De-novo design of cereblon (CRBN) effectors guided by natural hydrolysis products of thalidomide derivatives

Article abstract of DOI:10.1021/acs.jmedchem.9b00454

Targeted protein degradation via cereblon (CRBN), a substrate receptor of an E3 ubiquitin ligase complex, is an increasingly important strategy in various clinical settings, in which the substrate specificity of CRBN is altered via the binding of small-molecule effectors. To date, such effectors are derived from thalidomide and confer a broad substrate spectrum that is far from being fully characterized. Here, we employed a rational and modular approach to design novel and minimalistic CRBN effectors. In this approach, we took advantage of the binding modes of hydrolyzed metabolites of several thalidomide-derived effectors, which we elucidated via crystallography. These yielded key insights for the optimization of the minimal core binding moiety and its linkage to a chemical moiety that imparts substrate specificity. Based on this scaffold, we present a first active de-novo CRBN effector that is able to degrade the neo-substrate IKZF3 in the cell culture.

Full text of DOI:10.1021/acs.jmedchem.9b00454

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Article
De-novo design of cereblon (CRBN) effectors guided by
natural hydrolysis products of thalidomide derivatives
Christopher Heim, Dimanthi Pliatsika, Farnoush Mousavizadeh, Kerstin Bär,
Birte Hernandez Alvarez, Athanassios Giannis, and Marcus D Hartmann
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.9b00454 • Publication Date (Web): 28 Jun 2019
Downloaded from http://pubs.acs.org on June 29, 2019
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Journal of Medicinal Chemistry
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De-novo design of cereblon (CRBN) effectors guided by natural
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hydrolysis products of thalidomide derivatives
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Christopher Heim1, Dimanthi Pliatsika2, Farnoush Mousavizadeh2, Kerstin Bär1, Birte
Hernandez Alvarez1, Athanassios Giannis2*, Marcus D. Hartmann1*
1Department of Protein Evolution, Max Planck Institute for Developmental Biology, Max-Planck-Ring 5, 72076
Tübingen, Germany
2Faculty for Chemistry und Mineralogy, Institute of Organic Chemistry, University of Leipzig, Johannisallee 29,
04103 Leipzig, Germany
KEYWORDS. Amidosuccinimide, Immunomodulatory drug (IMiD), Proteolysis Targeting Chimera (PROTAC),
Ubiquitin-Proteasome Pathway, Rational Drug Design.
ABSTRACT: Targeted protein degradation via cereblon (CRBN), a substrate receptor of an E3 ubiquitin ligase
complex, is an increasingly important strategy in various clinical settings, in which the substrate specificity of CRBN
is altered via the binding of small-molecule effectors. To date, such effectors are derived from thalidomide and confer
a broad substrate spectrum that is far from being fully characterized. Here, we employed a rational and modular
approach to design novel and minimalistic CRBN effectors. In this approach we took advantage of the binding modes
of hydrolyzed metabolites of several thalidomide-derived effectors, which we elucidated via crystallography. These
yielded key insights for the optimization of the minimal core binding-moiety and its linkage to a chemical moiety that
imparts substrate specificity. Based on this scaffold, we present a first active de-novo CRBN effector that is able to
degrade the neo-substrate IKZF3 in cell culture.
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Introduction
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Classical immunomodulatory drugs (IMiDs) like thalidomide and its second and third generation analogs lenalidomide,
pomalidomide, avadomide (CC-122) and iberdomide (CC-220) have constantly emerged to new therapeutic areas. Originally
developed as a sedative1 and banned in 1961 for its teratogenic effects when used during pregnancy2-3, thalidomide and a number of
newly developed analogs are approved for the treatment of Multiple Myeloma (MM)4, Erythema nodosum5 and Myelodysplastic
syndrome (MDS)6-7. Because of their pleiotropic and especially anti-angiogenic properties, IMiDs have further been reported
effective in many off-label indications as for Hodgkin's lymphoma8-10, Light chain-associated (AL) amyloidosis11 and Acute
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myeloid leukemia (AML)12-13
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Figure 1. Chemical structures of thalidomide, lenalidomide, pomalidomide, avadomide and iberdomide with their glutarimide
moiety shown in red
Currently, IMiDs share a common glutarimide moiety (Figure 1), which is connected to a second moiety that is typically derived
from phthaloyl. Via the glutarimide moiety, they are able to bind to a tri-tryptophan pocket within the thalidomide binding
domain14-17 of cereblon (CRBN). CRBN is the substrate receptor of the Cullin RING E3 ubiquitin ligase CUL4-RBX1-DDB1-
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CRBN (CRL4CRBN
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and responsible for the recognition of endogenous substrates such as glutamine synthetase18, MEIS217 and
amyloid precursor protein (APP)19. In presence of IMiDs, however, its substrate specificity is changed. The solvent-exposed second
moiety, the protruding moiety that is unique to each IMiD, recruits novel substrates to the CRBN surface for ubiquitination. The
degradation of these neo-substrates accounts for most of the efficacy of IMiDs in multiple myeloma (IKZF1 and IKZF3)20, 5q-
deletion associated myelodysplastic syndrome (CK1α)21 and acute myeloid leukemia (GSPT1)22. Recently, it could also be shown
that the degradation of the neo-substrate SALL4 is linked to developmental malformations caused by thalidomide23-24
.
Crystal structures of the neo-substrate complexes CRBNlenalidomideCK1α, CRBNpomalidomideIKZF1, and
CRBNpomalidomideZNF692 provided first insights into the binding mode of neo-substrates25-26. Many of the identified neo-
substrates possess no obvious sequence homology, but they all exhibit a structurally analogous β-hairpin, which binds to the surface
around the IMiD-binding site, involving interactions with both surface residues and the IMiD itself. A potential prerequisite for the
recruitment is the presence of a glycine at the tip of this β-hairpin; this glycine was found to be conserved for many neo-substrates,
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like CK1α25 and GSPT122, and appears in the common C-X(2)-C-G motif24 in many transcription factors belonging to the C2H2 zinc
finger class, including IKZF325, IKZF125, ZFP9124 and SALL424. While IMiDs seem to generally recruit several members of the
C2H2 zinc finger family, only lenalidomide was proven to recruit CK1α, indicating that the protruding moiety imparts substrate
specificity.
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The CRBN-binding ability of IMiDs has further been exploited for targeted protein degradation in an approach called Proteolysis
Targeting Chimera (PROTAC), coined in 2001 by Craig Crews and co-workers27-28. PROTACs are bifunctional small molecules
with a binding moiety for a target protein linked to a binding moiety for an E3 ubiquitin ligase, thus inducing ubiquitination and
proteasomal degradation of the target protein. Recent examples of successful PROTACs target the estrogen29 and the androgen
receptor30 via a von-Hippel-Lindau (VHL) E3 ligase ligand, but also BET bromodomains via linkage to thalidomide as a CRBN
ligand31. Notably, also PROTACs with a VHL ligand on one, and thalidomide as CRBN ligand on the other end have been tested,
leading to unidirectional degradation of CRBN32. In general, also the PROTAC approach has high potential to target the
undruggable; a particularly illustrative example for this potential is exemplified by the recent discovery of PROTACs targeting the
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tau protein33-35
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To date, essentially all CRBN effectors – IMiDs and CRBN-based PROTACs – are derived from thalidomide and its derivatives.
However, the chemical space of CRBN ligands ranges far beyond thalidomide: In a first systematic characterization we have
previously revealed that a large variety of lactams and cyclic imides are potent binders, including several marketed drugs36.
Specifically, we have shown succinimide to exhibit higher affinity than glutarimide, using the single-domain bacterial CRBN
homologue from Magnetospirillum gryphiswaldense (MsCI4) in a FRET assay14. In this study, we set out to further explore and
exploit the chemical space of CRBN binding by designing effectors based on succinimide and glutarimide, which we characterize
with regard to their affinity, their structural binding mode, and their ability to induce proteasomal degradation of neo-substrates.
Guided by hydrolyzed metabolites of thalidomide and of three of our designs, we present novel minimalistic motifs that are able to
recruit and degrade neo-substrates, and may serve as E3-recruiting ligands for future PROTACs.
Results and Discussion
Biophysical and structural characterization of IMiD analogs and their hydrolysis products
As a starting point for new effectors we chose the classical IMiD scaffold. Based on the finding that succinimide is able to bind to
CRBN with a higher affinity than glutarimide (Ki values of 4.3 µM vs. 28 µM for MsCI436), we designed a first panel of derivatives
based on glutarimide and succinimide, in which we probed the effect of different substitutions in the phthaloyl moiety (Figure 2).
The respective compounds 2a-5b were prepared by the synthetic route shown in Scheme 1. The imides 3a and 3b were synthesized
from the commercially available Nα-(tert-butoxycarbonyl)-L-asparagine (1a) and Nα-(tert-butoxycarbonyl)-L-glutamine (1b)
respectively via an imide formation using N,N-carbodiimidazole (CDI) and 4-dimethylaminopyridine (4-DMAP) in THF as well as
a deprotection reaction in the presence of trifluoracetic acid (TFA). Compounds 4a-4d were obtained through a coupling between
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the imides 3a or 3b and the commercially available 3-nitrophthalic anhydride or 4-nitrophthalic anhydride. Catalytic hydrogenation
of the molecules 4a and 4b using 10 wt% Pd/C in EtOAc produced the derivatives 5a and 5b.
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Scheme 1. Reagents and conditions: (a) CDI, 4-DMAP, THF, reflux, 17-48 h; (b) TFA, RT, 30 min; (c) CDI, 4-DMAP, THF,
reflux, 5-20 h; (d) 10 wt. % Pd/C, EtOAc, RT, 20 h
The binding affinities of these compounds for MsCI4 were assessed in the FRET assay, starting with the glutarimide-based
compounds 4c and 4d in comparison to the commercial thalidomide derivatives lenalidomide and pomalidomide. Lenalidomide
(Ki = 3.1 µM) and pomalidomide (Ki = 0.8 µM), both carrying an additional amino group in the R2 position, show improved affinity
to MsCI4 as compared to thalidomide (Ki = 4.4 µM). When we exchanged this amino group for a nitro group (4c), we saw a
significant drop in affinity, into a range in which a precise value could not be obtained (Ki > 40 µM)36; moving this nitro group to
the R1 position (4d) had less impact on the affinity (Ki = 8.9 µM, Figure 2A). Similar effects were observed in a recent study that
was published during the preparation of this manuscript37. As both compounds, 4c and 4d, retained affinity to MsCI4, their overall
binding mode is supposedly conserved and comparable to thalidomide, which is consistent with previous studies showing that small
modifications on the protruding moiety have little influence on the overall affinity to CRBN17,37. The binding modes of
lenalidomide and pomalidomide have previously been reported to be virtually identical to thalidomide14,17
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For the succinimide-based compounds 4a, 4b, 5a and 5b, we obtained a similar picture as for the glutarimide-based compounds.
Although they showed overall weaker binding in the FRET assay (Figure 2B), substitutions in the R2 position are less favorable for
the affinity as substitutions in R1. For 4a and 5a, which have a nitro or an amino group in R2, respectively, we obtained Ki values of
> 40 µM. For 4b and 5b, which have the respective groups in the R1 position, the obtained Ki values are 11 µM and 12 µM,
respectively.
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determined as Ki values in the FRET assay.
In a next step, we determined the molecular binding determinants of the succinimide-based compounds via x-ray crystallography.
