2,4-Diamino-8-quinazoline carboxamides as novel, potent inhibitors of the NAD hydrolyzing enzyme CD38: Exploration of the 2-position structure-activity relationships

Article abstract of DOI:10.1016/j.bmc.2018.03.021

Starting from 4-amino-8-quinoline carboxamide lead 1a and scaffold hopping to the chemically more tractable quinazoline, a systematic exploration of the 2-substituents of the quinazoline ring, utilizing structure activity relationships and conformational constraint, resulted in the identification of 39 novel CD38 inhibitors. Eight of these analogs were 10–100-fold more potent human CD38 inhibitors, including the single digit nanomolar inhibitor 1am. Several of these molecules also exhibited improved therapeutic indices relative to hERG activity. A representative analog 1r exhibited suitable pharmacokinetic parameters for in vivo animal studies, including moderate clearance and good oral bioavailability. These inhibitor compounds will aid in the exploration of the enzymatic functions of CD38, as well as furthering the study of the therapeutic implications of NAD enhancement in metabolic disease models.

Full text of DOI:10.1016/j.bmc.2018.03.021

Accepted Manuscript
2
,4-Diamino-8-quinazoline carboxamides as novel, potent inhibitors of the
NAD hydrolyzing enzyme CD38: Exploration of the 2-position structure-activ-
ity relationships
David N. Deaton, Curt D. Haffner, Brad R. Henke, Michael R. Jeune, Barry G.
Shearer, Eugene L. Stewart, J. Darren Stuart, John C. Ulrich
PII:
S0968-0896(17)32375-1
https://doi.org/10.1016/j.bmc.2018.03.021
BMC 14259
DOI:
Reference:
To appear in:
Bioorganic & Medicinal Chemistry
Received Date:
Revised Date:
Accepted Date:
7 December 2017
1 March 2018
10 March 2018
Please cite this article as: Deaton, D.N., Haffner, C.D., Henke, B.R., Jeune, M.R., Shearer, B.G., Stewart, E.L.,
Stuart, J.D., Ulrich, J.C., 2,4-Diamino-8-quinazoline carboxamides as novel, potent inhibitors of the NAD
hydrolyzing enzyme CD38: Exploration of the 2-position structure-activity relationships, Bioorganic & Medicinal
Chemistry (2018), doi: https://doi.org/10.1016/j.bmc.2018.03.021
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2,4-Diamino-8-quinazoline carboxamides as novel, potent inhibitors of the NAD hydrolyzing enzyme CD38:
Exploration of the 2-position structure-activity relationships
*
,†
†
David N. Deaton, Curt D. Haffner, Brad R. Henke, Michael R. Jeune, Barry G. Shearer, Eugene L. Stewart,
†,‡
†
†
†
†,ǁ
J. Darren Stuart , John C. Ulrich
†,♯
†
GlaxoSmithKline Research and Development, 5 Moore Drive, P.O. Box 13398, Research Triangle Park, North
Carolina 27709 USA
‡
Current address: Opti-Mol Consulting LLC, Cary, NC 27513 USA
ǁ
Current address: Duke Molecular Physiology Center, 300 North Duke Street, Durham, NC 27701 USA
♯
Current address: Jacobs Technology, 111 Corning Road, Cary, NC 27518 USA
*
To whom correspondence should be addressed. Phone: 610-270-4425. E-mail: david.n.deaton@gsk.com.
KEYWORDS: Nicotinamide adenine dinucleotide, NAD, CD38, CD38 Inhibitor, NAD glycohydrolase
Abstract
Starting from 4-amino-8-quinoline carboxamide lead 1a and scaffold hopping to the chemically more tractable
quinazoline, a systematic exploration of the 2-substituents of the quinazoline ring, utilizing structure activity
relationships and conformational constraint, resulted in the identification of 39 novel CD38 inhibitors. Eight of
these analogs were 10-100-fold more potent human CD38 inhibitors, including the single digit nanomolar inhibitor
1am. Several of these molecules also exhibited improved therapeutic indices relative to hERG activity. A
representative analog 1r exhibited suitable pharmacokinetic parameters for in vivo animal studies, including
moderate clearance and good oral bioavailability. These inhibitor compounds will aid in the exploration of the
enzymatic functions of CD38, as well as furthering the study of the therapeutic implications of NAD enhancement
in metabolic disease models.
Introduction
1

Cluster of Differentiation 38 (CD38, EC 3.2.2.5) is a type II membrane glycoprotein, localized on chromosome 4
1
-2
and expressed in immune cells, such as T cells, B cells, and dendritic cells. CD38 has a single N-terminal
transmembrane domain with multiple asparagine-linked glycosylation sites at its extracellular C-terminal end. The
immunoglobulin Cluster of Differentiation 31 (CD31, PECAM-1) binds to CD38 and this interaction modulates
leukocyte migration.
CD38 is overexpressed on the surface of many hematological tumor cells and significant efforts have been made to
exploit the therapeutic implications of this finding for diagnosis and treatment. In 2015, the FDA approved
Genmab’s CD38 monoclonal antibody daratumumab (Darzalex™) for the treatment of multiple myeloma (plasma
3
cell myeloma). This therapeutic validation has intensified the interest in CD38.
Besides leukocytes, CD38 is also expressed in other tissues, including liver, intestine, kidney, pancreas, prostate,
4
muscle, bone, and brain. CD38 also has enzymatic activity in addition to its receptor functions. One of its
substrates is the redox cofactor nicotinamide adenine dinucleotide (NAD), which CD38 converts to cyclic ADP-
5
-6
ribose (cADPR) as well as ADP-ribose. cADPR is a second messenger that regulates cytosolic calcium fluxes.
Perhaps more importantly, CD38 reduces the concentration of NAD by functioning as a NAD glycohydrolase. By
decreasing the availability of this critical enzyme cofactor, CD38 modulates the activity of many NAD-dependent
proteins, including oxidoreductases, sirtuins, and the poly(ADP-ribose) polymerases (PARPs).
Inhibiting degradation of NAD via inhibition of CD38 is one approach to potentially increase NAD. Activation of
sirtuin1 increases insulin sensitivity, implying that elevation of NAD could have positive effects on metabolic
7
diseases. CD38 (-/-) mice have elevated NAD levels compared to wild-type littermates. Furthermore, the
8
knockout mice, when fed a high fat diet, are resistant to weight gain. They also have increased energy expenditure.
9
-10
Moreover, small molecule CD38 inhibitors elevate NAD levels in diet-induced obese mice.
This murine NAD paradigm is likely operational in humans as well, because the obese have decreased NAD levels,
yet exercise and caloric restriction, which both protect against metabolic diseases, elevate NAD levels. NAD
concentrations are also decreased in the elderly, while type II diabetes rates increase with age, and this inverse
11-12
relationship might be correlated.
Thus, treatment of metabolic diseases like obesity, where NAD levels are
2

abnormally low, with a CD38 inhibitor could have therapeutic utility, by increasing this important cofactor of
metabolic proteins.
Because of the potential therapeutic benefits of elevating NAD, the discovery of CD38 inhibitors has been pursued
1
3-18
by various researchers.
This laboratory has recently disclosed novel CD38 inhibitors, including the quinoline-8-
carboxamides exemplified by 1a (IC50 = 510 nM). Compound 1a had acceptable pharmacokinetics to be utilized
as a tool compound and raised NAD levels in a diet-induced obese mouse model; however, this compound had
modest potency and drug development liabilities such as hERG inhibition. CD38 protein X-ray co-crystal structures
of the quinoline-8-carboxamide series (e.g. PDB codes 4XJT and 4XJS) revealed additional space in the binding
pocket, near the 2-position of the quinoline, that could be occupied for potential increases in potency or alteration of
drug properties. Preliminary chemistry efforts at this position produced the pyrazole 1b (IC50 = 72 nM) with
increased CD38 inhibitory activity relative to its congener 1a. To increase the ease of synthesis and allow for rapid
structure-activity relationship (SAR) development at this position, the synthesis of quinazolines as a bioisosteric
core replacement for the quinoline were undertaken. This article describes GlaxoSmithKline’s detailed efforts to
explore the SAR at the 2-position of that quinazoline series.
Chemistry
The quinazoline CD38 inhibitors 1c-1ao were prepared as depicted in Scheme 1. Room temperature, base-
promoted, regioselective, ipso displacement of the 4-chloro group of 2,4-dichloroquinazoline 2 by 2-fluoro-6-
trifluoromethylbenzylamine 3 provided the 4-aminoquinazoline 4. Subsequent thermal, base-promoted, ipso
displacement of the 2-chloro moiety of the quinazoline 4 with amines 5a-5ai afforded the quinazoline nitriles 6a-
6
ai. Hydrolysis of the cyanides 6a-6ai with basic hydrogen peroxide gave the desired quinazoline carboxamides 1c-
ao.
1
Scheme 1. Synthesis of 2-substituted quinazoline carboxamides 1c-1ao
3

