1H-pyrazol-4-yl)quinazolin-4-yl)
amino)phenyl)urea (16)
Synthesized as described in general procedure 5.1.2 from intermediate 11 and 1-isocyanato-4-
1
methoxybenzene prepared as described in general procedure 5.1.1. Yield: 55%; H NMR (400
MHz, DMSO-d6)
1H), 8.45 (s, 1H), 8.13 (s, 1H), 7.98 (d,
7.3 Hz, 1H), 7.37 (d, = 8.8 Hz, 2H), 7.31 (d,
3H), 3.71 (s, 3H). 13C NMR (100 MHz, DMSO-d6)
δ
10.54 (s, 1H), 9.06 (s, 1H), 8.86 (s, 1H), 8.70 (s, 1H), 8.67 (d,
= 9.1 Hz, 1H), 7.93 (d, = 11.7 Hz, 2H), 7.42 (d,
= 7.9 Hz, 2H), 6.87 (d, = 8.8 Hz, 2H), 3.92 (s,
158.79, 154.82, 153.53, 153.19, 140.75,
J = 8.7 Hz,
J
J
J =
J
J
J
δ
139.11, 138.79, 137.51, 133.20, 129.99, 129.18, 125.27, 124.97, 120.75, 120.21(2 * CH),
118.19, 117.35, 115.18, 114.41(2 * CH), 114.36, 113.46, 112.64, 55.58, 39.30. LC-MS (ESI) m/z
466.18866 [M+H]+.
5.2.3
1-(4-(tert-butyl)phenyl)-3-(3-((7-(1-methyl-1H-pyrazol-4-yl)quinazolin-4-
yl)amino)phenyl)urea (17)
Synthesized as described in general procedure 5.1.2 from intermediate 11 and 1-(tert-butyl)-4-
isocyanatobenzene prepared as described in general procedure 5.1.1. Yield: 60%;1H NMR (400
MHz, DMSO-d6)
1H), 8.13 (s, 1H), 8.01 (s, 1H), 7.95 (s, 1H), 7.89 (d,
7.38 (d, = 8.6 Hz, 2H), 7.30 (d, = 7.7 Hz, 3H), 7.26 (d,
9H). 13C NMR (100 MHz, DMSO-d6)
158.03, 155.08, 152.96, 150.44, 144.53, 140.37, 139.98,
δ
9.80 (s, 1H), 8.74 (s, 1H), 8.63 (s, 1H), 8.57 (d,
= 8.6 Hz, 1H), 7.50 (d,
= 3.5 Hz, 1H), 3.91 (s, 3H), 1.26 (s,
J
= 6.3 Hz, 2H), 8.43 (s,
J
J
= 7.4 Hz, 1H),
J
J
J
δ
137.78, 137.49, 137.36, 129.63, 129.07, 125.82 (2 * CH), 124.39, 124.24, 121.97, 121.21,
118.39 (2 * CH), 116.52, 114.08, 113.56, 112.60, 39.239, 34.31, 31.68 (3 * CH3). LC-MS (ESI)
m/z 492.25507 [M+H]+.
5.2.4
1-(4-isopropylphenyl)-3-(3-((7-(1-methyl-1H-pyrazol-4-yl)quinazolin-4-
yl)amino)phenyl)urea (18)

Synthesized as described in general procedure 5.1.2 from intermediate 11 and 1-isocyanato-4-
isopropylbenzene prepared as described in general procedure 5.1.1. Yield: 60%;1H NMR (400
MHz, DMSO-d6)
1H), 7.96 (s, 1H), 7.88 (m, 2H), 7.45 (d,
18.9, 8.1 Hz, 2H), 7.11 (d, = 8.3 Hz, 2H), 3.88 (s, 3H), 2.78 (dt,
= 6.9 Hz, 6H). 13C NMR (100 MHz, DMSO-d6)
158.26, 154.62, 153.00, 149.01, 142.26,
δ
9.99 (s, 1H), 8.82 (s, 1H), 8.70 (s, 1H), 8.57 (mf, 2H), 8.40 (s, 1H), 8.09 (s,
= 7.4 Hz, 1H), 7.34 (d, = 8.3 Hz, 2H), 7.25 (dd,
= 13.7, 6.8 Hz, 1H), 1.14 (d,
J
J
J =
J
J
J
δ
140.46, 139.62, 138.18, 137.83, 137.41, 129.74, 129.10, 126.91(2 * CH), 124.66, 124.46,
121.07, 120.83, 118.68(2 * CH), 116.79, 114.42, 113.28, 112.86, 39.26, 33.19, 24.45. LC-MS
(ESI) m/z 478.15912 [M+H]+.
5.2.5 1-(3-((7-(1-methyl-1H-pyrazol-4-yl)quinazolin-4-yl)amino)phenyl)-3-(3-(trifluoromethyl)
phenyl)urea (19)
Synthesized as described in general procedure 5.1.2 from intermediate 11 and 1-isocyanato-3-
(trifluoromethyl)benzene prepared as described in general procedure 5.1.1. Yield: 65%;1H NMR
(400 MHz, DMSO-d6)
δ 9.76 (s, 1H), 9.04 (s, 1H), 8.87 (s, 1H), 8.56 (d, J = 8.2 Hz, 2H), 8.42
(s, 1H), 8.12 (s, 1H), 8.05 (d,
= 15.9, 7.9 Hz, 3H), 7.30 (t,
J
= 4.5 Hz, 2H), 7.95 (s, 1H), 7.88 (d,
= 6.1 Hz, 2H), 7.25 (d,
J
= 8.6 Hz, 1H), 7.52 (dd, J
13
J
J
= 8.0 Hz, 1H), 3.91 (s, 3H); C NMR
(100 MHz, DMSO-d6)
δ 157.99, 155.18, 152.90, 150.72, 140.96, 140.05, 139.85, 137.70,
137.32, 130.35, 130.11, 129.80, 129.60, 129.13, 125.98, 124.37, 124.17, 123.27, 122.18, 121.22,
118.50, 116.91, 114.51, 114.47, 114.40, 113.60, 112.93, 40.43, 39.21. LC-MS (ESI) m/z
504.16180 [M+H]+.