To this end, crystals of the MsCI4thalidomide complex were reproduced36 and subsequently used for soaking experiments, in
which the thalidomide molecules bound to MsCI4 may be displaced by the compound of interest. There are three chains, i.e. three
MsCI4thalidomide complexes, in the asymmetric unit (ASU) of these crystals, in which the bound thalidomide molecules can
potentially be displaced. However, as the three chains form different crystal contacts and differ slightly in their conformation, it is
possible that thalidomide is only replaced in one or two chains of the ASU, depending on the particular compound36. These
experiments yielded crystal structures for the four compounds tested, 4a, 4b, 5a and 5b, all showing the classical IMiD binding-
mode with the basal main-chain interactions of the succinimide amino-group with F77 and the carbonyl group with W79 of MsCI4.
Also the orientation of the protruding moieties of 4a, 5a and 5b are very similar to that of thalidomide, despite the different ring
size of glutarimide and succinimide. As a result, the hydrogen bond that is typically observed between the conserved N50 and a
carbonyl group of the phthaloyl moieties for the classical glutarimide-based compounds is also found for 5b.
However, the crystal structures also held surprises. They did not only reveal the binding modes of the pure compounds, but also that
of hydrolysis products of 4a, 4b and 5a with unambiguous electron density; only 5b was exclusively observed in non-hydrolyzed
form (Figures 3, 4). For 4a and 5a, hydrolyzed metabolites were only observed in one chain of the ASU, with the second chain
occupied with a non-hydrolyzed version and the third chain with thalidomide. For 4b, which showed the highest affinity with a Ki
of 11 µM, all three binding pockets in the ASU were occupied by a hydrolyzed metabolite, so the binding mode of non-hydrolyzed
4b could not be studied. In all cases, for the hydrolysis products of 4a, 4b and 5a, ring opening of the phthaloyl group had led to the
formation of a secondary amide and a carboxyl group, which are clearly resolved in FO-FC omit maps (Figure 3). Both of these form
important interactions with the binding pocket: Firstly, the secondary amide rescues the hydrogen bond to the conserved N50,
which is typically formed by a carbonyl of the phthaloyl moiety. Secondly, the additional carboxyl group replaces a conserved
water molecule previously coordinated by W99 and engages in direct hydrogen bonding with the W99 side chain. In contrary, the
primary amino group of hydrolyzed 5a and the solvent exposed nitro groups of hydrolyzed 4a and 4b do not show additional
interactions.
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Figure 3. Overview of the thalidomide binding mode and electron densities of thalidomide analogs and their hydrolysis products
bound to MsCI4. Left: Cartoon representation of thalidomide-bound MsCI4 with key residues of the binding pocket shown as
sticks. Right: FO - FC maps of CBG (PDB 6R0Q), 4a and hydrolyzed 4a (PDB 6R0S), hydrolyzed 4b (PDB 6R0V), 5a and
hydrolyzed 5a (PDB 6R0U) and 5b (PDB 6R11) in the MsCI4 binding pocket, contoured at 2σ.
Binding mode of CBG, a major hydrolysis product of thalidomide
Admittedly unintentionally, in addition to the hydrolysis products of 4a, 4b and 5a, we were able to characterize the binding mode
of a major hydrolyzed thalidomide metabolite. It is known that IMiDs can rapidly racemize in bodily fluids and water38, and
spontaneously hydrolyze under physiological conditions39-41. For thalidomide, being eliminated mainly by spontaneous hydrolysis
in blood and tissues, a half-life of about 5.5 to 7.3 h at the physiological pH of 7.4 was reported42. Among several proposed
hydrolysis products, the two main urinary metabolites are 2-phthalimidoglutaramic acid (~50%) and α-(2-Carboxybenzamido)
glutarimide (CBG) (~30%)43. Of these, CBG has an unmodified glutarimide moiety, and is also one of three major metabolites in
human plasma44. As CBG was also reported to possess higher TNF-α production-inhibitory activity (80%) than thalidomide (32%)
at concentrations of 3 µM44-45, it is of great pharmacological interest, but its mode of interaction remained elusive so far46. During
the course of this study, we obtained a crystal structure of CBG in complex with a humanized mutant form of MsCI4 (Figures 3, 4).
This structure was the result of a co-crystallization trial of this mutant with thalidomide, which aimed at the characterization of the
mutant protein. However, the mutant selectively bound CGB that was presumably present in traces in our crystallization setup with
thalidomide. This mutant has a number of non-conserved residues in the direct vicinity of the thalidomide binding pocket mutated
to the residues in the human protein, including the substitution F56H. While this residue is not directly involved in classical IMiD
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binding in MsCI4 or the human protein, it plays an important role in the binding of CBG: Together with N50, it coordinates one
oxygen of the carboxyl group in the CBG protruding moiety; the other oxygen of this carboxyl coordinates the conserved water
molecule bound to W99. However, in contrast to the hydrolysis products of the succinimide-based compounds 4a, 4b and 5a, the
amide moiety resulting from ring-opening is not found to be involved in defined hydrogen bonding – although superposing
approximately with the phthaloyl moiety of thalidomide, it does not form the canonical hydrogen bond with N50, as the latter is
engaged in the hydrogen bond to the carboxyl group.
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Figure 4. The binding mode of the initial compounds and their hydrolysis products inside the binding pocket. Thalidomide, 4a and
5a are shown with their respective hydrolysis products. Ring opening of the phtaloyl moiety that leads to the observed hydrolysis
product is indicated in red in the chemical drawings. 5b was exclusively found in non-hydrolyzed form, whereas 4b was
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exclusively found as a hydrolysis product. PDB codes are 4V2Y (thalidomide), 6R0Q (CBG), 6R0S (4a), 6R0U (5a), 6R11 (5b),
6R0V (4b).
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Rational design of novel succinimide effectors guided by hydrolyzed metabolites
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In a next step, we aimed to exploit our knowledge on the binding mode of hydrolyzed metabolites for the design of novel effectors.
The fact that the hydrolysis products were selected against their parent compounds in several co-crystallization or soaking
experiments suggested that they pose binders of similar, if not superior affinity. Comparing the binding modes of CBG and
hydrolyzed 4a, 4b and 5a, we further hypothesized that the difference seen for the hydrogen bonding of their amide linkers should
yield increased affinity for succinimide-based effectors. Consequently, we used this amide linker to connect different functional
groups as protruding moieties to succinimide as the binding moiety. To this aim, we prepared derivatives 7a-7h as shown in
Scheme 2, by treating imide 3a with the corresponding acyl chlorides 6a-6h and N, N-diisopropylethylamine (DIPEA) in THF.
o
Scheme 2. Reagents and conditions: (a) CDI, 4-DMAP, THF, reflux, 48h; (b) TFA, RT, 30 min; (c) 6a-h, DIPEA, THF, 0 C to
reflux, 2 h
All derivatives of this second panel were tested in the FRET assay. Only for compound 7e, with a 3,5-dinitrobenzol group, the
affinity was decreased under the detectable levels of the FRET assay. For the compounds with an isobutylene (7a), benzyl (7b) or
chlorobenzothiophene group (7g) as the protruding moiety, binding was well detectable but could not be quantified (Ki > 40 µM).
Better binding was observed for compounds with a styryl (7f) and dichloro-benzothiophene (7h) group, both showing Ki values of
20 µM (Figure 5A). Finally, the highest affinities were achieved with a 2,4,6-trichlorobenzol moiety (7c, Ki = 9 µM) and benzyloxy
group (7d, Ki = 4 µM), rendering 7d the highest-affinity binder in this study, with a Ki value comparable to unmodified
succinimide36 (Figure 5A).
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Figure 5. Compounds inspired by hydrolysis products of thalidomide analogs and their affinity data. (A) Second panel of
compounds, based on the 3-amidosuccinimide scaffold. (B) Third panel of compounds, inspired by 7d. * 20a could not be purified
to satisfactory levels for affinity testing.
We continued with characterizing the binding modes of compounds 7a-c and 7f via crystallography. For the best binder 7d we
performed a co-crystallization screen, which yielded a new crystal form diffracting to 1.1 Å resolution; the other compounds were
successfully evaluated in soaking experiments. All compounds revealed the expected binding mode as observed for the hydrolyzed
metabolites of 4a, 4b and 5a, with the succinimide moiety forming the canonical interaction within the binding pocket, and the
amide linker forming the hydrogen bond with N50. Besides these hydrogen bonds of the binding and linking moiety, no defined
interactions with the protein were found for the protruding moieties of any of the five compounds, including the best binder 7d.
Figure 6 shows a superposition of the compounds of this panel and their conserved binding mode in the aromatic cage.
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Figure 6. Binding modes of compounds from the second and third panel. (A) Superposition of all compounds bound to MsCI4. (B)
Detailed side and top view of bound effectors based on the 3-amidosuccinimide scaffold: 7a (yellow, PDB 6R1X), 7b (sand, PDB
6R12), 7c (pink, PDB 6R1K), 7d (green, PDB 6R1D), 7f (blue, PDB 6R13) and the water soluble 11a (cyan, PDB 6R18) and 12a
(light blue, PDB 6R1C), indicating interactions with N50. (C) Side and top view of compounds based on 3-amidoglutarimide, 16b
(brown, PDB 6R1W) and 20b (dark green, PDB 6R1A). Although the depicted instances for this scaffold do not show the
interaction with N50, this interaction was observed in one other instance for 16b.
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Based on these data, we hypothesized that the planar benzene connected via an oxygen as in 7d is favorable for the affinity.
Consequently, we designed a third panel of derivatives carrying this feature based on succinimide and glutarimide (Figure 5B). In
this panel, in addition to affinity improvement, we also sought to increase the solubility in water via additional polar or charged
groups. As described in Scheme 3, reaction of the intermediates 8, 13 and 17 with phosgene solution (15 wt% in toluene) in THF
yielded to the corresponding chloroformates 9, 14 and 18. Derivatives 11a, 11b, 16a, 16b, 20a and 20b were prepared by a
coupling reaction between the imides 3a or 3b and the aforementioned chloroformates 9, 14 and 18 using DIPEA in THF followed
by a deprotection using TFA. Treatment of the derivatives 11a and 11b with succinic anhydride and triethylamine (Et3N) in DMF
produced the molecules 12a and 12b, respectively.
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Scheme 3. Reagents and conditions: (a) Phosgene solution 15 wt. % in toluene, THF, 0 oC to RT, 20 h; (b) 3a or 3b, DIPEA, THF,
0 oC to reflux, 20 h; (c) TFA, DCM, 0 oC to RT, 2 h; (d) Et3N, DMF, 0 oC to RT, 20 h. Note that 13 and thereby also 14-16 are in
cis conformation.
Indeed, the final compounds with terminal amino groups were water-soluble (11a, 11b, 16a, 16b), while 12a and 12b, with a
terminal succinyl group, were highly soluble in bicarbonate buffer (> 200 mM). The compounds were subsequently tested in the
FRET assay, apart from 20a, which still contained impurities (see Methods). The assay indicated that all derivatives retained high
affinity for CRBN independent of binding moiety and planarity of the substituent; a clear preference for either binding moiety was
not recognizable.
In soaking experiments, we obtained crystal structures with the 5-ring members 11a, 12a, 20a, and the 6-ring members 16b and
20b, all forming the canonical interactions within the aromatic cage. As expected, the 5-ring effectors form the interaction of the
amide linker with N50, which is also observed in one instance for the 6-ring effector 16b. Further interactions of the protruding
moieties are not observed for any of the compounds. Consequently, the prolonged extensions are less resolved in the electron
density map, which is especially evident for 12a (Figure 7). As these prolonged compounds still retain high affinity, this confirms
that the amidosuccinimide scaffold can serve as universal CRBN-binding moiety allowing great chemical variability on the
protruding moiety.
