Design of Potent mRNA Decapping Scavenger Enzyme (DcpS) Inhibitors with Improved Physicochemical Properties To Investigate the Mechanism of Therapeutic Benefit in Spinal Muscular Atrophy (SMA)

Article abstract of DOI:10.1021/acs.jmedchem.7b00124

The C-5 substituted 2,4-diaminoquinazoline RG3039 (compound 1), a member of a chemical series that was identified and optimized using an SMN2 promoter screen, prolongs survival and improves motor function in a mouse model of spinal muscular atrophy (SMA).

Full text of DOI:10.1021/acs.jmedchem.7b00124

Subscriber access provided by University of Newcastle, Australia
Article
Design of Potent mRNA Decapping Scavenger Enzyme (DcpS)
Inhibitors with Improved Physicochemical Properties to Investigate the
Mechanism of Therapeutic Benefit in Spinal Muscular Atrophy (SMA)
Ariamala Gopalsamy, Arjun Narayanan, Shenping Liu, Mihir D. Parikh, Robert E. Kyne,
Jr., Olugbeminiyi O Fadeyi, Michael A Tones, Jonathan J. Cherry, Joseph F. Nabhan,
Gregory LaRosa, Donna N Petersen, Carol A. Menard, Timothy L Foley, Stephen Noell,
Yong Ren, Paula M. Loria, Jodi Maglich-Goodwin, Haojing Rong, and Lyn H. Jones
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.7b00124 • Publication Date (Web): 03 Mar 2017
Downloaded from http://pubs.acs.org on March 4, 2017
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a free service to the research community to expedite the
dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts
appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been
fully peer reviewed, but should not be considered the official version of record. They are accessible to all
readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered
to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published
in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just
Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor
changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers
and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors
or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
Journal of Medicinal Chemistry is published by the American Chemical Society. 1155
Sixteenth Street N.W., Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the course
of their duties.

Page 1 of 50
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Design of Potent mRNA Decapping Scavenger
Enzyme (DcpS) Inhibitors with Improved
Physicochemical Properties to Investigate the
Mechanism of Therapeutic Benefit in Spinal
Muscular Atrophy (SMA)
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Ariamala Gopalsamy,§* Arjun Narayanan,§ Shenping Liu,⊥ Mihir D. Parikh,⊥ Robert E. Kyne
Jr.,⊥ Olugbeminiyi Fadeyi,⊥ Michael A. Tones‡, Jonathan J. Cherry,‡ Joseph F. Nabhan,‡
Gregory LaRosa,‡ Donna N. Petersen,† Carol Menard,† Timothy L. Foley,† Stephen Noell,† Yong
Ren,† Paula M. Loria,† Jodi Maglich-Goodwin,§ Haojing Rong,# Lyn H. Jones§
§Medicine Design and ‡Rare Disease Research Unit, #Pharmacokinetics and Drug Metabolism,
Pfizer, 610 Main Street, Cambridge, Massachusetts 02139, USA
⊥Medicine Design and †Primary Pharmacology Group, Pfizer, Eastern Point Road, Groton,
Connecticut 06340, USA
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 2 of 50
1
2
3
4
ABSTRACT
5
6
7
8
The Cꢀ5 substituted 2,4ꢀdiaminoquinazoline RG3039 (compound 1), a member of a
chemical series that was identified and optimized using an SMN2 promoter screen, prolongs
survival and improves motor function in a mouse model of spinal muscular atrophy (SMA). It is
a potent inhibitor of the mRNA Decapping Scavenger Enzyme (DcpS), but the mechanism
whereby DcpS inhibition leads to therapeutic benefit is unclear. Compound 1 is a dibasic
lipophilic molecule that is predicted to accumulate in lysosomes. To understand if the in-vivo
efficacy is due to DcpS inhibition or other effects resulting from the physicochemical properties
of the chemotype, we undertook structure based molecular design to identify DcpS inhibitors
with improved physicochemical properties. Herein we describe the design, synthesis, in-vitro
pharmacological characterization of these DcpS inhibitors along with the in-vivo mouse CNS PK
profile of PFꢀDcpSi (compound 24), one of the analogs found to be efficacious in SMA mouse
model.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
KEYWORDS – SMA, Spinal Muscular Atrophy, DcpS, mRNA Decapping Scavenger Enzyme,
DcpS inhibitor, LipE, Lipophilic Efficiency
ACS Paragon Plus Environment

Page 3 of 50
Journal of Medicinal Chemistry
1
2
3
4
5
INTRODUCTION
6
7
8
9
Spinal muscular atrophy (SMA) is an autosomal recessive, inherited neurodegenerative
disease and a leading genetic cause of infant death. It is characterized by loss of alphaꢀmotor
neurons in the spinal cord leading to progressive atrophy and weakness of limb and trunk
muscles.1, 2 There is currently no cure for SMA.3 Humans carry two Survival Motor Neuron
(SMN) genes: SMN1 and SMN2. SMA is caused by deficiency of the SMN protein resulting
from homozygous deletion of SMN1, partly compensated for by the presence of the paralogous
gene SMN2. While both SMN1 and SMN2 encode an identical protein, full length protein
production occurs at much lower efficiency from SMN2 than from SMN1 due to the exclusion of
Exonꢀ7 from the majority of SMN2 transcripts leading to their degradation. An attractive
potential therapeutic approach is to increase the SMN2 promoter activity.4 This approach was
employed by Jarecki et al. to identify C5ꢀsubstituted 2,4ꢀdiaminoquinazolines5 from a high
throughput reporter gene assay using the SMN2 promoter to drive βꢀlactamase activity.6 Further
followꢀup was carried out using protein microarray scanning with a radiolabelled C5ꢀsubstituted
2,4ꢀdiaminoquinazoline probe that identified DcpS as a single binding protein. We recently
reported7 the results of a related protein microarray and affinity capture technique that confirmed
the exquisite selectivity of the 2,4ꢀdiaminoquinazoline template. DcpS thus became a potential
novel therapeutic target for modulating gene expression.8
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Eukaryotic mRNA turnover pathway begins with deadenylation followed by decapping
and 5’→3’ exonucleolytic degradation. This process results in accumulation of residual Nꢀ7
guanine cap from the degraded mRNA. The human decapping scavenger enzyme (DcpS)
catalyzes residual cap hydrolysis following mRNA degradation and prevents accumulation of
intermediates that might interfere with translation and normal processing of mRNAs.9ꢀ12 By
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 4 of 50
1
2
3
4
5
6
7
8
9
restoring this essential function, DcpS protects the cell from the potential toxic accumulation of
capped mRNA fragments and plays a regulatory role. DcpS belongs to the HIT family of
pyrophosphatases13 and its structure bound to either m7GpppG or m7GpppA reveals an
asymmetric dimer with a domainꢀswapped amino terminus.14 DcpS is a dimeric protein with two
distinct cap binding pockets. It exists as a symmetrical dimer with open conformation in the
ligandꢀfree state and adopts an asymmetric conformation when bound to ligand, with one site
open and other site closed. C5ꢀsubstituted 2,4ꢀdiaminoquinazolines identified from the reporter
screen potently inhibit DcpS and were found to occupy the cap binding pocket when coꢀ
crystallized with the enzyme. While its role in mRNA turnover is well established, DcpS is an
unprecedented drug discovery target.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
RG3039 (compound 1), one of the advanced C5ꢀsubstituted 2,4ꢀdiaminoquinazolines to
enter Phase 1 clinical development, is effective in prolonging the survival of SMA mice after
oral administration.15, 16 We appreciated that 1 is a lipophilic dibasic compound – the calculated
LogP of 5.3 and measured pKas of 8.1 and 7.8 confirmed this assertion. Compounds with these
physicochemical properties have the propensity to accumulate in tissues as the result of
partitioning into membranes and entrapment within the lysosomal compartment by virtue of
protonation in its acidic environment.17 Soꢀcalled lysosomotropic agents have been reported to
provide neuroprotection in a model of neurodegeneration involving activation of apoptosis.18
Additionally, lysosomotropism contributes to autophagy perturbation,19 and autophagic
dysregulation is a feature of SMA.20, 21 To understand if the in-vivo efficacy of 1 is due to a
DcpSꢀdependent mechanism or due to the physicochemical properties of the chemotype, we
sought to develop potent DcpS inhibitors devoid of lysosomotropism, with adequate
pharmacokinetics (and possessing the requisite CNS free drug levels) to test in an in-vivo SMA
ACS Paragon Plus Environment

