Discovery of Novel PDEδDegraders for the Treatment of KRAS Mutant Colorectal Cancer

Article abstract of DOI:10.1021/acs.jmedchem.0c00929

KRAS-PDEδprotein-protein interaction represents an appealing target for cancer therapy. However, fast release of high-affinity inhibitors from PDEδhampered drug binding affinity and antiproliferative activity. To overcome the limitations, the first proteolysis-targeting chimeric (PROTAC) small molecules targeting PDEδwere designed. By employment of PDEδinhibitor deltazinone (2) and cereblon ligand pomalidomide (6), a series of potent PROTAC PDEδdegraders were obtained. The most promising compound 17f efficiently induced PDEδdegradation and demonstrated significantly improved antiproliferative potency in KRAS mutant SW480 cells. Compound 17f also achieved significant tumor growth inhibition in the SW480 colorectal cancer xenograft model. This proof-of-concept study provided a new strategy to validate the druggability of KRAS-PDEδinteraction and offered an effective lead compound for the treatment of KRAS mutant cancer.

Full text of DOI:10.1021/acs.jmedchem.0c00929

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Article
Discovery of Novel PDE# Degraders for the
Treatment of KRAS Mutant Colorectal Cancer
Junfei Cheng, Yu Li, Xu Wang, Guoqiang Dong, and Chunquan Sheng
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.0c00929 • Publication Date (Web): 30 Jun 2020
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Discovery of Novel PDEδ Degraders for the Treatment of KRAS
Mutant Colorectal Cancer
Junfei Cheng†, Yu Li†, Xu Wang, Guoqiang Dong*, Chunquan Sheng*
Department of Medicinal Chemistry, School of Pharmacy, Second Military Medical
University, 325 Guohe Road, Shanghai 200433, People’s Republic of China
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ABSTRACT
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KRAS-PDEδ protein-protein interaction represents an appealing target for cancer
therapy. However, fast release of high-affinity inhibitors from PDEδ hampered drug
binding affinity and antiproliferative activity. To overcome the limitations, the first
proteolysis-targeting chimeric (PROTAC) small molecules targeting PDEδ were
designed. Employing PDEδ inhibitor deltazinone (2) and cereblon ligand
pomalidomide (6), a series of potent PROTAC PDEδ degraders were obtained. The
most promising compound 17f efficiently induced PDEδ degradation and
demonstrated significantly improved antiproliferative potency in KRAS mutant
SW480 cells. Compound 17f also achieved significant tumor growth inhibition in the
SW480 colorectal cancer xenograft model. This proof-of-concept study provided a
new strategy to validate the druggability of KRAS-PDEδ interaction and offered an
effective lead compound for the treatment of KRAS mutant cancer.
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INTRODUCTION
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RAS proteins regulate many cellular processes including cell proliferation and
differentiation and play a causal role in human cancers.1, 2 Mutational activation of
RAS is a common feature accounting for about 30% of human cancers.3 KRAS, the
most frequently mutated isoform of RAS, is commonly found in approximately 30%
of lung cancer, 45% of colorectal and 90% of pancreatic ductal carcinomas.2, 4, 5
Therefore, KRAS is well recognized as an appealing target for cancer therapy.6, 7
However, direct targeting KRAS by small molecules remains to be a highly
challenging task without drugs approved in the clinics.6, 8, 9
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The function of KRAS signaling critically depends on its proper localization at the
plasma membrane (PM).10 To get to the PM, KRAS binds to a shuttling factor called
PDEδ through its farnesylated hypervariable region.10 By this process, PDEδ prevents
KRAS from binding to endomembranes and thereby facilitates its diffusion
throughout the cell.11, 12 Subsequently, KRAS is released from PDEδ by release factor
Arl2 and shuttled to PM via vesicular transport.13 Based on the crucial role of PDEδ,
Waldmann’s group reported a series of small molecule KRAS-PDEδ inhibitors
binding to the prenyl binding pocket of PDEδ with low nanomolar affinity, such as
deltarasin (1, Figure 1), deltazinone (2) and deltasonamide (3).14-16 However, despite
potent binding in vitro (KD < 10 nM), compounds 1-3 only showed moderate
15
antitumor activity at micromolar concentrations.14,
The discrepancy between
molecular and cellular potency was also observed for KRAS-PDEδ inhibitors 4 and 5
discovered by our group.17, 18 The main reason was that Arl2 induced fast release of
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high-affinity inhibitors from PDEδ and finally reduced their antitumor efficacy
(Figure 2A).16 As a result, most PDEδ inhibitors failed to exert in vivo antitumor
activity. To overcome these inherent limitations, it is urgent to develop new strategies
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to target PDEδ more effectively.
NH
N
N
O
H
O
N
N
N
N
N
N
O
N
Deltazinone (2)
F
Deltarasin (1)
H
N
N
NH
N
N
O
N
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F
HN
O
O
S
O
O
N
H
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S
N
N
N
O
F
N
N
N
Cl
Deltasonamide (3)
F
5
Figure 1. Chemical structures of representative KRAS-PDEδ inhibitors.
Proteolysis-targeting chimera (PROTAC) represents an emerging therapeutic
strategy to eliminate disease-causing proteins.19, 20 Generally, PROTAC molecules are
bifunctional compounds with one end binding to the protein of interest (POI) and the
other end targeting to an E3 ligase, which were connected by a chemical linker
(Figure 2B). Upon ternary complex formation, the chimeric molecules hijack E3
ligase to induce POI poly-ubiquitination, leading to subsequent degradation by the
proteasome.21 This strategy has been successfully applied to a number of
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well-validated drug targets, such as bromodomain and extra-terminal (BET),
androgen receptor (AR), estrogen receptor (ER) bruton’s tyrosine kinase (BTK) and
Focal adhesion kinase (Fak).22-26 However, translating cellular degradative effects into
in vivo potency and ultimately clinical efficacy is a highly challenging task.27 There
are still very few examples of heterobifunctional degraders with favorable
physicochemical/pharmacokinetic properties and potent in vivo activity.28 Up to now,
only two PROTAC molecules, namely ARV-110 and ARV-471 developed by Arvinas,
have been progressed into clinical trials.29, 30 Moreover, despite numerous PROTAC
cases dealing with traditional drug targets,31 the investigations of degraders against
proteins that are considered to be undruggable by small molecule inhibitors are still
rare.28, 32
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Figure 2. Limitations of PDEδ inhibitors and PROTAC strategy for efficient PDEδ
targeting. (A) Release of high affinity inhibitors from PDEδ by Arl2. (B) Illustration
of the catalytic cycle of the ubiquitination and degradation of PDEδ.
