Discovery of tricyclic indoles that potently inhibit Mcl-1 using fragment-based methods and structure-based design

Article abstract of DOI:10.1021/jm501984f

Myeloid cell leukemia-1 (Mcl-1) is an antiapoptotic member of the Bcl-2 family of proteins that is overexpressed and amplified in many cancers. Overexpression of Mcl-1 allows cancer cells to evade apoptosis and contributes to the resistance of cancer cells to be effectively treated with various chemotherapies. From an NMR-based screen of a large fragment library, several distinct chemical scaffolds that bind to Mcl-1 were discovered. Here, we describe the discovery of potent tricyclic 2-indole carboxylic acid inhibitors that exhibit single digit nanomolar binding affinity to Mcl-1 and greater than 1700-fold selectivity over Bcl-xL and greater than 100-fold selectivity over Bcl-2. X-ray structures of these compounds when complexed to Mcl-1 provide detailed information on how these small-molecules bind to the target, which was used to guide compound optimization.

Full text of DOI:10.1021/jm501984f

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Article
Discovery of Tricyclic Indoles That Potently Inhibit Mcl-1
Using Fragment-Based Methods and Structure-Based Design
Jason P. Burke, zhiguo bian, Subrata Shaw, Bin Zhao, Craig M. Goodwin, Johannes
Belmar, Carrie F. Browning, Dominico Vigil, Anders Friberg, DeMarco Camper,
Olivia W. Rossanese, Taekyu Lee, Edward T Olejniczak, and Stephen W. Fesik
J. Med. Chem., Just Accepted Manuscript • Publication Date (Web): 06 Apr 2015
Downloaded from http://pubs.acs.org on April 7, 2015
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Discovery of Tricyclic Indoles That Potently Inhibit
Mcl-1 Using Fragment-Based Methods and
Structure-Based Design
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Jason P. Burke , Zhiguo Bian, Subrata Shaw, Bin Zhao, Craig M. Goodwin, Johannes Belmar,
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Carrie F. Browning, Dominico Vigil , Anders Friberg^, DeMarco V. Camper, Olivia W.
Rossanese, Taekyu Lee, Edward T. Olejniczak, and Stephen W. Fesik*
Department of Biochemistry, Vanderbilt University School of Medicine, 2215 Garland Avenue,
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07 Light Hall, Nashville, Tennessee 37232-0146, USA
KEYWORDS:
Fragment-based screening; apoptosis; cancer; Mcl-1; inhibitor; drug discovery
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Abstract
Myeloid cell leukemia-1 (Mcl-1) is an anti-apoptotic member of the Bcl-2 family of proteins that
is overexpressed and amplified in many cancers. Overexpression of Mcl-1 allows cancer cells to
evade apoptosis and contributes to the resistance of cancer cells to be effectively treated with
various chemotherapies. From an NMR-based screen of a large fragment library, several distinct
chemical scaffolds that bind to Mcl-1 were discovered. Here, we describe the discovery of potent
tricyclic 2-indole carboxylic acid inhibitors that exhibit single digit nanomolar binding affinity to
Mcl-1 and greater than 1700-fold selectivity over Bcl-xL and greater than 100 fold selectivity
over Bcl-2. X-ray structures of these compounds when complexed to Mcl-1 provide detailed
information on how these small-molecules bind to the target, which was used to guide compound
optimization.
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INTRODUCTION
The intrinsic apoptosis pathway is tightly regulated by a balance of pro-apoptotic and
anti-apoptotic proteins that respond to numerous extracellular and intracellular stresses,
including growth factor and oxygen deprivation, DNA damage, oncogene induction, metabolic
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changes, and the effects of cytotoxic drugs. In normal cells, these stresses induce
oligomerization of the pro-apoptotic proteins Bax and Bak at the mitochondrial outer membrane,
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which triggers cytochrome c release and caspase-dependent apoptosis. Anti-apoptotic Bcl-2
family proteins, including Bcl-2, Bcl-xL, Bcl-w, Bcl-A1, and Mcl-1, bind to and sequester pro-
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apoptotic members of the same family, leading to the inhibition of apoptosis. In cancers, anti-
apoptotic Bcl-2-family proteins are often upregulated causing the enhancement of cancer cell
survival in otherwise stressful conditions and contributes to increased resistance to anti-cancer
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therapeutics.