5.2.6
1-(3-bromophenyl)-3-(3-((7-(1-methyl-1H-pyrazol-4-yl)quinazolin-4-
yl)amino)phenyl)urea (20)
Synthesized as described in general procedure 5.1.2 from intermediate 11 and 1-bromo-3-
isocyanatobenzene prepared as described in general procedure 5.1.1. Yield: 62%;1H NMR (400
MHz, DMSO-d6)
1H), 8.44 (s, 1H), 8.13 (s, 1H), 7.98 (s, 1H), 7.96 (d,
7.49 (d,
NMR (100 MHz, DMSO-d6)
δ 10.29 (s, 1H), 9.18 (s, 1H), 9.10 (s, 1H), 8.66 (s, 1H), 8.63 (d, J = 8.7 Hz,
J
= 8.7 Hz, 1H), 7.92 (s, 1H), 7.87 (s, 1H),
13
J
= 8.0 Hz, 1H), 7.33 (m, 2H), 7.25 (m, 2H), 7.15 (d,
J
= 7.9 Hz, 1H), 3.92 (s, 3H). C
δ
158.54, 154.05, 152.82, 147.26, 141.84, 140.38, 140.11, 139.21,
138.70, 137.46, 131.15, 129.88, 129.23, 125.02, 124.71, 122.16, 120.89, 120.70, 119.44, 117.50,
117.32, 115.10, 113.47, 112.93, 39.27. LC-MS (ESI) m/z 514.12024 [M+H]+.

5.2.7
1-(4-chlorophenyl)-3-(3-((7-(1-methyl-1H-pyrazol-4-yl)quinazolin-4-
yl)amino)phenyl)urea (21)
Synthesized as described in general procedure 5.1.2 from intermediate 11 and 1-chloro-4-
isocyanatobenzene prepared as described in general procedure 5.1.1. Yield: 59%; 1H NMR (400
MHz, DMSO-d6)
1H), 8.41 (s, 1H), 8.09 (s, 1H), 7.95 (d,
8.8 Hz, 2H), 7.41 (d, = 7.4 Hz, 1H), 7.33 – 7.24 (m, 4H), 3.88 (s, 3H). C NMR (100 MHz,
DMSO-d6) 158.80, 153.51, 152.92, 145.66, 140.36, 139.20, 139.14, 138.82, 137.51, 130.00,
δ
10.52 (s, 1H), 9.25 (s, 1H), 9.18 (s, 1H), 8.68 (s, 1H), 8.63 (d,
J = 8.7 Hz,
J
= 8.8 Hz, 1H), 7.91 (s, 1H), 7.88 (s, 1H), 7.46 (d,
J =
13
J
δ
129.24, 129.06(2 * CH), 125.66, 125.30, 124.98, 120.74, 119.92(2 * CH), 118.14, 117.68,
115.35, 113.63, 112.62, 39.30. LC-MS (ESI) m/z 470.20343 [M+H]+.
5.2.8
1-(3-chlorophenyl)-3-(3-((7-(1-methyl-1H-pyrazol-4-yl)quinazolin-4-
yl)amino)phenyl)urea (22)
Synthesized as described in general procedure 5.1.2 from intermediate 11 and 1-chloro-4-
isocyanatobenzene prepared as described in general procedure 5.1.1. Yield: 60%; 1H NMR (400
MHz, DMSO-d6)
1H), 8.61 (d, = 8.6 Hz, 1H), 8.45 (s, 1H), 8.14 (s, 1H), 8.00 (s, 1H), 7.94 (d,
7.74 (s, 1H), 7.49 (d, = 7.6 Hz, 1H), 7.30 (m, 4H), 7.01 (d,
= 5.5, 1H), 3.92 (s, 4H). 13C NMR
(100 MHz, DMSO-d6) 163.15, 157.55, 146.45, 144.83, 144.17, 143.23, 142.26, 142.15,
δ
10.16 (s, 1H), 9.07 (d,
J
= 10.1 Hz, 1H), 8.99 (d,
J
= 8.0 Hz, 1H), 8.65 (s,
J
J
= 10.2 Hz, 2H),
J
J
δ
138.37, 135.59, 134.65, 134.53, 133.95, 129.62, 129.39, 126.59, 125.71, 122.61, 122.05, 121.71,
119.62, 118.05, 118.01, 117.84, 44.05.LC-MS (ESI) m/z 470.21063 [M+H]+.
5.2.9
1-(2-fluorophenyl)-3-(3-((7-(1-methyl-1H-pyrazol-4-yl)quinazolin-4-
yl)amino)phenyl)urea (23)
Synthesized as described in general procedure 5.1.2 from intermediate 11 and 1-fluoro-2-
isocyanatobenzene prepared as described in general procedure 5.1.1. Yield: 71%; 1H NMR (400
MHz, DMSO-d6)
8.16 (t, = 8.2 Hz, 1H), 8.13 (s, 1H), 8.01 (s, 1H), 7.95 (d,
1H), 7.36 – 7.28 (m, 2H), 7.27 – 7.19 (m, 1H), 7.14 (t, = 7.7 Hz, 1H), 7.00 (dd,
Hz, 1H), 3.91 (s, 3H). 13C NMR (100 MHz, DMSO-d6)
158.51, 154.04, 152.65, 152.44(d,
J=240 Hz), 147.37, 140.18, 139.30, 138.65, 137.45, 129.86, 129.25, 127.97 (d, J=10.4 Hz),
δ
10.31 (s, 1H), 9.44 (s, 1H), 8.71 (s, 1H), 8.68 – 8.62 (m, 2H), 8.44 (s, 1H),
= 10.3 Hz, 2H), 7.48 (d, = 6.8 Hz,
= 13.1, 6.9
J
J
J
J
J
δ

124.97, 124.92, 124.89, 124.75, 122.87 (d,
J = 7.5 Hz), 120.94, 120.91, 119.51, 117.40, 115.40
(d, = 19.0 Hz), 114.79, 113.06 (d,
J
J
= 18.8 Hz), 39.27. LC-MS (ESI) m/z 453.90192 [M+H]+.