Page 5 of 50
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
mouse model. Herein, we describe the structureꢀbased design of potent DcpS inhibitors with
improved physicochemical properties and report that therapeutic benefit in SMA is consistent
with a DcpSꢀrelated mechanism. We also describe the creation of a 2,4ꢀdiaminoquinoline
derivative that is closely related in structure to our lead equity, but which is DcpS inactive and
therefore serves as a useful negative control chemical probe of DcpS biology.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
RESULT AND DISCUSSIONS
Chemistry
Compounds 1-8 (Table 1) were synthesized as disclosed previously for the synthesis of 1,
as shown in Scheme I. 2,4ꢀdiamonquinazolines functionalized at the C5 position were prepared
by reaction of Boc protected piperidine alcohol 30 with 2,6ꢀdifluorobenzonitrile 31. Resulting
2ꢀfluoroꢀ6ꢀarylalkoxybenzonitrile 32 was cyclized to give intermediate 33 by heating with
guanidine carbonate in N,Nꢀdimethylacetamide at high temperature. The removal of the
protecting group under standard acidic conditions to generate intermediate 34, followed by
alkylation with various substituted benzyl bromides afforded corresponding 5ꢀsubstituted 2,4ꢀ
diaminoquinazolines 1-8 (Scheme 1). The aliphatic ether linked racemate compound 37 was
obtained in two steps from intermediate 31, F displacement followed by cyclization. The
racemate 37 was separated by chiral SFC to give single enantiomers 15 and 16 (Scheme 2).
The reverse ether analog 25 was synthesized from 2ꢀfluoroꢀ6ꢀmethylbenzonitrile 38.
Radical bromination of 38 afforded 2ꢀ(bromomethyl)ꢀ6ꢀfluorobenzonitrile 39. Displacement of
substituted benzyl bromide with 40 in presence of potassium carbonate afforded 41, which was
subsequently cyclized using guanidine carbonate to yield 25. The compound 25 was further
converted into crystalline hydrochloride salt 25a (Scheme 3). The synthesis of C5, C7
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 6 of 50
1
2
3
4
5
6
7
8
9
disubstituted 2,4ꢀdiamonquinazolines is shown in Scheme 4. 2,6ꢀdifluoroꢀ4ꢀmethylbenzonitrile
43 was the common intermediate for C7 substituted ring systems, which was synthesized by
Suzuki coupling of 42. Displacement with 30 followed by cyclization and deprotection provided
intermediate 46, which was alkylated with 2ꢀ(bromomethyl)ꢀ1,3ꢀdichlorobenzene 47 to afford 7ꢀ
methylꢀ5ꢀsubstituted 2,4ꢀdiaminoquinazoline 26.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
The synthesis of C7 substituted reverse ether analog 28 is shown in Scheme 5.
Bromination of aniline 48 afforded 49. The amino group was converted to cyano using
Sandmeyer reaction 50. Formylation using iPrMgCl at low temperature gave 51, which was
reduced under mild conditions to give primary alcohol 52 without any lactone formation through
intramolecular cyclization. The alkyl halide 53 was displaced with phenol 54 and then cyclized
to afford reverse ether analog 28.
Cellular Accumulation
The physicochemistry of DcpS inhibitors such as 1 is suggestive of lysosomal
accumulation which may impact autophagic phenotypes in SMA mouse models. In order to
investigate the cellular accumulation of the C5ꢀsubstituted 2,4ꢀdiaminoquinazolines, an
evaluation of compound 1 and close analogs (compound 2 and 3) (Fig 1) in HEK293T cells and
lymphoblasts was carried out in the presence and absence of either concanamycin A and
bafilomycin. These compounds block lysosomal acidification through selective inhibition of the
vacuolar type ATPase22 that establishes the low pH environment of the lysosome. Chloroquine,
a wellꢀestablished lysosomotrope, was included in these experiments as a positive control. As
expected, chloroquine accumulation by cells was inhibited by >98% by the presence of either
bafilomycin or concanamycin A. Similarly, significant accumulation of 1 and the other lipophilic
dibasic C5ꢀsubstituted diaminoquinazolines compound 2 and compound 3 was also observed and
ACS Paragon Plus Environment

Page 7 of 50
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
was also largely inhibited by either of the two inhibitors of lysosomal acidification (Fig 2). This
observation prompted us to investigate the requirement of the dibasic lipophilic moieties for
DcpS inhibition, to eventually avoid potential issues arising from lysosomotropismꢀrelated
effects. The design and synthesis of analogs with improved pharmaceutical properties along
with further biological profiling of both classes of DcpS inhibitors is described below.
Structure Based Design of DcpS Inhibitors
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
The structureꢀactivity relationship generated for C5ꢀsubstituted 2,4ꢀdiaminoquinazolines
in the earlier iteration that led to the identification of compound 1 utilized an SMN promoter
assay and lacked a targeted assay such as DcpS inhibition assay for inhibitor optimization. In the
absence of target information, structureꢀbased ligand design was not utilized at that time.
Currently, the 2,4ꢀdiaminoquinazoline pharmacophore that mimics the 7ꢀmethylguanine mRNA
cap structure is the only reported chemotype known to block cap binding to DcpS. The coꢀ
crystal structure of compound 2 (PDB ID code: 3BL7) shown in Fig 3 (Left) explains the
excellent complementarity exhibited by this scaffold. The template 2,4ꢀdiaminoquinazoline is
critical for activity since in its protonated state it invokes a network of hydrogen bonds. Gluꢀ185
forms a strong bidentate hydrogen bond to the N1 and C2ꢀNH2, which additionally forms a
strong hydrogen bond to the backbone carbonyl of Pro204. The flat bicyclic quinazoline
scaffold engages in productive π−π stacking with Trpꢀ175, CHꢀπ interaction with Leuꢀ206, and
an edgeꢀtoꢀface interaction with the π system of Tyrꢀ113, leading to excellent packing of the
quinazoline ring. From steric considerations, the C5 position of the quinazoline provides an
appropriate vector to extend, mimicking cap binding in the DcpS active site. However, the
region occupied by the Cꢀ5 substituent (2,6ꢀdichlorobenzyl substituted piperidinemethyl in 1) is
an elongated pocket (Fig 3 ꢀ Right). As seen from the structure of capꢀbound DcpS, this pocket
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 8 of 50
1
2
3
4
5
6
7
8
9
accommodates the phosphate groups and the base. This region of the pocket therefore seemed
amenable to investigate further structureꢀactivity relationships, and hence we targeted this region
to optimize the physicochemical properties of compound 1.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
From the structure activity data published for the SMN2 promoter driven reporter assay,
it appeared that the lipophilic achiral piperidine substituent in 1 resulted from the desire to
overcome cellular toxicity that was attributed to dihydrofolate reductase (DHFR) enzyme
inhibition as an offꢀtarget for the quinazoline chemotype. Based on the reported data, lipophilic
halogenated benzyl groups were pursued to improve potency in the SMN2 promoter assay (Table
1). We initiated profiling of a cohort of compounds which were active in the SMN2 promoter
assay in the DcpS (human DcpS) enzyme assay. Compounds were indeed very potent in this
biochemical assay.23, 24 However, successful medicinal chemistry programs often do not focus
exclusively on potency, but rather balance a myriad of features that include metabolic stability,
solubility and offꢀtarget selectivity for a favorable in vivo outcome. A more useful parameter in
the design process is lipophilic efficiency,25 which normalizes the potency achieved with the
lipophilicity of the molecule (LipE = pIC50 – LogP) thus serving as a useful descriptor of drugꢀ
like characteristics. High lipophilicity can drive poor metabolic stability, solubility and
polypharmacology, hence a higher LipE is preferable. Hence, towards our goal of reducing
lipophilicity, while retaining adequate DcpS potency, we generated LipE plots (Figure 4) of the
lipophilic piperidine analogs along with some nonꢀpiperidine analogs using DcpS inhibitory
activity. As seen from the LipE plot and Table 1, the lipophilic efficiency of 1 (LipE: 4.9) and
the closeꢀin analogs were not inferior to the simple unsubstituted 2,4ꢀdiaminoquinazoline
template (compound 9: LipE: 5.8) and the minimally substituted 5ꢀmethoxyꢀ2,4ꢀ
diaminoquinazoline (Compound 10: LipE: 6.6).
ACS Paragon Plus Environment

