Optimization of PDE3A Modulators for SLFN12-Dependent Cancer Cell Killing

Article abstract of DOI:10.1021/acsmedchemlett.9b00360

6-(4-(Diethylamino)-3-nitrophenyl)-5-methyl-4,5-dihydropyridazin-3(2H)-one, or DNMDP, potently and selectively inhibits phosphodiesterases 3A and 3B (PDE3A and PDE3B) and kills cancer cells by inducing PDE3A/B interactions with SFLN12. The structure-activity relationship (SAR) of DNMDP analogs was evaluated using a phenotypic viability assay, resulting in several compounds with suitable pharmacokinetic properties for in vivo analysis. One of these compounds, BRD9500, was active in an SK-MEL-3 xenograft model of cancer.

Full text of DOI:10.1021/acsmedchemlett.9b00360

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Letter
Optimization of PDE3A Modulators for SLFN12-Dependent Cancer Cell Killing
Timothy A Lewis, Luc de Waal, Xiaoyun Wu, Willmen Youngsaye, Antje
Wengner, Charlotte Kopitz, Martin Lange, Stefan Gradl, Manuel Ellermann,
Philip Lienau, Stuart L. Schreiber, Heidi Greulich, and Matthew Meyerson
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acsmedchemlett.9b00360 • Publication Date (Web): 18 Oct 2019
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Optimization of PDE3A Modulators for SLFN12-Dependent Cancer
Cell Killing
Timothy A. Lewis,*,† Luc de Waal,†,‡ Xiaoyun Wu,†,‡ Willmen Youngsaye,† Antje Wengner,§ Charlotte
Kopitz,§ Martin Lange,§ Stefan Gradl,§ Manuel Ellermann,§ Philip Lienau,§ Stuart L. Schreiber,† Heidi
Greulich,†,‡ Matthew Meyerson†,‡
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†
‡
Broad Institute of MIT and Harvard, Cambridge, Massachusetts 02142, United States. Dana-Farber Cancer Institute, Boston,
§
Massachusetts, 01255, United States. Bayer AG, Berlin, Germany.
ABSTRACT: 6-(4-(Diethylamino)-3-nitrophenyl)-5-methyl-4,5-dihydropyridazin-3(2H)-one, or DNMDP, potently and selectively
inhibits phosphodiesterases 3A and 3B (PDE3A and PDE3B) and kills cancer cells by inducing PDE3A/B interactions with SFLN12.
The structure-activity relationship (SAR) of DNMDP analogs was evaluated using a phenotypic viability assay, resulting in several
compounds with suitable pharmacokinetic properties for in vivo analysis. One of these compounds, BRD9500, was active in an SK-
MEL-3 xenograft model of cancer.
KEYWORDS: PDE3A, SLFN12, DNMDP, HeLa, SK-MEL-3
We previously reported on the results of a differential
viability screen, where a p53 mutant lung cancer cell line, NCI-
H1734, was killed by 6-(4-(diethylamino)-3-nitrophenyl)-5-
methyl-4,5-dihydropyridazin-3(2H)-one, or DNMDP, while a
second lung cancer cell line, A549, was not affected.1 Further
screening detected selective cell killing in 22 out of 766 cancer
cell lines, correlating with elevated expression of
phosphodiesterase 3A (PDE3A). While DNMDP selectively
inhibited PDE3, most PDE3 inhibitors had no cell killing effects
and in fact rescued cancer cells from DNMDP-induced death.
PDE3A immunoprecipitation experiments from HeLa cell
lysates showed PDE3A bound to Schlafen family member 122
(SFLN12) in the presence of DNMDP, but not in the presence
of trequinsin, a PDE3 inhibitor that does not kill cancer cells.
DNMDP-sensitive cell lines were found to express elevated
levels of both PDE3A and SLFN12.1 Since our report, others
have discovered that selected PDE3 inhibitors, some of which
are known to phenocopy DNMDP, kill a gastrointestinal
stromal tumor (GIST) cell line3 and a subset of primary ovarian
cancer cells.4
available starting material used for the synthesis of the inotropic
drug levosimenden.5 Compound 1 was acetylated, then nitrated
followed by acetyl hydrolysis. Reductive amination with
acetaldehyde produced both the mono and dialkylated amine,
(R)-DNMDP, as a 19:1 ratio of enantiomers as determined by
chiral SFC analysis. DNMDP was N-alkylated producing
racemic compound 6.