To address the limitations of conventional KRAS-PDEδ inhibitors, we envisioned
that PDEδ degraders might achieve robust and durable target suppression and exert
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better antitumor potency. Herein, we designed the first generation of PDEδ degraders
by connecting PDEδ inhibitor 2 and cereblon (CRBN) ligand pomalidomide (6).
Among them, PDEδ degrader 17f effectively induced PDEδ degradation and
demonstrated significantly improved antiproliferative potency against KRAS mutant
colorectal cancer cells both in vitro and in vivo. Thus, PROTAC 17f provided a new
chemical tool or lead compound for navigating the druggablility of KRAS-PDEδ
interaction.
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RESULTS AND DISCUSSION
Molecular Design. PDEδ-degrading PROTACs were designed based on PDEδ
inhibitor 2, which was proven to be highly selective without unspecific binding to
other proteins.14 Molecular modeling revealed that compound 2 adopted a
conformation fitting well with the prenyl binding pocket of PDEδ and formed key
hydrogen bonds with Arg61, Gln78 and Tyr149 (Figure 3A). The homobenzyl group
was exposed to the solvent region, providing a feasible site for E3 ligase ligand
tethering. Meanwhile, CRBN ligand pomalidomide (6, Figure 3B) was chosen
because of its reasonably drug-like physiochemical properties and broad utility in
PROTAC design.19, 20, 33 To facilitate the chemical synthesis, the homobenzyl moiety
in compound 2 was removed and the remaining amide group was used as the tethering
site for PROTAC design (Figure 3C). This structural adjustment facilitated linker
incorporation via amide bond formation without disrupting the critical hydrogen
bonds. As a result, a series of PROTACs with different length of alkyl or ethylene
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glycol (PEG) linkers were designed to give compounds 17a-h. In addition, compound
17i was synthesized as a negative control by methylation of the amino group of the
glutarimide in 6, with the purpose of blocking the binding to CRBN completely.21
Finally, in order to evaluate other attachment points for linker conjugation, an
alternative tethering position in phenyl ring of the CRBN ligand 6 was explored
(compound 17j).
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Figure 3. Design of PDEδ PROTACs (A) Predicted binding model of compound 2
with PDEδ (PDB: 5E8014). (B) Structure of CRBN ligand pomalidomide. (C) Design
strategy of PDEδ degraders.
Chemistry. The synthetic routes of the target compounds were outlined in Scheme 1.
Starting from commercially available 4-methylaniline (7), the key intermediate 12
was synthesized via five steps according to the reported protocols.14 Then,
condensation
of
4-fluoroisobenzofuran-1,3-dione
(13)
with
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2,6-dioxopiperidine-3-ammonium chloride (14) afforded compound 15, which was
reacted with various aliphatic amine to form intermediates 16a-h. After removing the
Boc-protecting group under acidic conditions, compounds 16a-h were coupled with
12 to yield compounds 17a-h.
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Scheme 1. Synthetic Route of Target Compounds 17a-ha
O
O
Et
O
O
Cl
O
OEt
NH2
N
N
N
N
9
NH
N
a, b
c
d
HN
e
N
7
10
8
O
O
O
OH
N
N
N
f
N
N
N
N
N
O
O
12
11
BocHN
Linker NH
F
O
O
O
NH2•HCl
O
h
g
O
N
O
N
O
NH
NH
N
O
BocHN
Linker NH2
H
O
O
O
O
O
F
16a-h
17a Linker = (CH2)3
17b Linker = (CH2)4
17c Linker = (CH2)5
17d Linker = (CH2)6
17e Linker = (CH2)7
17f Linker = (CH2)8
13
14
15
O
H
N
Linker NH
N
O
i, j
N
N
N
O
N
O
NH
O
O
17g Linker = CH2(CH2OCH2)2CH2
17h Linker = (CH2)2(CH2OCH2)3(CH2)2
17a-h
aReagents and conditions: (a) NaNO2, 6 M HCl, 0 oC; (b) Ethyl
o
2-chloro-3-oxobutanoate, NaOAc, EtOH: H2O = 7: 1, 0 C, 4 h, 70%; (c)
Pentane-2,4-dione, NaOEt, EtOH, r.t., 16 h, 93%; (d) Hydrazine hydrate, EtOH, 110
o
oC, 6 h, 78%; (e) Methyl 4-bromobutanoate, NaH, DMF, 0 C, 4 h, 80 %; (f) LiOH,
THF: MeOH: H2O = 3: 2: 1, 1 h, 81%; (g) NaOAc, AcOH, reflux, 12 h, 90%; (h)
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o
DIPEA, DMF, 90 C, 12 h, 40%-50%; (i) DCM, TFA, r.t., 0.5 h; (j) 12, HATU,
6
7
DIPEA, DMF, r.t., 2 h, 40%-50%.
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The synthesis of compounds 17i-j was depicted in Scheme 2. The key intermediate
18 was obtained by methylation from compound 15, followed by removing
Boc-protecting group under acidic conditions. Compound 17i was synthesized by
amide condensation reaction with intermediate 12. Condensation of
5-fluoroisobenzofuran-1,3-dione (19) with 14 gave compound 20, which was reacted
with 1-Boc-1,7-diaminoheptane to form intermediate 16i. After removing the
Boc-protecting group under acidic conditions, compound 16i was coupled with 12 to
yield compound 17j.
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Scheme 2. Synthetic Route of Target Compounds 17i-ja
BocHN
F
F
NH
O
O
O
7
a
b
N
O
N
O
N
O
NH
N
N
NH2
7
BocHN
O
O
O
O
O
O
15
18
16h
O
H
N
N
c, e
N
N
NH
O
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N
7
O
O
N
O
O
17i
O
O
O
H
N
BocHN
F
d
b
O
7
N
O
N
O
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NH
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NH
NH2
7
BocHN
O
O
O
O
O
16i
19
20
O
O
N
O
H
H
N
N
N
c, e
N
8
N
O
N
O
NH
O
17j
aReagents and conditions: (a) NaH, CH3I, DMF, 0 oC, 4 h, 80%;(b) DIPEA, DMF, 90
oC, 12 h, 40%-50%; (c) DCM, TFA, r.t., 0.5 h; (d) NaOAc, AcOH, reflux, 12 h, 90%;
(e) compound 12, HATU, DIPEA, DMF, r.t., 12 h, 40%-50%.