Page 9 of 50
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Simple aliphatic/alicyclic groups at the C5ꢀposition using synthetically expedient ether
linkages, as represented by analogs 14ꢀ18, resulted in moderate DcpS activity compared to the
piperidine analogs shown in Table 2. However, these analogs exhibited superior lipophilic
efficiency as shown in Table 2 and Fig 4. From the ligandꢀbound crystal structure of DcpS, the
Cꢀ5 substituents are placed in a hydrophobic environment and an aromatic ring seems to provide
the best complementarity. Based on this analysis, unsubstituted and halogenꢀsubstituted
aromatic ethers (compounds 11-13) were evaluated for DcpS inhibitory activity and were found
to be more active than the aliphatic analogs. However, this modification resulted in increased
lipophilicity, thus compromising the lipophilic efficiency. The corresponding reverse ether
analogs provided a slight boost in potency with an added advantage of reduced cLogP. The
replacement of an aromatic chloride with a nitrile often improves LipE,26 hence a nitrile group
was introduced in the aryl ether substituent to yield compound 24. Additionally, the replacement
of the nitrile with a bioisosteric27 pyridine derivative as in compound 25, retained the improved
LipE. The reverse ether analogs had an added advantage with respect to DHFR selectivity as
shown in Table 2.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Physicochemical Properties
As predicted, analogs with less lipophilic C5ꢀsubstituents showed higher metabolic
stability. These compounds also showed potential for good brain penetration as determined by
the MDR BA/AB ratio of <2.5 and good aqueous solubility.
In-vitro ADME and
physicochemical profiling of selected nonꢀlipophilic compounds in comparison to the original
lipophilic series (exemplified by derivative 1) is shown in Fig 5. Table 3 shows the
physicochemical properties improvement of representative examples 24 and 25 in comparison to
compound 1.
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 10 of 50
1
2
3
4
5
6
7
8
9
Having identified potent DcpS inhibitors with improved physicochemical properties, a
representative analog Compound 25, was evaluated for cellular accumulation in the presence and
absence of either concanamycin A or bafilomycin. In marked contrast to 1 and close analogs,
compound 25 showed dramatically reduced cellular accumulation. As a result, the concentration
of compound 25 in cells was too low to reliably determine the effect of bafilomycin or
concanamycin A (Fig 6).
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Pharmacokinetics (PK)
Towards the goal of understanding if in-vivo efficacy is due to a DcpSꢀdependent
mechanism and not the effect of the physicochemical properties of the chemotype, one of the
optimized analogs, compound 24 was selected for in-vivo PK investigation. In-vivo PK
evaluation of compound 24 was carried out in WT mice (FVB/N) to understand the free
compound exposure in plasma, brain and CSF (Table 4). Free drug levels in brain were ~2.5
fold lower than that unbound in plasma, indicating minimal brain impairment. The PK data
indicated that free drug levels in brain were above the required coverage based on an estimated
IC90 concentration for DcpS inhibition. With adequate CNS exposure achieved compound 24
was evaluated in the 2B/ꢀ SMA mouse model and found to be effective in ameliorating the
disease phenotype in these mice. This result is consistent with DcpS inhibition being the
mechanism of action underlying therapeutic benefit of the C5ꢀsubstituted 2,4ꢀ
diaminoquinazolines in SMA. These data and a comprehensive study of the biological effects of
DcpS inhibitors in cells in-vitro and in-vivo are the subject of another report.28
Design of matched inactive analogs of DcpS inhibition
Towards our ultimate goal of understanding if the efficacy observed in SMA mouse
models by administering 1 is due to DcpS inhibition or due to the physicochemical and/or
ACS Paragon Plus Environment

Page 11 of 50
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
general structural characteristics of the chemotype, we were in need of structurallyꢀsimilar but
DcpS inactive 2,4ꢀdiaminoquinazolines as tool compounds. While scouting for inactive tool
compounds is not a common strategy in a drug discovery paradigm, such tools are an important
part of successful target validation29 and have the potential to unearth unprecedented molecular
pathways. Closely matched active/inactive pairs of compounds were generated using our
structural knowledge of the interaction of the Cꢀ5 substituted 2,4ꢀdiaminoquinazolines with the
DcpS. As seen in Fig 8 (left panel), the quinazoline template occupies the binding pocket
making a favorable edge to face interaction with Tyr113 and any substitution from the 7ꢀposition
of the aromatic ring will not be accommodated (Fig 8, right panel). This prompted us to make
use of this spatial constraint to design 7ꢀsubstituted 2,4ꢀdiamino analogs as closeꢀin matched
inactive analogs.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
A subset of potent DcpS inhibitors (compounds 1, 2, 17, 24) from the C5ꢀsubstitued 2,4ꢀ
diaminoquinazolines were chosen to explore substituent effects in the 7ꢀposition. The methyl
substituent was chosen to maintain the electronics of the diaminoquinazoline ring while
providing a potentially significant steric clash with the protein. As shown in Tableꢀ5 all the 7ꢀ
methyl analogs (compound 26-29) were considerably less active compared to the 7ꢀH DcpS
inhibitors. As anticipated, irrespective of the length or nature of the Cꢀ5 substituents, 7ꢀMe
substituted 2,4ꢀdiaminoquinazolines possessed the desired loss of Dcps activity. These tool
compounds serve as useful probes in a DcpS toolkit for further target validation and were
extensively used in further in-vitro studies.28
CONCLUSION
Compound 1 is a dibasic lipophilic Cꢀ5ꢀdisubstituted 2,4ꢀdiaminoquinazoline analog that
prolongs survival and function in SMA mouse models. While the compound is a potent DcpS
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 12 of 50
1
2
3
4
5
6
7
8
9
inhibitor, in the absence of a direct link to the mechanistic role of DcpS inhibition for efficacy,
lysosomotropism was considered as a possible contributor to in-vivo efficacy. To shed more
light to this, we employed rational structureꢀbased design of potent DcpS inhibitors with
improved physicochemical properties with adequate CNS exposure to evaluate in-vivo.
Compound 24 was found to be efficacious in an SMA mouse model by prolonging survival and
improving function thereby ruling out lysosomotropism as the mechanism of efficacy.
Compound 24 (referred to now as PFꢀDcpSi) and related analogs with good physicochemical
properties, along with the matched pair of inactive analogs, provide key starting points for
understanding DcpS as a drug discovery target for spinal muscular atrophy.
EXPERIMENTAL SECTION
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
General information
Chemicals were typically obtained from SigmaꢀAldrich or Alfa Aesar and used as
received, unless noted otherwise. Solvents were commercial anhydrous grade, used as received.
Unless otherwise stated, reactions were run under a positive pressure of nitrogen at ambient
temperature. “Flash chromatography” refers to normal phase chromatography using a medium
pressure Biotage or ISCO system employing commercial preꢀpacked silica gel columns.
Analytical thin layer chromatography (TLC) was performed on 60 F254 glass plates preꢀcoated
with a 0.25ꢀmm thickness of silica gel purchased from EMD chemical Inc. TLC plates were
visualized with UV light.
13
19
1H , C (1H decoupled), and F (1H decoupled) nuclear magnetic resonance (NMR)
spectra were recorded on a Bruker or Varian spectrometer operating at 400, 101 and 376 MHz,
respectively, or a Bruker 500 MHz spectrometer operating at 500, 126 and 470 MHz,
respectively. Chemical shifts reported in parts per million ( δ ) relative to CDCl 1H NMR,
3 (
ACS Paragon Plus Environment