Scheme 1. Synthesis of (R)-DNMDP and Analogsa
O
O
O
O
NH
NH
NH
a
b
c
NH
O
N
O
N
3
N
N
N
N
H2
N
H2
N
H
H
1
NO2
2
NO2
4
O
O
O
NH
+
NH
N
e
N
N
N
d
DNMDP
N
N
N
H
NO2
NO2
NO2
5
(R)-DNMDP
6
aReagents and conditions: a) Ac2O (91%); b) 90% HNO3/H2SO4 (19%);
c) NaOH/H2O/MeOH (quant.); d) CH3CHO, NaBH(OAc)3/DCM (7%
combined); e) NaH, EtI/DMF (61%).
Although DNMDP is a very potent and highly
selective compound in a cellular cytotoxicity assay, it has
structural liabilities making it unsuitable for further
development: a diakylanilino group prone to metabolic
instability, and a potentially reactive nitro group. We sought to
discover analogs which maintained or improved cellular
activity while improving pharmacokinetic properties.
Furthermore, we needed potent and selective compounds to
better examine the relationship between PDE3 and SFLN12, a
protein of unknown function, in sensitive cancer cells. Without
sufficient quantities of SLFN12 to study in detail, our initial
SAR was driven using phenotypic screening, i.e., viability
assays.
We were concerned initially that the nitro group of
DNMDP might lead to promiscuous protein binding.
Reductive amination of 1 with acetaldehyde under standard
conditions gave des-nitro (R)-DNMDP (7) (Scheme 2). The
reaction was performed using racemic 1 as well. Modifying
reaction conditions allowed isolation of monoethyl analog 8.
Compound 1 was alkylated to make the morpholino analog 9
which was chlorinated adjacent to the morpholine ring (10).
Heterocycle 11 was prepared by condensation of 1 with
diformyl hydrazine.
The more active (R)-enantiomer of DNMDP had
previously been synthesized1 from (R)-6-(4-aminophenyl)-5-
methyl-4,5-dihydropyridazin-3(2H)-one (1), a commercially
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Scheme 2. Synthesis of (R)-des-Nitro DNMDP Analogsa
Scheme 4. Synthesis of 3-Amino Substituted Analogsa
3
O
O
O
O
4
5
6
7
8
9
NH
NH
NH
N
N
N
N
NH
N
a
N
b
a
or
N
H
DNMDP
1
1
N
N
N
N
N
8
7
9
HN
NH2
O
O
O
22
21
NH
NH
b
c
N
O
O
N
NH
NH
O
O
Cl
N
O
N
10
c
d
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
3
N
HN
O
H
N
NH2
23
NH
24
d
1
aReagents and conditions: a) H2, 10% Pd/C/MeOH (65%); b) Ac2O (90%);
c) H2, 10% Pd/C/MeOH (92%); d) toluene, reflux (54%).
N
N
11
aReagents and conditions: a) 7 - CH3CHO, NaBH3CN/MeOH (82%); 8 -
CH3CHO, then NaBH3CN/MeOH (8%); b) (BrCH2CH2)2O, K2CO3/DMF
(46%); c) NaOCl/HOAc (40%); d) CHONHNHCHO (73%).
Halogenation of acetamide 2 provided the chloride
(25) and bromide (26), respectively (Scheme 5). The bromide
underwent Sonogishira coupling with trimethylsilylacetylene
generating the protected alkyne which was deprotected with
fluoride ion to give 27 or hydrolyzed directly to ketone 28.
Diazotization of 1 followed by halogenation produced
the fluoro (12), chloro (13) and iodo (14) derivatives,
respectively (Scheme 3). Compound 13 (racemic) is a known
PDE3 inhibitor.6 The iodide 14 reacted at high temperature with
N-methyl piperazine to produce 15. Suzuki coupling with 14
followed by hydrogenation gave analogs with cyclic, acyclic,
sp2 and sp3 carbon substituents on the para position of the
phenyl ring (16-19). Heterocycle 20 was prepared similarly.