1
2
Reagents and conditions: a) iPr
2
NEt, 3, THF, RT, 81%; b) iPr
2
NEt, R R NH 5a-5ai, n-BuOH or 1,4-dioxane or
DMSO, 100 °C, 11-91%; c) NaOH or KOH, 30% H
2
O
2
, DMSO, 5-81%.
Many of the amines 5a-5k and 5m-5n were commercially available, while some amines 5l, 5s, 5z, and 5aa were
known in the literature. The rest of the amines 5o-5r, 5t-5y, and 5ab-5ai were synthesized as depicted in Schemes
2-3. Ketones 7a-7f were heated at reflux with N,N-dimethylformamide dimethyl acetal to produce β-amino-enones
8a-8f. Subsequent Michael addition with hydrazines, followed by cyclization, and dehydration afforded the
protected pyrazoles 9a-9g. Then, removal of the various protecting groups gave the free amines 5o-5r and 5t-5v.
Some ketones 7a and 7c were commercially available, but other ketones 7b and 7d-7f were prepared. For example,
protection of the aminoesters 10a-10b gave carbamates 11a-11b. Subsequent Michael addition of these species
11a-11b or commercially available compounds 10a-10f, 11c, and 11d to unsaturated esters 12a-12b, ketone 12c, or
nitrile 12d, then in situ Dieckmann or Thorpe-Ziegler cyclization afforded β-ketoesters 13a, 13d-13g, β-diketones
1
9-20
13h-13i, and β-ketonitriles 13j-13n.
The β-ketoesters 13a-13e could be decarboxylated under Krapcho
2
conditions to provide the ketones 7b and 7d-7f. Alternatively, the β-ketoesters 13f-13g, β-ketoesters 13h-13i ,and
1
β-ketonitriles 13j-13n could also be heated with hydrazine to give the protected pyrazolone 9j, 3-alkylpyrazoles 9l-
9
m, or 3-aminopyrazoles 9n-9r, then deprotected to give amines 5y and 5ac-5ai. In another method, the ketones
d-7e could be derived from the β-amino esters 14a-14b. First, monoalkylation of β-amino esters 14a or 14b with
7
ethyl 2-bromoacetate, followed by protection of the free secondary amines provided the carbamates 15a-15b. Then,
Dieckmann condensation provided the β-ketoesters 13b-13c. β-Diketones 16a-16b could be synthesized by Claisen
4

condensation of ketone 7c with esters or anhydrides. These diketones 16a-16b could be converted to pyrazoles 5w-
x as above via hydrazine addition to give pyrazoles 9h-9i, followed by deprotection.
5
Scheme 2. Synthesis of amines 5o-5r, 5t-5y, and 5ac-5ai
Reagents and conditions: a) (CH
3
)
2
NCH(OCH
/Pd-C, MeOH, 41-99%; d) PhCH
2d, NaH or LDA or NaHMDS, PhH or THF, -78-80 °C, 10-73%; f) NaCl, DMSO, H
CO Et, iPr NEt, dioxane 80 °C; CbzCl, 18-48%; h) LDA or NaHMDS, THF, -78 °C, 28%; i) LDA or
NaHMDS, THF, (RCO) O or RCO Et, -78 °C, 23-26%; j) 12d, NaOH, H O, 0 °C-25 °C; Me NOH, (tBuOC=O)
MeCN, 50 °C; KOtBu, MePh, 0 °C-25 °C, 9-33%.
3
)
2
, reflux, 12-91%; b) H
2
NNH
2
or H
NEt, CH
O, 120 °C; CbzCl, 28-89%;
2
NNHCH
3
, EtOH, 80 °C, 19-
66%; c) HCl, dioxane, MeOH or H
2
2
OCOCl, iPr
2
2
Cl
2
, 93-96%; e) 12a-
1
2
g) BrCH
2
2
2
2
2
2
4
2
O,
As shown in Scheme 3, the pyrazole aminoalcohol 5ab was synthesized from the ketoalcohol 17a. First, base-
promoted protection of the secondary alcohol 17a with tert-butyldiphenylsilyl chloride afforded the silylether 18a.
Then, Claisen condensation of the enolate of ketone 18a with diethyl oxalate 19a provided the diketoester 20a.
Aldol condensation of the β-diketoester 20a with Eschenmoser’s salt 21a, followed by elimination of dimethylamine
5

provided a methylene diketone, which was subjected in situ to conjugate addition by 4-methoxybenzylamine 22a
with subsequent cyclization to give the pyrrolidinone 23a. Then, condensation of the ketone 23a with hydrazine
gave the pyrazole 24a. Fluoride-mediated cleavage of the silyl ether protecting group of 24a provided the alcohol
25a. Subsequent reduction of the amide 25a afforded the pyrrolidine 9k. Finally, palladium hydroxide on carbon
catalyzed hydrogenolysis of the benzyl group of pyrazole 9k afforded the amine 5ab.
Scheme 3. Synthesis of pyrazole amine 5ab
Reagents and conditions: a) imidazole, TBDPSCl, DMF, 50 °C, 84%; b) 19a, NaOEt, EtOH, 60 °C, 96%; c) 21a,
MeCN, 0 °C to 25 °C; 22a, iPr
2
NEt, 0 °C to 25 °C, 58%; d) H
C, 29%; e), TBAF, THF, 25 °C, 77%; f) LiAlH , MePh, THF, 25 °C to 50 °C, 92%; g) H
C, 69%.
2
NNH
2
, NMP, 125 °C; K
2
CO
3
, EtOH, microwave, 110
°
°
4
2
/Pd(OH)
2
-C, MeOH, 50
Results and Discussion
6

The SAR among the quinazoline analogs are depicted in Table 1. Although the quinazolines were less difficult to
prepare than the corresponding quinolines, the 2-(4-aminomethylpyrazole)quinazoline carboxamide 1c (IC50 = 450
nM) was ~6-fold less potent than its corresponding quinoline 1b ( IC50 = 72 nM), possibly due to a higher penalty to
desolvate the additional nitrogen and/or alteration of the π-stacking energy of the quinazoline ring with the indole
1
89
ring of Trp, relative to the quinoline ring. Like the previously prepared quinolines, these quinazolines reversibly
bind at the active site and exhibit mixed inhibition of CD38. Searching for the optimal heterocycle, the 4-pyrazole
of 1c was replaced with the regioisomeric pyrazole 1d ( IC50 = 2300 nM), the triazole 1e ( IC50 = 4300 nM), the
imidazoles 1f ( IC50 = 1600 nM) and 1g ( IC50 = 6800 nM), the tetrazole 1h ( IC50 = 8000 nM), the oxazoles 1i ( IC50
=
4200 nM) and 1j ( IC50 = 3400 nM), and the isoxazole 1k ( IC50 = 5500 nM); however, none of these analogs
inhibited CD38 as well as the 4-pyrazole. Possibly, a hydrogen bond donor was important for binding as the
regioisomeric pyrazole 1d and the imidazole 1f retained the most activity relative to 1c. Lone pair repulsion and/or
increased desolvation costs might account for the further decrease in inhibition of the other analogs.
With the pyrazole 1c appearing to be the best heterocycle, the N-methyl derivative 1l ( IC50 = 1900 nM) was
prepared, but was ~4-fold less active. Only the 3-methylpyrazole 1m (IC50 = 620 nM) retained similar inhibitory
potency to the parent pyrazole 1c. Fortunately, the constrained 1,4,5,6-tetrahydropyrrolo[3,4-c]pyrazole derivative
1n (IC50 = 92 nM) was ~5-fold more potent, likely by lowering the entropic penalty of binding in the active site of
the enzyme or decreasing the desolvation cost. Searching for the best constraint, the 5-membered ring of 1n was
replaced with the regioisomeric six-membered rings 1o (IC50 = 630 nM) and 1p (IC50 = 1900 nM), as well as the
seven-membered ring 1q (IC50 = 19000 nM), but none of these analogs proved superior to 1n.
To further explore the binding pocket of the constrained pyrazole 1n, various methyl derivatives were synthesized to
identify optimal substitutions that would maximize binding interactions with the CD38 enzyme and potentially
provide improved potency. The (R)-6-methyl analog 1r (IC50 = 51 nM) was ~8-fold more potent than its (S)-
antipode 1s (IC50 = 410 nM). While the N-methyl derivatives 1t (IC50 = 82 nM, Sol. = 2 μg/mL) and 1u (IC50 = 160
nM) did not significantly alter CD38 inhibition, the 2-methyl-tetrahydropyrrolopyrazole analog 1u was 2-fold less
potent than its 1-methyl congener 1t. Methylation of the pyrazole nitrogen locks resonance tautomers and
eliminates a hydrogen bond donor, but also lowers the desolvation penalty and those opposing factors likely cancel
each other out, resulting in little change in potency, but a decrease in solubility relative to 1n (Sol. = 6 μg/mL).
7