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Biological Evaluations and Structure-Activity Relationships. Initially, the binding
affinity of all the designed PDEδ degraders were determined by fluorescence
polarization assay described previously.17, 18 As shown in Table 1, all the compounds
showed nanomolar affinity with KD values ranging from 9 to 216 nM. The alkyl
derivatives were more potent than the PEG analogues. The linker length dependence
played an important role in PDEδ binding and the binding affinity was significantly
increased with the extension of carbon chain. In particular, compound 17f with linker
length of 8 carbon atoms showed the best potency with PDEδ (KD = 9.0 nM), which
was comparable to positive control 2 (KD = 8.0 nM).
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Then, PDEδ degradation and cell growth inhibitory effect were tested in the cell
culture. KRAS mutation occurs in about 45% colorectal cancers, which is often
associated with poor prognosis and emergence of drug resistance.2, 7, 34 To assess the
antitumor potency of PDEδ degraders, KRAS mutant colorectal cancer SW480 and
HCT116 cells were selected for the assay. The SW480 cell line is homozygote for
oncogenic KRAS (G12V). HCT116 cells have one mutant (G13D) and one wild type
KRAS allele.35, 36 The degradative potency for PDEδ was evaluated in SW480 cells
according to western blotting analysis. As shown in Figure 4A, most PDEδ
PROTACs could efficiently induce PDEδ degradation at the concentration of 10 μM.
The degradation potency was increased with the extension of linker length.
Compound 17f with a diaminoheptane linker exerted the most potent degradation
efficiency with the DC50 value of 3.6 μM in SW480 cells (Figure 4B). As assessed by
the Caco2 assay, compound 17f showed low cell permeability and significant efflux
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(Table S1, Supporting Information), which might be the reason for its moderate
activity in SW480 cells. Interestingly, the degradation potency correlated well with
the binding affinity and antitumor activity.
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Table 1. PDEδ Degraders Derived from Pomalidomide with Various Linkers.
O
H
N
Linker NH
N
O
N
N
N
O
N
O
NH
O
O
17a-h
SW480
SW480
HCT116
Compds
Linker
KD (nM)
DC50 (μM) IC50 (μM) IC50 (μM)
(CH2)3
(CH2)4
(CH2)5
(CH2)6
(CH2)7
(CH2)8
17a
17b
17c
17d
17e
17f
17g
17h
2
216 ± 9.6
30 ± 3.8
29 ± 4.6
17 ± 2.1
14 ± 3.4
9.0 ± 1.3
160 ± 8.8
70 ± 6.4
8.0 ± 2.1
30
25
22
15
14
3.6
30
10
-
81 ± 8.2
76 ± 3.9
17 ± 1.8
15 ± 3.4
12 ± 1.4
8.0 ± 2.7
58 ± 9.8
26 ± 3.9
56 ± 4.3
68 ± 8.0
51 ± 2.4
32 ± 4.0
19 ± 1.0
20 ± 4.2
8.0 ± 3.6
25 ± 1.3
23 ± 0.6
74 ± 5.4
CH2(CH2OCH2)2CH2
(CH2)2(CH2OCH2)3(CH2)2
-
As illustrated in Table 1, all the compounds possessed moderate to good antitumor
activity against SW480 and HCT116 cells. Similar to the degradation potency, the
antitumor activity was also increased with the extension of linker length. Among
them, the most potent PDEδ degrader 17f also showed the best antitumor activity in
suppression of cell proliferation in SW480 (IC50 = 8 μM) and HCT116 cells (IC50 = 8
μM). As compared with PDEδ inhibitor 2, PDEδ degrader 17f showed significantly
better antitumor activity, indicating the distinct advantages of PDEδ degradation over
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inhibition. Furthermore, other possible tethering position in the phenyl ring of
pomalidomide was investigated. Compound 17j demonstrated comparable biological
activity to compound 17f (Table 2), suggesting that both tethering positions in
pomalidomide were favorable for the design of PDEδ degraders. PDEδ degradation
and antitumor activity were also evaluated in RAS-dependent (MiaPaca-2) and
independent (Panc-1) pancreatic cancer cells (Figure S1, Supporting Information).
Compound 17f could efficiently induce PDEδ degradation at 10 μM and demonstrated
moderate antitumor activity against RAS-dependent MiaPaca-2 cells. For
RAS-independent Panc-1 cells, compound 17f was less potent in PDEδ degradation.
Mechanism and Validation of PDEδ Degrader 17f. A time-course study was
performed to assess the kinetics of PDEδ degradation induced by compound 17f in
SW480 cells. As shown in Figure 4C, compound 17f reduced PDEδ protein level in a
time-dependent manner. PDEδ degradation was observed at time points as early as 1 h
after treatment with compound 17f. Meanwhile, compound 2 alone could not elicit the
degradation of PDEδ even at 30 μM (Figure 4D). To further explore the mechanism
of action of PDEδ degradation induced by compound 17f, compound 17i (Table 2) by
methylation of pomalidomide part was synthesized as a negative control. Although
compound 17i demonstrated potent binding affinity to PDEδ (KD = 30 nM), it failed
to induce PDEδ degradation in SW480 cells (Figure 4E). Compound 17h, behaved as
a PDEδ inhibitor, was more than 8-times less potent (IC50 = 70 μM) than 17f in cell
growth inhibition (Table 2, Figure 4F), further confirming that PDEδ degradation
was a better strategy than PDEδ inhibition in exerting antitumor activity. Similarly,
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CRBN ligand 6 alone could not induce PDEδ degradation. In contrast, the addition of
PDEδ inhibitor 2 or CRBN ligand 6 efficiently suppressed the PDEδ degradation
mediated by compound 17f (Figure 4E) in SW480 cells. Moreover, compound 6
reduced the antitumor activity of 17f against SW480 cells (Figure 4F) with the IC50
value increased from 8 μM to 71 μM. In addition, pretreatment with proteasome
inhibitor MG-132 could block the PDEδ degradation induced by compound 17f in
SW480 cells (Figure 4E). IKZF1/3 degradation is a typical off target effect of CRBN
PROTACs. Therefore, we further evaluated the selectivity of compound 17f by
monitoring degradation of IKZF1 and IKZF3. As shown in Figure 4G, compound 17f
had a limited effect on IKFZ degradation, indicating little or no off-target IKZF1 and
IKZF3 activity. All these observations confirmed the fact that compound 17f bound to
PDEδ and CRBN simultaneously, hijacked CRBN and subsequently induced PDEδ
degradation by ubiquitin-proteasome system.
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Table 2. PDEδ Degraders Linked from Different Positions of Pomalidomide.