Scheme 5. Synthesis of Acetylene and Acetyl Analogsa
O
O
O
NH
NH
NH
O
N
O
N
O
N
a or b
c
e
2
N
N
N
H
H
H
X
O
Scheme 3. Synthesis of (R)-DNMDP Analogsa
X
25 (X = Cl)
26 (X = Br)
X = TMS
X = H (27)
28
d
O
O
NH
NH
aReagents and conditions: a) Br2, CH2Cl2 (15%); b) NaOCl/HOAc (41%);
c) 28, TMSCCH, Pd2(dba)3, Et3N/DMF (80%); d) TBAF/THF (52%); e)
HCOOH/H2O (83%) .
N
a
b
N
2
X
N
N
12 (X = F)
13 (X = Cl)
14 (X = I)
15
Fluorinated analogs were prepared starting with 3,4-
difluoropropiophenone which was first alkylated with ethyl
bromoacetate then condensed with hydrazine to give 29
(Scheme 6). The 4-fluoro group was displaced by refluxing
morpholine to give 30. Racemic 29 and 30 were separated into
enantiomers with chiral SCF chromatography. Only one
enantiomer of both 29 and 30 was active against HeLa cells, and
the active enantiomer of 29 was converted to the active
enantiomer of 30. The absolute stereochemistry of the
separated enantiomers was determined by low yielding
reductive removal of the fluorine atom from 30 to give 9. The
compound 9 obtained was compared to racemic and (R)-9 by
chiral SCF chromatography to assign stereochemistry at the
chiral center. The dichloro analog of 29 was enantioselectively
synthesized via amide 25. The amine resulting from hydrolysis
was diazotized and converted to dichloro analog 31 (Scheme 6).
aReagents and conditions: a) NaNO2, HCl/water, then NaBF4 (12, 15%) or
CuCl2 (13, 77%) or KI (14, 49%); b) N-methyl piperazine, NMP, 160 °C
microwave (20%).
O
O
O
NH
NH
NH
b
a
c
N
N
N
14
R
R
N
16 (R = 1-propenyl)
17 (R = 1-cyclohexenyl)
18 (R = 1-propyl)
19 (R = cyclohexyl)
N
20
aReagents and conditions: a) Pd(PPh3)4, Na2CO3/H2O/THF, propene (16,
90%) or cyclohexene (17, 90%); b) H2, 10% Pd/C, MeOH (60% 18, 63%
19); c) 5-pyrimidine boronic acid, Pd(PPh3)4, Na2CO3/H2O/dioxane (68%)
Substituent effects on the phenyl ring were examined.
The nitro group of DNMDP was reduced and the amine product
(21) was acetylated (22) (Scheme 4). Reducing the nitro group
of 3 gave 23, subsequent heating produced benzimidazole 24.7
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Scheme 6. Synthesis of Dihalogenated Analogsa
Table 1. HeLa Cell Viability of DNMDP Analogsa
3
4
5
6
7
8
9
O
O
Compound
EC50 (nM) Compound EC50 (nM)
a
b
COOEt
DNMDP
(R)-DNMDP
6.9
3.8
18
19
16
7.7
F
F
F
F
1
5
6
>1000
1.1
20
21
13
240
O
O
NH
NH
N
c
N
>1000
8.8
3.3
71
36
13
4.5
>1000
410
33
22
23
24
(±)-29
(R)-29
(S)-29
(±)-30
(R)-30
(S)-30
31
>1000
>1000
>1000
64
22
>1000
2.8
1.6
>1000
4.5
N
F
(±)-7
(R)-7
8
(±)-9
(R)-9
10
11
12
13
14
O
F
F
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
29
30
aReagents and conditions: a) LiHMDS, THF, -78 °C, then BrCH2COOEt
(30%); b) hydrazine/EtOH (29%); c) morpholine, 130 °C (53%).
O
O
NH
NH
a
b
N
N
25
H2N
Cl
Cl
31
Cl
aReagents and conditions: a) NaOH/MeOH (98%); b) NaNO2/HCl then
CuCl2 dihydrate/CH3CN, 80 °C (58%).