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Figure 7. FO – FC maps of bound compounds shown in Figure 6. All compounds are clearly defined by their electron density maps
with the exception of the prolonged extension in 12a. Crystallographic structures were refined to resolutions between 1.1 Å and 1.8
Å, the maps are contoured at 2σ. PDB accession codes are 6R1X (7a), 6R12 (7b), 6R1K (7c), 6R1D (7d), 6R13 (7f), 6R18 (11a),
6R1C (12a), 6R1W (16b), 6R19 (20a), 6R1A (20b).
Degradation of neo-substrates
After assessing the biophysical and structural parameters of our designs, we tested their potential for the degradation of neo-
substrates in the MM-derived human cell line OPM-2. To this end, we have selected the established neo-substrates IKZF3, which is
targeted via a variety of IMiDs20, 47-49, and CK1α, which is so far only targeted via lenalidomide21, as two complementary targets.
We therefore treated OPM-2 cells for 24 h with the different compounds and assayed for the endogenous levels of both neo-
substrates; for comparison, the classical IMiDs thalidomide, lenalidomide and pomalidomide were included in the test set. As
anticipated, the results for the two neo-substrates were very different. None of the marketed IMiDs apart from lenalidomide was
able to reduce the levels of CK1α significantly21, but also none of our compounds showed any effect on CK1α. This indicates that
our compounds could neither supersede nor sufficiently mimic the interface for CK1α recruitment formed by lenalidomide,
substantiating the notion of a very narrow specificity window for this substrate21.
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However, the situation was completely different for IKZF3. Here, in addition to all classical IMiDs, multiple of our designs were
successful: Significant effects were observed for 5a, 7d and 7f, which are all compounds with a rather compact structure from our
first and second panel (Figure 8). The only successful compound from the first, phthaloyl-based panel, 5a, represents a direct
succinimide analog of pomalidomide. It reduced IKZF3 levels by almost 40%. Comparison of its structural binding mode to that of
thalidomide or pomalidomide already suggested that it may be a possible functional substitute for pomalidomide. The fact that the
other compounds of that panel did not show similar effects indicate that substitutions larger than an amino group in the R2 position
of the phthaloyl moiety, as for 4a and 4c, or any substitution in R1, as for 4b, 4d, and 5b, abolish IKZF3 recruitment.
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Figure 8. Compound-mediated IKFZ3 degradation in OPM-2 cells. (A) Immunoblot analysis of IKZF3 levels after treatment with
compounds 7d, 5a and 7f (100 μM) for 24 h, compared to DMSO (control) and lenalidomide (20 μM) (n=3). Lenalidomide at
100 μM reduced IKFZ3 levels effectively by 100% (not shown). (B) Averaged IKZF3 levels from the three independent
experiments, normalized against total protein loading control (Figure S2). The significance of the data was tested comparing IKZF3
levels in the presence of the compounds and DMSO (p > 0.05).
Most interesting is the analysis of the successful compounds of the second panel, 7d and 7f, which are not derived from classical
IMiDs, but from the hydrolysis products of the first panel. 7d, which is also the highest-affinity binder in this study, reduced IKZF3
levels by about 20%. Moreover, the analogous 7f, which is a weaker binder that only differs from 7d in one heavy atom in the
linker, reduced IKZF3 levels by even 40%. Both compounds are more flexible and elongated than 5a, so it appears impossible that
their protruding moieties adopt a conformation mimicking the classical IMiDs at the interface between CRBN and IKZF3. Their
terminal benzyl groups inevitably project further away from the thalidomide binding pocket, requiring another mode of interaction
with the zinc finger motif than characterized for classical IMiDs26. This interaction mode is also very sensitive to chemical changes:
Shortening of the linker by only one atom, as for 7b, further modifications on the benzyl group, as for 11a or 12a, or any other
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variant we designed in the second or third panel abolished IKZF3 recruitment, which is indicative for a highly specific interaction.
Therefore, 7d and 7f are the first representatives of a novel type of CRBN effectors with a recognition mode that is clearly distinct
from that of the classical thalidomide-based IMiDs.
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The development of CRBN effectors is a rapidly growing field, and novel IMiDs and PROTACS are reported frequently. To date,
essentially all of these compounds are based on the classical thalidomide scaffold, which significantly restrains the chemical space
available for the recruitment of neo-substrates. Here, following on from our previous characterization of CRBN-binding moieties,
we have probed the chemical space for the linking and protruding moiety, taking advantage of the structural binding modes of
hydrolyzed metabolites. Although not in the focus of this work, the apparent specificity of CRBN for these hydrolysis products,
especially for the major thalidomide metabolite CBG, may inspire further research towards the understanding of their
pharmacological relevance.
As a consensus from the binding modes of our initial designs and their hydrolysis products, we derived an amidosuccinimide
scaffold as a minimal binding and linking moiety. 3-Amidosuccinimide can be used to mount almost arbitrary chemistry as the
protruding moiety, while retaining affinities to CRBN in the range of classical IMiDs, rendering it an attractive CRBN-binding
moiety for future PROTACS.
However, the most relevant aspect of our study concerns the versatility of the 3-amidosuccinimide scaffold for the design of IMiD-
like CRBN effectors. As a minimal binding and linking moiety, it does not restrain the chemical space of protruding moieties,
allowing for the design of substrate-recruiting motifs that cannot be realized on a classical IMiD scaffold. In our attempt to probe
the effect of different protruding moieties mounted on the amidosuccinimide scaffold, two out of twelve compounds were able to
recruit the neo-substrate IKZF3 without further optimization. Although we do not have structural insight into the IKZF3-
recognition mode of these effectors, it is clearly incompatible with that of the classical IMiDs, which points at a large unexplored
chemical space for the recruitment of new therapeutic targets. For the ongoing characterization of the proteome druggable via the
IMiD approach, novel CRBN effectors are needed both for probing the space of neo-substrates, as well as for future
pharmacological exploitation. With this work, we contribute first steps towards the rational design of a post-thalidomide generation
of such effectors, towards unlocking the full potential of the IMiD approach.
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Experimental Section
Chemistry
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General Procedures
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Room temperature refers to 22 °C. Reagents and anhydrous solvents were transferred via oven-dried syringe or cannula. Flasks
were flame-dried under vacuum and cooled under a constant stream of argon. Anhydrous solvents (Toluene, Dioxane, DMF) were
purchased from Sigma-Aldrich (anhydrous over molecular sieves). THF was dried over potassium. All other chemicals were
purchased from ABCR, Acros, Alfa Aesar, Fluorochem, Merck, Sigma-Aldrich, and TCI Europe at highest commercially available
purity and used without further purification. Compound 2b and 3b were prepared according to the procedures reported by Capitosi
et al.50 Compounds 8, 13 and 17 were prepared according to the procedures previously reported51-54. Thin layer chromatography
(TLC) was performed on Merck silica gel 60 F 254 TLC aluminium sheets and visualized by ultraviolet light (254 nm) and/or with
ceric ammonium molybdate, potassium permanganate or ninhydrine staining solution. Flash column chromatography was
performed on Acros silica gel 35-70, 60 Å, using a forced flow of eluent (method of Still). Yields refer to chromatographically
purified and spectroscopically pure compounds. NMR spectra were recorded on a Varian Mercury plus 400 (operating at 400 MHz
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for H and 100 MHz for 13C acquisitions), and a Varian Mercury plus 300 (operating at 300 MHz for H and 75 MHz for 13C
acquisitions). Chemical shifts are reported in ppm with the solvent resonance as the internal standard (d1-chloroform: 7.26 (1H
NMR), 77.16 (13C NMR); d6-dimethylsulfoxide: 2.50 (1H NMR), 39.52 (13C NMR); (d4-methanol: 3.31 (1H NMR), 49.00 (13C
NMR); (d6-acetone: 2.05 (1H NMR), 29.84, 206.26 (13C NMR)). Coupling constants J are reported in Hertz (Hz). Multiplicities are
indicated by s = singlet, d = doublet, t = triplet, q = quartet, sep = septet, dd = doublet of doublet, dt = doublet of triplet, m =
multiplet, br = broad resonance. High-resolution mass spectra were obtained on a Bruker Daltonics ESI-FT-ICR-MS APEX II. IR
spectra were measured on an ATI/MATTSON Genesis FT-IR as thin film (in CCl4) or KBr-disk. Absorbance frequencies are
reported in reciprocal centimetres (cm−1). Melting points were measured on a Boetius-micro hot stage and are uncorrected. The
purity of compounds was analysed by detecting UV absorbance at 254 nm using a Poroshell 120 EC-C18 column on a 1260 Infinity
II system (Agilent Technologies, Inc.) (Figure S1). All compounds showed > 95% purity with the exception of 7a (87.6 %), 7b
(91.5 %), 7c (88.2 %). 12a (89.4 %), 12b (88.2 %) and 20a (<50 %).
General Procedure A
Preparation of the compounds 4a-d. To a solution of 3a or 3b (1.0 equiv.) in anhydrous THF (4.60 ml/mmol), 4-nitrophthalic
anhydride (1.5 equiv.) in anhydrous THF (0.5 ml/mmol) was added. CDI (1.7 equiv.), Et3N (2.9 equiv.) and catalytic amounts of 4-
DMAP were added and the reaction mixture was heated to reflux for 5 h. The solvent was evaporated and the resulting crude oil
was purified by column chromatography to give 4a-d.
General Procedure B
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Reduction of the nitro group. To a solution of 4a or 4b (1.0 equiv.) in EtOAc (100 ml/mmol) 10 wt. % Pd/C (27 mol. %) was
added. The flask was degassed and let to stir under hydrogen atmosphere for 20 h at RT. After completion of the reaction (TLC
control) the mixture was filtered over Celite and washed with copious amounts of acetone. Afterwards the solvent was evaporated
and 5a or 5b respectively were obtained.
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General Procedure C
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Preparation of the compounds 7a-h (amide bond formation). To a solution of the TFA-salt 3a (1.0 equiv.) in THF (5 ml/mmol)
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DIPEA (2.0 equiv.) was added. The mixture was cooled down to 0 C, the acyl chloride 6a-h (1.0 equiv.) was added and the
reaction mixture was left to stir under reflux for 2 h. Upon completion, the reaction was diluted with EtOAc and washed with 3 M
HCl and brine. The organic layer was dried over Na2SO4 ant the solvent was evaporated in vacuo. The resulting crude product was
purified by flash column chromatography to give 7a-h.
General procedure D
Phosgenation, carbamate formation, Boc deprotection. To a 15% wt solution of COCl2 in toluene (1.3 equiv.) was added dry THF
(0.58 ml/mmol) under argon. The solution was cooled to 0 oC and a solution of 8, 13 or 17 (1.0 equiv.) in dry THF (0.6 ml/mmol)
o
was added over 10 min. The reaction mixture was stirred at 0 C for 15 min and then warmed up to room temperature and stirred
for two more hours. After completion of the reaction, the volatiles were completely evaporated under reduced pressure to give the
chloroformates 9, 14 or 18 respectively, which were used to the next step without further purification. To a stirred suspension of the
o
TFA-salt 3a or 3b (1.03 equiv.) in dry THF (2 ml/mmol), DIPEA (0.35 ml/mmol) was added at 0 C. Then, a solution of the
chloroformate 9, 14 or 18 (1.0 equiv.) in dry THF (0.35 ml/mmol) was added gradually to the suspension. The reaction was warmed
up to room temperature, stirred for 30 min at RT and then refluxed overnight. After completion of the reaction, the solvent was
evaporated and the crude product was purified by column chromatography giving the carbamates 10a-b, 15a-b or 19 respectively.