Amplification of the gene encoding the anti-apoptotic protein Myeloid cell leukemia-1
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(Mcl-1) is one of the most common genetic aberrations in human cancers. Mcl-1
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overexpression is associated with high tumor grade and poor survival in lung , breast , prostate ,
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pancreatic , ovarian , and cervical cancers , as well as melanoma and leukemia . Mcl-1
activity has also been implicated in the resistance to multiple therapies, including microtubule-
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targeting agents like paclitaxel and vincristine, which are widely prescribed for cancer patients.
Thus, neutralizing the function of Mcl-1 holds promise as an effective strategy to restore
apoptotic signaling in cancer cells and enhance the response to currently approved
chemotherapeutics. Despite this compelling rationale, no clinical therapeutic agent selectively
targeting Mcl-1 is currently available.
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Mcl-1 mediates its effects primarily through protein-protein interactions and is therefore
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considered difficult to target with small molecules. However, small molecule inhibitors that
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potently bind to the related family members Bcl-2 and Bcl-xL have been discovered.
Among
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them, are the Bcl-2/Bcl-xL dual inhibitor ABT-263 and the selective Bcl-2 inhibitor ABT-199,
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both of which are in clinical trials.
These inhibitors were reported to bind to their target
proteins with 2-3 orders of magnitude higher affinities than the best Mcl-1 inhibitors reported to
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date in the literature.
To discover Mcl-1 inhibitors, we have previously conducted a fragment-based screen of
approximately 15,000 compounds using NMR and reported on the discovery of high-affinity (Ki
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100 nM) inhibitors that bind to Mcl-1 starting from two classes of hits identified in the screen.
The best inhibitor in this series was an optimized 2-indole carboxylic acid derivative 1 that binds
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to Mcl-1 with a K of 55 nM.
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Several additional structurally distinct small molecules were identified in the fragment
screen with similar or higher binding affinities and/or ligand efficiency (LE). One of these series
contains a tricyclic indole 2-carboxylic acid core, as found in the fragment hit 2. Here we
describe the discovery of very potent Mcl-1 inhibitors that were derived from this fragment hit.
These compounds have single digit nanomolar binding affinity and high selectivity for Mcl-1
over Bcl-xL and Bcl-2. Crystal structures of this series of inhibitors when bound to Mcl-1
provide detailed information on the molecular interactions that stabilize complex formation.
Structure of Compound 1 & 2
RESULTS AND DISCUSSION
Synthesis. The general synthetic approach used for synthesizing tricyclic 2-indole carboxylic
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acids derivatives is illustrated in Scheme 1 - 3. The preparation of the tricyclic analogs based on
the fragment hit 2 that are listed in Table 1 is depicted in Scheme 1. The appropriate bicyclic
hydrazine 10 was reacted with a selected α-ketoester 11 to give the tricyclic indole-2-carboxylic
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acid 3-8 via Fisher indole reaction
followed by saponification.
a
Scheme 1
The synthetic route illustrated in Scheme 2 was developed to prepare the compounds
depicted in Table 2. Using the Fischer indole condition depicted in Scheme 1, the selected
hydrazine 10 was cyclized with a selected α-ketoacid 12 to give a mixture of corresponding
tricyclic indole diester 13a and indole acid 13b. Formation of the tricyclic indole diester 13a
was improved by extending the reaction time, however, the reaction suffered from a poor yield.