7.1
310
8.8
32
33
34
35
>1000
>1000
>1000
>1000
15
16
17
Isosteric
replacement
of
chiral
6-methyl
2.2
dihydropyridazinones by achiral 5,5-dimethylpyrazolones has
maintained PDE3/4 activities in other systems.9 Synthesis of
this heterocycle began with ethyl 4-nitrobenzoyl acetate which
was dimethylated, condensed with hydrazine, and reduced to
produce aniline 32 (Scheme 8). Conversion of the amino group
of 32 to the diethylamino (33), morpholino (34) and chloro (35)
groups was done similarly to the corresponding
dihydropyridazinones.
a n ≥ 3, std. error ≤ 20%
Several active compounds, (R)-7, (R)-30, and 31, were
tested in HeLa cells at effective doses and treated with varying
levels of trequinsin (PDE IC50 0.25 nM10). As was seen
previously with DNMDP,1 dose dependent cell rescue was
observed indicating a similar mechanism of action.
We previously reported that the (R)-enantiomer of
DNMDP is 200-500 times more active, depending upon the cell
line tested, than the (S)-enantiomer.1 This stereochemical
dependence was seen in four new examples, where the (R)-
enantiomer was more active than the (S)-enantiomer or the
racemate (7, 9, 29, 30).
Removing one ethyl group from DNMDP (5) had
little effect on potency, while N-ethylating the
dihydropyridazinone ring (6) diminished activity. Removing
the nitro group from DNMDP did not have a deleterious effect
(7). Reduction of the nitro group of DNMDP (21) reduced
activity and acylation of the resulting amine (22) removed
activity.
The diethylamino group of (R)-des-nitro DNMDP (7)
could be replaced by a morpholine ring (9). The N-methyl-
piperazine analog (15) displayed much less activity. The
presence of a fluorine atom adjacent to the morpholine nitrogen
(30), as found in the antibiotic linezolid,11 improved activity in
the racemic and (R)-examples, while the (S)-enantiomer was
still inactive. Substitution of fluorine with chlorine maintained
activity (10). Replacement of the diethylamino group with
heterocycles on (R)-des-nitro DNMDP (7) lead to both active
(20) and inactive (24, 11) compounds.
Scheme 7. Synthesis of Dimethylpyrazolonesa
O
O
O
O
O
O
a
b
O2N
O2N
O
O
O
NH
NH
NH
d, e, or f
c
N
N
N
O2N
H2N
R
32
33 (R = Et2N)
34 (R = morpholino)
35 (R = Cl)
aReagents and conditions: a) NaH, MeI/THF (40%); b) hydrazine/EtOH
(64%); c) H2, Pd/C/EtOH (94%); d) CH3CHO, NaBH3CN/MeOH (56%); e)
(BrCH2CH2)2O, K2CO3/DMF (33%); f) NaNO2, HCl/water, then CuCl
dihydrate/CH3CN, 80 °C (72%).
Cellular SAR analysis was performed by treating
DNMDP-sensitive HeLa cells with compounds for 3 days at a
dose range of 1 nM through 10 M, with a counterscreen using
DNMDP-insensitive A549 cells (Tables 1-2). No compounds
tested against A549 cells displayed activity at concentrations up
to 10 uM (data not shown)
Replacing the diethylamino group of des-nitro
DNMDP (7) with halogens chloro (13) and iodo (14) was
permitted, while the fluoro analog was significantly less active
(12). As was the case with a 4-morpholino group, adding an
additional halogen at the 3-position improved activity (29/12,
31/13). Substitution with carbon substituents (16-19) retained
cellular activity.
The dimethyl pyrrolidinones (32-35) were inactive.
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aIC50s are an average of two values
1
2
3
4
5
6
7
8
Intermediate acetamides were tested (Table 2).
Contrary to the case with the diethylamino analogs (7 and
DNMDP), the des-nitro analog 2 had no activity whereas the
nitrated analog 3 was very potent. Electron withdrawing
substituents improved activity, but an amino group was not
tolerated (25).
Attempts to find other molecular targets of our
compounds were unsuccessful. A Millipore Kinase Profiler
screen of DNMDP found no inhibition (conc. 10 M) against
234 kinases.1 A Eurofins Lead Profiling screen of compound 9
(conc. 10 M, 68 assays) found no interactions with a non-
kinase targets (Supporting Information Table 2).