Similar to analog 1m, the 3-methyl derivative 1v (IC50 = 42 nM) was tolerated and led to a slight increase in
inhibitory activity. Finally, the 4-methyl analogs 1w (IC50 = 2500 nM) and 1x (IC50 = 330 nM) were both
detrimental to inhibitory binding, with the (R)-isomer ~8-fold better tolerated by the CD38 binding site than its (S)-
antipode.
Since the 3- and 6- positions of the 1,4,5,6-tetrahydropyrrolo[3,4-c]pyrazole were the best positions for enhancing
binding, additional optimization was focused at these positions. The (R)-6-ethyl analog 1y (IC50 = 71 nM) exhibited
similar potency as its (R)-6-methyl congener 1r, while the (S)-6-ethyl antipode 1z (IC50 = 370 nM) was less active,
similar to the (S)-6-methyl analog 1s. Also, the 3-ethyl derivative 1aa (IC50 = 83 nM) was tolerated, but did not
offer any advantage in potency relative to its 3-methyl analog 1v. The 3-trifluoromethyl analog 1ab (IC50 = 4100
nM) was a substantially less potent inhibitor of CD38, which is likely due to its increased size relative to the 3-
methyl analog 1v.
In an attempt to increase the aqueous solubility of these inhibitors, a series of small hydrophilic substitutions at the
3-position were prepared. The 1,2,5,6-tetrahydropyrrolo[3,4-c]pyrazol-3(4H)-one 1ac (IC50 = 1600 nM) was
detrimental to enzyme inhibition, but the 3-amino 1ad (IC50 = 110 nM), the 3-carboxamido 1ae (IC50 = 140 nM),
and the enantiomers alcohols 1af (IC50 = 110 nM) and 1ag (IC50 = 230 nM) were modest CD38 inhibitors with only
a slight decrease in potency relative to the 3-methyl analog 1v. Because the size of the pyrazolone 1ac can be
accomodated in the active site, lone pair-lone pair repulsion, change in the electronic character of the ring, and/or
desolvation effects might account for its poor inhibition. Pleasingly, the alcohols 1af (Sol. = 12 μg/mL) and 1ag
(
Sol. = 11 μg/mL) were slightly more soluble than there parent analog 1n (Sol. = 6 μg/mL).
Disubstitution of the template with optimal groups was also pursued. The (R)-3,6-dimethyl derivative 1ah (IC50
1 nM) slightly increased activity relative to monosubstituted analogs 1r and 1v. As expected, its (S)-antipode 1ai
IC50 = 170 nM) was less potent. Furthermore, the (R)-3-amino-6-methyl derivative 1aj (IC50 = 45 nM) also
=
2
(
improved inhibitory activity compared to the 3-amino analog 1ad.
Desiring to eliminate the chiral center to avoid chiral chromatography, derivatives with geminal disubstitution were
also synthesized. Fortuitously, and despite the (S)-3-methyl group reducing CD38 potency, the 3,3-dimethyl
derivative 1al (IC50 = 20 nM) was as active as the (R)-6-methyl analog 1aj. The spiro derivatives 1am (IC50 = 4.0
8

nM), 1an (IC50 = 7.8 nM), and 1ao (IC50 = 17 nM) were also prepared with the cyclopropyl 1am and cyclobutyl
analogs 1an being more optimal for enzyme inhibition. Additionally reducing the planarity of the 1,4,5,6-
tetrahydropyrrolo[3,4-c]pyrazole as demonstrated in these derivatives might decrease the crystal lattice energy of the
solids, resulting in faster dissolution and potentially enhancing solubility.
Table 1. Human CD38 enzyme inhibition data.
1
R
a
1
R
a
#
X
IC50 ± S.D.
nM
#
X
IC50 ± S.D.
nM
1
1
a
CH
CH
510 ± 100
1v
N
N
42 ± 22
b
c
72 ± 13
450 ± 81
2300 ± 37
1w
1x
1y
2500 ± 120
330 ± 43
71 ± 0.0
1
1
N
N
N
N
d
9

1
1
1
e
f
N
N
N
4300 ± 210
1600 ± 610
6800 ± 4800
1z
N
N
N
370 ± 18
83 ± 5.4
1aa
1ab
g
4100 ± 200
1
1
1
h
N
N
N
8000 ± 2400
4200 ± 200
3400 ± 1800
1ac
1ad
1ae
N
N
N
1600 ± 320
110 ± 29
140 ± 13
i
j
1
1
k
N
N
5500 ± 1600
1900 ± 120
1af
N
N
110 ± 12
230 ± 34
l
1ag
b
1
1
m
n
N
N
620 ± 10
92 ± 7.5
1ah
1ai
N
N
21
170 ± 28
10

1
1
1
1
1
1
o
p
q
r
s
N
N
N
N
N
N
N
630 ± 21
1900 ± 390
19000 ± 1800
51 ± 3.3
1aj
N
N
N
N
N
N
45 ± 0.0
310 ± 5.0
20 ± 5.5
1ak
1al
1am
1an
1ao
4.0 ± 3.6
7.8 ± 0.76
17 ± 0.54
410 ± 100
t
82 ± 22
1
u
160 ± 19
a
S.D. = standard deviation; mean ± S.D. All inhibitors were tested with N ≥ 4 unless overwise noted.
b
N = 1.
Utilizing the coordinates from this laboratory’s previously published CD38 protein co-crystal structures, inhibitors
10,22
1ah and 1am were docked into the active site.
Depictions of the predicted binding complex of CD38 and these
compounds are shown in Figures 1 and 2. As with similar CD38 inhibitors, these analogs contain a carboxamide
group coplanar to the quinazoline ring and form an intramolecular hydrogen bond between the protonated basic
quinazoline N1 nitrogen and a lone pair of the carbonyl oxygen of the carboxamide. The nitrogen forms hydrogen
1
bonds with the carboxylate side chains of the protein residues Glu and Asp. The indole ring of Trp forms a
46
155
189
11

π-π face to face stacking interaction with the quinazoline ring. This interaction is facilitated by the carboxamide
withdrawing electron density from the quinoline ring, increasing its acceptance of electron density from the electron
rich indole ring. Moreover, a triple π- π -π stacking interaction exists between the adenine ring of the substrate, the
176
indole ring of Trp, and the 4-benzyl ring, whose conformation is facilitated by its two ortho substituents. The 4-
221
amino group of these compounds forms a hydrogen bond with the side chain hydroxyl oxygen of Thr. The
1,4,5,6-tetrahydropyrrolo[3,4-c]pyrazole occupies a pocket with its N2-nitrogen forming a hydrogen bond with the
152
guanidine side chain of Arg. The 6-methyl and cyclopropyl groups of their respective compounds form
185
hydrophobic interactions with the side chain of Ile, while the 3-methyl moiety interacts with the side chain of
1
57
Leu. Moreover, the docking results show that compounds such as 1am form a water-mediated hydrogen bond
1
76
between the 3-amino group of the tetrahydropyrrolopyrazole and the backbone nitrogen of Trp. This additional
interaction may explain the improved CD38 potency for many of the 1,4,5,6-tetrahydropyrrolo[3,4-c]pyrazole-3-
amines. Most importantly, these models provided insight into the binding modes of this class of CD38 inhibitors
and aid in rationalizing much of the SAR observed in the quinazoline carboxamide inhibitor series.
2
26
Figure 1. Docking model of 1ah in the hCD38 E Q mutant protein bound with ADPR (from PDB code: 4XJT). The hCD38 carbons are
colored green, with the inhibitor carbons colored blue and ADPR carbons colored magenta. Hydrogen bonds are depicted as yellow dashed lines.
This figure was generated using PyMOL (The PyMOL Molecular Graphics System, version 1.7.6.6, Schrödinger, LLC).
The quinoline carboxamide 1a was a potent human ether-a-go-go-related gene (hERG) inhibitor (hERG pIC50
=
2
.4), offering no therapeutic index between its CD38 inhibition and its hERG liability. In contrast, analogs 1r
3
6
(
hERG pIC50 = 5.5) and 1v (hERG pIC50 = 4.9) were less potent hERG inhibitors with better CD38 inhibition,
12

relative to 1a. Although 2-substitution decreased potential QTc liabilities, those derivatives were detrimental to
cytochrome p450 inhibition. Quinoline carboxamide 1a was a modest 3A4 inhibitor (3A4 pIC50 = 4.9), while
inhibitors 1r (3A4 pIC50 = 6.8) and 1v (3A4 pIC50 = 6.2) were substantially more potent, limiting their development
potential due to possible drug-drug interactions.
The physiochemical properties and pharmacokinetic parameters of a representative inhibitor 1r were also
24
determined. Its artificial membrane permeability was high (147 nm/sec), while its aqueous solubility in fasted
25
state-simulated intestinal fluid (FaSSIF) was low (FaSSIF = 0.034 mg/mL). The iv in vivo half-life of 1r in mice
(
2.0 mg/kg dose) was moderate (t1/2 = 1.4 h), as were the clearance (C = 37 mL/min/kg) and steady state volume of
l
distribution (VSS = 2.7 L/kg). Despite its low solubility, 1r exhibited good oral bioavailability in mice (F = 69%).
with an oral dose normalized area under the curve exposure equal to 327 ng/hr/mL (30 mg/kg dose).
2
26
Figure 2. Docking model of 1am in the hCD38 E Q mutant protein bound with ADPR (from PDB code: 4XJT). The hCD38 carbons are
colored green, with the inhibitor carbons colored cyan and ADPR carbons colored magenta. Hydrogen bonds are depicted as yellow dashed lines.
This figure was generated using PyMOL (The PyMOL Molecular Graphics System, version 1.7.6.6, Schrödinger, LLC).
Conclusion
In summary, starting from the moderately potent quinoline carboxamide 1a, replacement of the 2-methyl subtituent
was investigated in the more chemically tractable quinazoline template. Human CD38 inhibitors were iteratively
prepared, by employing feedback from SAR and utilizing predicted binding modes of inhibitors for CD38. These
efforts led to the identification of several low nanomolar CD38 inhibitors. For example, quinazoline 1r is an order
of magnitude more potent than the starting quinoline 1a with good oral bioavailability and sufficient in vivo half-life
13