Therefore, the indole acid 13b in the mixture was converted to the methyl ester 13c using
TMSCHN then both indole esters 13a and 13c were carried to the subsequent reaction without
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isolation. The alcohol 14 was prepared by selective reduction of the extended ethyl ester at the
3-position of the indole using BH -THF. Mitsunobu condensation (as used in our previous
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report ) was used to couple a selected phenol to yield the phenyl ether 15. Subsequent
saponification of the ester gave the desired carboxylic acid moiety in compounds 16-21, 23, 26-
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3. The sulfide groups in the tricyclic indole acids 19 and 23 were oxidized to the corresponding
sulfoxide 24 or sulfone 22 and 25 using m-CPBA.
a
Scheme 2
Tricyclic 6-Cl-2-indole carboxylic acid 34 and 35 were prepared as outlined in Scheme 3.
-Br-3-Cl-aniline 38 was converted to the indole 39 utilizing the modified Japp−Klingemann
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reaction condition. Selective BH reduction followed by a Mitsunobu reaction gave the indole
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ester 41. Allylation at the indole nitrogen gave compound 42, which underwent a radical
cyclization to yield the tricyclic indole ester containing the piperidine C-ring. Subsequent
saponification gave the desired acid 34. The intermediate 41 was converted to the pinacol borate
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3, which was oxidized to give the phenol 44. The morpholine C-ring was constructed using di-
bromoethane followed by saponification to produce the 6-Cl-indole acid 35.
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Tricyclic Fragment Optimization. Among the initial hits obtained in the screen, we identified
fragments containing a tricyclic indole 2-carboxylic acid that bind to Mcl-1 (Table 1). The
thiazine-containing tricyclic fragment 2 displaces a FITC-labeled BAK peptide from Mcl-1 with
a K of 35 µM in an FPA assay. SAR of the fragment core was investigated by using small
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tricyclic indoles with substitutions on rings A-C (Table 1) that were either purchased or
synthesized as described in Scheme 1.
Table 1.
In general, all of the tricyclic indole acids exhibited similar binding affinity for Mcl-1
compared to the initial hit 2 with ligand efficiencies (LE) ≥ 0.35. Both compounds 3 and 4,
containing C-ring moieties showed significantly enhanced binding affinity when compared to the
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unsubstituted indole 2-carboxylic acid 8 . Improved affinity was also observed by substituting a
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methyl group at the R (compound 5, 6) or R (compound 2) positions compared to the
unsubstituted fragments 3 and 4, suggesting that additional hydrophobic protein binding pockets
may be accessible from these positions. In addition, C-ring analogs containing a thiazine moiety
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3 and 5) or morpholine (7) were potent and preferred over the piperidine analogs 4 and 6. These
results suggest that modifications in the C-ring are not only tolerated but can make additional
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interactions with Mcl-1 that result in improvements in binding affinity.
To determine how these tricyclic indole fragments bind to Mcl-1, we performed NMR-
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based structural studies using compound 2 bound to double-labeled ( N, C) Mcl-1. Several key
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NOEs were observed between the indole phenyl ring B and A227, M231, and F270 of Mcl-1. In
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addition, the thiazine methyl group at R exhibited weak NOEs involving T266 (Figure 1A). This
pattern of intermolecular protein/ligand NOEs strongly suggests that compound 2 and similar
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analogs bind in the upper part of the second hydrophobic pocket , P2, utilized by peptides that
bind to Mcl-1. In peptides, this pocket is occupied by a highly conserved Leu residue.
The NMR-derived structural information was used to generate a model in which the
carboxylic acid moiety of compound 2 on ring A points toward R263. In addition, the indole
phenyl on ring B points towards A227 and M231 with ring C sitting next to the adjacent
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hydrophobic pocket (P3) (Figure 1B). This orientation places the indole 3-position (R ) to point
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into the lower P2 pocket. Thus, the model structure predicted that the R position is in an ideal
location for extending deep into the pocket.
Figure 1.