O
H
H
N
N
N
N
N
CRBN Ligand
8
N
O
SW480
DC50 (μM)
SW480
IC50 (μM)
KD (nM)
Compds
17f
CRBN Ligand
O
N
O
9.0 ± 1.3
8.0 ± 2.1
3.6
8.0 ± 2.7
7.0 ± 3.1
70 ± 9.8
NH
O
O
O
N
O
17j
5
-
NH
O
O
O
N
O
17i
30 ± 4.6
N
O
O
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Figure 4. Screen and characterization of potent PDEδ degraders. (A) PDEδ levels in
SW480 cells upon 24 h drug treatment at 10 μM. (B, D) PDEδ levels in SW480 cells
treated with 2 or 17f at various concentrations for 24 h. (C) PDEδ levels in SW480
cells treated with 17f at 10 μM for indicated time points. (E) SW480 cells were
pretreated with the negative control 17i, or CRBN ligand 6, or PDEδ inhibitor 2 , or
proteasome inhibitor MG-132, followed by treated with DMSO or PDEδ degrader
17f. (F) Cell growth inhibition activity of the PDEδ inhibitor 2, negative control 17i
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and PDEδ degrader 17f in the absence or presence of 6 in SW480 cells. (G) IKZF1/3
levels in SW480 cells treated with 6 or 17f at various concentrations for 24 h.
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Compound 17f inhibits RAS signalling and induces the apoptosis of KRAS
mutant SW480 cells. Given the enhanced antitumor potency induced by PDEδ
degradation, the best PDEδ degrader 17f was selected to assess the effect on RAS
signaling downstream. RAS proteins regulate MAPK and mTOR pathways associated
with cell growth, proliferation and differentiation.14 After 5 min stimulation with
EGF, SW480 cells were treated with compound 17f for 12 h. Compound 2 was used
as the control. The phosphorylation of extracellular signal-regulated kinase (Erk) and
Akt were determined by western blotting assay. As shown in Figure 5, compound 17f
effectively reduced Akt and Erk phosphorylation at 5 μM in SW480 cells and
decreased p-Akt and p-Erk levels in a dose-dependent manner. In contrast, compound
2 was still ineffective in reducing Akt and Erk phosphorylation at 20 μM. The results
confirmed that PDEδ degradation induced by 17f significantly downregulated the
levels of both p-Akt and p-Erk. Furthermore, we also investigated the ability of
compound 17f in inducing apoptosis in SW480 cells. As illustrated in Figure 6A,
PDEδ degrader 17f caused significant induction of apoptosis in SW480 cells at a
concentration of 20 μM. The percentage of apoptotic cells was 38.31%, which was
much higher than that of PDEδ inhibitor 2 (11.1%). Moreover, the apoptotic rate
reached to 65.53% when SW480 cells were treated with 17f at a concentration of 40
μM. Finally, immunofluorescence staining (Figure 6B) showed that compound 17f
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caused significant PDEδ degradation and induced a distribution of endogenous RAS
to endomembranes. These results validated the significant advantages of PDEδ
degrader at the cellular level.
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Figure 5. Compound 17f inhibits RAS signalling. Phosphorylation levels of Erk and
Akt in SW480 cells stimulated with EGF (125 ng/mL, 5 min). From top to bottom:
phosphorylated Akt on S473 (p-Akt), total level of Akt1 (t-Akt1), phosphorylated Erk
on Thr202 and Tyr204 (p-Erk), total level of Erk (t-Erk), and loading control
(GAPDH). *** P value < 0.001.
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Figure 6. Compound 17f inhibits RAS signalling and initiates the apoptosis of KRAS
mutant SW480 cells. (A) Flow cytometry analysis of the apoptosis induction by 17f in
SW480 cells. (B) Immunostaining of SW480 with anti-PDEδ (green) and
antipan-RAS (red) after treatment with compound 17f at 10 μM.
In Vivo Antitumor Efficacy and Preliminary PK Study of Compound 17f. To
access the therapeutic potential of PDEδ degraders, compound 17f was selected to
investigate in vivo antitumor potency in SW480 xenograft nude mice models. The
SW480 colorectal cancer xenograft model was prepared using the reported
procedure.37 After the tumor volume reached to 100 mm3 in each group, compound
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17f and positive control were administered intraperitoneally (IP) once a day for 12
consecutive days. As depicted in Figure 7A, PDEδ degrader 17f (50 mg/kg) achieved
significant tumor growth inhibition with tumor growth inhibition (TGI) values of
43%. In contrast, IP dosing with PDEδ inhibitor 2 (50 mg/kg) failed to exert
significant tumor growth inhibition (TGI = 24%) as compared with the vehicle group.
Thus, degrader 17f demonstrated significantly better antineoplastic effects in vivo
than that of inhibitor 2. Moreover, no significant body weight loss and no obvious
adverse effects were observed during the study (Figure 7B), suggesting the low
toxicity of compound 17f. In order to further clarify the mechanism of 17f in vivo,
immunofluorescence assays were performed to detect the expression of PDEδ, RAS
and p-Akt in tumor tissues. Consistently, treatment with compound 17f led to
significant decrease in PDEδ and p-Akt proteins in vivo (Figure 7C). Notably,
compound 2 showed no effect on PDEδ protein levels and slightly decreased p-Akt
protein levels. The in vivo results highlighted the distinct advantages of PDEδ
degradation over PDEδ inhibition and provided an efficient strategy for the treatment
of KRAS mutant cancer.
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Furthermore, pharmacokinetic (PK) studies of compound 17f were evaluated in
Sprague-Dawley (SD) rats. After IP administration dosing at 50 mg/kg, the
concentrations of compound 17f in plasma were analyzed. As shown in Figure 7C,
the half-life of 17f was approximately 5.1 h and the peak concentration Cmax was 564
ng/mL. Despite its relatively large size (MW = 723), compound 17f could be
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effectively absorbed and achieved a sufficient plasma exposure in rats, with the area
under the curve (AUC) value of 4710 h*ng/mL.
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Figure 7. In vivo efficacy of compound 17f. (A) Growth curve of implanted SW480
xenograft in nude mice. Data are expressed as the mean ± standard deviation. Not
significant (n.s.) P value > 0.05; * P value < 0.05. (B) Body weight loss in SW480
xenograft experiments. (C) The immunofluorescence analysis of PDEδ, RAS and
p-Akt protein expression in transplanted nude mice. (D) PK parameters of compound
17f in rats. Data are expressed as the mean ± standard deviation.