Page 13 of 50
Journal of Medicinal Chemistry
1
2
7.26 ppm; 1 3 C NMR, 77.00 ppm), DMSOꢀd6 (1H NMR, 2.50 ppm; 1 3 C NMR, 39.52 ppm),
CD3OD (1H NMR, 3.31 ppm; 1 3 C NMR; 49.00 ppm), D2O (1H NMR 4.79 ppm), CD2Cl2 (13C
NMR 54.00 ppm) The following abbreviations are used for peak multiplicities: s = singlet, d =
doublet, t = triplet, m = multiplet, br = broadened, dd = doublet of doublets, dt = doublet of
triplets. Low resolution mass spectroscopy was carried out using 1) liquid chromatography−mass
spectrometry (LCMS) on a Waters Acquity UPLC instrument using atmospheric pressure
chemical ionization (APCI) or electrospray ionization (ESI), or 2) by gas chromatographyꢀmass
spectroscopy (GCMS) on an Agilent 5975 series instrument with electron ionization (EI). High
resolution mass measurements were carried out on an Agilent TOF 6200 series instrument with
ESI. The purity of final compounds as measured by HPLC was at least above 95%.
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
All procedures involving animals were conducted as per the study protocols approved by
Pfizer or other Institutional Animal Care and Use Committee and, in compliance with applicable
institutional guidelines, state & national regulations and the Guide for Care and Use of
Laboratory Animals.
Compounds 1ꢀ 8 were prepared as described in Reference 5.
(S)-5-((1-Methoxypropan-2-yl)oxy)quinazoline-2,4-diamine (15) and (R)-5-((1-
Methoxypropan-2-yl)oxy)quinazoline-2,4-diamine (16). Compound 37 (140 mg) was
separated by chiral SFC (ChiralPak AD (250x30mm, 20 ꢀm); 40% EtOH (0.05% NH3 in H2O);
80 mL/min) to give two peaks arbitrarily assigned: 15 (peak 1, RT = 7.958 min, 39 mg, 28 %) as
1
a yellow solid: LC/MS (ESI) m/z 249.0 [M+H]+; H NMR (400 MHz, METHANOLꢀd4) δ 7.34
(t, J=8.20 Hz, 1H), 6.78 (d, J=7.80 Hz, 1H), 6.60 (d, J=8.20 Hz, 1H), 4.63ꢀ4.74 (m, 1H), 3.40ꢀ
13
3.69 (m, 2H), 3.32 (s, 2H), 1.31 (d, J=6.24 Hz, 2H); C NMR (101 MHz, METHANOLꢀd4) δ
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 14 of 50
1
2
3
4
5
6
7
8
9
163.0, 160.2, 156.1, 153.3, 133.3, 115.7, 104.4, 102.2, 74.8, 74.3, 58.1, 15.1; HRMS m/z calcd
for C12H16N4O2 [M+H]+: 249.1346. Found: 249.1354 and 16 (peak 2, RT = 9.19 min, 39 mg,
28%) as a white solid: LC/MS (ESI) m/z 248.9 [M+H]+; 1H NMR (400 MHz, METHANOLꢀd4)
δ 7.35 (t, J=8.20 Hz, 1H), 6.78 (d, J=8.20 Hz, 1H), 6.60 (d, J=8.20 Hz, 1H), 4.63ꢀ4.74 (m, 1H),
3.43ꢀ3.62 (m, 2H), 3.32 (s, 3H), 1.30 (d, J=6.24 Hz, 3H); 13C NMR (101 MHz, METHANOLꢀd4)
δ 163.0, 160.1, 156.1, 153.2, 133.3, 115.6, 104.5, 102.2, 74.8, 74.3, 58.1, 15.1; HRMS m/z calcd
for C12H16N4O2 [M+H]+: 249.1346. Found: 249.1353.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
5-(((Tetrahydro-2H-pyran-4-yl)oxy)methyl)quinazoline-2,4-diamine
(18).
The
procedure used to prepare compound 25 was used to prepare compound 18; using commercially
available tetrahydroꢀ2Hꢀpyranꢀ4ꢀol and 2ꢀ(bromomethyl)ꢀ6ꢀfluorobenzonitrile (39) as reagents in
1
the initial nucleophilic displacement step followed by cyclization: H NMR (400 MHz, DMSOꢀ
d6) δ 7.37ꢀ7.47 (m, 1H), 7.30 (br. s., 2H), 7.23 (d, J=8.59 Hz, 1H), 7.03 (d, J=6.63 Hz, 1H), 6.01
(br. s., 2H), 4.78 (s, 2H), 3.77ꢀ3.86 (m, 2H), 3.74 (td, J=4.59, 8.78 Hz, 1H), 3.37ꢀ3.42 (m, 2H),
13
1.90ꢀ1.93 (m, 2H), 1.37ꢀ1.57 (m, 2H); C NMR (101 MHz, DMSOꢀd6) δ 163.1, 160.6, 155.4,
134.0, 132.0, 126.4, 124.8, 111.1, 73.2, 69.5, 65.2, 32.5; HRMS (ESI) m/z: calculated for
C14H18N4O2 [M + H]+ 275.1503; observed 275.1509.
3-((2,4-Diaminoquinazolin-5-yl)methoxy)-5-fluorobenzonitrile (24). Compound 24
was prepared similarly to 25 using commercially available 3ꢀfluoroꢀ5ꢀhydroxybenzonitrile and 2ꢀ
(bromomethyl)ꢀ6ꢀfluorobenzonitrile (39) as reagents in the nucleophilic displacement step
followed by cyclization, compound was purified by reverse phase HPLC using a gradient of
water and acetonitrile (95:5 to 5:95) to give 24 (7 mg, 51% yield over two steps) as a yellow
1
color TFA salt: H NMR (400 MHz, DMSOꢀd6) δ 13.13 (br. s., 2H), 8.13 (br. s., 2H), 7.87 (t,
ACS Paragon Plus Environment

Page 15 of 50
Journal of Medicinal Chemistry
1
2
J=7.81 Hz, 1H), 7.59ꢀ7.67 (m, 2H), 7.50ꢀ7.59 (m, 3H), 5.73 (s, 2H); 19F NMR (376 MHz,
3
4
5
6
7
8
13
DMSOꢀd6) δ ꢀ73.77 (s, 3F, TFA), ꢀ108.59 (s, 1F); C NMR (101 MHz, DMSOꢀd6) δ 163.45 (t,
1
JCF=7.63 Hz), 163.0 (d, JCF=244.64 Hz), 159.6 (q, JCF=12.18 Hz), 154.1, 141.9, 135.4, 134.2,
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
126.4, 118.0, 116.3, 113.7 (d, JCF=10.42 Hz), 112.8 (d, JCF=27.48 Hz), 109.3 (d, JCF=24.63 Hz),
109.0, 69.4; HRMS (ESI) m/z: calculated for C16H12FN5O [M + H]+ 310.1099; observed
310.1107.
5-(((2-Methylpyridin-3-yl)oxy)methyl)quinazoline-2,4-diamine
dihydrochloride
(25a). To a solution of 41 (33.0 g, 136 mmol) in DMA (350 mL) was added guanidine carbonate
(49.1 g, 272 mmol). The reaction mixture was stirred at 140oC for 18 hours. The reaction mixture
was poured over ice, the precipitated solid was collected by filtration and washed with water,
dried in vacuo to afford 5ꢀ(((2ꢀmethylpyridinꢀ3ꢀyl)oxy)methyl)quinazolineꢀ2,4ꢀdiamine (33.2 g,
86.6%) as yellow solid as free base. The solid was dissolved using water and conc. HCl, mixture
was lyophilized on the high vacuum to afford 25 (40.9 g, 98.4%) as a pink solid: LC/MS (ESI)
1
m/z 282.1 [M+H]+; H NMR (400 MHz, DMSOꢀd6) δ 8.07 (d, J=4.68 Hz, 1H), 7.60 (d, J=8.20
Hz, 1H), 7.45 (d, J=7.81 Hz, 1H), 7.20ꢀ7.30 (m, 2H), 7.17 (d, J=7.02 Hz, 1H), 6.78 (br. s., 2H),
13
6.08 (br. s., 2H), 5.45 (s, 2H), 2.36 (s, 3H); C NMR (101 MHz, DMSOꢀd6) δ162.4, 160.0,
154.9, 151.4, 147.8, 141.1, 131.9, 131.6, 126.2, 123.9, 122.1, 119.4, 110.1, 69.6, 19.3; HRMS
(ESI) m/z: calculated for C15H15N5O [M + H]+ 282.1349; observed 282.1351.
5-((1-(2,6-Dichlorobenzyl)piperidin-4-yl)methoxy)-7-methylquinazoline-2,4-diamine
(26). To a solution of 46 (150 mg, 0.522 mmol) and triethylamine (106 mg, 1.04 mmol) in DMF
(2 mL) was added 47 (138 mg, 0.574 mmol), the reaction stirred at 60 °C for 16 h. The solution
was poured over iceꢀwater and precipitated solid was collected by filtration to afford 26 (130 mg,
55.8 %) as an off white solid: LC/MS (ESI) m/z 445.9 [M+H]+;1H NMR (400 MHz, DMSOꢀd6)
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 16 of 50
1
2
3
4
5
6
7
8
9
δ 7.47 (d, J=8.03 Hz, 2H), 7.34 (t, J=8.03 Hz, 1H), 7.12 (br. s., 2H), 6.60 (s, 1H), 6.39 (s, 1H),
5.93 (br. s., 2H), 3.96 (d, J=6.53 Hz, 2H), 3.69 (s, 2H), 2.78ꢀ2.96 (m, 2H), 2.28 (s, 3H), 2.22 (t,
J=10.79 Hz, 3H), 1.75 (d, J=12.05 Hz, 2H), 1.13ꢀ1.35 (m, 2H); 13C NMR (101 MHz, DMSOꢀd6)
δ 162.6, 162.5, 157.2, 156.4, 146.7, 136.6, 134.4, 130.3, 129.1, 111.2, 107.3, 98.5, 74.1, 56.8,
53.1, 35.1, 29.0, 22.3; HRMS m/z calcd for C22H25Cl2N5O [M+H]+: 446.1509. Found: 446.1508
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
5-((1-(2-Fluorobenzyl)piperidin-4-yl)methoxy)-7-methylquinazoline-2,4-diamine
(27). Compound 27 was prepared similarly to 26 using commercially available 1ꢀ(bromomethyl)ꢀ
2ꢀfluorobenzene and 7ꢀmethylꢀ5ꢀ(piperidinꢀ4ꢀylmethoxy)quinazolineꢀ2,4ꢀdiamine (46) as
1
reagents in the nucleophilic displacement step: LC/MS (ESI) m/z 396.1 [M+H]+; H NMR (400
MHz, DMSOꢀd6) δ 7.42 (t, J=6.78 Hz, 1H), 7.28ꢀ7.37 (m, 1H), 7.07ꢀ7.23 (m, 4H), 6.61 (s, 1H),
6.39 (s, 1H), 5.93 (br. s., 2H), 3.98 (d, J=6.02 Hz, 2H), 3.36 (s, 2H), 2.88 (d, J=11.04 Hz, 2H),
2.29 (s, 3H), 2.03 (t, J=10.79 Hz, 2H), 1.86 (br. s., 1H), 1.76 (d, J=12.05 Hz, 2H), 1.28ꢀ1.44 (m,
13
1
2H); C NMR (101 MHz, DMSOꢀd6) 162.5 (d, JCF=247.51 Hz), 162.1, 161.2, 156.9, 155.6,
3
3
2
143.0, 132.0 (d, JCF=5.03 Hz), 129.4 (d, JCF=8.32 Hz), 125.2 (d, JCF=14.77 Hz), 124.6 (d,
4JCF=3.5 Hz),, 117.2, 115.5 (d, 2JCF=23.96 Hz), 103.8, 99.7, 73.5, 55.3, 53.0, 35.6, 29.2, 22.3; 19F
NMR (376 MHz, DMSOꢀd6) δ ꢀ118.10 (s, 1F); HRMS m/z calcd for C22H26FN5O [M+H]+:
396.2194. Found: 396.2201.
3-((2,4-Diamino-7-methylquinazolin-5-yl)methoxy)-5-fluorobenzonitrile (28). To a
solution of 55 (1.47 g, 5.171 mmol) in DMA (16 mL) was added guanidine carbonate (1.86 g,
o
10.3 mmol) at 10oC. The reaction mixture was heated at 140 C for 4 h, quenched by pouring
over iceꢀwater and resulting solid filtered and washed with water. The residue was purified by
chromatography over silica gel eluting with gradient of DCM and MeOH (1:0 to 10:1) to give
the crude 28 (560 mg) as a brown solid. The solid was recrystallized from EtOH, dried on high
ACS Paragon Plus Environment