Fragment Merging with the Tricyclic Scaffold. We previously discovered that a substantial
boost in affinity could be obtained by merging fragments that bind in the upper part of the P2
pocket to fragments that predominantly bind deeper into the pocket than Leu of the BH3
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peptide. On the basis of the fragment SAR (Table 1), NMR-derived structural information, and
molecular modeling, we designed and synthesized a new series of Mcl-1 inhibitors by merging
molecules that bind deep into the P2 pocket to the tricyclic indoles. The binding affinity and
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selectivity of these compounds for binding to Mcl-1 are shown in Table 2. In an earlier study ,
we found that both a 1-naphthyl and a 3,5-dimethyl-4-chloro-phenyl fragment bound to the lower
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P2 pocket and gave similar affinities for compounds containing both upper and lower P2 pocket
binders. To generate the initial SAR in this series, a 1-naphthyl group as the lower P2 pocket
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binding group was connected to the R position of tricyclic 2-indole carboxylic acid cores
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bearing thiazine, piperidine or a morpholine C-ring moiety, using either a three- or four-atom
linker. As expected, the merged compounds (16-21) exhibited markedly enhanced binding
affinities compared to the parent compounds (2-7) that lack a lower pocket binding group. In
particular, compounds containing a four-atom linker (19, 20 and 21) showed more than 2 orders
of magnitude increased affinity for Mcl-1 compared to the initial fragment 2. The three-atom
linker is not preferred, as compounds 16, 17 and 18 exhibited >10-fold higher K values
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compared to the corresponding four-atom linker analogs. Compounds containing different C-ring
moieties had improved affinity from the original fragment and showed a parallel SAR trend with
respect to linker length.
Table 2.
Structure of compounds 36 and 37
SAR of the C-ring Unit in the Merged Compounds. The influence of C-ring composition on
binding to Mcl-1 was evaluated by preparing compounds 19-25 that contain heterocyclic units
with varying ring sizes and functional groups. Similar to that observed for the fragments, the 6-
membered C-ring moiety bearing S- (19) or O- (21) were slightly preferred to the compounds
containing a carbon analog 20. The 7-membered thiazepine C-ring unit was well tolerated with
compound 23 exhibiting near equal potency to the thiazine analog 19. We also explored the
possibility of incorporating polar functional groups within the C-ring component. Interestingly,
both sulfoxide and sulfone oxidation states (22, 24, 25) showed higher affinity to Mcl-1 than
their parent sulfides (19 or 23). These observed trends in the SAR for the C-ring moiety were
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hypothesized to be advantageous for modulating the drug-like properties of ligands. For
example, pharmaceutical agents containing a sulfone functional group generally exhibit more
desirable properties, such as higher aqueous solubility and better metabolic stability, than the
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corresponding sulfide. Compound 25, the most potent Mcl-1 inhibitor (K = 61 nM) in this
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30 nM) , which contains the unsubstituted indole core lacking the C-ring moiety. These
observations strongly suggest that introduction of a proper C-ring group in the series can enhance
not only the binding affinity for Mcl-1 but also provides a pharmacophore unit which can be
utilized to optimize the pharmaceutical properties of the molecules without sacrificing the
potency of the parent ligand.
Optimization of the P2 Pocket Anchor Group. To further explore the SAR of the tricyclic
indoles, compounds 26-30 were prepared by tethering lower pocket binding units that were
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identified in our earlier work along with new chemical moieties to the thiazine containing
tricyclic indole fragment 3 using optimized four-atom linkers. Bicyclic aromatic P2 binding
groups, such as 1′-(5,6,7,8-tetrahydronaphthyl) and 1′-(4′-Cl-naphthyl) contained in compounds
26 and 27, exhibited comparable potency to the 1-naphthyl analog 19. Conversely, compound
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compared to its regioisomer 26 and the 1-naphthyl analog 19, indicating the importance of the
attachment position for optimal binding. Compound 29 which contains a 3′-Me-4′-Cl-phenyl
group displays a 240-fold improved potency over fragment 3, and compound 30 bearing a 3′,5′-
di-Me-4′-Cl-phenyl moiety exhibited the most potent inhibitory activity against Mcl-1 with a Ki
of 71 nM, representing a 720-fold improvement in affinity compared to the core unit 3.