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CONCLUSION
6
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In summary, the first-in-class PDEδ degraders were identified by the PROTAC
approach derived from conventional PDEδ inhibitors. The most promising compound
17f simultaneously bound to both PDEδ and CRBN and efficiently induced PDEδ
degradation. It significantly induced apoptosis, downregulated EGF-induced Erk and
Akt phosphorylation in KRAS mutant SW480 cells. As compared to PDEδ inhibitor
2, degrader 17f showed improved in vitro and in vivo antitumor activity. More
importantly, degrader 17f achieved significant tumor growth inhibition in SW480
colorectal cancer xenograft model, which provided important evidence to validate the
druggability of KRAS-PDEδ interaction. Taken together, this proof-of-concept study
highlighted the advantages of PDEδ degradation in target validation and novel
antitumor drug discovery. Further structural optimization studies to improve the
degradation potency and antitumor efficacy of lead compound 17f are in progress.
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EXPERIMENTAL SECTION
Chemistry. General Methods. All starting materials were commercially available and
analytical pure. 1H-NMR and 13C-NMR spectra were recorded on Bruker AVANCE II
300 or AVANCE II 600 spectrometer (Bruker Company, Germany), using TMS as an
internal standard and DMSO-d6 as solvents. Chemical shifts (δ values) and coupling
constants (J values) are given in ppm and Hz, respectively. The mass spectra were
recorded on an Esquire 3000 LC-MS mass spectrometer. Silica gel thin-layer
chromatography was performed on precoated plates GF-254 (Qingdao Haiyang
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Chemical, China). Silica gel column chromatography was performed with Silica gel
60G (Qingdao Haiyang Chemical, China). Purity of the compound was analyzed by
HPLC (Agilent Eclipse Plus C18 5 μM 4.6 × 250 mm), and final compounds exhibited
the purity greater than 95%.
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Methyl
4-(3,4-dimethyl-7-oxo-2-(p-tolyl)-2,7-dihydro-6H-pyrazolo[3,4-d]
pyridazin-6-yl) butanoate (11). The compound 9 (3.5 g, 12 mmol) was dissolved in
20 ml ethanol and placed in microwave tube with hydrazine hydrate (1.8 g, 36 mmol).
o
After 6 hours at 110 C, a large number of yellow solids were precipitated, filtered
and washed to give Intermediate 10 (2.4 g, 78% yield) which was synthesized
according to literature methods.14 Intermediate 10 (0.5 g, 1.9 mmol) was dissolved in
o
DMF (3 mL) with NaH (0.12 g, 2.85 mmol) at 0 C and stirred for 0.5 h. Then, the
methyl 3-bromopropanoate (0.66 g, 3.8 mmol) was added slowly and the mixture was
stirred for 6 h at room temperature. Then the mixture was poured into brine (50 mL).
The white precipitate was then filtered and dried to give the rude product. The crude
product was purified by column chromatography (DCM/MeOH = 100:1) to afford
pure product 11 (0.22 g, 80% yield) as a white solid. 1H-NMR (DMSO-d6, 600 MHz)
δ: 7.49 (d, J = 8.49 Hz, 2H), 7.43 (d, J = 8.13 Hz, 2H), 4.08 (t, J = 6.72 Hz, 2H), 3.57
(s, 3H), 2.60 (s, 3H), 2.52 (s, 3H), 2.43 (s, 3H), 2.35 (t, J = 7.07 Hz, 2H), 1.98-1.93
(m, 2H).
4-(3,4-Dimethyl-7-oxo-2-(p-tolyl)-2,7-dihydro-6H-pyrazolo[3,4-d]pyridazin-6-yl)
butanoic acid (12). To a solution of intermediate 11 (0.22 g, 0.65 mmol) in
THF-MeOH-H2O solvent system (18 mL, THF: MeOH: H2O = 3: 2: 1) was slowly
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added aqueous LiOH (1 M, 55 mg, 1.3 mmol). The mixture was stirred for 1 h at
room temperature. Then, the solvent was removed under the reduced pressure and the
pH of the solution was adjusted to 2 with 2 M HCl. The white precipitate was then
filtered and dried to give intermediate 12 (0.17 g, 80% yield) as a white solid.
1H-NMR (DMSO-d6, 600 MHz) δ: 12.06 (s, 1H), 7.49 (d, J = 8.38 Hz, 2H,), 7.43 (d, J
= 8.38 Hz, 2H,), 4.07 (t, J = 6.82 Hz, 2H,), 2.60 (s, 3H), 2.52 (s, 3H), 2.43 (s, 3H),
2.26 (t, J = 7.41 Hz, 2H), 1.90-1.96 (m, 2H).
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2-(2,6-Dioxopiperidin-3-yl)-4-fluoroisoindoline-1,3-dione (15). A solution of
4-fluoroisobenzofuran-1,3-dione (0.5 g, 3 mmol), 2,6-Dioxopiperidine-3-ammonium
chloride (0.5 g, 3 mmol) and NaOAc (0.3 g, 3.6 mmol) in AcOH (50 mL) was reflux
for 12 h. The reaction mixture was poured into water (300 mL) and extracted with
EtOAc (3  150 mL). The organic phases were combined, dried with anhydrous
sodium sulfate and concentrated under the reduced pressure. The crude product was
purified by column chromatography (DCM/MeOH = 100:1) to give intermediate 15
as a white solid (0.74 g, 90% yield). 1H-NMR (600 MHz, DMSO-d6) δ: 11.15 (s, 1H),
7.93-7.97 (m, 1H), 7.79 (d, J = 7.31 Hz, 1H), 7.73 (t, J = 8.86 Hz, 1H), 5.16 (dd, J =
13.16, 5.35 Hz, 1H), 2.93-2.86 (m, 1H), 2.63-2.58 (m, 1H), 2.56-2.47 (m, 1H),
2.09-2.04 (m, 1H). Compound 20 was synthesized according to the similar procedure
described for 15.
tert-Butyl (3-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino) propyl)
carbamate (16a). A solution of intermediate 15 (0.2 g, 0.7 mmol) and tert-butyl
(3-aminopropyl)carbamate (0.14 g, 0.8 mmol) in DMF (2 mL) was treated with
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DIPEA (0.18 g, 1.4 mmol) under 90 C for 12 h. The reaction mixture was poured
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into water (20 mL) and extracted with EtOAc (3  15 mL). The combined organic
phases were dried and concentrated under the reduced pressure. The crude product
was purified by column chromatography (DCM/MeOH = 100:1) to give intermediate
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1
16a as a yellow solid (0.12 g, 40% yield). H-NMR (300 MHz, DMSO-d6) δ: 11.10
(s, 1H), 7.61-7.54 (m, 1H), 7.08 (d, J = 8.74 Hz, 1H), 7.02 (d, J = 7.12 Hz, 1H), 6.93
(t, J = 5.18 Hz, 1H), 6.67 (t, J = 5.18 Hz, 1H), 5.05 (dd, J = 12.62, 5.18 Hz, 1H),
3.36-3.26 (m, 2H), 3.05-2.95 (m, 2H), 2.91-2.81 (m, 1H), 2.65-2.58 (m, 1H),
2.57-2.52 (m, 1H), 2.08-2.00 (m, 1H), 1.73-1.59 (m, 2H), 1.38 (s, 9H). The
compounds 16b-i were synthesized according to a similar procedure described for
16a.