Table 2. HeLa Cell Viability of Acylated Aminesa
Previously we used mass spectrometry proteomic
analysis to demonstrate that DNMDP binds to PDE3A,
inducing PDE3A binding to SLFN12, which results in cell
killing whereas non-toxic PDE3 inhibitors do not induce
PDE3A/SLFN12 complex formation.1 To observe compound-
induced PDE3/SLFN12 interaction with newer compounds we
transfected HeLa cells with a plasmid that expresses SLFN12
fused with a V5 epitope tag. We then treated the cells with
DNMDP or (R)-30 (10 M). After 8 hours of compound
treatment the cells were lysed and endogenous PDE3A protein
was immunoprecipitated using an anti-PDE3A antibody.
O
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
HN
O
N
NH
X
Compound
X
H
NO2
NH2
Cl
Br
CCH
Ac
EC50 (nM)
>1000
4.9
>1000
24
2
3
23
25
26
27
28
SLFN12
co-immunoprecipitation
was
analyzed
by
21
65
130
immunoblotting with an anti-V5 antibody to detect the
SLFN12-V5 fusion protein. The SLFN12-V5 was clearly
detected with the anti-V5 antibody for both DNMDP and (R)-
30, indicating that both compounds stabilize the PDE3A-
SLFN12 interaction (Figure 1).
Cell death with DNMDP occurs via apoptosis by an
as of yet determined mechanism.1 A recent article reports
PDE3A/SLFN12 interaction being induced by high
concentration of 17--estradiol, leading to HeLa cell death
which can be rescued by the PDE3 inhibitors cilostazole and
trequinsin, as seen with DNMDP-induced HeLa cell death.15
The stabilization of SFLN12 leads to a decrease in translation
of anti-apoptotic proteins leading to cell death.15
a n ≥ 3, std. error ≤ 20%
We extended our cellular testing beyond HeLa cells to
the DNMDP-sensitive NCI-H2122 (lung cancer) and
COLO741 (melanoma) cell lines, as well as the DNMDP-
insensitive HCT116 (colorectal cancer) and IMR90 (normal
lung fibroblast) cell lines. The results mirror those using HeLa
and A549 cells; active compounds are only slightly less active
against H2122 and COLO741 cell lines, and all the compounds
are inactive against the HCT116 and IMR-90 cells (Supporting
Information Table 1).
While the mechanism of action of our compounds
involves inducing PDE3/SLFN12 interaction, we nonetheless
tested some of our compounds for biochemical inhibition of
PDE3A and PDE3B (Table 3).12 No major selectivity for one
isozyme over the other was observed, and the HeLa activity
generally mirrored the biochemical results. Halogenation of the
phenyl ring (9 to (R)-30) increased PDE3A/B biochemical
inhibition ten-fold. The cellular SAR generally agreed with
reported SAR of PDE3 inhibition13 with the (R)-enantiomers
being more potent.14 However, as was seen previously with
commercially available PDE3 inhibitors, some DNMDP
analogs, e.g 2, were potent PDE3 inhibitors with no cellular
activity.
The increase in cellular potency relative to
biochemical activity for active compounds (HeLa EC50s 2.4-15
fold lower than PDE3A/B average IC50s) is consistent with
neomorphic modulation, due to complex formation with
SLFN12 in this case (see below).
Figure 1. Detection of PDE3A/SLFN12 Binding
The in vitro pharmacokinetic properties of selected
active compounds were determined (Table 4). Overall the
properties were good, and all compounds were highly soluble
(>70 M). Removal of the nitro group from DNMDP (7) and
replacing the diethylamino group with a morpholine (9)
improved microsomal stability. Addition of a fluorine atom
(30) was well tolerated. Halogenated analogs (13) were stable
to microsomes as was one heterocyclic analog (20). Other
carbon substituted analogs (16, 18) were unstable to
microsomes. Plasma protein binding of the analogs generally
correlated with cLogP, with the more polar compounds
Table 3. PDE3 Inhibition and HeLa Viabilitya
IC50 (nM)
PDE3A PDE3B
EC50, HeLa
(nM)
Compound
DNMDP
25
120
10
100
260
27
6.9
13
1.6
9
(R)-30
31
8
14
4.5
15
2
25
2000
24
8
3500
16
10
310
>1000
24
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displaying less protein binding. Plasma stability, mouse and
human, was >80% at 5 hours for all compounds tested.
well tolerated without critical body weight loss (>10%) or
toxicities (Supporting Information Figure 1).