Page 17 of 50
Journal of Medicinal Chemistry
1
2
1
vacuum to give 28 (296 mg, 18%) as a light brown solid: LC/MS (ESI) m/z 323.9 [M+H]+; H
3
4
5
6
7
NMR (400 MHz, CD3OD) δ 7.41 (s, 1H), 7.29ꢀ7.35 (m, 1H), 7.25 (d, J=8.03 Hz, 1H), 7.19 (s,
1H), 7.13 (s, 1H), 5.45 (s, 2H), 2.42 (s, 3H); 13C NMR (101 MHz, DMSOꢀd6) δ 163.0 (d,
1JCF=245.88 Hz), 162.6, 160.6, 159.6 (d, JCF=12.93 Hz), 155.7, 141.9, 131.6, 126.0 (d, JCF=26.67
Hz), 118.0 (d, JCF=5.49 Hz), 116.3, 113.7 (d, JCF=12.98 Hz), 112.8 (d, JCF=25.46 Hz), 109.3 (d,
JCF=24.46 Hz), 108.4, 71.0. 21.6; 19F NMR (376 MHz, DMSOꢀd6) δ ppm ꢀ108.5 (s, 1 F); HRMS
m/z calcd for C17H14N5O [M+H]+: 324.1255. Found: 324.1261.
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
7-Methyl-5-((tetrahydro-2H-pyran-4-yl)methoxy)quinazoline-2,4-diamine
(29).
Compound 29 was prepared similarly to 26 using commercially available (tetrahydroꢀ2Hꢀpyranꢀ
4ꢀyl)methanol and 2,6ꢀdifluoroꢀ4ꢀmethylbenzonitrile (43) as reagents in the nucleophilic
1
displacement step followed by cyclization: LC/MS (ESI) m/z 289.2 [M+H]+; H NMR (400
MHz, CD3OD) δ 6.74 (s, 1H), 6.54 (s, 1H), 3.93ꢀ4.14 (m, 4H), 3.52 (t, J=11.04 Hz, 2H), 2.39 (s,
13
3H), 2.12ꢀ2.31 (m, 1H), 1.81 (d, J=13.05 Hz, 2H), 1.52 (dq, J=4.52, 12.38 Hz, 2H); C NMR
(101 MHz, DMSOꢀd6) δ 162.07, 161.25, 156.84, 155.62, 142.97, 117.18, 103.84, 99.73, 73.52,
67.03, 34.65, 29.79, 22.31; HRMS m/z calcd for C15H20N4O2 [M+H]+: 289.1659. Found: 289.167
2-Fluoro-6-((1-methoxypropan-2-yl)oxy)benzonitrile (36). To a stirred suspension of
sodium hydride (0.8 g, 20 mmol) in THF (20 mL) was added 35 (0.9 g, 10 mmol) at 0oC and
reaction stirred at same temperature for 1h. A solution of 2,6ꢀdifluorobenzonitrile 31 (1.39 g, 10
mmol) in THF (25 mL) was added at 0oC and reaction mixture stirred for 12h at room
temperature. The reaction mixture was poured over crushed iceꢀwater, and extracted with ethyl
acetate. The organic layer was washed with brine, dried over MgSO4, filtered and concentrated
in vacuum. The residue was purified by silica gel flash chromatography eluting with a gradient
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 18 of 50
1
2
1
3
4
5
6
7
8
9
of petroleum ether and EtOAc (10: 1 to 3:1) to afford 36 (1.2 g, 60%) as an oil: H NMR (400
MHz, CDCl3) δ 7.40ꢀ7.57 (m, 1H), 6.86 (d, J=8.59 Hz, 1H), 6.78 (t, J=8.39 Hz, 1H), 4.54ꢀ4.75
(m, 1H), 3.66 (dd, J=6.44, 10.34 Hz, 1H), 3.57 (dd, J=4.29, 10.54 Hz, 1H), 3.44 (s, 3H), 1.39 (d,
J=6.24 Hz, 3H).
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
5-((1-Methoxypropan-2-yl)oxy)quinazoline-2,4-diamine (racemic) (37). A mixture of
36 (1.2 g, 6 mmol) and guanidine carbonate (2.16 g, 12 mmol) in DMA (12 mL) was heated at
140oC for 12 h. The reaction mixture was cooled to room temperature and crude product was
purified by reverse phase HPLC using a gradient of water and acetonitrile (95:5 to 5:95) to give
racemic 37 (140 mg, 10 %) as a white solid: LC/MS (ESI) m/z 249.0 [M+H]+.
2-(Bromomethyl)-6-fluorobenzonitrile (39). To a mixture of 38 (45.0 g, 333 mmol) and
NBS (53.3 g, 300 mmol) in carbon tetrachloride (450 mL) was added AIBN (2.73 g, 16.6 mmol).
The reaction was heated at reflux for 18 h, allowed to cool at room temperature and quenched
with saturated sodium thiosulfate. The reaction was extracted with DCM; organic layer was dried
over sodium sulfate, filtered and concentrated in vacuo. The residue was purified by silica gel
flash chromatography eluting with a gradient of petroleum ether and ethyl acetate (10: 0 to 2:8)
1
to afford 39 (42.5 g, 59.6%) as a white solid. H NMR (400 MHz, CDCl3) δ ppm 7.62 (m, 1H),
7.36 (d, J=6.2 Hz, 1 H), 7.17 (t, J=8.20 Hz, 1 H) 4.63 (2, 2H)
2-Fluoro-6-(((2-methylpyridin-3-yl)oxy)methyl)benzonitrile (41). To a mixture of 39
(42.2 g, 197 mmol) and potassium carbonate (68.1 g, 493 mmol) in acetonitrile (400 mL) was
added 40 (21.5 g, 197 mmol). The mixture was stirred at room temperature for 18h, filtered and
the filter cake was washed with ethyl acetate (300 mL). The residue was purified by silica gel
flash chromatography eluting with a gradient of petroleum ether and ethyl acetate (5:1 to 3:1) to
ACS Paragon Plus Environment