To a solution of 10a-b, 15a-b or 19 (1.0 equiv.) in DCM (4.5 ml/mmol), TFA (30.0 equiv) was added at 0 oC. The reaction solution
was warmed up to room temperature and stirred for 2 h. After completion of the reaction, the volatiles were completely evaporated
to give 11a-b, 16a-b or 20 respectively.
General procedure E
Preparation of 12a-b (amide bond formation). Derivatives 11a-b (1.0 equiv.) was dissolved in DMF (2.8 ml/mmol) and Et3N (2.2
equiv.) was added at 0°C. After 20 min of stirring, succinic anhydride (1.0 equiv.) was added and the reaction mixture was stirred at
RT overnight. After the end of the reaction, the solvent was evaporated the corresponding acid 12a-b was afforded via flash column
chromatography.
Tert-butyl (S)-(2,5-dioxopyrrolidin-3-yl)carbamate (2a)
A mixture of N-Boc-L-asparagin (2 g, 8.61 mmol, 1 equiv.), CDI (1.39 g, 8.61 mmol, 1 equiv.) and catalytic amounts 4-DMAP in
anhydrous THF (21.5 mL) was stirred and heated to reflux for 48 h. After the end of the reaction, the solvent was evaporated and
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the crude product was purified by column chromatography (n-hexane/ethyl acetate, 1:1 v/v) to give 2a (1.54 g, 6.75 mmol, 78%) as
a colorless solid. TLC (n-hexane/EtOAc, 3:7 v/v): Rf = 0.48; m. p.: 160-162 oC; IR (KBr): ṽmax 3376.75, 3361.32, 1709.59, 1521.56,
1171.54 cm-1; 1H NMR (400 MHz, DMSO-d6) δ 11.18 (s, 1H), 7.42 (d, J = 8.1 Hz, 1H), 4.33 – 4.24 (m, 1H), 2.85 (dd, J = 17.5, 9.4
Hz, 1H), 2.44 (dd, J = 17.5, 5.7 Hz, 1H), 1.37 (s, 9H); 13C NMR (101 MHz, DMSO) δ 178.2, 176.5, 155.2, 78.8, 50.3, 36.4, 28.2;
HRMS ESI (m/z): [M+Na]+ Calcd for C9H14N2O4Na+: 237.08513, found: 237.08513.
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2,5-Dioxopyrrolidin-3-aminium 2,2,2-trifluoroacetate (3a)
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Compound 3a (1.54 g, 6.75 mmol, 1 equiv.) was dissolved in TFA (7.8 ml, 101.25 mmol, 15 equiv.) and stirred for 30 min. The
excess of the acid was removed in vacuo and the resulting product was dried under vacuum to give 3a (1.54 g, 6.75 mmol, quant) as
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an off-white solid. TLC (EtOAc): Rf = 0.0; m. p.: 109-114 C; IR (KBr): ṽmax 3446.17, 1665.23, 1617.02, 1593.88, 1207,22,
1181.19, 1149.37, 1137.8, 723.175 cm-1; 1H NMR (400 MHz, DMSO-d6) δ 11.71 (s, 1H), 8.61 (s, 3H), 4.39 – 4.26 (m, 1H), 2.95 (d,
J = 26.9 Hz, 1H), 2.61 (dd, J = 17.7, 5.5 Hz, 1H); 13C NMR (101 MHz, DMSO-d6)δ 175.29, 175.14, 158.69 (q, J = 31.8 Hz),
117.08 (q, J = 298.4 Hz), 48.53, 34.73; 19F NMR (377 MHz, DMSO-d6) δ -73.84; HRMS ESI (m/z): [M+H]+ Calcd for
+
C4H7N2O2 : 115.05075, found: 115.05142.
2-(2,5-Dioxopyrrolidin-3-yl)-4-nitroisoindoline-1,3-dione (4a)
Compound 4a was synthesized according to general procedure A using 3b (0.50 g, 2.19 mmol), 3-nitrophthalic anhydride (655.7
mg, 3.29 mmol), CDI (603.2 mg, 3.72 mmol), Et3N (0.89 ml, 6.35 mmol) and catalytic amounts of 4-DMAP. After purification via
flash column chromatography (n-hexane/EtOAc, 1:1 v/v) compound 4a (412 mg, 1.43 mmol. 65%) was isolated as a colorless
solid. TLC (n-hexane/EtOAc, 1:3 v/v): RF = 0.72; m. p.: 129 - 131 oC; IR (KBr): ṽmax 3442.31, 1722.12, 1541.81, 1396,21 cm-1; 1H
NMR (400 MHz, acetone-d6) δ 10.55 (br s, 1H), 8.35 – 8.28 (m, 1H), 8.28 – 8.21 (m, 1H), 8.22 – 8.15 (m, 1H), 5.47 (dd, J = 9.7,
5.9 Hz, 1H), 3.21 (dd, J = 18.0, 9.7 Hz, 1H), 3.05 (dd, J = 18.0, 5.9 Hz, 1H); 13C NMR (101 MHz, acetone-d6) δ 175.2, 174.9,
165.9, 163.3, 146.0, 137.4, 134.6, 129.7, 128.0, 124.1, 49.5, 35.2; HRMS ESI (m/z): [M+Na]+ Calcd for C12H7N3O6Na+:
312.02325, found: 312.04880
2-(2,5-Dioxopyrrolidin-3-yl)-5-nitroisoindoline-1,3-dione (4b)
Compound 4b was synthesized according to general procedure A using 3a (0.5 g, 2.19 mmol), 4-nitrophthalic anhydride (656 mg,
3.29 mmol), CDI (603 mg, 3.72 mmol), Et3N (0.89 ml, 6.35 mmol) and catalytic amounts of 4-DMAP. After purification via flash
column chromatography (n-hexane/EtOAc, 1:1 v/v) compound 4b (483 mg, 1.67 mmol. 76%) was isolated as a colorless solid.
TLC (n-hexane/EtOAc, 1:3 v/v): RF = 0.61; m. p.: 203-204 oC; IR (KBr): ṽmax 3489.56, 1787.69, 1538.92, 1399.1, 1349.93, 722.211
cm-1; 1H NMR (400 MHz, acetone-d6) δ 10.55 (br s, 1H), 8.75 (dd, J = 8.2, 2.0 Hz, 1H), 8.62 (dd, J = 2.0, 0.5 Hz, 1H), 8.21 (dd, J =
8.2, 0.5 Hz, 1H), 5.50 (dd, J = 9.7, 5.9 Hz, 1H), 3.23 (dd, J = 18.0, 9.7 Hz, 1H), 3.06 (dd, J = 18.0, 5.9 Hz, 1H);13C NMR (101
MHz, acetone-d6) δ 175.2, 174.9, 166.3, 166.0, 153.1, 137.2, 134.1, 130.8, 125.8, 119.3, 49.5, 35.3; HRMS ESI (m/z):
[M+Na+MeOH]+ Calcd for C13H11N3O7Na+: 344.04947, found: 344.04985.
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2-(2,6-Dioxopiperidin-3-yl)-4-nitroisoindoline-1,3-dione (4c)
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Compound 4c was synthesized according to general procedure A using 3b (3.11 g, 12.9 mmol), 3-nitrophthalic anhydride (3.73 g,
19.3 mmol), CDI (3.55 g, 21.9 mmol) Et3N (5.2 ml, 37.3 mmol) and catalytic amounts of 4-DMAP. After purification via flash
column chromatography (n-hexane/EtOAc, 1:1-1:4 v/v) compound 4c (3.48 g, 11.5 mmol, 89%) was isolated as a colorless solid.
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TLC (n-hexane/EtOAc, 3:7 v/v): RF = 0.40; H NMR (400 MHz, DMSO-d6) δ 11.17 (s, 1H), 8.34 (dd, J = 8.1, 0.7 Hz, 1H), 8.23
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(dd, J = 7.5, 0.7 Hz, 1H), 8.15 – 8.08 (m, 1H), 5.19 (dd, J = 12.9, 5.4 Hz, 1H), 2.88 (ddd, J = 17.3, 13.9, 5.4 Hz, 1H), 2.61 (dt, J =
17.0, 2.7 Hz, 1H), 2.56 – 2.44 (m, 1H), 2.08 (dtd, J = 12.9, 5.3, 2.1 Hz, 1H).; 13C NMR (101 MHz, DMSO) δ 172.8, 169.6, 165.3,
162.6, 144.5, 136.9, 133.1, 129.0, 127.4, 122.6, 49.5, 30.9, 21.8; HRMS ESI (m/z): [M+Na+MeOH]+ Calcd for C14H13N3O7Na+:
358.06512, found: 358.06703; Spectral data were consistent with the literature55.
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2-(2,6-Dioxopiperidin-3-yl)-5-nitroisoindoline-1,3-dione (4d)
Compound 4d was synthesized according to general procedure A using 3b (4.17 g, 17.2 mmol), 4-nitrophthalic anhydride (4.99 g,
25.9 mmol), CDI (4.73 g, 29.1 mmol) Et3N (6.97 ml, 50.00 mmol) and catalytic amounts of 4-DMAP. After purification via flash
column chromatography (n-hexane/EtOAc, 1:1-1:4 v/v) compound 4d (2.35 g, 7.8 mmol, 45 %) was isolated as a colorless solid.
TLC (n-Hexane/EtOAc, 3:7 v/v): Rf = 0.50; 1H NMR (400 MHz, DMSO-d6) δ 11.18 (s, 1H), 8.67 (dd, J = 8.2, 2.0 Hz, 1H), 8.56 (d,
J = 2.0 Hz, 1H), 8.19 (d, J = 8.2 Hz, 1H), 5.23 (dd, J = 12.9, 5.4 Hz, 1H), 2.90 (ddd, J = 17.1, 13.9, 5.4 Hz, 1H), 2.63 (ddd, J = 17.1,
4.3, 2.3 Hz, 1H), 2.58 – 2.47 (m, 1H), 2.10 (dtd, J = 12.9, 5.2, 2.2 Hz, 1H); 13C NMR (101 MHz, DMSO) δ 172.82, 169.61, 165.63,
165.36, 151.79, 135.80, 132.60, 130.17, 125.09, 118.46, 49.57, 30.95, 21.91; HRMS ESI (m/z): [M+Na+MeOH]+ Calcd for
C14H13N3O7Na+: 358.06512, found: 358.06642; Spectral data were consistent with the literature50.
4-Amino-2-(2,5-dioxopyrrolidin-3-yl)isoindoline-1,3-dione (5a)
Compound 5a was synthesized according to general procedure B using 4a (0.30 g, 1.04 mmol), and 10 wt. % Pd/C (0.30 g, 0.28
mmol). Compound 5a (0.25 g, 0.95 mmol, 92%) as a yellow-green solid. TLC (n-hexane/EtOAc, 1:3 v/v): Rf = 0.61; m. p.: 292 -
295 oC; IR (KBr): ṽmax 3474.13, 1727.91, 1702.84, 1632.45 cm-1; 1H NMR (400 MHz, acetone-d6) δ 10.45 (br s, 1H), 7.50 (dd, J =
8.4, 7.1 Hz, 1H), 7.07 (dd, J = 10.7, 7.7 Hz, 2H), 6.17 (s, 1H), 5.34 (dd, J = 9.7, 5.9 Hz, 1H), 3.18 (dd, J = 17.9, 9.7 Hz, 1H), 3.02
(dd, J = 17.9, 5.9 Hz, 1H); 13C NMR (101 MHz, acetone-d6) δ 175.7, 175.5, 170.9, 170.9, 147.8, 136.4, 133.4, 122.5, 122.4, 112.4,
48.7, 35.6; HRMS ESI (m/z): [M+Na]+ Calcd for C12H9N3O4Na+: 282.04908, found: 282.04853.