5.2.10
1-(3-fluorophenyl)-3-(3-((7-(1-methyl-1H-pyrazol-4-yl)quinazolin-4-
yl)amino)phenyl)urea (24)
Synthesized as described in general procedure 5.1.2 from intermediate 11 and 1-fluoro-3-
isocyanatobenzene prepared as described in general procedure 5.1.1. Yield: 76%; 1H NMR (400
MHz, DMSO-d6)
δ
9.79 (s, 1H), 8.92 (s, 1H), 8.83 (s, 1H), 8.61 – 8.52 (m, 2H), 8.44 (s, 1H),
= 1.6 Hz, 1H), 7.89 (dd, = 8.7, 1.7 Hz, 1H), 7.54 – 7.48
= 8.2, 4.4 Hz, 3H), 7.12 (dd, = 8.2, 1.2 Hz, 1H), 6.79 (td, = 8.3, 2.0 Hz,
164.03, 159.81 (d, J = 367.4 Hz), 155.14,
8.13 (s, 1H), 8.02 (s, 1H), 7.94 (d,
J
J
(m, 2H), 7.29 (qd,
J
13
J
J
1H), 3.91 (s, 3H). C NMR (100 MHz, DMSO-d6)
δ
152.77, 150.64, 142.02 (d, J = 11.4 Hz), 140.06, 139.95, 137.74, 137.36, 130.75 (d, J = 9.8 Hz),
129.62, 129.12, 124.37, 124.20, 122.12, 121.23, 116.79, 114.28, 114.24, 113.59, 112.78, 108.54
(d, J = 21.3 Hz), 105.20 (d, J = 26.6 Hz), 39.24. LC-MS (ESI) m/z 454.16642 [M+H]+.
5.2.11
1-(4-fluorophenyl)-3-(3-((7-(1-methyl-1H-pyrazol-4-yl)quinazolin-4-
yl)amino)phenyl)urea (25)
Synthesized as described in general procedure 5.1.2 from intermediate 11 and 1-fluoro-4-
isocyanatobenzene prepared as described in general procedure 5.1.1. Yield: 73%; 1H NMR (400
MHz, DMSO-d6)
8.12 (s, 1H), 8.06 (d,
= 8.0 Hz, 1H), 7.15 – 7.06 (m, 2H), 7.03 (d,
3H). 13C NMR (100 MHz, DMSO-d6)
159.30, 157.99 (d,
152.09, 140.71, 140.19, 138.09, 137.61, 136.50 (d, = 2.3 Hz), 130.28, 129.63, 129.36, 125.98,
125.61, 120.34, 120.06 (d, = 7.7 Hz), 118.37, 115.83, 115.60, 115.03, 114.45 (d, = 20.1 Hz),
114.07, 111.82, 39.35. LC-MS (ESI) m/z 454.18188 [M+H]+.
δ
11.08 (s, 1H), 9.40 (d,
J = 16.2 Hz, 1H), 8.83-8.72 (m, 3H), 8.47 (s, 1H),
J
= 7.3 Hz, 1H), 7.94-7.87 (m, 2H), 7.5-43 (m, 1H), 7.37 (s, 1H), 7.19 (t,
J
J
= 7.8 Hz, 1H), 6.66 (d,
J
= 6.7 Hz, 1H), 3.93 (s,
δ
J = 300.6 Hz), 153.13, 152.15,
J
J
J
5.2.12
1-(3-fluoro-4-methylphenyl)-3-(3-((7-(1-methyl-1H-pyrazol-4-yl)quinazolin-4-
yl)amino)phenyl)urea (26)
Synthesized as described in general procedure 5.1.2 from intermediate 11 and 1-fluoro-4-
1
isocyanato-2-methylbenzene prepared as described in general procedure 5.1.1. Yield: 70%; H
NMR (400 MHz, DMSO-d6) δ 10.52 (s, 1H), 9.21 (s, 1H), 9.17 (s, 1H), 8.71 (s, 1H), 8.66 (d, J =

8.7 Hz, 1H), 8.45 (s, 1H), 8.13 (s, 1H), 7.98 (d,
(d, = 10.1 Hz, 2H), 7.36 – 7.26 (m, 2H), 7.16 (t,
(s, 3H), 2.16 (s, 3H); 13C NMR (100 MHz, DMSO-d6)
J
= 8.8 Hz, 1H), 7.94 (d,
= 8.6 Hz, 1H), 7.02 (d,
160.91 (d, J = 240.1 Hz), 158.77,
J
= 14.6 Hz, 2H), 7.45
J
J
J = 8.1 Hz, 1H), 3.92
δ
153.57, 152.91, 140.35, 139.61 (d, J = 11.2 Hz), 139.10, 138.86, 137.51, 131.80 (d, J = 6.5 Hz),
129.99, 129.23, 125.27, 124.95, 120.75, 118.28, 117.63, 117.21, 117.04, 115.30, 114.035(d, J=3
Hz), 113.58, 112.66, 105.10 (d, J = 27.4 Hz), 39.30, 13.975 (d,
468.21515 [M+H]+.