Page 19 of 50
Journal of Medicinal Chemistry
1
2
3
4
1
afford 41 (33.0 g, 69.1%) as a yellow solid. H NMR (400 MHz, CDCl3) δ ppm 8.18 (d, J=6.2
5
Hz, 1H), 7.65 (m, 1H), 7.5 (d, J=7.80 Hz, 1 H), 7.23 (t, J=8.2 Hz, 1H), 7.22ꢀ7.07 (m, 2H), 5.26
6
7
8
9
(s, 2H), 2.57 (s, 2H)
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
2,6-Difluoro-4-methylbenzonitrile (43). To a solution of 42 (1 g, 4.59 mmol) in
dioxane/water (5:1, 12 mL, degassed) was added methylboronic acid (412 mg, 6.88 mmol),
tetrakis(triphenylphosphine)palladium(0) (530 mg, 0.459 mmol) and potassium carbonate (1.9 g,
14 mmol). The mixture was stirred at 110oC for 3 h under nitrogen. The suspension was
concentrated in vacuo and residue was purified by silica gel flash chromatography eluting with a
gradient of petroleum ether and ethyl acetate (10:1 to 3:1) to give 43 (0.37 g, 52.7%) as a white
solid: 1H NMR (400 MHz, CDCl3) δ 6.87 (d, J=8.53 Hz, 2H), 2.45 (s, 3H).
t-Butyl 4-((2-cyano-3-fluoro-5-methylphenoxy)methyl)piperidine-1-carboxylate (44).
A solution of 30 (0.50 g, 2.35 mmol) in DMF (5 mL) was added dropwise to a suspension of
sodium hydride (0.141 g, 3.53 mmol, 60% in mineral oil) in DMF (2.5 mL) at 0 °C, reaction
gradually warmed up to room temperature and stirred for 1 h. A solution of 43 (360 mg, 2.35
mmol) in DMF (2.5 mL) was cooled to 0 °C, and the alcohol mixture was added dropwise to the
benzonitrile solution and stirred for 3 h. The solution was poured over iceꢀwater. The
precipitated solid was collected by filtration and residue was purified by silica gel flash
chromatography using petroleum ether and ethyl acetate (1:1) to give 44 (0.3 g, 36.6%) as a
colourless oil: LC/MS (ESI) m/z 371.2 [M+Na]; 1H NMR (400 MHz, CDCl3) δ 6.60 (d, J=9.03
Hz, 1H), 6.53 (s, 1H), 4.18 (br. s., 2H), 3.89 (br. s., 2H), 2.76 (br. s., 2H), 2.39 (s, 3H), 2.05 (br.
s., 1H), 1.87 (d, J=12.05 Hz, 2H), 1.47 (s, 9H), 1.26 (dd, J=4.02, 12.05 Hz, 2H)
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 20 of 50
1
2
3
4
5
6
7
8
9
t-Butyl-4-(((2,4-diamino-7-methylquinazolin-5-yl)oxy)methyl)piperidine-1-
carboxylate (45). A mixture of 44 (0.295 g, 0.847 mmol) and guanidine carbonate salt (0.305 g,
1.69 mmol) in DMA (5 mL) was heated at 140oC for 18 h. The solution was poured over iceꢀ
water. The precipitated solid was collected by filtration to afford 45 (0.25 g, 76.2 %) as a white
solid: LC/MS (ESI) m/z 387.9 [M+H]+; 1H NMR (400 MHz, DMSOꢀd6) δ 7.09 (br. s., 2H), 6.60
(s, 1H), 6.40 (s, 1H), 5.91 (br. s., 2H), 3.99 (m, 4H), 2.79 (m, 2H), 2.29 (s, 3H), 2.07 (m, 1H),
1.76 (d, J=12.05 Hz, 2H), 1.41 (s, 9H), 1.06ꢀ1.31 (m, 2H)
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
7-Methyl-5-(piperidin-4-ylmethoxy)quinazoline-2,4-diamine (46). A solution of HCl
in dioxane (4M, 8 mL) was added to a solution of 45 (0.250 g, 0.645 mmol) in MeOH (8 mL).
The solution was stirred at room temperature for 3 h under nitrogen. The precipitate was
collected by filtration to afford 46 (0.165 g, 89 %) as a yellow solid; LC/MS (ESI) m/z 287.6
1
[M+H]+; H NMR (400 MHz, CD3OD) δ 6.93 (s, 1H), 6.84 (s, 1H), 4.25 (d, J=6.53 Hz, 2H),
3.50 (d, J=12.55 Hz, 2H), 3.03ꢀ3.19 (m, 2H), 2.49 (s, 3H), 2.35ꢀ2.45 (m, 1H), 2.13 (d, J=13.55
Hz, 2H), 1.53ꢀ1.76 (m, 2H)
2-Bromo-6-fluoro-4-methylaniline (49). To a solution of 48 (30 g, 240 mmol) in acetic
acid (240 mL) was added NBS (48 g, 270 mmol) in portions over 30 minutes at room
temperature. The reaction turned from brown to red in color and was stirred for another 90 min.
Water was added, and the mixture extracted into ethyl acetate (2 times). The combined organics
were washed sequentially with 10% aqueous sodium carbonate, brine, dried over sodium sulfate,
filtered and evaporated in vacuum. The residue was purified by column chromatography over
silica gel which was eluted using a gradient of petroleum ether and ethyl acetate (1:0 to 3:1) to
give 49 (28.4 g, 58%) as brown oil, which solidified on standing. 1H NMR (400 MHz, CDCl3) δ
7.03 (s, 1H), 6.78 (d, J=11.54 Hz, 1H), 3.96 (br. s., 2H), 2.23 (s, 3H)
ACS Paragon Plus Environment