tert-Butyl (4-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)butyl)
1
carbamate (16b). H-NMR (600 MHz, DMSO-d6) δ: 11.08 (s, 1H), 7.59-7.56 (m,
1H), 7.10 (d, J = 8.94 Hz, 1H), 7.02 (d, J = 7.45 Hz, 1H), 6.83 (t, J = 5.96 Hz, 1H),
6.55 (t, J = 5.47 Hz, 1H), 5.05 (dd, J = 12.92, 4.97 Hz, 1H), 3.32-3.27 (m, 2H),
2.97-2.92 (m, 2H), 2.90-2.87 (m, 1H), 2.62-2.56 (m, 1H), 2.55-2.51 (m, 1H),
2.05-2.00 (m, 1H), 1.59-1.51 (m, 2H), 1.48-1.41 (m, 2H), 1.37 (s, 9H).
tert-Butyl
(5-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)
pentyl)carbamate (16c). 1H-NMR (600 MHz, DMSO-d6) δ: 11.09 (s, 1H), 7.60-7.56
(m, 1H), 7.10 (d, J = 8.26 Hz, 1H), 7.02 (d, J = 8.26 Hz, 1H), 6.78 (t, J = 5.08 Hz,
1H), 6.53 (t, J = 5.72 Hz, 1H), 5.05 (dd, J = 12.54, 5.53 Hz, 1H), 3.30-3.25 (m, 2H),
2.93-2.88 (m, 2H), 2.88-2.84 (m, 1H), 2.62-2.56 (m, 1H), 2.56-2.51 (m, 1H),
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2.05-2.00 (m, 1H), 1.60-1.53 (m, 2H), 1.44-1.38 (m, 2H), 1.36 (s, 9H), 1.33-1.29
(m,2H).
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tert-Butyl (6-((2-(2,6-dioxopiperidin-3-yl)-1,3- dioxoisoindolin-4-yl)amino)hexyl)
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1
carbamate (16d). H-NMR (600 MHz, DMSO-d6) δ: 11.04 (s, 1H), 7.61-7.57 (m,
1H), 7.10 (d, J = 8.52 Hz, 1H), 7.03 (d, J = 7.01 Hz, 1H), 6.78 (t, J = 5.45 Hz, 1H),
6.54 (t, J = 5.96 Hz, 1H), 5.06 (dd, J = 13.12, 5.62 Hz, 1H), 3.32-3.27 (m, 2H),
2.92-2.89 (m, 2H), 2.88-2.85 (m, 1H), 2.63-2.57 (m, 1H), 2.57-2.52 (m, 1H),
2.06-2.00 (m, 1H), 1.60-1.54 (m, 2H), 1.37 (s, 9H), 1.37-1.35 (m, 2H), 1.35-1.32 (m,
2H), 1.31-1.27 (m, 2H).
tert-Butyl (8-((2-(2,6-dioxopiperidin-3-yl)-1,3- dioxoisoindolin-4-yl)amino)octyl)
1
carbamate (16e). H-NMR (600 MHz, DMSO-d6) δ: 11.07 (s, 1H), 7.58-7.55 (m,
1H), 7.08 (d, J = 8.51 Hz, 1H), 7.00 (d, J = 7.25 Hz, 1H), 6.73 (t, J = 5.36 Hz, 1H),
6.51 (t, J = 5.68 Hz, 1H), 5.03 (dd, J = 13.24, 5.36 Hz, 1H), 3.30-3.25 (m, 2H),
2.87-2.83 (m, 2H), 2.61-2.58 (m, 1H), 2.57-2.55 (m, 1H), 2.55-2.51 (m, 1H),
2.04-1.99 (m, 1H), 1.58-1.52 (m, 2H), 1.35 (s, 9H), 1.33-1.21 (m, 10H).
tert-Butyl
(2-(2-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)
1
amino)ethoxy)ethoxy)ethyl)carbamate (16f). H-NMR (300 MHz, DMSO-d6) δ:
11.10 (s, 1H), 7.58 (t, J = 8.29 Hz, 1H), 7.15 (d, J = 8.29 Hz, 1H), 7.04 (d, J = 6.74
Hz, 1H), 6.75 (t, J = 5.18 Hz, 1H), 6.61 (t, J = 5.18 Hz, 1H), 5.06 (dd, J = 12.96, 5.18
Hz, 1H), 3.66-3.39 (m, 10H), 3.13-2.99 (m, 2H), 2.98-2.79 (m, 1H), 2.67-2.52 (m,
2H), 2.09-2.00 (m, 1H), 1.36 (s, 9H).
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tert-Butyl
(3-(2-(2-(3-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin
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8
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-4-yl)amino)propoxy) ethoxy)ethoxy)propyl)carbamate (16g). 1H-NMR (300
MHz, DMSO-d6) δ: 11.10 (s, 1H), 7.58 (t, J = 7.94 Hz, 1H), 7.10 (d, J = 8.58 Hz,
1H), 7.02 (d, J = 6.67 Hz, 1H), 6.75 (t, J = 5.08 Hz, 1H), 6.67 (t, J = 5.72 Hz, 1H),
5.05 (dd, J = 13.03, 5.08 Hz, 1H), 3.59-3.42 (m, 10H), 3.42-3.40 (m, 4H), 3.00-2.90
(m, 2H), 2.90-2.80 (m, 1H), 2.66-2.54 (m, 2H), 2.08-1.99 (m, 1H), 1.88-1.75 (m, 2H),
1.66-1.51 (m, 2H), 1.36 (s, 9H).