J = 3 Hz); LC-MS (ESI) m/z
5.2.13
1-(4-chloro-3-fluorophenyl)-3-(3-((7-(1-methyl-1H-pyrazol-4-yl)quinazolin-4-
yl)amino)phenyl)urea (27)
Synthesized as described in general procedure 5.1.2 from intermediate 11 and 2-chloro-1-fluoro-
1
4-isocyanatobenzene prepared as described in general procedure 5.1.1. Yield: 67%; H NMR
(400 MHz, DMSO-d6)
δ
10.45 (s, 1H), 9.31 (s, 1H), 9.19 (s, 1H), 8.69 (s, 1H), 8.65 (d,
J
= 8.6
= 5.8 Hz, 1H), 7.47 (d,
158.70,
Hz, 1H), 8.44 (s, 1H), 8.13 (s, 1H), 7.97 (m, 2H), 7.91 (s, 1H), 7.82 (d,
J
13
J
= 7.6 Hz, 1H), 7.39 – 7.25 (m, 4H), 3.92 (s, 3H); C NMR (100 MHz, DMSO-d6)
δ
153.71, 152.95, 152.70 (d,
J
= 240 Hz), 146.24, 140.19, 138.98, 137.49, 129.95, 129.23, 125.19,
= 6.7 Hz), 118.62 (d, = 2.0 Hz), 117.67, 117.32
= 21.7 Hz), 115.32, 113.64, 112.73, 39.29. LC-MS (ESI) m/z 488.17548 [M+H]+.
124.87, 120.79, 119.68, 119.48, 118.70 (d,
(d,
J
J
J
5.2.14 1-(3-fluorophenyl)-3-(3-((7-(thiophen-2-yl)quinazolin-4-yl)amino)phenyl)urea (28)
Synthesized as described in general procedure 5.1.2 from intermediate 13 and 1-fluoro-3-
isocyanatobenzene prepared as described in general procedure 5.1.1. Yield: 53%; 1H NMR (400
MHz, DMSO-d6)
(s, 1H), 8.08 – 8.01 (m, 2H), 7.80 (d,
(m, 2H), 7.33 – 7.24 (m, 3H), 7.12 (d,
DMSO-d6) 162.83 (d,
140.50, 139.97, 139.95, 139.68, 130.76 (d,
124.30, 124.08, 123.93, 116.88, 114.35, 114.30 (d,
105.21 (d,
= 26.5 Hz). LC-MS (ESI) m/z 456.14355 [M+H]+.
δ 9.88 (s, 1H), 8.92 (s, 1H), 8.83 (s, 1H), 8.66-8.58 (m, 2H), 8.24 (s, 1H), 8.10
J
= 4.6 Hz, 1H), 7.72 (dd,
J
= 4.9, 2.9 Hz, 1H), 7.54 – 7.48
13
J
= 8.1 Hz, 1H), 6.82-6.74 (m, 1H); C NMR (100 MHz,
δ
J
= 240.6 Hz), 158.07, 155.21, 152.76, 150.47, 142.01 (d,
= 9.8 Hz), 129.15, 128.05, 127.06, 126.85, 125.18,
= 2.5 Hz), 112.86, 108.55 (d, = 21.2 Hz),
J = 11.4 Hz),
J
J
J
J
5.2.15 1-(4-fluorophenyl)-3-(3-((7-(thiophen-2-yl)quinazolin-4-yl)amino)phenyl)urea (29)

Synthesized as described in general procedure 5.1.2 from intermediate 13 and 1-fluoro-4-
isocyanatobenzene prepared as described in general procedure 5.1.1. Yield: 58%; 1H NMR (400
MHz, DMSO-d6)
= 2.9, 1.3 Hz, 1H), 8.09 – 8.01 (m, 2H), 7.97 (s, 1H), 7.78 (dd,
5.0, 2.9 Hz, 1H), 7.46-7.40 (m, 3H), 7.29-7.20 (m, 2H), 7.09 (t,
MHz, DMSO-d6) 158.92,157.38 (d, J = 164.7 Hz), 154.98, 153.01, 149.72, 140.41, 140.27,
δ
9.92 (s, 1H), 8.73 (s, 1H), 8.69 (s, 1H), 8.60 (d,
J
= 7.6 Hz, 2H), 8.23 (dd,
J
J
= 5.1, 1.3 Hz, 1H), 7.70 (dd,
J
=
J
= 8.9 Hz, 2H); 13C NMR (100
δ
139.85, 139.74, 136.45 (d, J = 2.3 Hz), 129.13, 128.10, 126.82, 125.33, 124.40, 124.20, 123.35,
120.30 (d, J = 7.7 Hz) (2 * CH), 116.84, 115.72 (d, J = 22.2 Hz) (2 * CH), 114.47, 114.22,
112.93. LC-MS (ESI) m/z 456.11871 [M+H]+.
5.2.16 1-(3-fluorophenyl)-3-(3-((7-(4-methoxyphenyl)quinazolin-4-yl)amino)phenyl)urea (30)
Synthesized as described in general procedure 5.1.2 from intermediate 14 and 1-fluoro-3-
isocyanatobenzene prepared as described in general procedure 5.1.1. Yield: 79%; 1H NMR (400
MHz, DMSO-d6)
Hz, 1H), 8.00 (s, 2H), 7.86 (d,
(d, = 8.7 Hz, 3H), 6.78 (td,
162.83 (d, = 240.6 Hz), 160.45, 158.71, 154.00, 152.86, 146.79, 145.41, 142.06 (d,
Hz), 140.19, 139.15, 130.78, 130.69, 129.22, 128.96 (2 * CH), 125.92, 124.77, 121.26, 117.50,
115.15, 115.09 (2 * CH), 114.21 (d, = 2.4 Hz), 113.51, 113.43, 108.49 (d, = 21.3 Hz), 105.11
(d,
= 26.6 Hz), 55.73; LC-MS (ESI) m/z 480.19550 [M+H]+.