Page 21 of 50
Journal of Medicinal Chemistry
1
2
3
4
2-Bromo-6-fluoro-4-methylbenzonitrile (50). Compound 49 (5 g, 20.0 mmol) was
5
6
o
suspended in a mixture of water (10.5 mL) and glacial AcOH (17.5 mL) at 20 C, concentrated
7
8
9
o
H2SO4 (3.7 mL) was added dropwise at the same temperature. The mixture was cooled to 0 C
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
and a solution of sodium nitrite (2.0 g, 28.99 mmol) in water (3.7 mL) was added dropwise with
vigorous stirring. In another 500 mL roundꢀbottomed flask, copper(II) sulfate pentahydrate (7.65
g, 30.6 mmol) was dissolved in water (20 mL), and solution of KCN (8.0 g, 122.9 mol) in water
o
(20 mL) was added dropwise slowly at 20 C. NaHCO3 (17.5 g, 208.32 mmol) and toluene (20
mL) were added and the reaction was placed in an oil bath preheated to 55 oC. To this flask, the
above prepared solution of the diazonium salt was added dropwise with vigorous stirring over 30
minutes and heating continued for additional 30 min. The reaction was basified with NaOH
(pH=11). The reaction was diluted with DCM, filtered over celite, organics extracted and washed
with water and brine, dried over Na2SO4, filtered and evaporated in vacuum. The residue was
purified by chromatography over silica gel eluting with gradient of petroleum ether and ethyl
acetate (1:0 to 3:1) to give 50 (2.3 g, 40%) as a light brown solid. 1H NMR (400 MHz, CDCl3) δ
7.33 (s, 1H), 7.00 (d, J=9.03 Hz, 1H), 2.43 (s, 3H)
2-Fluoro-6-formyl-4-methylbenzonitrile (51). To a solution of 50 (2.2 g, 10 mmol) in
o
THF (15 mL) was added iꢀPrMgCl (6.2 mL, 2M) drop wise at ꢀ40 C, reaction continued for
o
o
additional 3 h while maintaining internal temperature between ꢀ40 C to ꢀ30 C. DMF (2.25 g,
30.8 mmol) was added drop wise over 20 min and reaction warmed to 20oC for 1 h. The reaction
mixture was cooled to ꢀ10 oC, quenched with slow addition of 2M aqueous HCl (22 mL) over 20
min and stirring continued at ꢀ10 oC for 30 min. Ethyl acetate was added, organic layer extracted,
washed with brine, dried over Na2SO4 and concentrated. The residue was purified by
chromatography over silica gel eluting with gradient of petroleum ether and ethyl acetate (1:0 to
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 22 of 50
1
2
3
4
1
5:1) to give 51 (1.37 g, 82%) as a yellow solid: H NMR (400 MHz, CDCl3) δ 10.25 (s, 1H),
5
7.66 (s, 1H), 7.32 (d, J=9.03 Hz, 1H), 2.53 (s, 3H)
6
7
8
9
2-Fluoro-6-(hydroxymethyl)-4-methylbenzonitrile (52). To a solution of 51 (1.27 g,
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
7.784 mmol) in EtOAc (30 mL, dried over Na2SO4) was added NaBH(OAc)3 (3.3 g, 15.6 mmol)
o
o
in portions at 20 C, after stirring for 30 min, reaction was heated to 40 C for 2 h. The reaction
o
mixture was cooled to 10 C and quenched with water, aqueous phase extracted with EtOAc (2
times), combined organic layers dried over Na2SO4, filtered and concentrated in vacuum. The
residue was purified by chromatography over silica gel eluting with gradient of petroleum ether
and EtOAc (10:1 to 1:1) to give 52 (1.1 g, 86%) as a yellow solid: 1H NMR (400 MHz, CDCl3) δ
7.25 (d, J=9.03 Hz, 1H), 6.95 (d, J=9.54 Hz, 1H), 4.87 (d, J=4.52 Hz, 2H), 2.44 (s, 3H)
2-(Bromomethyl)-6-fluoro-4-methylbenzonitrile (53). To a solution of compound 52
(24.4 g, 147.73 mmol) in DCM (500 mL) was added carbon tetrabromide (162 g, 488 mmol) and
o
triphenylphosphine (58.1 g, 222 mmol) at 10 C. The reaction mixture was stirred at room
temperature for 18 h. The reaction solution was concentrated in vacuum to give 53 (238 g crude,
contained about 33.6 g product, 100%) as brown oil, which was used for the next step directly
without further purification.
2-((3-Cyano-5-fluorophenoxy)methyl)-6-fluoro-4-methylbenzonitrile (55). To
a
solution of 54 (20.2 g, 147 mmol) in acetonitrile (400 mL) was added K2CO3 (61.1 g, 442 mmol)
o
and 53 (238 g, crude, contained about 33.6 g product, 147.33 mmol) at 10 C. The light brown
suspension was stirred at 10oC for 18 h. The reaction mixture was concentrated in vacuum to
remove acetonitrile. The residue was diluted with water and extracted with DCM. The organic
layer was dried over Na2SO4, filtered and the filtrate was concentrated in vacuum. The residue
ACS Paragon Plus Environment

Page 23 of 50
Journal of Medicinal Chemistry
1
2
3
4
was purified by chromatography over silica gel eluted using a gradient of petroleum ether and
5
6
1
EtOAc (10:1 to 3:1) to afford 55 (28.2 g, 67.6%) as a light brown solid: H NMR (400 MHz,
7
8
9
CDCl3) δ 7.23 (s, 1H), 7.02ꢀ7.12 (m, 3H), 6.98 (dd, J=2.26, 9.79 Hz, 1H), 5.20 (s, 2H), 2.42ꢀ2.53
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
(m, 3H)
Cellular accumulation assay: HEK293T cells or lymphoblasts were plated at 1 x 106
cells per well of a 6ꢀwell plate and incubated overnight before being treated for 2 h with either
Concanamycin A (100 nM) or Baflamycin (50 nM) or an equivalent concentration of DMSO as
control. Thereafter, either Chloroquine (100 nM), or one of the diaminoquinazoline compounds
(10 nM) or an equivalent DMSO as control were added to the cells and incubated for a further
2.5 h after which a fresh solution of drug or DMSO was applied for a further 2.5 h incubation.
The medium was aspirated; cells were washed with PBS, scraped from plates, suspended and
counted before being pelleted, the supernatant removed and the pellet solubilized in acetonitrile
for determination of compound concentration.
ASSOCIATED CONTENT
Supporting Information. The Supporting Information is available free of charge on the ACS
Publications website at DOI: 10.1021/acs.jmedchem.xxxxxxx.
Molecule formula strings (CSV)
AUTHOR INFORMATION
Corresponding Author
Eꢀmail: ariamala.gopalsamy@pfizer.com. Phone: (617) 674ꢀ7075
Notes
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 24 of 50
1
2
3
4
The authors declare no competing financial interest.
5
6
ACKNOWLEDGEMENT
7
8
9
We thank Ms. Catherine M. Ambler, Pharmaceutical Sciences, Pfizer, Groton, CT for
formulation help and Dr. Eddine Saiah, Ms. Rebeca RamosꢀZayas for early project enablement.
ABBREVIATIONS USED
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
DcpS – mRNA Decapping Scavenger Enzyme; SMA – Spinal Muscular Atrophy; SMN1 and
SMN2 – Survival Motor Neuron genes 1 and 2; LipE – Lipophilic Efficiency; ADME –
Absorption, Distribution, Metabolism and Excretion; HLM – human Liver Microsome; AcOH –
Acetic Acid; DCE – 1,2ꢀdichloroethane; DCM – dichloromethane; DMF – N,Nꢀ
dimethylformamide; DMAP – 4ꢀdimethylamino pyridine; DMSO – dimethylsulfoxide; DMA –
N,Nꢀdimethylacetamide; EtOH – ethanol; EtOAc – ethyl acetate; NBS – Nꢀbromosuccinimide;
MgSO4 – magnesium sulfate; Na2S2O4 – sodium sulfate; THF – tetrahydrofuran; SFC – Super
critical fluid chromatography
REFERENCES
1.
Nurputra, D. K.; Lai, P. S.; Harahap, N. I. F.; Morikawa, S.; Yamamoto, T.; Nishimura,
N.; Kubo, Y.; Takeuchi, A.; Saito, T.; Takeshima, Y.; Tohyama, Y.; Tay, S. K. H.; Low, P. S.;
Saito, K.; Nishio, H. Spinal muscular atrophy: From gene discovery to clinical trials. Ann. Hum.
Genet. 2013, 77, 435ꢀ463.
2.
Monani, U. R.; De Vivo, D. C. Neurodegeneration in spinal muscular atrophy: From
disease phenotype and animal models to therapeutic strategies and beyond. Future Neurol. 2014,
9, 49ꢀ65.
3.
Calder, A. N.; Androphy, E. J.; Hodgetts, K. J. Small molecules in development for the
treatment of spinal muscular atrophy. J. Med. Chem. 2016, 59, 10067ꢀ10083.
ACS Paragon Plus Environment