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4-(3,4-Dimethyl-7-oxo-2-(p-tolyl)-2,7-dihydro-6H-pyrazolo[3,4-d]pyridazin-6-yl)-
N-(3-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)propyl)butana
mide (17a). The intermediate 16a (0.2 g, 0.47 mmol) was dissolved in DCM (2 mL)
and then TFA (1 mL) was added to the reaction. The reaction was stirred for 1 h at
room temperature. Then, the reaction mixture was evaporated under the reduced
pressure and the crude product was dissolved in DMF (2 mL) with HATU (0.27 g, 0.7
mmol), DIPEA (0.18 g, 1.4 mmol) and compound 12 (0.24 g, 0.7 mmol). After 2 h,
the reaction mixture was poured into water (20 mL) and extracted with EtOAc (3  15
mL). The combined organic phases were dried and concentrated under the reduced
pressure. The crude product was purified by column chromatography (DCM/MeOH =
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100:1) to give intermediate 17a as a yellow solid (0.12 g, 40% yield). H-NMR (600
MHz, DMSO-d6) δ: 11.08 (s, 1H, imino), 7.90 (t, J = 5.69 Hz, 1H, imino), 7.57-7.53
(m, 1H, aromatic), 7.47 (d, J = 8.19 Hz, 2H, aromatic), 7.42 (d, J = 8.19 Hz, 2H,
aromatic), 7.08 (d, J = 8.70 Hz, 1H, aromatic), 6.99 (d, J = 6.91 Hz, 1H, aromatic),
6.66 (t, J = 6.14 Hz, 1H, imino), 5.05-5.00 (m, 1H, hypomethyl), 4.03 (t, J = 7.17 Hz,
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2H, methylene), 3.33-3.28 (m, 2H, methylene), 3.12-3.07 (m, 2H, methylene),
2.90-2.82 (m, 1H, methylene), 2.61-2.55 ((m, 1H, methylene; s, 3H, methyl),
2.54-2.50 (m, 1H, methylene), 2.50 (s, 3H, methyl), 2.41 (s, 3H, methyl), 2.11 (t, J =
7.68 Hz, 2H, methylene), 2.04-1.98 (m, 1H, methylene), 1.95-1.89 (m, 2H,
methylene), 1.68-1.62 (m, 2H, methylene). 13C-NMR (DMSO-d6, 150 MHz) δ: 173.3
(C=O), 172.0 (C=O), 170.6 (C=O), 169.3 (C=O), 167.8 (C=O), 155.6 (C=O), 146.8
(C-aromatic), 141.6, 141.5, 139.8 (C-aromatic), 137.9, 136.7 (C-aromatic), 136.3
(C-aromatic), 132.7 (C-aromatic), 130.4 (C-aromatic), 126.2 (C-aromatic), 117.6
(C-aromatic), 110.8 (C-aromatic), 109.6 (C-aromatic), 49.02, 49.0, 36.4, 33.1, 31.4,
29.5, 29.2, 25.0, 22.6, 21.2 (C-methyl), 19.9 (C-methyl), 12.3 (C-methyl). HR-MS
(ESI+): m/z calcd for [M+H]+ C34H37N8O6: 653.2831, found: 653.2832. HPLC purity
98.9%.
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Compounds 17b-i were synthesized according to a similar procedure described for
17a.
4-(3,4-Dimethyl-7-oxo-2-(p-tolyl)-2,7-dihydro-6H-pyrazolo[3,4-d]pyridazin-6-yl)-
N-(4-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)butyl)butanami
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de (17b). H-NMR (600 MHz, DMSO-d6) δ: 11.11 (s, 1H, imino), 7.85 (t, J = 5.53
Hz, 1H, imino), 7.57-7.53 (m, 1H, aromatic), 7.49 (d, J = 9.22 Hz, 2H, aromatic),
7.44 (d, J = 8.61 Hz, 2H, aromatic), 7.09 (d, J = 8.53 Hz, 1H, aromatic), 6.97 (d, J =
7.13 Hz, 1H, aromatic), 6.55 (t, J = 5.81 Hz, 1H, imino), 5.06-5.02 (m, 1H,
hypomethyl), 4.02 (t, J = 6.87 Hz, 2H, methylene), 3.32-3.27 (m, 2H, methylene),
3.10-3.05 (m, 2H, methylene), 2.91-2.83 (m, 1H, methylene), 2.63-2.56 (m, 1H,
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methylene; s, 3H, methyl), 2.55-2.53 (m, 1H methylene), 2.51 (s, 3H, methyl), 2.43 (s,
3H, methyl), 2.11 (t, J = 7.13 Hz, 2H, methylene), 2.05-1.99 (m, 1H, methylene),
1.95-1.88 (m, 2H, methylene), 1.60-1.53 (m, 2H, methylene), 1.49-1.43 (m, 2H,
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methylene). C-NMR (DMSO-d6, 150 MHz) δ: 173.3 (C=O), 171.9 (C=O), 170.6
(C=O), 169.4 (C=O), 167.8 (C=O), 155.6 (C=O), 146.9 (C-aromatic), 141.6, 141.5,
139.9 (C-aromatic), 137.9, 136.7 (C-aromatic), 136.3 (C-aromatic), 132.6
(C-aromatic), 130.4 (C-aromatic), 126.2 (C-aromatic), 117.7 (C-aromatic), 117.6
(C-aromatic), 110.8 (C-aromatic), 109.4 (C-aromatic), 49.1, 49.0, 42.0, 38.5, 33.1,
31.4, 26.9, 26.6, 25.2, 22.6, 21.2 (C-methyl), 19.8 (C-methyl), 12.3 (C-methyl).
HR-MS (ESI+): m/z calcd for [M+H]+ C35H39N8O6: 667.2987, found: 667.2987. HPLC
purity 95.2%.
4-(3,4-Dimethyl-7-oxo-2-(p-tolyl)-2,7-dihydro-6H-pyrazolo[3,4-d]pyridazin-6-yl
)-N-(5-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)pentyl)butana
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mide (17c). H-NMR (600 MHz, DMSO-d6) δ: 11.10 (s, 1H), 7.82 (t, J = 5.59 Hz,
1H), 7.58-7.54 (m, 1H), 7.62 (d, J = 8.13 Hz, 2H), 7.44 (d, J = 8.13 Hz, 2H), 7.09 (d,
J = 8.70 Hz, 1H), 6.99 (d, J = 6.91 Hz, 1H), 6.52 (t, J = 6.02 Hz, 1H), 5.07-5.02 (m,
1H), 4.03 (t, J = 7.14 Hz, 2H), 3.32-3.25 (m, 2H), 3.07-3.01 (m, 2H), 2.91-2.84 (m,
1H), 2.63-2.57 (m, 4H), 2.56-2.52 (m, 1H), 2.52 (s, 3H), 2.43 (s, 3H), 2.10 (t, J = 7.58
Hz, 2H), 2.07-2.00 (m, 1H), 1.96-1.88 (m, 2H), 1.61-1.54 (m, 2H), 1.46-1.40 (m, 2H),
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1.38-1.31 (m, 2H). C-NMR (DMSO-d6, 150 MHz) δ: 173.3, 171.9, 170.6, 169.4,
167.8, 155.6, 146.9, 141.6, 141.5, 139.8, 137.9, 136.7, 136.3, 132.6, 130.4, 126.2,
117.6, 117.6, 110.8, 109.4, 49.1, 49.0, 42.3, 38.8, 33.1, 31.4, 29.3, 28.8, 25.1, 24.2,
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22.6, 21.2, 19.8, 12.3. HR-MS (ESI+): m/z calcd for [M+H]+ C36H41N8O6: 681.3144,
found: 681.3148. HPLC purity 95.2%.