1
2
3
4
5
6
7
8
9
150
Vehicle
Table 4. Pharmacokinetic In Vitro Properties of Select
(R)-30_10 mg/kg 2QD
Compoundsa
Microsomal
100
(R)-30_20 mg/kg 2QD
Plasma
Protein
Binding
Plasma
Stability
(% remaining
at 5 h)
(R)-30_50 mg/kg QD
stability
(%
remaining at
1 h)
50
Compound
(% bound)
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
0
h
95
89
m
23
26
99
102
82
h
m
92
90
42
55
52
81
54
h
85
104
87
80
93
m
87
96
105
101
96
0
10
20
30
40
50
DNMDP
87
90
47
57
56
87
56
Days after tumor cell inoculation
7
9
Figure 2. Anti-tumor efficacy of (R)-30 in the SK-MEL-3 tumor
model in NMRI nude mice
94
(±)-30
(±)-29
13
109
108
81
Plasma levels of (R)-30 were monitored throughout
the xenograft experiment (Figure 3). With a dose of 50 mg/kg
QD the unbound plasma level of (R)-30 at 24 h was >100-fold
the EC50 against SK-MEL-3 cells, and at doses of 10 and 20
mg/kg 2QD the EC50 was covered for >30-fold at 24 h (Figure
3).
92
105
93
104
86
108
20
83
ah = human, m = mouse, values are average of three values
Compound (R)-30, a.k.a. BRD9500, was profiled to
determine its suitability for animal studies. Compound (R)-30
does not inhibit other phosphodiesterases tested (Supporting
Information Table 3). No inhibition in a cytochrome P450
panel (CYPs 1A2, 2C8, 2C9, 2D6, and 3A4) was detected when
tested at high concentrations. Compound (R)-30 is soluble (ca.
1 mM), permeable (Caco2 A-B 279 nm/s; B-A 198 nm/s, efflux
ratio 0.71) and stable in solution at various pH levels (1, 7, and
10) for extended time periods. An Ames test was negative and
no hERG interaction was detected at high concentration. (R)-
30 showed high plasma levels in mice after iv (1 mg/kg) as well
as po (2 mg/kg) dosing over eight hours making it a valuable
candidate for in vivo xenograft testing (Supporting Information
Table 4).
HeLa proved to be a convenient and robust cell line
for viability assays, but for xenograft experiments we chose to
work with a cancer cell line more relevant to therapeutic
opportunities. Viability assays were performed under identical
procedures using the SK-MEL-3 melanoma cell line with eight
compounds (Table 5). Gratifyingly, the SK-MEL-3 EC50 values
were similar to those of HeLa.
Figure 3. Unbound plasma levels of (R)-30 on day 45 after final
dose to SK-MEL-3 bearing NMRI nude mice. Curves for lower
dose 2QD simulated.
In summary, starting with our initial screening hit
DNMDP, compounds were optimized using a cellular viability
assay, making them not only more potent, but improving upon
their pharmacological properties. The increase in cellular
potency correlated with an increase in PDE3 inhibition, though
most known PDE3 inhibitors do not kill HeLa cells. Our
advanced compound, (R)-30/BRD9500, induced PDE3A/
SLFN12 binding in HeLa cells and was active with oral dosing
in a mouse xenograft model of melanoma. Current studies are
focused on determining the mechanism of action of small
molecule modulators of PDE3 and SLFN12 leading to cancer
cell death.