δ
10.44 (s, 1H), 9.19 (s, 1H), 9.09 (s, 1H), 8.75-8.67 (m, 2H), 8.05 (d,
= 8.6 Hz, 2H), 7.51 (t, = 9.2 Hz, 2H), 7.37-7.26 (m, 3H), 7.12
= 8.5, 2.4 Hz, 1H), 3.84 (s, 3H); 13C NMR (100 MHz, DMSO-d6)
= 11.4
J = 8.8
J
J
J
J
δ
J
J
J
J
J
5.2.17 1-(4-fluorophenyl)-3-(3-((7-(4-methoxyphenyl)quinazolin-4-yl)amino)phenyl)urea (31)
Synthesized as described in general procedure 5.1.2 from intermediate 14 and 1-fluoro-4-
isocyanatobenzene prepared as described in general procedure 5.1.1. Yield: 76%; 1H NMR (400
MHz, DMSO-d6)
8.00 – 7.93 (m, 2H), 7.85 (d,
(m, 4H), 3.83 (s, 3H); 13C NMR (100 MHz, DMSO-d6)
δ
9.84 (s, 1H), 8.75 (s, 1H), 8.72 (s, 1H), 8.68 – 8.59 (m, 2H), 8.04 (s, 1H),
= 8.6 Hz, 2H), 7.55 – 7.44 (m, 3H), 7.33-7.24 (m, 2H), 7.17-7.05
160.18, 158.09, 157.745 (d, J=237
J
δ
Hz), 155.25, 153.01, 150.59, 144.45, 140.22, 140.02, 136.46 (d, J = 2.4 Hz), 131.30, 129.10,
128.88, 128.84 (2 * CH), 125.19, 124.24, 124.19, 120.32 (d, J = 7.7 Hz) (2 * CH), 116.58,
115.72 (d, J = 22.2 Hz) (2 * CH), 115.00 (2 * CH), 114.19, 112.70, 55.69. LC-MS (ESI) m/z
480.19580 [M+H]+.

5.2.18
1-(3-fluorophenyl)-3-(3-((7-(4-(methylsulfonyl)phenyl)quinazolin-4-
yl)amino)phenyl)urea (32)
Synthesized as described in general procedure 5.1.2 from intermediate 12 and 1-fluoro-3-
isocyanatobenzene prepared as described in general procedure 5.1.1. Yield: 62%; 1H NMR (400
MHz, DMSO-d6)
(m, 7H), 7.54-7.45 (m, 2H), 7.37-7.27 (m, 3H), 7.12 (d,
(s, 3H). 13C NMR (100 MHz, DMSO-d6)
172.49, 162.81 (d,
152.85, 144.05, 143.51, 141.99 (d, = 11.4 Hz), 141.18, 140.20, 138.87, 130.75 (d,
129.29, 128.77, 128.25, 126.53, 125.22, 123.04, 117.68, 115.46, 114.70, 114.23 (d,
δ
9.26 (s, 1H), 9.15 (s, 1H), 8.80 (d,
J
= 8.5 Hz, 1H), 8.76 (s, 1H), 8.14 – 8.07
= 8.1 Hz, 1H), 6.8-6.72 (m, 1H), 3.29
= 240.6 Hz), 158.86, 154.18,
J
δ
J
J
J
J
= 9.8 Hz),
= 2.4 Hz),
113.62, 108.54 (d,
[M+H]+.
J = 21.2 Hz), 105.13 (d, J = 26.5 Hz), 43.92. LC-MS (ESI) m/z 528.15308
5.2.19
1-(4-fluorophenyl)-3-(3-((7-(4-(methylsulfonyl)phenyl)quinazolin-4-
yl)amino)phenyl)urea (33)
Synthesized as described in general procedure 5.1.2 from intermediate 12 and 1-fluoro-4-
isocyanatobenzene prepared as described in general procedure 5.1.1. Yield: 66%; 1H NMR (400
MHz, DMSO-d6)
7.5 Hz, 4H), 8.10 – 8.02 (m, 5H), 7.54 – 7.45 (m, 4H), 7.33 – 7.24 (m, 2H), 7.12 (t,
2H), 3.29 (s, 4H); 13C NMR (100 MHz, DMSO-d6)
157.76 (d, J=237 Hz), 158.15, 155.59,
δ
9.92 (s, 1H), 8.75 (s, 1H), 8.71 (d,
J
= 6.4 Hz, 2H), 8.64 (s, 1H), 8.16 (t,
J =
J
= 8.9 Hz,
δ
153.02, 150.49, 144.03, 142.99, 140.86, 140.22, 139.84, 136.40 (d, J = 2.4 Hz), 129.15, 128.70
(2 *CH), 128.17 (2 *CH), 126.23, 125.61, 124.64, 120.38 (d, J = 7.7 Hz, 2 * CH), 116.73,
115.73 (d, J = 22.2 Hz, 2 * CH), 115.39, 114.42, 112.86, 43.95. LC-MS (ESI) m/z 528.15308
[M+H]+. LC-MS (ESI) m/z 528.11365 [M+H]+.
5.2.20
1-(3-fluorophenyl)-3-(3-((6-(1-methyl-1H-pyrazol-4-yl)quinazolin-4-
yl)amino)phenyl)urea (34)
Synthesized as described in general procedure 5.1.2 from intermediate 15 and 1-fluoro-3-
isocyanatobenzene prepared as described in general procedure 5.1.1. Yield: 71%; 1H NMR (400
MHz, DMSO-d6)
1H), 8.26 (d, = 8.6 Hz, 1H), 8.13 (s, 1H), 7.95 (s, 1H), 7.86 (d,
11.8 Hz, 1H), 7.47 – 7.24 (m, 4H), 7.12 (d,
= 7.7 Hz, 1H), 6.77 (s, 1H), 3.92 (s, 3H); 13C NMR
δ
11.26 (s, 1H), 9.53 (s, 1H), 9.43 (s, 1H), 9.03 (s, 1H), 8.81 (s, 1H), 8.36 (s,
J
J
= 8.6 Hz, 1H), 7.51 (d,
J =
J

(100 MHz, DMSO-d6)
Hz), 140.45, 139.39, 137.84, 137.13, 133.24, 133.10, 130.75 (d,
122.29, 121.05, 119.16, 118.66, 116.36, 114.78, 114.47, 114.14 (d,
δ
162.815 (d, J=239 Hz), 159.57, 152.91, 151.21, 142.03 (d,
= 9.8 Hz), 129.41, 129.23,
= 2.3 Hz), 108.49 (d,
J
= 26.6 Hz), 39.31. LC-MS (ESI) m/z 454.20142 [M+H]+.