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4-(3,4-Dimethyl-7-oxo-2-(p-tolyl)-2,7-dihydro-6H-pyrazolo[3,4-d]pyridazin-6-yl
)-N-(6-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)hexyl)butana
mide (17d). 1H-NMR (600 MHz, DMSO-d6) δ: 11.07 (s, 1H imino), 7.75 (t, J = 5.56
Hz, 1H imino), 7.57-7.51 (m, 1H, aromatic), 7.46 (d, J = 8.42 Hz, 2H, aromatic), 7.41
(d, J = 8.16 Hz, 2H, aromatic), 7.05 (d, J = 8.50 Hz, 1H, aromatic), 6.97 (d, J = 6.99
Hz, 1H, aromatic), 6.50 (t, J = 5.81 Hz, 1H, imino), 5.06-5.00 (m, 1H, hypomethyl),
4.02 (t, J = 6.73 Hz, 2H, methylene), 3.28-3.23 (m, 2H, methylene), 3.03-2.98 (m, 2H,
methylene), 2.91-2.82 (m, 1H, methylene), 2.62-2.55 (m, 1H, methylene; s, 3H,
methyl), 2.54-2.50 (m, 1H, methylene), 2.49 (s, 3H, methyl), 2.41 (s, 3H, methyl),
2.08 (t, J = 7.42 Hz, 2H, methylene), 2.05-1.98 (m, 1H, methylene), 1.94-1.86 (m, 2H,
methylene), 1.59-1.51 (m, 2H, methylene), 1.41-1.35 (m, 2H, methylene), 1.34-1.29
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(m, 4H, methylene). C-NMR (DMSO-d6, 150 MHz) δ: 173.3 (C=O), 171.7 (C=O),
170.5 (C=O), 169.4 (C=O), 167.8 (C=O), 155.6 (C=O), 146.9 (C-aromatic), 141.6,
141.4, 139.8 (C-aromatic), 137.8, 136.7 (C-aromatic), 136.4 (C-aromatic), 132.6
(C-aromatic), 130.4 (C-aromatic), 126.2 (C-aromatic), 117.6 (C-aromatic), 110.8
(C-aromatic), 109.5 (C-aromatic), 49.1, 49.0, 42.2, 38.8, 33.1, 31.4, 29.5, 29.1, 26.6,
26.5, 25.2, 22.6, 21.2 (C-methyl), 19.9 (C-methyl), 12.3 (C-methyl). HR-MS (ESI+):
m/z calcd for [M+H]+ C37H43N8O6: 695.3300, found: 695.3304.
4-(3,4-dimethyl-7-oxo-2-(p-tolyl)-2,7-dihydro-6H-pyrazolo[3,4-d]pyridazin-6-yl)-
N-(7-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)heptyl)butana
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mide (17e). 1H-NMR (600 MHz, DMSO-d6) δ: 11.10 (s, 1H, imino), 7.78 (t, J = 5.42
Hz, 1H, imino), 7.56 (dd, J = 8.51, 7.3 Hz, 1H, aromatic), 7.49 (d, J = 8.24 Hz, 2H,
aromatic), 7.44 (d, J = 8.13 Hz, 2H, aromatic), 7.07 (d, J = 8.52 Hz, 1H, aromatic),
6.99 (d, J = 6.91 Hz, 1H, aromatic), 6.51 (t, J = 5.72 Hz, 1H, imino), 5.06 (dd, J =
12.94, 5.52 Hz, 1H, hypomethyl), 4.04 (t, J = 7.01 Hz, 2H, methylene), 3.30-3.25 (m,
2H, methylene), 3.05-3.00 (m, 2H, methylene), 2.93-2.85 (m, 1H, methylene),
2.63-2.59 (m, 1H, methylene), 2.59 (s, 3H, methyl), 2.56-2.51 ((m, 1H, methylene; s,
3H, methyl), 2.43 (s, 3H, methyl), 2.10 (t, J = 7.32 Hz, 2H, methylene), 2.06-2.00 (m,
1H, methylene), 1.96-1.89 (m, 2H, methylene), 1.61-1.53 (m, 2H, methylene),
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1.42-1.36 (m, 2H, methylene), 1.35-1.26 (m, 6H, methylene). C-NMR (DMSO-d6,
150 MHz) δ: 173.3 (C=O), 171.7 (C=O), 170.5 (C=O), 169.4 (C=O), 167.8 (C=O),
155.6 (C=O), 146.9 (C-aromatic), 141.6, 141.4, 139.8 (C-aromatic), 137.8, 136.7
(C-aromatic), 136.3 (C-aromatic), 132.6 (C-aromatic), 130.4 (C-aromatic), 126.2
(C-aromatic), 117.6 (C-aromatic), 110.8 (C-aromatic), 109.5 (C-aromatic), 49.1, 49.0,
42.3, 38.9, 33.1, 31.5, 29.5, 29.1, 28.9, 26.8, 26.7, 25.2, 22.6, 21.2 (C-methyl), 19.9
(C-methyl), 12.3 (C-methyl). HR-MS (ESI+): m/z calcd for [M+H]+ C38H45N8O6:
709.3457, found: 709.3455. HPLC purity 99.4%.
4-(3,4-Dimethyl-7-oxo-2-(p-tolyl)-2,7-dihydro-6H-pyrazolo[3,4-d]pyridazin-6-yl
)-N-(8-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)octyl)butana
mide (17f). 1H-NMR (600 MHz, DMSO-d6) δ: 11.09 (s, 1H, imino), 7.76 (t, J = 5.62
Hz, 1H, imino), 7.56 (dd, J = 8.41, 7.01 Hz, 1H, aromatic), 7.48 (d, J = 8.44 Hz, 2H,
aromatic), 7.43 (d, J = 8.44 Hz, 2H, aromatic), 7.06 (d, J = 8.51 Hz, 1H, aromatic),
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