in mice to be employed as a tool compound to investigate CD38 biology. Furthermore, inhibitor 1am is ~160-fold
more potent than 1a, while maintaining a good ligand efficiency (LE = 0.31) and lowering the chromatographic log
D (1a Chrom Log D = 5.1, 1am Chrom Log D = 3.9). Since GSK decided to exit the obesity therapeutic area and
terminate the CD38 inhibitor project, no pharmacokinetics or pharmacodynamic studies were performed on the best
analogs in this series, such as 1am. Nevertheless, these inhibitors should aid the investigation of the enzymatic roles
of CD38 and help disclose the potential of NAD elevation in metabolic diseases intervention.
Experimental Section
All commercial chemicals and solvents were reagent grade and were used without further purification unless
otherwise specified. The following solvents and reagents have been abbreviated: tetrahydrofuran (THF), diethyl
ether (Et
TFA), N,N-dimethylformamide (DMF), methanol (MeOH), dimethoxyethane (DME), N-methylpyrrolidine (NMP),
acetonitrile (MeCN), chloroform (CHCl ), phosphorous oxychloride (POCl ), magnesium sulfate (MgSO ),
triethylamine (Et N), 2-propanol (iPrOH), N,N-diisopropylethylamine (iPr NEt), sodium hydroxide (NaOH), t-
butylmethylether (TBME), acetic acid (AcOH or HOAc), ethanol (EtOH), di-tert-butyldicarbonate (BOC O),
sodium sulfate (Na SO ), N,N-dimethylacetamide (DMA), sodium bicarbonate (NaHCO ), potassium carbonate
), 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxid hexafluorophosphate
HATU), azobisisobutyronitrile (AIBN), 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES),
2 2 2
O), dimethyl sulfoxide (DMSO), ethyl acetate (EtOAc), dichloromethane (CH Cl ), trifluoroacetic acid
(
3
3
4
3
2
2
2
4
3
(
2 3
K CO
(
dithiothreitol (DTT). All reactions except those in aqueous media were carried out with the use of standard
techniques for the exclusion of moisture. Reactions were monitored by thin-layer chromatography (TLC) on 0.25
mm silica gel plates (60F-254, E. Merck) and visualized with UV light, iodine, iodoplatinate, potassium
permanganate, cerium molybdate, or 5% phosphomolybdic acid in 95% ethanol. Final compounds were typically
purified either by flash chromatography on silica gel (E. Merck 40-63 mm) or ISCO Combiflash pump and fraction
collection system utilizing prepacked silica gel. Analytical purity was assessed by reversed phase high-performance
liquid chromatography (RP-HPLC) using an Agilent 1100 system equipped with a diode array spectrometer (λ range
190-400 nm). The stationary phase was a Keystone Scientific BDS Hypersil C-18 column (5 µm, 4.6 mm x 200
mm). The mobile phase employed 0.1% aqueous TFA with MeCN as the organic modifier and a flow rate of 1.0
1
) in minutes and % purity. H NMR spectra were recorded
mL/min. Analytical data are reported as retention time (t
R
14

on either a Varian UnityPlus-400 MHz or a Bruker Avance III 400 MHz NMR spectrometer. Chemical shifts are
reported in parts per million (ppm, δ units). Coupling constants are reported in units of hertz (Hz). Splitting patterns
are designated as s, singlet; d, doublet; t, triplet; q, quartet; p, pentet; h, hextet; m, multiplet; or br, broad. Low-
resolution mass spectra (MS) were recorded on a Waters SQD. The UPLC analysis was conducted utilizing a
Phenomenex Kinetex 1.7 µm 2.1 x 50mm XB-C18 column at 40 °C. The gradient employed was: mobile phase A:
water + 0.2% v/v formic acid mobile phase B: MeCN + 0.15% v/v FA. High-resolution MS were recorded on a
Waters (Micromass) LCT time-of-flight mass spectrometer. Mass spectra were obtained under electrospray
ionization (ESI), atmospheric pressure chemical ionization (APCI), or fast atom bombardment (FAB) methods.
Combustion analyses were performed by Robertson Microlit Laboratories, Inc. (Ledgewood, NJ).
2-Chloro-4-((2-fluoro-6-(trifluoromethyl)benzyl)amino)quinazoline-8-carbonitrile 4
N,N-Diisopropylethylamine (3.3 mL, 18.75 mmol) was added to 2,4-dichloroquinazoline-8-carbonitrile 2 (1.40 g,
.25 mmol) in tetrahydrofuran (59.2 mL) at room temperature, followed by 2-fluoro-6-trifluoromethylbenzylamine 3
1.267 g, 6.56 mmol) and the solution was stirred at room temperature for sixteen hours. The reaction mixture was
6
(
poured into ether, washed with saturated sodium bicarbonate, dried over magnesium sulfate, filtered, and
concentrated. The residue was purified by silica gel chromatography, eluting with ethyl acetate:hexanes (1:1) to
give 2-chloro-4-((2-fluoro-6-(trifluoromethyl)benzyl)amino)quinazoline-8-carbonitrile 4 (2.02 g, 5.04 mmol, 81 %
1
yield). H NMR (400 MHz, CD
3
SOCD
3
) δ 9.26 (br s, 1H), 8.63 (d, 1H, J = 8 Hz), 8.36 (d, 1H, J = 7 Hz), 7.72-7.60
(
m, 4H), 4.83 (br s, 2H); LC-MS (LC-ES) M+H = 381.
2-(((1H-pyrazol-4-yl)methyl)amino)-4-((2-fluoro-6-(trifluoromethyl)benzyl)amino)quinazoline-8-carbonitrile
6a
15

N,N-Diisopropylethylamine (0.25 mL, 1.442 mmol) was added to 2-chloro-4-((2-fluoro-6-
trifluoromethyl)benzyl)amino)quinazoline-8-carbonitrile 4 (109.8 mg, 0.288 mmol) in 1,4-dioxane (4.6 mL) and
dimethyl sulfoxide (0.92 mL) at room temperature, followed by (1H-pyrazol-4-yl)methanamine dihydrochloride 5a
74 mg, 0.433 mmol) and the solution was heated to 100 °C and stirred for sixteen hours. The reaction mixture was
(
(
poured into ether, washed with saturated sodium bicarbonate, dried over magnesium sulfate, filtered, and
concentrated. The residue was purified by silica gel chromatography, eluting with ethyl acetate:hexanes (4:1) to
give 2-(((1H-pyrazol-4-yl)methyl)amino)-4-((2-fluoro-6-(trifluoromethyl)benzyl)amino)quinazoline-8-carbonitrile
1
a (99.9 mg, 0.215 mmol, 74.6 % yield). H NMR (400 MHz, CD
6
8
7
3
SOCD
3
) δ 12.56 (br s, 1H), 8.39 (br s, 0.4H),
.29 (d, 1H, J = 7 Hz), 8.17 (br s, 0.6H), 8.01-7.91 (m, 1H), 7.78-7.58 (m, 4H), 7.55 (br s, 1H), 7.48-7.36 (m, 1H),
.10-7.00 (m, 1H), 4.92-4.68 (m, 2H), 4.38 (d, 2H, J = 5 Hz); LC-MS (LC-ES) M+H = 442.
2-(((1H-pyrazol-4-yl)methyl)amino)-4-((2-fluoro-6-(trifluoromethyl)benzyl)amino)quinazoline-8-carboxamide
1c
Sodium hydroxide (5.0 M, 0.14 mL, 0.679 mmol) was added to 2-(((1H-pyrazol-4-yl)methyl)amino)-4-((2-fluoro-6-
trifluoromethyl)benzyl)amino)quinazoline-8-carbonitrile 6a (99.9 mg, 0.226 mmol) in dimethyl sulfoxide (1.9 mL)
(
at room temperature, followed by 35% hydrogen peroxide (0.24 mL, 2.72 mmol) and the solution was stirred for
forty hours at room temperature. The reaction mixture was poured into ether, washed with water, dried over
magnesium sulfate, filtered, and concentrated. The residue was purified by silica gel chromatography, eluting with
methanol:ethyl acetate (1:9) to give 2-(((1H-pyrazol-4-yl)methyl)amino)-4-((2-fluoro-6-
1
trifluoromethyl)benzyl)amino)quinazoline-8-carboxamide 1c (69.3 mg, 0.143 mmol, 63.3 % yield). H NMR (400
(
16