J = 11.4
J
J
J =
21.1 Hz), 105.03 (d,
5.2.25
1-(4-fluorophenyl)-3-(3-((6-(1-methyl-1H-pyrazol-4-yl)quinazolin-4-
yl)amino)phenyl)urea (35)
Synthesized as described in general procedure 5.1.2 from intermediate 15 and 1-fluoro-4-
isocyanatobenzene prepared as described in general procedure 5.1.1. Yield: 73%; 1H NMR (400
MHz, DMSO-d6)
(m, 3H), 7.76 (d,
(s, 3H); 13C NMR (100 MHz, DMSO-d6)
δ
9.74 (s, 1H), 8.74 (d,
= 8.6 Hz, 1H), 7.52-7.41 (m, 3H), 7.35-7.22 (m, 2H), 7.17-7.01 (m, 2H), 3.92
157.765 (d, J=237 Hz), 157.95, 154.11, 153.03,
J = 18.8 Hz, 3H), 8.54 (s, 1H), 8.27 (s, 1H), 8.10 – 8.00
J
δ
148.50, 140.27, 139.84, 136.90, 136.40 (d, J = 2.4 Hz), 131.25, 131.08, 129.15, 128.76, 128.65,
121.83, 120.37 (d, J = 7.7 Hz) (2 *CH), 117.92, 116.85, 115.72 (d, J = 22.2 Hz) (2 *CH), 115.61,
114.39, 112.96, 39.21; LC-MS (ESI) m/z 454.18842 [M+H]+.
5.2.26
4-methoxy-N-(3-((7-(1-methyl-1H-pyrazol-4-yl)quinazolin-4-
yl)amino)phenyl)benzamide (36)
Synthesized as described in general procedure 5.1.3 from intermediate 11 and commercially
available 4-methoxybenzoic acid. Yield: 79 %; 1H NMR (400 MHz, DMSO-d6)
10.53 (s, 1H),
δ
10.24 (s, 1H), 8.72-8.65 (m, 2H), 8.46 (s, 1H), 8.30 (s, 1H), 8.13 (s, 1H), 8.03-7.9 (m, 4H), 7.60-
13
7.51 (m, 2H), 7.39 (t,
J
= 8.0 Hz, 1H), 7.07 (d,
J
= 8.7 Hz, 2H), 3.92 (s, 3H), 3.84 (s, 3H); C
NMR (100 MHz, DMSO-d6)
δ
165.39, 162.36, 158.75, 153.77, 140.03, 138.96, 138.72, 137.50,
130.09 (2* CH), 129.95, 128.94, 127.27, 125.18, 124.92, 120.81, 119.27, 118.71, 118.67,
117.56, 116.05, 114.03 (2* CH), 112.75, 55.87, 39.30; LC-MS (ESI) m/z 451.2209 [M+H]+
5.22.7 3-fluoro-N-(3-((7-(1-methyl-1H-pyrazol-4-yl)quinazolin-4-yl)amino)phenyl)benzamide
(37)
Synthesized as described in general procedure 5.1.3 from intermediate 11 and commercially
available 3-fluoro benzoic acid. Yield: 73 %; 1H NMR (400 MHz, DMSO-d6)
10.14 (s, 1H), 8.68-8.58 (m, 2H), 8.43 (s, 1H), 8.35 (s, 1H), 8.13 (s, 1H), 7.97 – 7.90 (m, 2H),
δ 10.45 (s, 1H),

13
7.88-7.76 (m, 2H), 7.65 – 7.52 (m, 3H), 7.49-7.34 (m, 2H), 3.91 (s, 3H); C NMR (100 MHz,
DMSO-d6) 164.60 (d, = 2.4 Hz), 162.71, 162.34 (d, = 244.3 Hz), 158.30, 154.62, 149.04,
139.47, 138.20, 137.62 (d, = 6.8 Hz), 137.40, 130.99 (d, = 8.0 Hz), 129.74, 128.94, 124.69,
124.52, 124.38 (d, = 2.7 Hz), 121.08, 120.83, 119.15, 118.91 (d, = 21.1 Hz), 116.99, 115.60,
114.98 (d,
= 22.9 Hz), 113.26, 39.26; LC-MS (ESI) m/z 459.2507 [M+H]+
δ
J
J
J
J
J
J
J
5.2.28 3-bromo-N-(3-((7-(1-methyl-1H-pyrazol-4-yl)quinazolin-4-yl)amino)phenyl)benzamide
(38)
Synthesized as described in general procedure 5.1.3 from intermediate 11 and commercially
1
available 3-bromobenzoic acid. Yield: 42 %; H NMR (400 MHz, DMSO-d6)
δ 10.43 (s, 1H),
9.81 (s, 1H), 8.61-8.53 (m, 2H), 8.43 (s, 1H), 8.35 (s, 1H), 8.17 (s, 1H), 8.13 (s, 1H), 8.01 – 7.93
(m, 2H), 7.89 (d, = 8.6 Hz, 1H), 7.81 (d, = 8.0 Hz, 1H), 7.64 (d, = 8.0 Hz, 1H), 7.55-7.45
(m, 2H), 7.40-7.34 (m, 1H), 3.91 (s, 3H); 13C NMR (100 MHz, DMSO-d6)
164.41, 157.99,
J
J
J
δ
155.26, 150.92, 139.90, 139.39, 137.69, 137.52, 137.35, 134.72, 131.08, 130.69, 129.61, 128.89,
127.34, 124.35, 124.16, 122.33, 122.10, 121.25, 118.75, 116.49, 115.19, 113.59, 39.24; LC-MS
(ESI) m/z 499.2083 [M+H]+
5.2.29
N-(4-isopropylphenyl)-3-((7-(1-methyl-1H-pyrazol-4-yl)quinazolin-4-
yl)amino)benzamide (42)
Synthesized as described in general procedure 5.1.3 from intermediate 41 and commercially
available 4-isopropylaniline. Yield: 68 % 1H NMR (400 MHz, DMSO-d6)
10.18 (s, 1H), 9.90
(s, 1H), 8.62 – 8.50 (m, 2H), 8.41 (s, 1H), 8.33 (s, 1H), 8.21 – 8.08 (m, 2H), 7.94 (s, 1H), 7.88
(d, = 8.9 Hz, 1H), 7.72-7.63 (m, 3H), 7.51 (t, = 7.9 Hz, 1H), 7.19 (d, = 8.3 Hz, 2H), 3.88 (s,
3H), 2.84 (dt, = 13.8, 6.9 Hz, 1H), 1.17 (d,
= 6.9 Hz, 6H); 13C NMR (100 MHz, DMSO-d6)
δ
J
J
J
J
J
δ
165.73, 157.90, 155.20, 150.92, 144.18, 139.93, 137.81, 137.38, 137.32, 135.92, 129.65, 128.85,
126.75 (2 * CH), 125.54, 124.51, 124.06, 122.84, 122.34, 122.00, 121.22, 120.90 (2 * CH),