5-Amino-2-(2,5-dioxopyrrolidin-3-yl)isoindoline-1,3-dione (5b)
Compound 5b was synthesized according to general procedure B using 4a (0.30 g, 1.0 mmol), and 10 wt. % Pd/C (0.30 g, 0.3
mmol). Compound 5b (0.26 g, 1.0 mmol, 97%) as a yellow-green solid. TLC (n-hexane/EtOAc, 1:3 v/v): RF = 0.68; m. p.: 259 -
260 oC; IR (KBr): ṽmax 3473.17, 3354.57, 1701.87, 1628.59, 1613.16, 1405.85 cm-1; 1H NMR (300 MHz, acetone-d6) δ 10.40 (br s,
1H), 7.54 (d, J = 8.2 Hz, 1H), 7.07 (d, J = 1.8 Hz, 1H), 6.97 (dd, J = 8.2, 2.1 Hz, 1H), 6.00 (br s, 2H), 5.32 (dd, J = 9.7, 6.0 Hz,
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1H), 3.16 (dd, J = 17.9, 9.7 Hz, 1H), 2.98 (dd, J = 17.9, 6.0 Hz, 1H) 13C NMR (75 MHz, acetone-d6) δ 175.8, 175.6, 155.9, 135.6,
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N-(2,5-dioxopyrrolidin-3-yl)-3-methylbut-2-enamide (7a)
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Compound 7a was synthesized according to general procedure C using 3a (0.10 g, 0.44 mmol), DIPEA (0.15 ml, 0.88 mmol) and 3,
3-dimethylacryloyl chloride 6a (51.9 mg, 0.44 mmol). After purification via flash column chromatography (n-hexane/EtOAc, 5:5
v/v) compound 7a (56.8 mg, 0.29 mmol, 66%) was isolated as a colorless solid. TLC (n-hexane/EtOAc, 3:7 v/v): Rf = 0.18; m. p.:
181-188 oC; IR (KBr): ṽmax 3354.57, 1729.83, 1715.37, 1624.73, 1530.24, 1363.43, 1261.22, 1193.72, 1177.33 cm-1; 1H NMR (300
MHz, acetone-d6) δ 10.04 (s, 1H), 7.63 (s, 1H), 5.72 (s, 1H), 4.54 (ddd, J = 9.3, 7.5, 5.8 Hz, 1H), 2.97 (dd, J = 17.6, 9.3 Hz, 1H),
2.71 (dd, J = 17.6, 5.8 Hz, 1H), 2.11 (d, J = 1.2 Hz, 3H), 1.82 (d, J = 1.2 Hz, 3H); 13C NMR (75 MHz, acetone-d6) δ 176.0, 172.1,
167.3, 152.3, 118.6, 51.1, 37.2, 27.1, 19.7; HRMS ESI (m/z): [M+Na]+ Calcd for C9H12N2O3Na+: 219.07456, found: 219.07401.
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N-(2,5-dioxopyrrolidin-3-yl)-2-phenylacetamide (7b)
Compound 7b was synthesized according to general procedure C using 3a (0.10 g, 0.44 mmol), DIPEA (0.15 ml, 0.876 mmol) and
phenylacetyl chloride 6b (67.7 mg, 0.44 mmol). After purification via flash column chromatography (n-hexane/EtOAc, 1:1 v/v)
compound 7b (75.9 mg, 0.33 mmol, 75%) was isolated as a colorless solid. TLC (n-hexane/EtOAc, 3:7 v/v): Rf = 0.15; m. p.: 181-
184 oC; IR (KBr): ṽmax 3502.1, 3259.11, 1731.76, 1715.35, 1654.62, 1542.77, 1358.6, 1196.61, 1172.51 cm-1; 1H NMR (400 MHz,
acetone-d6) δ 10.02 (br s, 1H), 7.81 (br s, 1H), 7.35 – 7.18 (m, 5H), 4.57 (ddd, J = 9.3, 7.5, 5.8 Hz, 1H), 3.56 (s, 2H), 2.96 (dd, J =
17.6, 9.4 Hz, 1H), 2.66 (dd, J = 17.6, 5.8 Hz, 1H); 13C NMR (101 MHz, acetone-d6) δ 176.3, 175.0, 170.7, 135.7, 129.2, 128.3,
126.6, 50.4, 42.2, 36.2; HRMS ESI (m/z): [M+Na]+ Calcd for C12H12N3O6Na+: 255.07456, found: 255.07401.
2,4,6-Trichloro-N-(2,5-dioxopyrrolidin-3-yl)benzamide (7c)
Compound 7c was synthesized according to general procedure C using 3a (0.10 g, 0.438 mmol), DIPEA (0.15 ml, 0.88 mmol) and
2,4,6-trichlorobenzoyl chloride 6c (106.8 mg, 0.44 mmol). After purification via flash column chromatography (n-hexane/EtOAc,
5:5 v/v) compound 7c (81.7 mg, 0.25 mmol, 58%) was isolated as a colorless solid. TLC (n-hexane/EtOAc, 3:7 v/v): RF = 0.47; m.
p.: 210-212 oC; IR (KBr): ṽmax 3450.03, 3249.47, 1717.3, 1640.16, 1582.31, 1547.59 cm-1; 1H NMR (400 MHz, DMSO-d6) δ 11.35
(s, 1H), 9.37 (d, J = 7.5 Hz, 1H), 7.75 (s, 1H), 4.59 (ddd, J = 9.3, 7.6, 5.3 Hz, 1H), 3.01 (dd, J = 17.6, 9.4 Hz, 1H), 2.59 (dd, J =
17.6, 5.3 Hz, 1H); 13C NMR (101 MHz, DMSO) δ 176.5, 176.3, 163.3, 134.9, 134.3, 132.3, 128.1, 50.0, 35.9; HRMS ESI (m/z):
[M+Na]+ Calcd for C11H7Cl3N2O3Na+: 342.94199, found: 342.94145.
Benzyl (2,5-dioxopyrrolidin-3-yl)carbamate (7d)
Compound 7d was synthesized according to general procedure C using 3a (0.10 g, 0.44 mmol), DIPEA (0.15 ml, 0.876 mmol) and
benzyl chloroformate 6d (74.7 mg, 0.44 mmol). After purification via flash column chromatography (n-hexane/EtOAc, 5:5 v/v)
compound 7d (105.9 mg, 0.43 mmol, 98%) was isolated as a colorless solid. TLC (n-hexane/EtOAc, 3:7 v/v): Rf = 0.44; m. p.: 93-
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1H), 7.41 – 7.27 (m, 5H), 7.08 (br s, 1H), 5.10 (s, 2H), 4.63 – 4.53 (m, 1H), 3.05 (dd, J = 17.6, 9.4 Hz, 1H), 2.74 (dd, J = 17.6, 5.5
Hz, 1H); 13C NMR (101 MHz, acetone-d6) δ 177.5, 175.8, 156.9, 137.8, 129.2, 128.7, 67.1, 52.0, 37.1; HRMS ESI (m/z): [M+Na]+
Calcd for C12H12N2O4Na+: 271.06948, found: 271.06893.
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N-(2,5-dioxopyrrolidin-3-yl)-3,5-dinitrobenzamide (7e)
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Compound 7e was synthesized according to general procedure C using 3a (0.1 g, 0.44 mmol), DIPEA (0.15 ml, 0.88 mmol) and
3,5-dinitrobenzoyl chloride 6e (0.10 g, 0.44 mmol). After purification via flash column chromatography (n-hexane/EtOAc, 5:5 v/v)
compound 7e (0.10 g, 0.34 mmol, 76%) was isolated as a colorless solid. TLC (n-hexane/EtOAc, 3:7 v/v): Rf = 0.28; m. p.: 238-239
oC; IR (KBr): ṽmax 3447.16, 3389.28, 1717.3, 1659.45, 1540.85, 1348, 1188.9 cm-1; 1H NMR (400 MHz, acetone-d6) δ 10.24 (br s,
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1H), 9.15 (d, J = 7.1 Hz, 1H), 9.12 – 9.06 (m, 3H), 4.99 – 4.91 (m, 1H), 3.20 – 3.11 (m, 1H), 2.93 (dd, J = 17.8, 5.8 Hz, 1H); 13
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NMR (101 MHz, acetone) δ 176.7, 175.7, 163.7, 149.7, 137.6, 128.3, 122.1, 51.8, 36.8; HRMS ESI (m/z): [M+Na]+ Calcd for
C11H8N4O7Na+: 331.02907, found: 331.02842.
N-(2,5-dioxopyrrolidin-3-yl)cinnamamide (7f)
Compound 7f was synthesized according to general procedure C using 3a (0.10 g, 0.44 mmol), DIPEA (0.15 ml, 0.88 mmol) and
cinnamoyl chloride 6f (73.0 mg, 0.44 mmol). After purification via flash column chromatography (n-hexane/EtOAc, 1:1 v/v)
compound 7f (102 mg, 0.42 mmol, 96%) was isolated as a colorless solid. TLC (n-hexane/EtOAc, 3:7 v/v): Rf = 0.19; m. p.: 213-
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214 C; IR (KBr): ṽmax 3431.71, 1718.26, 2654.62, 1617.02, 1522.52, 1191.79 cm-1; H NMR (300 MHz, acetone-d6) δ 10.11 (s,
1H), 8.01 (d, J = 6.2 Hz, 1H), 7.71 – 7.33 (m, 6H), 6.71 (d, J = 15.8 Hz, 1H), 4.76 – 4.63 (m, 1H), 3.10 – 2.97 (m, 1H), 2.80 (dd, J
= 17.6, 5.9 Hz, 1H); 13C NMR (75 MHz, acetone-d6) δ 177.3, 175.9, 166.4, 141.5, 136.0, 130.5, 129.7, 128.6, 121.5, 51.5, 37.2;
HRMS ESI (m/z): [M+Na]+ Calcd for C13H12N2O3Na+: 267.07456, found: 267.07401.
3-Chloro-N-(2,5-dioxopyrrolidin-3-yl)benzo[b]thiophene-2-carboxamide (7g)
Compound 7g was synthesized according to general procedure C using 3a (100 mg, 0.44 mmol), DIPEA (0.15 ml, 0.88 mmol) and
3-chlorobenzo[b]thiophen-2-carbonyl chloride 6g (101 mg, 0.44 mmol). After purification via flash column chromatography (n-
hexane/EtOAc, 1:1 v/v) compound 7g (128 mg, 0.41 mmol, 94%) was isolated as a colorless solid. TLC (n-hexane/EtOAc, 3:7
v/v): Rf = 0.46; m. p.: 215-218 oC; IR (KBr): ṽmax 3445.21, 2932.23, 1729.83, 1636.3, 1534.1 cm-1; 1H NMR (400 MHz, acetone-d6)
δ 10.18 (br s, 1H), 8.47 (br d, J = 6.6 Hz, 1H), 8.10 – 7.98 (m, 1H), 8.00 – 7.90 (m, 1H), 7.66 – 7.55 (m, 2H), 4.99 (ddt, J = 10.4,
7.2, 5.2 Hz, 1H), 3.14 (ddd, J = 17.7, 9.5, 3.5 Hz, 1H), 2.92 (dd, J = 17.7, 6.0 Hz, 1H); 13C NMR (101 MHz, acetone-d6) δ 176.8,
175.8, 161.4, 138.7, 137.7, 133.0, 128.7, 126.7, 124.0, 123.8, 120.6, 51.8, 37.1; HRMS ESI (m/z): [M+Na]+ Calcd for
C13H9ClN2O3SNa: 303.991201, found: 303.99146; [2M+Na]+ Calcd for C26H18Cl2N4O6S2Na+: 638.99425, found: 638.99370.