MHz, CD
3
SOCD
H), 7.14-7.02 (m, 1H), 4.96-4.68 (m, 2H), 4.48-4.26 (m, 2H); LC-MS (LC-ES) for C21
.56 min, 98.4% purity.
3
) δ 12.61 (br s, 1H),10.73 (br s, 1H), 8.34 (d, 1H, J = 7 Hz), 8.22 (d, 1H, J = 8 Hz), 8.18-7.40 (m,
8
0
H
17
F
4
N
7
O, M+H = 460; t =
R
2-(((1H-Pyrazol-5-yl)methyl)amino)-4-((2-fluoro-6-(trifluoromethyl)benzyl)amino)quinazoline-8-carbonitrile
6b
N,N-Diisopropylethylamine (0.14 mL, 0.828 mmol) was added to 2-chloro-4-((2-fluoro-6-
trifluoromethyl)benzyl)amino)quinazoline-8-carbonitrile 4 (105.1 mg, 0.276 mmol) in 1,4-dioxane (2.6 mL) at
(
room temperature, followed by (1H-pyrazol-5-yl)methanamine 5b (40 mg, 0.414 mmol) and the solution was heated
to 100 °C and stirred for sixteen hours. The reaction mixture was poured into ether, washed with saturated sodium
bicarbonate, dried over magnesium sulfate, filtered, and concentrated. The residue was purified by silica gel
chromatography, eluting with ethyl acetate:hexanes (4:1) to give 2-(((1H-pyrazol-5-yl)methyl)amino)-4-((2-fluoro-
1
-(trifluoromethyl)benzyl)amino)quinazoline-8-carbonitrile 6b (95.6 mg, 0.206 mmol, 74.5 % yield). H NMR (400
6
MHz, CD
3
3
SOCD ) δ 12.58-12.48 (m, 1H), 8.44-8.16 (m, 2H), 8.04-7.92 (m, 1H), 7.70-7.46 (m, 4H), 7.40-7.28 (m,
1H), 7.14-6.98 (m, 1H), 6.40-6.06 (m, 1H), 4.74 (s, 2H), 4.56 (s, 2H); LC-MS (LC-ES) M+H = 442.
2-(((1H-Pyrazol-5-yl)methyl)amino)-4-((2-fluoro-6-(trifluoromethyl)benzyl)amino)quinazoline-8-carboxamide
1d
Sodium hydroxide (5.0 M, 0.13 mL, 0.650 mmol) was added to 2-(((1H-pyrazol-5-yl)methyl)amino)-4-((2-fluoro-6-
trifluoromethyl)benzyl)amino)quinazoline-8-carbonitrile 6b (95.6 mg, 0.217 mmol) in dimethyl sulfoxide (1.8 mL)
(
17

at room temperature, followed by 35% hydrogen peroxide (0.23 mL, 2.60 mmol) and the solution was stirred for
four hours at room temperature. The reaction mixture was poured into ether, washed with water, dried over
magnesium sulfate, filtered, and concentrated. The residue was purified by silica gel chromatography, eluting with
methanol:ethyl acetate (1:9) to give 2-(((1H-pyrazol-5-yl)methyl)amino)-4-((2-fluoro-6-
1
trifluoromethyl)benzyl)amino)quinazoline-8-carboxamide 1d (58.9 mg, 0.122 mmol, 56.2 % yield). H NMR (400
(
MHz, CD
3
SOCD
H), 8.22 (d, 1H, J = 8 Hz), 7.74-7.30 (m, 6H), 7.16-7.02 (m, 1H), 6.24-6.10 (m, 1H), 4.77 (br s, 2H), 4.64-4.36 (m,
H); LC-MS (LC-ES) for C21 O M+H = 460; t = 0.59 min, 100% purity.
3
) δ 12.66 & 12.53 (br s, 1H),10.71 & 10.54 (br s, 1H), 8.34 (d, 1H, J = 7 Hz), 8.32 & 8.17 (br s,
1
2
H
17
F
4
N
7
R
2-(((1H-1,2,4-Triazol-3-yl)methyl)amino)-4-((2-fluoro-6-(trifluoromethyl)benzyl)amino)quinazoline-8-
carbonitrile 6c
N,N-Diisopropylethylamine (0.26 mL, 1.476 mmol) was added to 2-chloro-4-((2-fluoro-6-
(
trifluoromethyl)benzyl)amino)quinazoline-8-carbonitrile 4 (112.4 mg, 0.295 mmol) in 1,4-dioxane (4.5 mL) at
room temperature, followed by (1H-1,2,4-triazol-3-yl)methanamine hydrochloride 5c (60 mg, 0.443 mmol) and the
solution was heated to 100 °C and stirred for sixteen hours. The reaction mixture was poured into ether, washed
with saturated sodium bicarbonate, dried over magnesium sulfate, filtered, and concentrated. The residue was
purified by silica gel chromatography, eluting with ethyl acetate to give 2-(((1H-1,2,4-triazol-3-yl)methyl)amino)-4-
1
(2-fluoro-6-(trifluoromethyl)benzyl)amino)quinazoline-8-carbonitrile 6c (86.9 mg, 0.187 mmol, 63.2 % yield). H
(
3 3
NMR (400 MHz, CD SOCD ) δ 13.65 (br s, 1H), 8.31 (br s, 2H) 7.98 (d, 1H, J = 7 Hz), 7.88-7.52 (m, 5H), 7.08 (br
s, 1H), 4.74 (br s, 2H), 4.62 (br s, 2H); LC-MS (LC-ES) M+H = 443.
2-(((1H-1,2,4-Triazol-3-yl)methyl)amino)-4-((2-fluoro-6-(trifluoromethyl)benzyl)amino)quinazoline-8-
carboxamide 1e
18

Sodium hydroxide (5.0 M, 0.12 mL, 0.589 mmol) was added to 2-(((1H-1,2,4-triazol-3-yl)methyl)amino)-4-((2-
fluoro-6-(trifluoromethyl)benzyl)amino)quinazoline-8-carbonitrile 6c (86.9 mg, 0.196 mmol) in dimethyl sulfoxide
(
1.64 mL) at room temperature, followed by 35% hydrogen peroxide (0.21 mL, 2.357 mmol) and the solution was
stirred for sixteen hours at room temperature. The reaction mixture was poured into ethyl acetate, washed with
saturated sodium chloride, dried over magnesium sulfate, filtered, and concentrated. The residue was purified by
silica gel chromatography, eluting with methanol:ethyl acetate (1:9) to give 2-(((1H-1,2,4-triazol-3-
yl)methyl)amino)-4-((2-fluoro-6-(trifluoromethyl)benzyl)amino)quinazoline-8-carboxamide 1e (73.7 mg, 0.152
1
mmol, 77 % yield). H NMR (400 MHz, CD
3
SOCD
3
) δ 13.74 (br s, 1H), 10.73 & 10.30 (br s, 1H), 8.34 (d, 1H, J =
8
Hz), 8.46-8.14 (m, 2H), 7.94-7.24 (m, 6H), 7.09 (br s, 1H), 4.84-4.46 (m, 4H); LC-MS (LC-ES) for C20
16 4 8
H F N O
M+H = 461; t = 0.86 min, 100.0% purity.
R
2
6
-(((1H-Imidazol-4-yl)methyl)amino)-4-((2-fluoro-6-(trifluoromethyl)benzyl)amino)quinazoline-8-carbonitrile
d
N,N-Diisopropylethylamine (0.24 mL, 1.389 mmol) was added to 2-chloro-4-((2-fluoro-6-
trifluoromethyl)benzyl)amino)quinazoline-8-carbonitrile 4 (105.8 mg, 0.278 mmol) in 1,4-dioxane (1.3 mL) and
dimethyl sulfoxide (1.3 mL) at room temperature, followed by (1H-imidazol-4-yl)methanamine dihydrochloride 5d
71 mg, 0.417 mmol) and the solution was heated to 100 °C and stirred for sixteen hours. The reaction mixture was
(
(
poured into ether, washed with water, dried over magnesium sulfate, filtered, and concentrated. The residue was
purified by silica gel chromatography, eluting with methanol:ethyl acetate (1:24) to give 2-(((1H-imidazol-4-
yl)methyl)amino)-4-((2-fluoro-6-(trifluoromethyl)benzyl)amino)quinazoline-8-carbonitrile 6d (73.1 mg, 0.157
19