Page 25 of 50
Journal of Medicinal Chemistry
1
2
3
4
4.
Cherry, J. J.; Kobayashi, D. T.; Lynes, M. M.; Naryshkin, N. N.; Tiziano, F. D.;
5
6
Zaworski, P. G.; Rubin, L. L.; Jarecki, J. Assays for the identification and prioritization of drug
7
8
9
candidates for spinal muscular atrophy. Assay Drug Dev. Technol. 2014, 12, 315ꢀ341.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
5.
Thurmond, J.; Butchbach, M. E. R.; Palomo, M.; Pease, B.; Rao, M.; Bedell, L.; Keyvan,
M.; Pai, G.; Mishra, R.; Haraldsson, M.; Andresson, T.; Bragason, G.; Thosteinsdottir, M.;
Bjornsson, J. M.; Coovert, D. D.; Burghes, A. H. M.; Gurney, M. E.; Singh, J. Synthesis and
biological evaluation of novel 2,4ꢀdiaminoquinazoline derivatives as SMN2 promoter activators
for the potential treatment of spinal muscular atrophy. J. Med. Chem. 2008, 51, 449ꢀ469.
6.
Jarecki, J.; Chen, X.; Bernardino, A.; Coovert, D. D.; Whitney, M.; Burghes, A.; Stack,
J.; Pollok, B. A. Diverse smallꢀmolecule modulators of SMN expression found by highꢀ
throughput compound screening: Early leads towards a therapeutic for spinal muscular atrophy.
Hum. Mol. Genet. 2005, 14, 2003ꢀ2018.
7.
Hett, E. C.; Kyne, R. E., Jr.; Gopalsamy, A.; Tones, M. A.; Xu, H.; Thio, G. L.; Nolan,
E.; Jones, L. H. Selectivity determination of a small molecule chemical probe using protein
microarray and affinity capture techniques. ACS Comb. Sci. 2016, 18, 611ꢀ615.
8.
Singh, J.; Salcius, M.; Liu, S.ꢀW.; Staker, B. L.; Mishra, R.; Thurmond, J.; Michaud, G.;
Mattoon, D. R.; Printen, J.; Christensen, J.; Bjornsson, J. M.; Pollok, B. A.; Kiledjian, M.;
Stewart, L.; Jarecki, J.; Gurney, M. E. DcpS as a therapeutic target for spinal muscular atrophy.
ACS Chem. Biol. 2008, 3, 711ꢀ722.
9.
Gu, M.; Fabrega, C.; Liu, S.ꢀW.; Liu, H.; Kiledjian, M.; Lima, C. D. Insights into the
structure, mechanism, and regulation of scavenger mRNA decapping activity. Mol. Cell 2004,
14, 67ꢀ80.
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 26 of 50
1
2
3
4
10.
Liu, H.; Kiledjian, M. Decapping the message: A beginning or an end. Biochem. Soc.
5
6
Trans. 2006, 34, 35ꢀ38.
7
8
9
11.
Milac, A. L.; Bojarska, E.; Wypijewska del Nogal, A. Decapping scavenger (DcpS)
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
enzyme: Advances in its structure, activity and roles in the capꢀdependent mRNA metabolism.
Biochim. Biophys. Acta, Gene Regul. Mech. 2014, 1839, 452ꢀ462.
12.
Li, Y.; Kiledjian, M. Regulation of mRNA decapping. Wiley Interdiscip. Rev.: RNA
2010, 1, 253ꢀ265.
13.
Liu, H.; Rodgers, N. D.; Jiao, X.; Kiledjian, M. The scavenger mRNA decapping enzyme
DcpS is a member of the HIT family of pyrophosphatases. EMBO J. 2002, 21, 4699ꢀ4708.
14.
Chen, N.; Walsh, M. A.; Liu, Y.; Parker, R.; Song, H. Crystal structures of human DcpS
in ligandꢀfree and m7GDPꢀbound forms suggest a dynamic mechanism for scavenger mrna
decapping. J. Mol. Biol. 2005, 347, 707ꢀ718.
15.
Van Meerbeke, J. P.; Gibbs, R. M.; Plasterer, H. L.; Miao, W.; Feng, Z.; Lin, M.ꢀY.;
Rucki, A. A.; Wee, C. D.; Xia, B.; Sharma, S.; Jacques, V.; Li, D. K.; Pellizzoni, L.; Rusche, J.
R.; Ko, C.ꢀP.; Sumner, C. J. The DcpS inhibitor RG3039 improves motor function in SMA mice.
Hum. Mol. Genet. 2013, 22, 4074ꢀ4083.
16.
Gogliotti, R. G.; Cardona, H.; Singh, J.; Bail, S.; Emery, C.; Kuntz, N.; Jorgensen, M.;
Durens, M.; Xia, B.; Barlow, C.; Heier, C. R.; Plasterer, H. L.; Jacques, V.; Kiledjian, M.;
Jarecki, J.; Rusche, J.; DiDonato, C. J. The DcpS inhibitor RG3039 improves survival, function
and motor unit pathologies in two SMA mouse models. Hum. Mol. Genet. 2013, 22, 4084ꢀ4101.
17.
Black, W. C.; Percival, M. D. The consequences of lysosomotropism on the design of
selective cathepsin K inhibitors. ChemBioChem 2006, 7, 1525ꢀ1535.
ACS Paragon Plus Environment

Page 27 of 50
Journal of Medicinal Chemistry
1
2
3
4
18.
Wang, Y.; Gao, A.; Xu, X.; Dang, B.; You, W.; Li, H.; Yu, Z.; Chen, G. The
5
6
7
8
neuroprotection of lysosomotropic agents in experimental subarachnoid hemorrhage probably
involving the apoptosis pathway triggering by cathepsins via chelating intralysosomal iron. Mol.
Neurobiol. 2015, 52, 64ꢀ77.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
19.
autophagy perturbation. PLoS One 2013, 8, e82481.
20. Garcera, A.; Bahi, N.; Periyakaruppiah, A.; Arumugam, S.; Soler, R. M. Survival motor
Ashoor, R.; Yafawi, R.; Jessen, B.; Lu, S. The contribution of lysosomotropism to
neuron protein reduction deregulates autophagy in spinal cord motoneurons in vitro. Cell Death
Dis. 2013, 4, e686.
21.
models of spinal muscular atrophy. Mol. Cell. Neurosci. 2014, 61, 133ꢀ140.
22. Drose, S.; Altendorf, K. Bafilomycins and concanamycins as inhibitors of vꢀATPases and
pꢀATPases. J. Exp. Biol. 1997, 200, 1ꢀ8.
23. Xu, H.; Gopalsamy, A.; Hett, E. C.; Salter, S.; Aulabaugh, A.; Kyne, R. E.; Pierce, B.;
Custer, S. K.; Androphy, E. J. Autophagy dysregulation in cell culture and animals
Jones, L. H. Cellular thermal shift and clickable chemical probe assays for the determination of
drugꢀtarget engagement in live cells. Org. Biomol. Chem. 2016, 14, 6179ꢀ6183.
24.
Hett, E. C.; Xu, H.; Geoghegan, K. F.; Gopalsamy, A.; Kyne, R. E.; Menard, C. A.;
Narayanan, A.; Parikh, M. D.; Liu, S.; Roberts, L.; Robinson, R. P.; Tones, M. A.; Jones, L. H.
Rational targeting of activeꢀsite tyrosine residues using sulfonyl fluoride probes. ACS Chem.
Biol. 2015, 10, 1094ꢀ1098.
25.
FreemanꢀCook, K. D.; Hoffman, R. L.; Johnson, T. W. Lipophilic efficiency: The most
important efficiency metric in medicinal chemistry. Future Med. Chem. 2013, 5, 113ꢀ115.
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 28 of 50
1
2
3
4
26.
Jones, L. H.; Summerhill, N. W.; Swain, N. A.; Mills, J. E. Aromatic chloride to nitrile
5
6
7
8
transformation: Medicinal and synthetic chemistry. MedChemComm 2010, 1, 309ꢀ318.
27. Meanwell, N. A. Synopsis of some recent tactical application of bioisosteres in drug
design. J. Med. Chem. 2011, 54, 2529ꢀ2591.
28. Cherry, J. J.; DiDonato, C. J.; Androphy, E. J.; Calo, A.; Potter, K.; Custer, S. K.; Du, S.;
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Foley, T. L.; Gopalsamy, A.; Reedich, E. J.; Gordo, S. M.; Gordon, W.; Hosea, N.; Jones, L. H.;
Krizay, D. K.; LaRosa, G.; Li, H.; Mathur, S.; Menard, C. A.; Patel, P.; RamosꢀZayas, R.; Rietz,
A.; Rong, H.; Zhang, B.; Tones, M. A. Characterization of the in vitro and in vivo effects of 2,4ꢀ
diaminoquinazoline inhibitors of the decapping scavenger enzyme DcpS: Contextꢀspcific
modulation of SMN transcript levels. Manuscript submitted.
29.
Bunnage, M. E.; Gilbert, A. M.; Jones, L. H.; Hett, E. C. Know your target, know your
molecule. Nat. Chem. Biol. 2015, 11, 368ꢀ372.
ACS Paragon Plus Environment

Page 29 of 50
Journal of Medicinal Chemistry
1
2
3
4
FIGURES AND TABLES
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 1. C5ꢀsubstituted 2,4ꢀdiaminoquinazoline DcpS Inhibitors
ACS Paragon Plus Environment