3,4-Dichloro-N-(2,5-dioxopyrrolidin-3-yl)benzo[b]thiophene-2-carboxamide (7h)
Compound 7h was synthesized according to general procedure C using 3a (100 mg, 0.44 mmol), DIPEA (0.15 ml, 0.88 mmol) and
3,4-dichlorobenzo[b]thiophene-2-carbonyl chloride 6h (116 mg, 0.44 mmol). After purification via flash column chromatography
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(n-hexane/EtOAc, 1:1 v/v) compound 7h (63.6 mg, 0.27 mmol, 62%) was isolated as a light-yellow solid. TLC (n-hexane/EtOAc,
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3:7 v/v): RF = 0.46; m. p.: 159-162 C; IR (KBr): ṽmax 3396.03, 3192.58, 1135.62, 1620.88, 1532.17, 1193.72.755.244 cm-1; 1H
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NMR (300 MHz, DMSO-d6) δ 11.35 (s, 1H), 9.10 (d, J = 7.8 Hz, 1H), 8.10 (dd, J = 7.9, 1.2 Hz, 1H), 7.62 – 7.48 (m, 2H), 4.83 –
4.73 (m, 1H), 2.99 (dd, J = 17.6, 9.3 Hz, 1H), 2.67 (dd, J = 17.5, 5.6 Hz, 1H); 13C NMR (75 MHz, DMSO-d6) δ 177.1, 176.4, 160.3,
139.6, 133.7, 130.6, 128.4, 128.1, 123.1, 118.5, 50.4, 36.1, 30.8; HRMS ESI (m/z): [M+Na]+ Calcd for C13H8Cl2N2O3SNa+:
364.95304, found: 364.95249.
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Tert-butyl (4-((((2,5-dioxopyrrolidin-3-yl)carbamoyl)oxy)methyl)benzyl)carbamate (10a)
Compound 10a was synthesized according to general procedure D. To a 15% wt solution of COCl2 in toluene (12.3 ml, 17.29
mmol), a solution of 8 (3.15g, 13.3 mmol) in THF was added. After evaporation of the volatiles compound 9 was produced. By
using 3a (2.8 g, 12.4 mmol), DIPEA (4.0 ml, 23.0 mmol) and the chloroformate 9 (3.6 g, 12.0 mmol) compound 10a was
synthesized. After purification via flash column chromatography (DCM/MeOH 0-2% v/v) compound 10a (4.3 g, 11.4 mmol, 95%)
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was isolated as a white solid. TLC (DCM/MeOH, 5:0.3 v/v): Rf = 0.81; m. p.: 80-82 C; IR (KBr): ṽmax 3412, 1718, 1528, 1267,
1170 cm-1; 1H NMR: (300 MHz, CDCl3): δ 9.32 (s, 1H), 7.21 (q, J = 8.0 Hz, 4H), 6.20 (s, 1H), 5.14 (s, 1H), 5.02 (s, 2H), 4.24 (s,
3H), 2.90 (d, J = 8.8 Hz, 1H), 2.71 (d, J = 17.6 Hz, 1H), 1.43 (s, 9H); 13C NMR (100 MHz, CDCl3): δ 28.5, 36.7, 44.3, 51.3, 67.3,
79.9, 127.5, 128.6, 134.9, 139.4, 156.3, 175.2, 177.0; HRMS ESI (m/z): [M+Na]+: Calcd for C18H23N3O6Na+: 400.14850, found:
400.14782.
Tert-butyl (4-((((2,6-dioxopiperidin-3-yl)carbamoyl)oxy)methyl)benzyl)carbamate (10b)
Compound 10b was synthesized according to general procedure D. To a 15% wt solution of COCl2 in toluene (12.3 ml, 17.3
mmol), a solution of 8 (3.15 g, 13.3 mmol) in THF was added. After evaporation of the volatiles compound 9 was produced. By
using 3b (3.0 mg, 12.4 mmol), DIPEA (4.0 ml, 23 mmol) and the chloroformate 9 (3.6 g, 12.0 mmol) compound 10b was
synthesized. After purification via flash column chromatography (DCM/MeOH 0-2% v/v) compound 10b (4.23 g, 10.8 mmol,
90%) was isolated as a white solid. TLC (n-hexane/EtOAc, 3:7 v/v): RF= 0.16; m. p.: 154.4 °C; IR (KBr): ṽmax 3412.42, 3100.01,
2977.55, 2927.41, 2853.17, 1698.98, 1524.45 cm-1; 1H NMR: (300 MHz, CDCl3): δ 8.19 (s, 1H), 7.35 – 7.27 (m, 4H), 5.65 (s, 1H),
5.11 (s, 2H), 4.90 (s, 1H), 4.44 – 4.20 (m, 3H), 2.90 – 2.47 (m, 3H), 1.96 – 1.83 (m, 1H), 1.45 (s, 9H); 13C NMR: (75 MHz, CDCl3):
δ 171.1, 171.0, 156.1, 155.9, 139.2, 135.0, 128.5, 127.6, 77.2, 67.0, 52.2, 44.4, 31.2, 28.4, 25.3; HRMS ESI (m/z): [M+Na]+ Calcd
for C19H25N3O6Na+: 414.16410, found: 414.16326.
(4-((((2,5-Dioxopyrrolidin-3-yl)carbamoyl)oxy)methyl)phenyl)methanaminium 2,2,2-trifluoroacetate (11a)
Compound 11a was synthesized according to general procedure D using 10a (3.8 g, 10 mmol) and TFA (23 ml, 300 mmol). Upon
completion, the volatiles were completely evaporated to give 11a (3.91 g, 10 mmol, quant.) as a white solid. TLC (DCM/MeOH,
5:2 v/v): Rf = 0.26; m. p.: 197-200 °C; IR (KBr): ṽmax 3450, 1780, 1719, 1526, 1203, 1180 cm-1; 1H NMR: (400 MHz, DMSO-d6): δ
7.47 (s, 4H), 5.15 (s, 2H), 4.46 (dd, J = 9.3, 5.9 Hz, 1H), 4.13 (s, 2H), 3.02 (dd, J = 17.8, 9.3 Hz, 1H), 2.68 (dd, J = 17.8, 5.9 Hz,
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1H); 13C NMR (100 MHz, DMSO-d6): δ 179.5, 177.9, 139.3, 134.2, 130.1, 129.5, 67.2, 52.4, 44.0, 37.3, 27.72; 19F NMR (282
MHz, DMSO-d6) δ -73.61.; HRMS ESI (m/z): [M+Na]+: Calcd for C13H15N3O4Na+: 278.11353, found: 278,11345.
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Compound 11b was synthesized according to general procedure D using (3.91 g, 10 mmol) and TFA (23 mg, 300 mmol). Upon
completion, the volatiles were completely evaporated to give 11b (4.05 g, 10 mmol, quant) as a white solid. TLC (DCM/MeOH,
5:2 v/v): RF= 0.3; m. p.: 58-60 °C; IR (KBr): ṽmax 3419.17, 3240.79, 3094.23, 1698.98, 1532.17, 1251.58, 1204.33, 1136.83 cm-1;
1H NMR: (300 MHz, DMSO): δ 7.54 – 7.38 (m, 4H), 5.24 – 5.11 (m, 2H), 4.45 – 4.35 (m, 1H), 4.12 (s, 2H), 2.87 – 2.61 (m, 2H),
2.24 – 1.94 (m, 2H); 13C NMR: (100 MHz, DMSO): δ 173.4, 172.4, 157.1, 138.1, 132.6, 128.7, 128.0, 65.6, 51.3, 42.6, 30.7, 24.4;
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19F NMR (282 MHz, DMSO-d6) δ -73.70.; HRMS (m/z): [M+H]+ Calcd for C14H18N3O4 : 292.12973, found: 292.12885.
4-((4-((((2,5-Dioxopyrrolidin-3-yl)carbamoyl)oxy)methyl)benzyl)amino)-4-oxobutanoic acid (12a)
Compound 12a was synthesized according to general procedure E using 11a (175 mg, 0.45 mmol), succinic anhydride (44.7 mg,
0.45 mmol) and Et3N (0.14 ml, 0.98 mmol). After purification via flash column chromatography (EtOAc/IPA/H2O 4:2:1)
compound 12a (128 mg, 0.34 mmol, 76%) was isolated as a white solid. TLC (EtOAc/iPA/H2O, 4:3:2 v/v): Rf= 0.45; m. p.: 110 oC;
IR (KBr): ṽmax 3747.98, 3739.3, 2922.59, 1649.8, 1555.31, 1539.88, 1520.6, 1511.92 cm-1; 1H NMR (300 MHz, DMSO-d6) δ 8.53
(t, J = 5.8 Hz, 1H), 7.86 (d, J = 8.2 Hz, 1H), 7.25 (q, J = 8.1 Hz, 4H), 4.99 (s, 2H), 4.42 – 4.32 (m, 1H), 4.23 (d, J = 5.9 Hz, 2H),
2.94 – 2.83 (m, 1H), 2.53 – 2.43 (m, 1H), 2.37 – 2.25 (m, 4H); 13C NMR (101 MHz, DMSO-d6) δ 178.1, 176.5, 176.0, 172.6, 155.9,
-
139.7, 135.2, 128.1, 127.3, 65.8, 50.7, 41.9, 36.3, 32.1, 32.0; HRMS ESI (m/z): [M-H]- Calcd for C17H18N3O7 : 376.11447, found:
376.11646.
4-((4-((((2,6-Dioxopiperidin-3-yl)carbamoyl)oxy)methyl)benzyl)amino)-4-oxobutanoic acid (12b)
Compound 12b was synthesized according to general procedure E using 11b (121 mg, 0.3 mmol), succinic anhydride (30.0 mg, 0.3
mmol) and Et3N (0.092 ml, 0.66 mmol). After purification via flash column chromatography (EtOAc/IPA/H2O 4:2:1) compound
12a (117 mg, 0.3 mmol, quant) was isolated as a white solid. TLC (EtOAc/iPA/H2O, 4:3:2 v/v): Rf= 0.60; m. p.: 129 oC; IR (KBr):
ṽmax 3443.28, 1712.48, 1700.91, 1555.31, 1539.88, 1522.52, 1202.4 cm-1; 1H NMR (400 MHz, DMSO-d6) δ 10.80 (s, 1H), 8.49 (s,
1H), 7.59 (d, J = 8.6 Hz, 1H), 7.38 – 7.16 (m, 4H), 5.02 (s, 2H), 4.36 – 4.17 (m, 3H), 2.73 (ddd, J = 18.1, 11.8, 7.1 Hz, 1H), 2.47 (d,
J = 3.7 Hz, 1H), 2.32 (dq, J = 12.3, 7.2, 6.5 Hz, 3H), 1.95 (ddt, J = 17.6, 13.4, 5.2 Hz, 2H); 13C NMR: (101 MHz, DMSO-d6): δ
173.1, 172.5, 172.3, 156.2, 139.5, 135.4, 127.9, 127.3, 65.5, 50.9, 41.9, 31.9, 31.8, 31.7, 31.0, 24.4; HRMS ESI (m/z): [M-H]-
-
Calcd for C18H20N3O7 : 390.13013, found: 390.13109.