1
mmol, 56.6 % yield). H NMR (400 MHz, CD
.02-7.92 (m, 1H), 7.70-6.80 (m, 7H), 4.86-4.68 (m, 2H), 4.51 (d, 0.8H, J = 6 Hz), 4.45 (d, 1.2H, J = 6 Hz); LC-MS
LC-ES) M+H = 442.
3 3
SOCD ) δ 11.82 (br s, 0.6H), 11.71 (br s, 0.4H), 8.38-8.14 (m, 2H),
8
(
2-(((1H-Imidazol-4-yl)methyl)amino)-4-((2-fluoro-6-(trifluoromethyl)benzyl)amino)quinazoline-8-
carboxamide 1f
Sodium hydroxide (5.0 M, 0.10 mL, 0.497 mmol) was added to 2-(((1H-imidazol-4-yl)methyl)amino)-4-((2-fluoro-
6-(trifluoromethyl)benzyl)amino)quinazoline-8-carbonitrile 6d (73.1 mg, 0.166 mmol) in dimethyl sulfoxide (1.4
mL) at room temperature, followed by 35% hydrogen peroxide (0.17 mL, 1.987 mmol) and the solution was stirred
for sixteen hours at room temperature. The residue was purified by reverse phase HPLC eluting with
acetonitrile:water (10:90 to 50:50) to give 2-(((1H-imidazol-4-yl)methyl)amino)-4-((2-fluoro-6-
1
trifluoromethyl)benzyl)amino)quinazoline-8-carboxamide 1f (46.3 mg, 0.096 mmol, 57.8 % yield). H NMR (400
(
MHz, CD
3
SOCD
H), 7.40-6.76 (m, 3H), 4.90-4.70 (m, 2H), 4.54-4.30 (m, 2H); LC-MS (LC-ES) for C21
.51 min, 100% purity.
3
) δ 11.85 (br s, 1H), 10.80-10.58 (m, 1H), 8.34 (d, 1H, J = 7 Hz), 8.32-8.10 (m, 2H), 7.70-7.42 (m,
5
0
H
17
F
4
N
7
O M+H = 460; t
R
=
2
6
-(((1H-Imidazol-2-yl)methyl)amino)-4-((2-fluoro-6-(trifluoromethyl)benzyl)amino)quinazoline-8-carbonitrile
e
N,N-Diisopropylethylamine (0.30 mL, 1.726 mmol) was added to 2-chloro-4-((2-fluoro-6-
trifluoromethyl)benzyl)amino)quinazoline-8-carbonitrile 4 (131.4 mg, 0.345 mmol) in 1,4-dioxane (1.6 mL) and
(
20

dimethyl sulfoxide (1.6 mL) at room temperature, followed by (1H-imidazol-2-yl)methanamine dihydrochloride 5e
88 mg, 0.518 mmol) and the solution was heated to 100 °C and stirred for sixteen hours. The reaction mixture was
(
poured into ether, washed with water, dried over magnesium sulfate, filtered, and concentrated. The residue was
purified by silica gel chromatography, eluting with methanol:ethyl acetate (1:32) to give 2-(((1H-imidazol-2-
yl)methyl)amino)-4-((2-fluoro-6-(trifluoromethyl)benzyl)amino)quinazoline-8-carbonitrile 6e (113.1 mg, 0.243
1
mmol, 70.5 % yield). H NMR (400 MHz, CD
3
SOCD ) δ 11.62 (br s, 1H), 8.40-8.26 (m, 2H), 7.99 (d, 1H, J = 7
3
Hz), 7.74-7.34 (m, 4H), 7.16-6.88 (m, 2H), 6.78 (br s, 1H), 4.73 (br s, 2H), 4.60 (br s, 2H); LC-MS (LC-ES) M+H =
442.
2-(((1H-Imidazol-2-yl)methyl)amino)-4-((2-fluoro-6-(trifluoromethyl)benzyl)amino)quinazoline-8-
carboxamide 1g
Sodium hydroxide (5.0 M, 0.15 mL, 0.769 mmol) was added to 2-(((1H-imidazol-2-yl)methyl)amino)-4-((2-fluoro-
6-(trifluoromethyl)benzyl)amino)quinazoline-8-carbonitrile 6e (113.1 mg, 0.256 mmol) in dimethyl sulfoxide (2.1
mL) at room temperature, followed by 35% hydrogen peroxide (0.27 mL, 3.07 mmol) and the solution was stirred
for eighteen hours at room temperature. The residue was purified by reverse phase HPLC eluting with
acetonitrile:water (10:90 to 50:50) to give 2-(((1H-imidazol-2-yl)methyl)amino)-4-((2-fluoro-6-
1
trifluoromethyl)benzyl)amino)quinazoline-8-carboxamide 1g (76.3 mg, 0.158 mmol, 61.6 % yield). H NMR (400
(
MHz, CD
Hz), 7.84-7.34 (m, 5H), 7.10 (br s, 1H), 6.98 (br s, 1H), 6.79 (br s, 1H), 4.76 (br s, 2H), 4.50 (br s, 2H); LC-MS
LC-ES) for C21 O M+H = 460; t = 0.87 min, 98.0% purity.
3 3
SOCD ) δ 11.76 (br s, 1H), 10.74 (br s, 1H), 8.34 (d, 1H, J = 7 Hz), 8.34-8.24 (m, 1H), 8.24 (d, 1H, J = 7
(
H
17
F
4
N
7
R
2-(((1H-Tetrazol-5-yl)methyl)amino)-4-((2-fluoro-6-(trifluoromethyl)benzyl)amino)quinazoline-8-carbonitrile
trifluoroacetate 6f
21

N,N-Diisopropylethylamine (0.30 mL, 1.743 mmol) was added to 2-chloro-4-((2-fluoro-6-
trifluoromethyl)benzyl)amino)quinazoline-8-carbonitrile 4 (132.7 mg, 0.349 mmol) in 1,4-dioxane (1.6 mL) and
(
dimethyl sulfoxide (1.6 mL) at room temperature, followed by (1H-tetrazol-5-yl)methanamine hydrochloride 5f (71
mg, 0.523 mmol) and the solution was heated to 100 °C and stirred for sixteen hours. The residue was purified by
reverse phase HPLC eluting with acetonitrile:water (10:90 to 75:25) to give 2-(((1H-tetrazol-5-yl)methyl)amino)-4-
(
(2-fluoro-6-(trifluoromethyl)benzyl)amino)quinazoline-8-carbonitrile trifluoroacetate 6f (152.2 mg, 0.259 mmol,
1
4.4 % yield). H NMR (400 MHz, CD
7
7
3
SOCD ) δ 8.78 (br s, 1H), 8.37 (d, 2H, J = 8 Hz), 8.18-7.94 (m, 2H), 7.82-
3
.50 (m, 4H), 7.26-7.08 (m, 1H), 4.87 (br s, 2H), 4.82-4.50 (m, 2H); LC-MS (LC-ES) M+H = 444.
2-(((1H-Tetrazol-5-yl)methyl)amino)-4-((2-fluoro-6-(trifluoromethyl)benzyl)amino)quinazoline-8-
carboxamide trifluoroacetate 1h
Sodium hydroxide (5.0 M, 0.16 mL, 0.819 mmol) was added to 2-(((1H-tetrazol-5-yl)methyl)amino)-4-((2-fluoro-6-
(
trifluoromethyl)benzyl)amino)quinazoline-8-carbonitrile trifluoroacetate 6f (152.2 mg, 0.273 mmol) in dimethyl
sulfoxide (2.3 mL) at room temperature, followed by 35% hydrogen peroxide (0.29 mL, 3.28 mmol) and the
solution was stirred for sixteen hours at room temperature. The residue was purified by reverse phase HPLC eluting
with acetonitrile:water (25:75 to 75:25) to give 2-(((1H-tetrazol-5-yl)methyl)amino)-4-((2-fluoro-6-
(
trifluoromethyl)benzyl)amino)quinazoline-8-carboxamide trifluoroacetate 1h (133.4 mg, 0.220 mmol, 81 % yield).
1
H NMR (400 MHz, CD
8 Hz), 8.36 (d, 1H, J = 8 Hz), 8.15 (br s, 1H), 7.72-7.54 (m, 3H), 7.52 (t, 1H, J = 4 Hz), 5.03 (d, 2H, J = 4 Hz),
.71 (br s, 2H); LC-MS (LC-ES) for C19 O M+H = 462; t = 0.60 min, 100.0% purity.
3 3
SOCD ) δ 13.22 (br s, 1H), 10.03 (br s, 1H), 9.79 (br s, 1H), 8.65 (br s, 1H), 8.48 (d, 1H, J
=
4
H
15
F
4
N
9
R
22

4-((2-Fluoro-6-(trifluoromethyl)benzyl)amino)-2-((oxazol-2-ylmethyl)amino)quinazoline-8-carbonitrile 6g
N,N-Diisopropylethylamine (0.26 mL, 1.505 mmol) was added to 2-chloro-4-((2-fluoro-6-
trifluoromethyl)benzyl)amino)quinazoline-8-carbonitrile 4 (114.6 mg, 0.301 mmol) in 1,4-dioxane (1.4 mL) and
dimethyl sulfoxide (1.4 mL) at room temperature, followed by oxazol-2-ylmethanamine hydrochloride 5g (61 mg,
.452 mmol) and the solution was heated to 100 °C and stirred for sixty-six hours. The reaction mixture was poured
(
0
into ether, washed with water, dried over magnesium sulfate, filtered, and concentrated. The residue was purified by
silica gel chromatography, eluting with ethyl acetate:hexanes (1:1) to give 4-((2-fluoro-6-
(
trifluoromethyl)benzyl)amino)-2-((oxazol-2-ylmethyl)amino)quinazoline-8-carbonitrile 6g (64.8 mg, 0.111 mmol,
1
7.0 % yield). H NMR (400 MHz, CD
3
7
3
SOCD ) δ 8.45-8.26 (m, 2H), 8.04-7.84 (m, 2H), 7.72-7.52 (m, 4H), 7.16-
3
.02 (m, 2H), 4.80-4.68 (m, 2H), 4.68-4.54 (m, 2H); LC-MS (LC-ES) M+H = 443.
4-((2-Fluoro-6-(trifluoromethyl)benzyl)amino)-2-((oxazol-2-ylmethyl)amino)quinazoline-8-carboxamide 1i
Sodium hydroxide (5.0 M, 0.09 mL, 0.439 mmol) was added to 4-((2-fluoro-6-(trifluoromethyl)benzyl)amino)-2-
(oxazol-2-ylmethyl)amino)quinazoline-8-carbonitrile 6g (64.8 mg, 0.146 mmol) in dimethyl sulfoxide (1.2 mL) at
(
room temperature, followed by 35% hydrogen peroxide (0.15 mL, 1.758 mmol) and the solution was stirred for
sixteen hours at room temperature. The reaction mixture was poured into ether, washed with water, dried over
magnesium sulfate, filtered, and concentrated. The residue was purified by silica gel chromatography, eluting with
methanol:ethyl acetate (1:49) to give 4-((2-fluoro-6-(trifluoromethyl)benzyl)amino)-2-((oxazol-2-
1
ylmethyl)amino)quinazoline-8-carboxamide 1i (46.6 mg, 0.096 mmol, 65.6 % yield). H NMR (400 MHz,
23