113.54, 39.25, 33.35, 24.41 (2 * CH3); LC-MS (ESI) m/z 463.2256 [M+H]+
5.2.30 N-(3-fluorophenyl)-3-((7-(1-methyl-1H-pyrazol-4-yl)quinazolin-4-yl)amino)benzamide
(43)

Synthesized as described in general procedure 5.1.3 from intermediate 41 and commercially
available 3-fluoroaniline. Yield: 52 %; 1H NMR (400 MHz, DMSO-d6)
10.45 (s, 1H), 9.91 (s,
1H), 8.61 – 8.51 (m, 2H), 8.41 (s, 1H), 8.35 (s, 1H), 8.18 (d, = 8.1 Hz, 1H), 8.11 (s, 1H), 7.94
(s, 1H), 7.88 (d, = 8.6 Hz, 1H), 7.74 (d, = 11.8 Hz, 1H), 7.68 (d, = 7.6 Hz, 1H), 7.59 – 7.51
(m, 2H), 7.4—7.32 (m, 1H), 6.95-6.86 (m, 1H), 3.88 (s, 3H); 13C NMR (100 MHz, DMSO-d6)
166.24, 162.48 (d, = 240.9 Hz), 157.88, 155.19, 150.94, 141.37 (d, = 11.0 Hz), 140.03,
= 9.5 Hz), 129.65, 128.97, 125.80, 124.53, 124.05, 122.89,
= 2.7 Hz), 113.54, 110.55 (d, = 21.1 Hz), 107.37 (d,
δ
J
J
J
J
δ
J
J
137.83, 137.39, 135.50, 130.70 (d,
122.36, 121.97, 121.21, 116.40 (d,
J
J
J
J =
26.3 Hz), 39.25; LC-MS (ESI) m/z 439.2066 [M+H]+
5.2.31 N-(4-fluorophenyl)-3-((7-(1-methyl-1H-pyrazol-4-yl)quinazolin-4-yl)amino)benzamide
(44)
Synthesized as described in general procedure 5.1.3 from intermediate 41 and commercially
available 4-fluoroaniline. Yield: 59 % 1H NMR (400 MHz, DMSO-d6)
1H), 8.64 – 8.54 (m, 2H), 8.44 (s, 1H), 8.38 (s, 1H), 8.20 (d, = 7.8 Hz, 1H), 8.14 (s, 1H), 7.97
(s, 1H), 7.91 (d, = 8.3 Hz, 1H), 7.86 - 7.77 (m, 2H), 7.72 (d, = 7.5 Hz, 1H), 7.60 – 7.51 (m,
1H), 7.21 (t,
= 8.8 Hz, 2H), 3.91 (s, 3H); 13C NMR (100 MHz, DMSO-d6)
= 240.1 Hz), 157.89, 155.19, 150.93, 139.99, 137.81, 137.38, 135.95 (d,
129.64, 128.91, 125.65, 124.51, 124.06, 122.83, 122.60 (d,
δ 10.35 (s, 1H), 9.94 (s,
J
J
J
J
δ
165.87, 158.71 (d,
J
J = 2.5 Hz), 135.68,
J
= 7.8 Hz) (2 * CH), 122.36, 121.98,
121.22, 115.63 (d,
J
= 22.2 Hz) (2 * CH), 113.55, 39.24; LC-MS (ESI) m/z 439.2048 [M+H]+
5.2.31
1-(3-fluorophenyl)-3-(4-((7-(1-methyl-1H-pyrazol-4-yl)quinazolin-4-
yl)amino)phenyl)urea (45)
Synthesized as described in general procedure 5.1.2. The synthetic scheme is exactly same as
that of 24, except that 1,4-benzenediamine was used instead of 1,3-benzenediamine. Yield: 71
%; 1H NMR (400 MHz, DMSO-d6)
(d, = 8.6 Hz, 1H), 8.48 (s, 1H), 8.14 (s, 1H), 8.08 (d,
(m, 2H), 7.56 (d, = 8.9 Hz, 2H), 7.51 (d, = 11.9 Hz, 1H), 7.34-7.26 (m, 1H), 7.13 (d,
Hz, 1H), 6.81-6.75 (m, 1H), 3.93 (s, 3H). 13C NMR (100 MHz, DMSO-d6)
162.83 (d,
240.6 Hz), 159.09, 152.93, 152.19, 142.09 (d, = 11.4 Hz), 141.51, 140.16, 138.14, 137.61,
131.37, 130.74 (d, = 9.7 Hz), 130.26, 125.97, 125.51, 125.42 (2 * CH), 120.36, 118.55 (2 *
δ
11.08 (s, 1H), 9.28 (s, 1H), 9.19 (s, 1H), 8.80 (s, 1H), 8.69
= 9.0 Hz, 1H), 7.88 (s, 1H), 7.67-7.61
= 7.9
J
J
J
J
J
δ
J =
J
J

CH), 114.95, 114.14 (d,
J = 2.4 Hz), 111.81, 108.45 (d, J = 21.1 Hz), 105.04 (d, J = 26.5 Hz),
39.35; LC-MS (ESI) m/z 454.16473 [M+H]+
5.3 Aurora Kinase B enzymatic assay procedure
Kinase activity was measured in a microfluidics assay that monitors the separation of a
phosphorylated product from substrate. The assay was run using a 12-sipper chip on a Caliper
EZ Reader II (PerkinElmer®, Walthman, USA) with separation buffer (100
EDTA, 0.015% Brij-35, 0.1% CR-3 [PerkinElmer®, Walthman, USA]). In 96-well
polypropylene plates (Greiner, Frickenhausen, Germany) compound stocks (20 mM in DMSO-
d6) were diluted into kinase buffer (20 mM MOPS, 0.01% Brij-35, 5% glycerol, 2 mM DTT,
30 mM MgCl2 and 1mg/mL BSA) in 12-point ½log dilutions (2 mM–6.32 nM). After, 1ꢀμL was
ꢀmM HEPES, 10ꢀmM
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
transferred into a 384-well polypropylene assay plate (Greiner, Frickenhausen, Germany). The
Aurora B enzyme (Invitrogen™, Grand Island, USA) was diluted in kinase buffer to a
concentration of 2
ꢀ
nM and 5
ꢀµL of the enzyme mixture was transferred to the assay plate. The
inhibitors/FLT3 enzyme were incubated for 60
ꢀ
minutes with minor shaking. A substrate mix was