((1s,4s)-4-((Tert-butoxycarbonyl)amino)cyclohexyl)methyl (2,5-dioxopyrrolidin-3-yl)carbamate (15a)
Compound 15a was synthesized according to general procedure D. To a 15% wt solution of COCl2 in toluene (12.3 ml, 17.3
mmol), a solution of 13 (3.05 g, 13.3 mmol), in THF was added. After evaporation of the volatiles, compound 14 was produced. By
using 3a (2.8 mg, 12.4 mmol), DIPEA (4 ml, 23 mmol) and the chloroformate 14 (3.5 g, 12 mmol) compound 15a was synthesized.
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After purification via flash column chromatography (DCM/MeOH 0-2% v/v) compound 15a (3.9 mg, 10.44 mmol, 87%) was
isolated as a white solid. TLC (DCM/MeOH, 5:1 v/v): Rf = 0.71; m. p.: 95-97 °C; IR (KBr): ṽmax 3381, 1715, 1523, 1366, 1257,
1172 cm-1; 1H NMR: (400 MHz, CDCl3): δ 8.97 (s, 1H), 5.77 (s, 1H), 4.76 (s, 1H), 4.40 (s, 1H), 4.00 (s, 2H), 3.74 (s, 1H), 3.12 (dd,
J = 18.8, 10.0 Hz, 1H), 2.88 (d, J = 18.0 Hz, 1H), 1.82 – 1.56 (m, 6H), 1.47 (s, 9H), 1.31 (s, 2H); 13C NMR (101 MHz, CDCl3): δ
177.0, 175.2, 156.5, 155.3, 79.2, 69.1, 51.3, 46.3, 36.7, 35.2, 29.2, 28.4, 24.3; HRMS ESI (m/z): [M+Na]+: Calcd for
C17H27N3O6Na+: 392.17976, found: 392.18342.
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((1s,4s)-4-((Tert-butoxycarbonyl)amino)cyclohexyl)methyl (2,6-dioxopiperidin-3-yl)carbamate (15b)
Compound 15b was synthesized according to general procedure D. To a 15% wt solution of COCl2 in toluene (12.3 ml, 17.3
mmol), a solution of 13 (3.05 g, 13.3 mmol) in THF was added. After evaporation of the volatiles, compound 14 was produced. By
using 3b (3.0 mg, 12.4 mmol), DIPEA (4 ml, 23 mmol) and the chloroformate 14 (3.5 mg, 12 mmol) compound 15b was
synthesized. After purification via flash column chromatography (DCM/MeOH 0-2% v/v) compound 15b (2.99 g, 7.8 mmol, 65%)
was isolated as a white solid. TLC (DCM/MeOH, 5:2 v/v): Rf = 0.76; m. p.: 133-137 °C; IR (KBr): ṽmax 3379, 1703, 1523, 1247,
1198, 1171 cm-1; 1H NMR: (400 MHz, CDCl3): δ 8.33 (s, 1H), 5.57 (s, 1H), 4.50 (d, J = 130.1 Hz, 2H), 3.85 (d, J = 97.0 Hz, 3H),
2.91 – 2.32 (m, 3H), 1.91 – 1.13 (m, 18H); 13C NMR (101 MHz, CDCl3): δ 171.4, 171.3, 156.5, 155.4, 79.5, 69.0, 53.4, 46.4, 35.5,
31.3, 29.3, 28.5, 25.3, 24.3; HRMS ESI (m/z): [M+Na]+: Calcd for C18H29N3O6Na+: 406.19541, found: 406.19381.
((1s,4s)-4-((((2,5-Dioxopyrrolidin-3-yl)carbamoyl)oxy)methyl)cyclohexyl)methanaminium 2,2,2-trifluoroacetate (16a)
Compound 16a was synthesized according to general procedure D using 15a (3.7 g, 10 mmol) and TFA (23 ml, 300 mmol). Upon
completion, the volatiles were completely evaporated to give 16a (3.83 g, 10 mmol, quant) as a white solid. TLC (DCM/MeOH, 5:1
v/v): Rf = 0.21; m. p.: 74-76 °C; IR (KBr): ṽmax 3447, 1783, 1717, 1629, 1529, 1267, 1203, 1182, 1138 cm-1; 1H NMR: (400 MHz,
DMSO-d6): δ 11.23 (s, 1H), 7.90 – 7.71 (m, 4H), 4.53 – 4.21 (m, 1H), 3.98 – 3.86 (m, 3H), 3.16 (s, 1H), 2.88 (dd, J = 17.5, 9.4 Hz,
1H), 2.45 (d, J = 5.7 Hz, 1H), 1.87 – 1.35 (m, 9H); 13C NMR (75 MHz, DMSO): δ 178.3, 176.6, 156.4, 66.6, 50.9, 48.1, 36.6, 33.5,
+
26.4, 23.6.; 19F NMR (282 MHz, DMSO-d6) δ -73.69; HRMS (m/z): [M+2H]+: Calcd for C12H20N3O4 : 270.14483, found:
270.14076.
((1s,4s)-4-((((2,6-Dioxopiperidin-3-yl)carbamoyl)oxy)methyl)cyclohexyl)methanaminium 2,2,2-trifluoroacetate (16b)
Compound 16b was synthesized according to general procedure D using 15b (3.8 g, 10 mmol) and TFA (23 ml, 300 mmol). Upon
completion, the volatiles were completely evaporated to give 16b (3.97 g, 10.0 mmol, quant) as a white solid. TLC (DCM/MeOH,
5:1 v/v): RF = 0.20; m. p.: 189-191 °C; IR (KBr): ṽmax 3454, 1747, 1724, 1677, 1622, 1546, 1248, 1201, 1188, 1137 cm-1; 1H NMR:
(400 MHz, DMSO-d6): δ 10.78 (s, 1H), 7.80 (s, 3H), 7.44 (s, 1H), 4.28 (s, 1H), 3.94 (dd, J = 16.4, 5.5 Hz, 3H), 3.19 (s, 1H), 2.73 (s,
1H), 2.00 – 1.40 (m, 11H); 13C NMR (101 MHz, DMSO): 173.4, 172.9, 156.8, 66.4, 51.2, 48.2, 33.6, 31.4, 26.4, 24.8, 23.6; 19F
+
NMR (282 MHz, DMSO-d6) δ -73.96.; HRMS (m/z): [M+2H]+: Calcd for C13H22N3O4 : 284.16048, found: 284.15961.
Tert-butyl (2S)-2-((((2,6-dioxopiperidin-3-yl)carbamoyl)oxy)methyl)pyrrolidine-1-carboxylate (19b)
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Compound 19b was synthesized according to general procedure D. To a 15% wt solution of COCl2 in toluene (12.3 ml, 17.3
mmol), a solution of 17 (2.68 g, 13.3 mmol) in THF was added. After evaporation of the volatiles, compound 18 was produced. By
using 3b (3.0 g, 12.4 mmol), DIPEA (4 ml, 23 mmol) and the chloroformate 18 (3.16 g, 12 mmol) compound 19b was synthesized.
After purification via flash column chromatography (DCM/MeOH 0-2% v/v) compound 19b (3.07 g, 8.64 mmol, 72%) was
isolated as a white solid. TLC (DCM/MeOH, 5:0.5 v/v): Rf = 0.42; m. p.: 81-83 °C; IR (KBr): ṽmax 3437, 1698, 1540, 1403,
1249,1201, 1171 cm-1; 1H NMR: (400 MHz, CDCl3): δ 8.30 (s, 1H), 5.59 (s, 1H), 4.46 – 3.82 (m, 4H), 3.52 – 3.10 (m, 2H), 2.94 –
2.33 (m, 3H), 2.03 – 1.77 (m, 5H), 1.46 (s, 9H); 13C NMR (101 MHz, CDCl3): δ 171.2, 156.1, 154.5, 65.7, 55.7, 52.2, 46.7, 46.5,
31.2, 28.5, 27.8, 25.3, 23.7, 22.9; HRMS ESI (m/z): [M+Na]+: Calcd for C16H25N3O6Na+: 378.16411, found: 378.16862.
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(2S)-2-((((2,6-Dioxopiperidin-3-yl)carbamoyl)oxy)methyl)pyrrolidin-1-ium 2,2,2-trifluoroacetate (20b)
Compound 20b was synthesized according to general procedure D using 19b (3.55 g, 10 mmol) and TFA (23 ml, 300 mmol). Upon
completion, the volatiles were completely evaporated to give 20b (3.69 g, 10 mmol, quant.) as a white solid. TLC (DCM/MeOH,
5:0.5 v/v): RF = 0.19; m. p.: 149-151 °C; IR (KBr): ṽmax 3479, 1705, 1541, 1253, 1204, 1135 cm-1; 1H NMR: (400 MHz, DMSO): δ
10.82 (s, 1H), 9.24 (s, 1H), 8.74 (s, 1H), 7.62 (d, J = 8.5 Hz, 1H), 4.48 – 4.04 (m, 3H), 3.76 (s, 1H), 3.19 (s, 2H), 2.74 (ddd, J =
18.2, 12.7, 6.1 Hz, 1H), 2.12 – 1.81 (m, 5H), 1.79 – 1.57 (m, 1H); 13C NMR (101 MHz, DMSO): δ 173.4, 172.7, 156.0, 63.8, 58.2,
51.3, 45.8, 31.4, 26.9, 24.7, 23.7.; 19F NMR (282 MHz, DMSO-d6) δ -74.36; HRMS (m/z): [M+H]+: Calcd for
+
C11H18N3O4 : 256.12918, found: 256.12905.
Cloning, expression and protein purification
WT MsCI4 was cloned, expressed and purified as previously described56. In an additional construct (humanized MsCI4) surface
exposed residues were mutated to mimic hCRBN. The gene carrying the mutations A52H, M54Y, F56H, R68N, A72R, I87V and
L89Q was codon optimized for expression in E. coli and synthesized (eurofins). Humanized MsCI4 was cloned via BamHI and
XhoI restriction sites into pETHis_1a; expression and purification was performed as described for WT MsCI4.
FRET assay
FRET-based binding assay was performed using WT MsCI4 as described previously56. Compounds were dissolved in DMSO with
the exception of 11a/b, 16a/b, 20a/b, which were water-soluble, and compounds 12a/b that were dissolved in bicarbonate buffer.
Due to remaining impurities, 20a was not tested in this assay and only used for structural studies. For compounds 4a, 4c, 5a, 7a, 7b,
7e and 7g, binding was observed but could not be quantified, as saturation at higher concentrations was not appreciable in their
binding curves. Ki values are summarized in Table S1.
Crystallography
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For crystallization, MsCI4 and humanized MsCI4 protein solution was concentrated to 17 mg/mL and mixed with either 3 mM
thalidomide (MsCI4 and humanized MsCI4) or 7d (MsCI4). Crystallization trials for humanized MsCI4thalidomide, which turned
out to be humanized MsCI4CBG, and MsCI47d were performed via the vapor diffusion technique at 294 K in sitting-drops.
400 nL of protein solution were mixed with 400 nL of reservoir solution with the Honeybee 963 robot (Genomic Solutions Ltd).