3 3
CD SOCD ) δ 10.68 (br s, 0.4H),10.05 (br s, 0.6H), 8.34 (d, 1H, J = 7 Hz), 8.35-8.18 (m, 2H), 7.97 (br s, 1H), 7.82-
7
.44 (m, 5H), 7.16-7.04 (m, 2H), 4.84-4.48 (m, 4H); LC-MS (LC-ES) for C21
00.0% purity.
16 4 6 2 R
H F N O M+H = 461; t = 0.67 min,
1
4-((2-Fluoro-6-(trifluoromethyl)benzyl)amino)-2-((oxazol-5-ylmethyl)amino)quinazoline-8-carbonitrile 6h
N,N-Diisopropylethylamine (0.29 mL, 1.680 mmol) was added to 2-chloro-4-((2-fluoro-6-
trifluoromethyl)benzyl)amino)quinazoline-8-carbonitrile 4 (127.9 mg, 0.336 mmol) in 1,4-dioxane (1.5 mL) and
dimethyl sulfoxide (1.5 mL) at room temperature, followed by oxazol-5-ylmethanamine hydrochloride 5h (68 mg,
.504 mmol) and the solution was heated to 100 °C and stirred for sixteen hours. The reaction mixture was poured
(
0
into ether, washed with water, dried over magnesium sulfate, filtered, and concentrated. The residue was purified by
silica gel chromatography, eluting with methanol:ethyl acetate (1:1) to give 4-((2-fluoro-6-
(
trifluoromethyl)benzyl)amino)-2-((oxazol-5-ylmethyl)amino)quinazoline-8-carbonitrile 6h (118.9 mg, 0.255 mmol,
1
6 % yield). H NMR (400 MHz, CD
7
7
3
SOCD
3
) δ 8.50-8.18 (m, 2H), 8.32 (d, 1H, J = 8 Hz), 7.99 (d, 1H, J = 7 Hz),
.84-7.52 (m, 4H), 7.18-6.80 (m, 2H), 4.84-4.68 (m, 2H), 4.58 (d, 2H, J = 5 Hz); LC-MS (LC-ES) M+H = 443.
4-((2-fluoro-6-(trifluoromethyl)benzyl)amino)-2-((oxazol-5-ylmethyl)amino)quinazoline-8-carboxamide 1j
Sodium hydroxide (5.0 M, 0.16 mL, 0.806 mmol) was added to 4-((2-fluoro-6-(trifluoromethyl)benzyl)amino)-2-
(oxazol-5-ylmethyl)amino)quinazoline-8-carbonitrile 6h (118.9 mg, 0.269 mmol) in dimethyl sulfoxide (2.2 mL) at
(
room temperature, followed by 35% hydrogen peroxide (0.28 mL, 3.23 mmol) and the solution was stirred for
sixteen hours at room temperature. The reaction mixture was poured into ether, washed with water, dried over
24

magnesium sulfate, filtered, and concentrated. The residue was purified by silica gel chromatography, eluting with
methanol:ethyl acetate (1:32) to give 4-((2-fluoro-6-(trifluoromethyl)benzyl)amino)-2-((oxazol-5-
1
ylmethyl)amino)quinazoline-8-carboxamide 1j (99.9 mg, 0.206 mmol, 77 % yield). H NMR (400 MHz,
3 3
CD SOCD ) δ 10.63 (br s, 0.4H),10.45 (br s, 0.6H), 8.42-8.18 (m, 3H), 8.36 (dd, 1H, J = 7, 1 Hz), 7.84-7.54 (m,
5
H), 7.16-6.96 (m, 2H), 4.86-4.72 (m, 2H), 4.66-4.46 (m, 2H); LC-MS (LC-ES) for C21
.72 min, 100% purity.
16 4 6 2 R
H F N O M+H = 461; t =
0
4-((2-Fluoro-6-(trifluoromethyl)benzyl)amino)-2-((isoxazol-3-ylmethyl)amino)quinazoline-8-carbonitrile 6i
N,N-Diisopropylethylamine (0.26 mL, 1.483 mmol) was added to 2-chloro-4-((2-fluoro-6-
trifluoromethyl)benzyl)amino)quinazoline-8-carbonitrile 4 (112.9 mg, 0.297 mmol) in 1,4-dioxane (1.4 mL) and
dimethyl sulfoxide (1.4 mL) at room temperature, followed by isoxazol-3-ylmethanamine hydrochloride 5i (60 mg,
.445 mmol) and the solution was heated to 100 °C and stirred for four hours. The reaction mixture was poured into
(
0
ether, washed with water, dried over magnesium sulfate, filtered, and concentrated. The residue was purified by
silica gel chromatography, eluting with ethyl acetate:hexanes (2:3) to give 4-((2-fluoro-6-
(
trifluoromethyl)benzyl)amino)-2-((isoxazol-3-ylmethyl)amino)quinazoline-8-carbonitrile 6i (97.9 mg, 0.201 mmol,
1
7.9 % yield). H NMR (400 MHz, CD
6
3
SOCD ) δ 8.82-8.74 (m, 1H), 8.48-8.18 (m, 2H), 7.99 (d, 1H, J = 7 Hz),
3
7.88-7.54 (m, 4H), 7.16-7.04 (m, 1H), 6.68 (br s, 0.6H), 6.41 (br s, 0.4H), 4.74 (d, 2H, J = 2 Hz), 4.63 (br s, 2H);
LC-MS (LC-ES) M+H = 443.
4-((2-Fluoro-6-(trifluoromethyl)benzyl)amino)-2-((isoxazol-3-ylmethyl)amino)quinazoline-8-carboxamide 1k
25

Sodium hydroxide (5.0 M, 0.13 mL, 0.664 mmol) was added to 4-((2-fluoro-6-(trifluoromethyl)benzyl)amino)-2-
(isoxazol-3-ylmethyl)amino)quinazoline-8-carbonitrile 6i (97.9 mg, 0.221 mmol) in dimethyl sulfoxide (1.8 mL) at
(
room temperature, followed by 35% hydrogen peroxide (0.23 mL, 2.66 mmol) and the solution was stirred for
seventeen hours at room temperature. The reaction mixture was poured into ether, washed with water, dried over
magnesium sulfate, filtered, and concentrated. The residue was purified by silica gel chromatography, eluting with
ethyl acetate to give 4-((2-fluoro-6-(trifluoromethyl)benzyl)amino)-2-((isoxazol-3-ylmethyl)amino)quinazoline-8-
1
carboxamide 1k (65.2 mg, 0.135 mmol, 60.8 % yield). H NMR (400 MHz, CD
3
SOCD ) δ 10.65 (br s, 0.4H),10.21
3
(
br s, 0.6H), 8.79 (s, 1H), 8.42-8.18 (m, 1H), 8.36 (d, 1H, J = 7 Hz), 8.24 (d, 1H, J = 7 Hz), 7.90-7.48 (m, 5H), 7.11
br s, 1H), 6.56-6.38 (m, 1H), 4.76 (d, 2H, J = 3 Hz), 4.68-4.48 (m, 2H); LC-MS (LC-ES) for C21 M+H =
(
16 4 6 2
H F N O
R
461; t = 0.82 min, 100% purity.
2-(((1H-Pyrazol-5-yl)methyl)(methyl)amino)-4-((2-fluoro-6-(trifluoromethyl)benzyl)amino)quinazoline-8-
carbonitrile 6j
N,N-Diisopropylethylamine (0.26 mL, 1.509 mmol) was added to 2-chloro-4-((2-fluoro-6-
(
trifluoromethyl)benzyl)amino)quinazoline-8-carbonitrile 4 (114.9 mg, 0.302 mmol) in 1,4-dioxane (1.4 mL) and
dimethyl sulfoxide (1.4 mL) at room temperature, followed by N-methyl-1-(1H-pyrazol-5-yl)methanamine
dihydrochloride 5j (83 mg, 0.453 mmol) and the solution was heated to 100 °C and stirred for sixteen hours. The
reaction mixture was poured into ether, washed with water, dried over magnesium sulfate, filtered, and concentrated.
The residue was purified by silica gel chromatography, eluting with ethyl acetate:hexanes (4:1) to give 2-(((1H-
pyrazol-5-yl)methyl)(methyl)amino)-4-((2-fluoro-6-(trifluoromethyl)benzyl)amino)quinazoline-8-carbonitrile 6j
1
85.7 mg, 0.179 mmol, 59.2 % yield). H NMR (400 MHz, CD
(
3
SOCD
3
) δ 12.57 (br s, 1H), 8.38 (br s, 1H), 8.32 (d,
1
H, J = 8 Hz), 7.98 (d, 1H, J = 7 Hz), 7.66-7.54 (m, 4H), 7.07 (t, 1H, J = 8 Hz), 6.30-5.98 (m, 1H), 4.94-4.82 (m,
H), 4.81 (d, 2H, J = 3 Hz), 3.14 (s, 3H); LC-MS (LC-ES) M+H = 456.
2
26