prepared containing ATP (Ambresco®, Solon, USA) and 5FAM tagged peptide (peptide #21, 5-
FAM-LRRASLG-CONH2, PerkinElmer®, Walthman, USA) dissolved in kinase buffer, and
5
ꢀµL of the substrate mix was added to the assay plate. Running concentrations were as follows:
ATP (190 µM), peptide (1.5 µM), compound 12-point ½log dilutions (0.2 mM–0.632 nM). For
ꢀ
ꢀ
ꢀ
positive control, no inhibitor was added. For negative control, no enzyme was added. For
running control, quizartinib was utilized. The plate was run until 10–20% conversion based on
the positive control wells. The following separation conditions were utilized: upstream voltage
−500V; downstream voltage, −1900V; chip pressure −0.8. Percent inhibition was measured for
each well comparing starting peptide to phosphorylated product peaks relative to the baseline.
Dose response curves, spanning the IC50 dose, were generated in GraphPad Prisim 6 and fit to an
exponential one-phase decay line and IC50 values were obtained from the half-life value of the
curve. IC50 values were generated in duplicate and error was calculated from the standard
deviation between values.
5.4 Cellular assays

The growth inhibition of 60 cell lines was obtained by submitting the compound to National
Cancer Institute’s NCI60 panel. MCF-7 cells were cultured in RPMI-1640 medium with 5% FBS
in an incubator maintained at 37 ˚C and 5% CO2. The media was changed on alternate days.
After reaching confluency, the media was aspirated, cells were washed with PBS, detached using
0.25% trypsin and centrifuged. The collected cells were seeded in 96-well microtiter plates at a
density of 5000 cells/well. The cells were allowed to adhere to plate overnight. The test
compounds, vehicle control and positive controls were added 24 hours after the cells were plated
and allowed to incubate. Following 24 hours of incubation, the media was aspirated, and the cells
were washed with PBS. 40 µL of the media and 10 µL of 5 mg/mL of MTT solution prepared in
PBS was added to the cells and incubated for 4 hours at 37 °C and 5% CO2. After incubation,
150 µL of DMSO was added to each well and the cell viability was measured at 570 nM by an
ELISA plate reader.
5.5 Molecular modeling
Aurora Kinase B crystal structure was retrieved from RCSB Protein Data Bank. The protein was
prepared using Protein Preparation Wizard module of Maestro2018-2 software suite. Bond
orders were assigned properly and missing hydrogens were added at this stage. The prepared
protein structure was used for grid preparation and further docking studies. A grid was defined
around the ATP binding site covering the hinge region, DFG residues and the residues of the αC-
helix facing the ATP pocket. Using Autodock Vina, the ligand was docked into the grid. The
results were visualized using Maestro 2018-2 suite. The docked protein-ligand complex was
simulated using the Desmond package bundled with Maestro software received from D.E. Shaw
Research with an academic license. A triclinic shaped box was defined around the complex with
any atom of the complex at least 10 A away from the boundary of the box. The box was solvated
with SPC water molecules. NaCl was added to neutralize the system and to adjust the pH to
physiological values. The prepare system was equilibrated in NVT and NPT ensembles. The
converged system was subjected to molecular dynamics with default parameters. The results
were visualized using Maestro 2018-2 suite.
Acknowledgements

This work was supported by the Arkansas Research Alliance research grant 271/G1-53308-01
awarded to HYL.
Declaration of competing interest
The authors declare no conflict of interest
Author contributions
The idea was conceived by NRL, BF and HYL. The synthesis was done by NRL and NG. The
assays were performed by LZ and BB. The article was written by NRL and BF, and edited by all.
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