Table 5. Viability with SK-MEL-3 Cells
Compound
DNMDP
13
EC50 (nM)
Compound
EC50 (nM)
12
29
15
9
684
11
(R)-30
3
1.3
4.2
31
25
5.6
31
a n = 3, std. error < 10%
The anti-tumor activity of compound (R)-30 was
evaluated in tumor xenografts derived from SK-MEL-3
melanoma cells that were subcutaneously inoculated into
female NMRI nude mice (Figure 2). (R)-30 was applied orally
at 10 and 20 mg/kg twice daily (2QD) and at 50 mg/kg once per
day (QD). We observed inhibition of tumor growth upon all (R)-
30 treatments, achieving strongest anti-tumor activity at 50
mg/kg QD with a T/Crel.area of 0.09 and T/Cweight of 0.16 (p <
0.001 and p < 0.05 vs vehicle, respectively). All treatments were
ASSOCIATED CONTENT
Supporting Information
The Supporting Information is available free of charge on the ACS
Publications website at …..
Synthesis and characterization of all compounds, biolog-
ical assay procedures, and in vivo experiment details
(PDF)
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regulatory cardiac troponin C-troponin I complex. Biochemistry 2008,
47, 7485-7495.
1
7) Zhao, H.; Fu, H.; Qiao, R. Copper-catalyzed direct amination of
ortho-functionalized haloarenes with sodium azide as the amino source.
J. Org. Chem. 2010, 75, 3311-3316.
8) Ochiai, K.; Takita, S.; Kojima, A.; Eiraku, T.; Ando, N.; Iwase, K.;
Kishi, T.; Ohinata, A.; Yageta, Y.; Yasue, T.; Adams, D.R.; Kohno, Y.
4,4-Dimethylpyrrazolone replacement: Phosphodiesterase inhibitors.
Part 4: design, synthesis and structure-activity relationships of dual
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3
4
5
6
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AUTHOR INFORMATION
Corresponding Author
*E-mail: tlewis@broadinstitute.org
ORCID
Timothy A. Lewis: 0000-0001-9748-9575
Stuart L. Schreiber: 0000-0003-1922-7558
PDE3/4-inhibitory
fused
bicyclic
heteroaromatic-4,4-
dimethylpyrazolones. Bioorg. Med. Chem Lett. 2012, 22, 5833-8.
9) Cignarella, G.; Barlocco, D.; Pinna, G.A.; Loriga, M.; Curzu, M.
M.; Tofanetti, O.; Germini, M.; Cazzulani, P.; Cavalletti, E. Synthesis
and biological evaluation of substituted benzo[h]-cinnolinones and 3H-
benzo[6,7]cyclohepta[1,2-c]pyridazinones: higher homologs of the
antihypertensive and antithrombotic 5H-indeno[1,2-c]pyridazinones J.
Med. Chem. 1989, 32, 2277–2282
10) Ruppert, D.; Weithmann, K. U. HL 725, an extremely potent
inhibitor of platelet phosphodiesterase and induced platelet aggregation
in vitro. Life Sci. 1982, 31, 2037-43.
11) Barbachyn, M. R.; Ford, C. W. Oxazolidinone structure-activity
relationships leading to linezolid. Angew. Chem. Int. Ed. Eng. 2003,
42, 2010-2023.
12) In vitro PDE3 assays were performed at BPS Bioscience, San
Diego, CA, www.bpsbioscience.com.
13) Moos, W.H.; Humblet, C.C.; Sircar, I.; Rithner, C.; Weisharr, R.E.;
Bristol, J.A.; McPhail, A.T. Cardiotonic agents: 8. Selective inhibitors
of adenosine 3’,5’-cyclic phosphate phosphodiesterase III.
Elaboraboration of a five-point model for positive inotropic activity. J.
Med. Chem. 1987, 30, 1963-1972.
14) Owings, F.F.; Fox, M.; Kowalski, C.J.; Baine, N.H. An
enantioselective synthesis of SK&F 93505, a key intermediate for
preparing cardiotonic agents. J. Org. Chem. 1991, 56, 1963-1966.
15) Li, D.; Chen, J.; Ai, Y.; Gu, X.; Li, L.; Che, D.; Jiang, Z.; Li, L.;
Chen, S.; Huang, H.; Wang, J.; Cai, T.; Cao, Y.; Qi, X.; Wang, X.
Estrogen-related hormones induce apoptosis by stabilizing Schlafen-12
protein turnover. Mol. Cell 2019, 75, 1103-1116.