2-(((1H-Pyrazol-5-yl)methyl)(methyl)amino)-4-((2-fluoro-6-(trifluoromethyl)benzyl)amino)quinazoline-8-
carboxamide 1l
Sodium hydroxide (5.0 M, 0.11 mL, 0.565 mmol) was added to 2-(((1H-pyrazol-5-yl)methyl)(methyl)amino)-4-((2-
fluoro-6-(trifluoromethyl)benzyl)amino)quinazoline-8-carbonitrile 6j (85.7 mg, 0.188 mmol) in dimethyl sulfoxide
(
1.6 mL) at room temperature, followed by 35% hydrogen peroxide (0.20 mL, 2.258 mmol) and the solution was
stirred for four hours at room temperature. The reaction mixture was poured into ether, washed with water, dried
over magnesium sulfate, filtered, and concentrated. The residue was purified by silica gel chromatography, eluting
with methanol:ethyl acetate (1:12) to give 2-(((1H-pyrazol-5-yl)methyl)(methyl)amino)-4-((2-fluoro-6-
1
trifluoromethyl)benzyl)amino)quinazoline-8-carboxamide 1l (7.2 mg, 0.014 mmol, 7.7 % yield). H NMR (400
(
MHz, CD
3
SOCD
.25 (d, 1H, J = 8 Hz), 7.66-7.52 (m, 5H), 7.12 (t, 1H, J = 8 Hz), 6.08 (br s, 1H), 4.96-4.64 (m, 2H), 4.83 (d, 2H, J =
Hz), 3.10 (br s, 3H); LC-MS (LC-ES) for C22 O M+H = 474; t = 0.95 min, 100% purity.
3
) δ 12.76 & 12.58 (br s, 1H), 10.58 (br s, 1H), 8.36 (d, 1H, J = 8 Hz), 8.34 (br t, 1H, J = 3 Hz),
8
3
H
19
F
4
N
7
R
4-((2-Fluoro-6-(trifluoromethyl)benzyl)amino)-2-(((3-methyl-1H-pyrazol-4-yl)methyl)amino)quinazoline-8-
carbonitrile 6k
N,N-Diisopropylethylamine (0.25 mL, 1.446 mmol) was added to 2-chloro-4-((2-fluoro-6-
(
trifluoromethyl)benzyl)amino)quinazoline-8-carbonitrile 4 (110.1 mg, 0.289 mmol) in 1,4-dioxane (1.3 mL) and
dimethyl sulfoxide (1.3 mL) at room temperature, followed by (3-methyl-1H-pyrazol-4-yl)methanamine
dihydrochloride 5k (80 mg, 0.434 mmol) and the solution was heated to 100 °C and stirred for four hours. The
27

reaction mixture was poured into ether, washed with water, dried over magnesium sulfate, filtered, and concentrated.
The residue was purified by silica gel chromatography, eluting with ethyl acetate:hexanes (4:1) to give 4-((2-fluoro-
6-(trifluoromethyl)benzyl)amino)-2-(((3-methyl-1H-pyrazol-4-yl)methyl)amino)quinazoline-8-carbonitrile 6k
1
121.5 mg, 0.253 mmol, 88 % yield). H NMR (400 MHz, CD
(
3
SOCD
3
) δ 12.27 & 12.18 (br s, 1H), 8.38 & 8.15 (br
s, 1H), 8.29 (d, 1H, J = 7 Hz), 7.96 (d, 1H, J = 7 Hz), 7.76-7.40 (m, 5H), 7.04 (t, 1H, J = 8 Hz), 4.86 & 4.71 (br s,
2H), 4.34 (br s, 2H), 2.28 & 2.20 (br s, 3H); LC-MS (LC-ES) M+H = 456.
4-((2-Fluoro-6-(trifluoromethyl)benzyl)amino)-2-(((3-methyl-1H-pyrazol-4-yl)methyl)amino)quinazoline-8-
carboxamide 1m
Sodium hydroxide (5.0 M, 0.16 mL, 0.800 mmol) was added to 4-((2-fluoro-6-(trifluoromethyl)benzyl)amino)-2-
(
((3-methyl-1H-pyrazol-4-yl)methyl)amino)quinazoline-8-carbonitrile 6k (121.5 mg, 0.267 mmol) in dimethyl
sulfoxide (4.9 mL) at room temperature, followed by 35% hydrogen peroxide (0.28 mL, 3.20 mmol) and the
solution was stirred for sixteen hours at room temperature. The reaction mixture was poured into ether, washed with
water, dried over magnesium sulfate, filtered, and concentrated. The residue was purified by silica gel
chromatography, eluting with methanol:ethyl acetate (1:24) to give 4-((2-fluoro-6-(trifluoromethyl)benzyl)amino)-2-
(
((3-methyl-1H-pyrazol-4-yl)methyl)amino)quinazoline-8-carboxamide 1m (74.8 mg, 0.150 mmol, 56.3 % yield).
1
H NMR (400 MHz, CD
3
SOCD
8 Hz), 7.76-7.28 (m, 7H), 7.07 (br s, 1H), 4.94-4.70 (m, 2H), 4.40-4.22 (m, 2H), 2.19 (s, 3H); LC-MS (LC-ES) for
O M+H = 474; t = 0.53 min, 100% purity.
3
) δ 12.35 & 12.23 (br s, 1H), 10.72 (br s, 1H), 8.34 (d, 1H, J = 7 Hz), 8.21 (d, 1H, J
=
C
22
H
19
F
4
N
7
R
2-(4,6-dihydropyrrolo[3,4-c]pyrazol-5(1H)-yl)-4-({[2-fluoro-6-(trifluoromethyl)phenyl]methyl}amino)-8-
quinazolinecarbonitrile 6l
28

N,N-Diisopropylethylamine (0.10 mL, 0.581 mmol) was added to 2-chloro-4-({[2-fluoro-6-
trifluoromethyl)phenyl]methyl}amino)-8-quinazolinecarbonitrile 4 (55.3 mg, 0.145 mmol) in 1,4-dioxane (1.3 mL)
(
2
6
at room temperature, followed by 1,4,5,6-tetrahydropyrrolo[3,4-c]pyrazole 5l (0.04 mL, 0.160 mmol) and the
solution was heated to 100 °C and stirred for four hours. The reaction mixture was poured into ether, washed with
water, dried over magnesium sulfate, filtered, and concentrated. The residue was purified by silica gel
chromatography, eluting with ethyl acetate:hexanes (7:3) to give 2-(4,6-dihydropyrrolo[3,4-c]pyrazol-5(1H)-yl)-4-
(
{[2-fluoro-6-(trifluoromethyl)phenyl]methyl}amino)-8-quinazolinecarbonitrile 6l (59.1 mg, 0.124 mmol, 85 %
1
yield). H NMR (400 MHz, CD
3
SOCD
3
) δ 12.69 (br d, 1H, J = 4 Hz), 8.54-8.46 (m, 1H), 8.36 (d, 1H, J = 7 Hz),
8.01 (d, 1H, J = 7 Hz), 7.68-7.60 (m, 3H), 7.59 (s, 1H), 7.11 (br t, 1H, J = 8 Hz), 4.89 (s, 2H), 4.66-4.54 (m, 4H);
LC-MS (LC-ES) M+H = 454.
2-(4,6-Dihydropyrrolo[3,4-c]pyrazol-5(1H)-yl)-4-({[2-fluoro-6-(trifluoromethyl)phenyl]methyl}amino)-8-
quinazolinecarboxamide 1n
Sodium hydroxide (5.0 M, 0.06 mL, 0.312 mmol) was added to 2-(4,6-dihydropyrrolo[3,4-c]pyrazol-5(1H)-yl)-4-
(
{[2-fluoro-6-(trifluoromethyl)phenyl]methyl}amino)-8-quinazolinecarbonitrile 6l (59.1 mg, 0.104 mmol) in
dimethyl sulfoxide (0.87 mL) at room temperature, followed by 35% hydrogen peroxide (0.11 mL, 1.250 mmol) and
the solution was stirred for sixteen hours at room temperature. The reaction mixture was poured into ether, washed
29