Diffraction-quality crystals of humanized MsCI4CBG were obtained in a condition containing 20 % PEG 3350 and 0.2 M
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ammonium acetate, and crystals of MsCI47d were obtained using 15 % PEG 6K and 5 % Glycerol. For all other compounds,
crystals were prepared in hanging drop experiments by mixing 1 µL 0.4 M (NH4)2HPO4 ground solution with 1 µL
MsCI4thalidomide solution. Crystals grown in this set-up were transferred to 3 µL ground solution spiked with individual
compounds for soaking. After 36 h, crystals were cryoprotected by streaking through a 50 % PEG 3350 solution. Crystals were
flash-cooled in liquid nitrogen and diffraction data was collected at 100 K and a wavelength of 1 Å on beamline X10SA at the
Swiss Light Source using a PILATUS 6M-F hybrid pixel detector (Dectris Ltd.). Data was processed and scaled using XDS57.
Structures of soaking experiments were solved based on the MsCI4thalidomide coordinates (PDB 4v2y). The co-crystal structures
of the complexes with CBG and 7d were of other crystal forms and were solved using molrep58 with 4v2y as a search model,
locating four and two chains in the ASU, respectively. All structures were rebuilt using Coot59 and the integrated suite Lidia for
chemical structures and generation of restraints. The models were finalized via cyclic modeling in Coot and refinement using
REFMAC5. Molecular figures were generated using PyMOL60. Data collection and refinement statistics are summarized in Table 1.
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Cell Culture and Western Blot
OPM-2 cells were routinely cultivated in 90% RPMI 1640 + 10% FBS and 1% Penicillin/Streptomycin. Cells were split to 0.3·106
cells/mL 2 days prior to experiments. 4 mL of culture per well were pipetted in a sterile environment. Stock solutions of
lenalidomide were prepared at concentrations of 80 mM and 400 mM, stock solutions of test compounds were prepared at a
concentration of 400 mM. For the assay, 10 µL of stock solutions were added to 4 mL cell culture. DMSO controls were prepared
in the same manner, leading to a final DMSO concentration of 0.25 %. After 24 h of incubation, cell solution was centrifugated at
500 g. After one washing step with ice cold PBS, cell pellets were resuspended in 35 µL of lysis buffer (20 mM Hepes, 175 mM
NaCl, 1% NP40, 2mM MgCl2) on ice and supplemented with 0.5 µL Benzonase. Samples were resolved by Mini-PROTEAN TGX
gels (BIO-RAD) and activated under UV light, before transfer to low-fluorescence PVDF membranes, according to the
manufacturer’s protocol. Membranes were imaged before blocking in 5 % milk in PBS-T and incubating with primary antibodies
anti-IKZF3 (15103S, Cell Signaling Technology, Inc.) and anti-CK1α (ab108296, Abcam plc). After overnight incubation,
horseradish peroxidase conjugated secondary antibodies goat anti-rabbit (111-035-144, Jackson ImmunoResearch) and goat anti-
mouse (115-035-003, Jackson ImmunoResearch) were used at 1:20.000 dilutions for detection of bands by chemiluminescence
(ECL Vilbert). Protein bands were detected and integrated using the BioRad analysis suite. Endogenous protein levels were
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normalized using Stain-Free Technology (Figure S2) and analyzed by one way ANOVA according to Holm-Sidak method
integrated into SigmaPlot. The linearity of anti-IKZF3 mAb was tested via serial dilutions of cell extracts (Figure S3).
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7
MsCI4 · 4a
MsCI4 · 5b
MsCI4 · 4b
MsCI4 · 5a
MsCI4 · 7a
MsCI4 · 7b
MsCI4 · 7c
MsCI4 · 7d
MsCI4 · 7f
MsCI4 · 11a
MsCI4 · 12a
MsCI4 · 16b
MsCI4 · 20a
MsCI4 · 20b
MsCI4 · CBG
Data Collection
Space group
Unit cell
P212121
P212121
P212121
P212121
P212121
P212121
P212121
P21
P212121
P212121
P212121
P212121
P212121
P212121
P21
56.46, 58.82,
88.23
56.75, 58.82,
88.57
56.95, 59.58,
89.05
56.66, 58.71,
88.01
56.33, 58.68,
89.27
56.42, 58.80,
88.23
56.33, 58.68,
89.27
31.63, 52.39,
59.29
56.87, 58.55,
88.28
56.63, 59.59,
88.90
56.66, 59.02,
88.59
56.53, 59.44,
88.36
56.56, 59.23,
88.43
8
a, b, c (Å)
56.56, 59.3, 88.0 61.7, 59.1, 61.7
9
α, β, γ (°)
90, 90, 90
90, 90, 90
90, 90, 90
90, 90, 90
90, 90, 90
90, 90, 90
90, 90, 90
90.0, 95.8, 90.0
90, 90, 90
90, 90, 90
90, 90, 90
90, 90, 90
90, 90, 90
90, 90, 90
90, 105.6, 90
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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
44.1 - 1.55 (1.64 44.28 - 1.6 (1.7 -44.53 - 1.7 (1.8 - 47.64 - 1.75 49.03 - 1.8 (1.86 48.93 - 1.73
49.03 - 1.85 39.2 - 1.1 (1.16 - 44.14 - 1.65
44.45 - 1.35 49.12 - 1.5 (1.59 47.62 - 1.35
49.21 - 1.45
(1.50 - 1.45)
49.18 – 1.54
(1.64-1.54)
49.17 - 1.50
(1.59 – 1.50)
Resolution range, Å
- 1.55)
1.6)
1.7)
(1.85 - 1.75)
- 1.8)
(1.84 - 1.73)
(1.96 - 1.85)
1.1)
(1.75 - 1.65)
(1.43 - 1.35)
- 1.5)
(1.43 - 1.35)
Redundancy
12.7 (11.8)
99.8 (98.9)
9.0 (98.2)
12.9 (12.9)
99.9 (99.4)
5.3 (82.8)
12.4 (12.8)
99.0 (98.2)
7.3 (94.6)
12.8(12.8)
99.6 (97.7)
6.6 (98.5)
12.86(12)
99.6 (97.6)
7.2 (89.0)
100 (84.6)
22.12(2.39)
12.5 (12.0)
98.8 (92.4)
14.2 (88.8)
99.6 (85.7)
10.55 (1.73)
12.9 (13.0)
99.8 (98.6)
6.1 (102.8)
99.9 (78.1)
21.46 (2.25)
6.3(5.3)
99.1 (95.1)
5.3 (39.4)
12.9 (13.2)
99.9 (99.6)
6.9 (81.5)
12.7 (12.4)
99.9 (99.3)
7.4 (96.3)
12.6 (12.6)
99.5 (97.2)
11.4 (78.6)
99.8 (86.5)
14.29 (2.32)
12.6 (12.7)
99.8 (99.1)
7.9 (98.8)
12.8 (12.9)
99.8 (98.5)
9.5 (89.4)
12.6 (12.1)
99.5 (96.7)
13.1 (81.5)
99.7 (83.8)
10.28 (1.71)
3.4(3.1)
99.0 (97.5)
8.0 (88.0)
99.8 (55.6)
9.70(1.15)
Completeness %
R merge
%
CC(1/2)
99.9 (85.4)
15.09 (1.78)
100 (90.4)
25.88 (2.73)
99.9 (87.8)
20.22 (2.02)
100 (87.6)
20.47 (2.27)
99.8 (92.4)
17.67 (3.64)
99.9 (86.6)
20.72 (2.35)
99.9 (83.2)
17.62 (1.97)
99.9 (84.0)
16.63 (1.85)
99.8 (86.2)
14.83 (2.11)
I/s(I)
Refinement
Number of reflections (total/test)
No. of atoms
Protein
43370 (4254)
2871
39841 (3904)
2787
33803 (3354)
2767
30301 (2985)
2709
28122 (2771)
2544
30582 (2948)
2778
28122 (2771)
2675
77822 (7458)
2082
36182 (3558)
2464
66738 (6544)
2977
48282 (4739)
2995
66083 (6516)
3057
53344 (5211)
3074
44026 (4112)
2928
68399 (6707)
3985
2572
2519
2504
2523
2338
2521
2468
1768
2229
2619
2665
2638
2634
2617
3480
Solvent
223
173
185
134
175
187
123
276
166
281
252
338
339
232
421
Ligand
76
95
78
52
31
70
84
38
69
77
78
81
101
79
84
R work
R free
%
%
0.17
0.19
0.18
0.19
0.17
0.17
0.18
0.12
0.20
0.16
0.21
0.16
0.17
0.18
0.16
0.20
0.22
0.23
0.23
0.22
0.23
0.22
0.15
0.22
0.19
0.24
0.20
0.20
0.21
0.20
Ligand in chain
A
B
C
thalidomide
4a
hydrolyzed 4b
hydrolyzed 4b
hydrolyzed 4b
thalidomide
hydrolyzed 5a
5a
5b
5b
-
7a
7a
-
thalidomide
7b
7c
7c
7c
thalidomide
thalidomide
10a
thalidomide
12a
thalidomide
16b
thalidomide
20a
thalidomide
thalidomide
20b
Co-crystal with
7d
Co-crystal with
CBG
7f
-
hydrolyzed 4a
thalidomide
thalidomide
thalidomide
16b
thalidomide
PDB ID
6R0S
6R0V
6R0U
6R11
6R1X
6R12
6R1K
6R1D
6R13
6R18
6R1C
6R1W
6R19
6R1A
6R0Q
Table 1 Data collection and refinement statistics
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Page 29 of 35
Journal of Medicinal Chemistry
Supporting Information
1
2
3
4
Figure S1. HPLC traces of tested compounds
5
6
7
8
9
Figure S2. Western blot quantification and normalization IKZF3
Figure S3. Concentration response curve for IKZF3 mAb
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
Table S1. Molecular formula strings and Ki values of tested compounds.
Crystal structures have been deposited in the Protein Data Bank (PDB) under the accession codes 6R0S (4a), 6R0V (4b), 6R0U
(5a), 6R11 (5b), 6R1X (7a), 6R12 (7b), 6R1K (7c), 6R1D (7d), 6R13 (7f), 6R18 (11a), 6R1C (12a), 6R1W (16b), 6R19 (20a),
6R1A (20b), 6R0Q (CBG). Authors will release the atomic coordinates and experimental data upon article publication.
Corresponding Authors
*AG: email, giannis@uni-leipzig.de.
*MDH: email, marcus.hartmann@tuebingen.mpg.de.
ACKNOWLEDGMENTS
We thank Andrei Lupas for continuing support, Reinhard Albrecht for assistance with crystallization and crystallographic data
collection, and the staff of beamline X10SA of the Swiss Light Source (PSI, Villigen, Switzerland) for excellent technical support.
This work was supported by institutional funds of the Max Planck Society.
ABBREVIATIONS USED
4-DMAP, 4-dimethylaminopyridine; AML, acute myeloid leukemia; APP, amyloid precursor protein; ASU, asymmetric unit; BET,
Bromodomain and extraterminal domain; CBG, α-(2-Carboxybenzamido) glutarimide; CDI, N,N-carbodiimidazole;CK1α, casein
kinase 1α; CRBN, cereblon; CRL4CRBN, CUL4-RBX1-DDB1-CRBN; DIPEA, N, N-diisopropylethylamine; Et3N, triethylamine;
EtOAc, ethyl acetate; FBS, fetal bovine serum; GSPT1, eukaryotic peptide chain release factor GTP-binding subunit ERF3A;
IKZF1, IKAROS family zinc finger 1; IKZF3, zinc finger protein Aiolos; IMiD, immunomodulatory drug; MDS, myelodysplastic
syndrome; MM, multiple myeloma; MsCI4, CRBN homologue from Magnetospirillum gryphiswaldense; PROTAC, proteolysis
targeting chimera; SALL4, sal-like protein 4; VHL, von-Hippel-Lindau
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