16) Lewis, T.A.; de Waal, L.; Youngsaye, W.; Gechijian, L.; Hickey,
M.; Faloon, P.; Mikse, O.; Dandapani, S.; Wong, K.; Tolliday, N.;
Munoz, B.; Palmer, M.; Greulich, H.; Meyerson, M.L.; Schreiber, S.L.
A candidate cell-selective anticancer agent. Probe Reports from the
NIH Molecular Libraries Program [Internet]. Bethesda (MD): National
Center for Biotechnology Information (US); 2010-2013 Dec 12
[updated 2014 Sep 18].
Author Contributions
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T.A.L., W.Y., and S.G. designed and synthesized compounds,
L.d.W., X.W., and M.L. tested compounds, P.L. supervised
pharmacokinetic studies, A.W. and C.K. performed the in vivo
study, T.A.L., S.L.S., H.G., and M.M. directed the project and
wrote the manuscript which has been approved by all the authors.
Funding Sources
The authors would like to thank the US National Institutes of
Health’s Molecular Libraries Program Centers Network (MLPCN)
(grant number 3U45HG005032-05S1 to S.L.S.)16 and Bayer for
financial support.
ACKNOWLEDGMENTS
We are grateful to Steven Johnson (Broad Institute) for in vitro
pharmacokinetic data.
ABBREVIATIONS
PDE3, phosphodiesterase 3; SLFN12, Schlafen family member 12;
DNMDP,
6-(4-(diethylamino)-3-nitrophenyl)-5-methyl-4,5-
dihydropyridazin-3(2H)-one; NMP, N-methyl-2-pyrrolidinone;
TMSCCH, trimethylsilylacetylene; dba, dibenzylideneacetone;
TBAF, tetrabutylammonium fluoride; LiHMDS, lithium
hexamethyldisilazane
REFERENCES
1) de Waal, L.; Lewis, T.A.; Rees, M.G.; Tsherniak, A.; Wu, X.; Choi,
P.S.; Gechijian, L.; Hartigan, C.; Faloon, P.W.; Hickey, M.J.; Tolliday,
N.; Carr, S.A.; Clemons, P.A.; Munoz, B.; Wagner, B.K.; Shamji, A.F.;
Koehler, A.N.; Schenone, M.; Burgin, A.B.; Schreiber, S.L.; Greulich,
H.; Meyerson, M. Identification of cancer-cytotoxic modulators of
PDE3A by predictive chemogenomics. Nat. Chem. Biol. 2016, 12, 102-
8.
2) Puck, A.; Aigner, R.; Modak, M.; Cejka, P.; Blaas, D.; Stöckl, J.
Expression and regulation of Schlafen (SLFN) family members in
primary human monocytes, monocyte-derived dendritic cells and T
cells. Results Immunol. 2015, 5, 23-32.
3) Vandenberghe, P.; Hagué, P.; Hockman, S.C.; Manganiello, V.C.;
Demetter, P.; Erneux, C.; Vanderwinden, J.M. Phosphodiesterase 3A:
a new player in development of interstitial cells of Cajal and a
prospective target in gastrointestinal stromal tumors (GIST).
Oncotarget 2017, 8, 41026-41043.
4) Nazir, M.; Senkowski, W.; Nyberg, F.; Blom, K.; Edqvist, P.H.;
Jarvius, M.; Andersson, C.; Gustafsson, M.G.; Nygren, P.; Larsson R.;
Fryknäs, M. Targeting tumor cells based on Phosphodiesterase 3A
expression. Exp. Cell Res. 2017, 361, 308-315.
5) Szilágyi, S.; Pollesello, P.; Levijoki, J.; Kaheinen, P.; Haikala, H.;
Edes, I.; Papp, Z. The effects of levosimendan and OR-1896 on
isolated hearts, myocyte-sized preparations and phosphodiesterase
enzymes of the guinea pig. Eur. J. Pharmacol. 2004, 486, 67-74.
6) Robertson, I. M.; Baryshnikova, O. K.; Li, M. X.; Sykes, B. D.
Defining the binding site of levosimendan and its analogues in a
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O
O
NH
NH
N
N
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N
N
NO2
O
F
DNMDP
BRD9500
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EC50 12 nM (SK-MEL-3)
racemic
unstable to microsomes
EC50 1 nM (SK-MEL-3)
enantiomerically pure
active in xenograft
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