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To further investigate the effect of C-ring variations for compounds containing the 3,5′-
di-Me-4′-Cl-phenyl group, compounds 30-33 were prepared. These inhibitors exhibited a parallel
SAR trend compared to the corresponding 1-naphthyl analogs 19-21 and 23 with a 1.5–4.3 fold
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improvement in binding affinity. The thiazepine analog 33 displayed a K value (40 nM) which
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nM), which lacks a C-ring moiety.
X-Ray structures of the Tricyclic Compounds. To guide further compound optimization, X-
ray co-crystal structures of 17 and 24 complexed with Mcl-1 were obtained (Figure 2A-C). Both
compounds 17 and 24 adopt similar binding poses as our earlier benzthiophene analog (PDB:
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HW3). In both structures, the naphthyl group penetrated into the deepest part of the
hydrophobic pocket and induced a pronounced bend in helix 4 (α4) that likely helps to anchor
the unit at this binding sub-site. In addition, the naphthyl group was positioned near F270, which
causes favorable edge-to-face CH/π aromatic interactions with the phenyl group of this residue.
The top of α4 sits close to the ligand causing the loop connecting helix 4-5 to be pulled towards
the ligand allowing the carboxylic acid group of the ligand to engage in ion pairing interactions
with the guanidinium moiety of R263.
Figure 2A-C
Interestingly, when the co-crystal structures of 17 and 24 are superimposed, the indole
cores and naphthyl groups sit in almost the same position. However, the C-ring units adopt
different binding conformations, and the larger thiazepane-oxide ring of 24 causes a pucker that
localizes parts of the ring to be closer to A227 and T266 of Mcl-1 (Figure 2A). This may
contribute to hydrophobic burial and could explain the slight improvement in binding affinity
observed for this inhibitor. Finally, the X-ray structures predict that substitutions at the 6-
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position of the indole with small hydrophobic groups could fill the space between the ligands and
helix 3 (α3) to gain additional binding affinity (Figure 2B and C).
Tricyclic Indole Core optimization by Substitutions of the B-Ring. From our X-ray
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structures and previously reported SAR , we hypothesized that a substantial increase in binding
affinity to Mcl-1 could be obtained by a substitution at the 6-position of the indole of the 6,5-
fused heterocyclic carboxylic acid core. In a previously described series of compounds, a 6-Cl-
indole addition (as exemplified in compound 1) exhibited greater than a 5-fold enhanced potency
2
8
compared to the parent (37). To determine if this trend was the same for the tricyclic series, two
6
-Cl-tricyclic indoles 34 and 35, containing piperidine and morpholine C-ring moieties
respectively, were evaluated. Indeed, these modifications resulted in a >10-fold increase in
binding affinities for both compounds, when compared to the des chloro analogs 31, 32. The
affinity gain is likely the result of filling the pocket indicated by the arrow in Figure 2 (B,C). Our
best inhibitor, 34, displayed a remarkable 36-fold higher potency over its des-Cl parent (31) and
had a K of 3 nM and LE = 0.40. These results strongly suggest that binding contributions from
i
the C-ring moiety and the 6-Cl group are additive and can even be synergistic for some
combinations.
Mcl-1 Selectivity. Excessive inhibition of Bcl-xL in platelets causes mechanism-based, dose-
24,36,37
dependent thrombocytopenia in preclinical studies
and in clinical trials of the dual Bcl-
2
4
2/Bcl-xL inhibitor ABT-263. Therefore, we sought to obtain selective Mcl-1 inhibitors that do
not inhibit Bcl-xL to avoid these undesired adverse effects. To evaluate the selectivety of our
series, binding affinities to the anti-apoptotic proteins Bcl-xL and Bcl-2 were measured in an
FPA assay (Table 2). In all cases, the tricyclic 2-indole carboxylic acids (19-35) containing the
4
-atom linker exhibited only weak affinity for both Bcl-xL (K ≥ 1.9 μM) and Bcl-2 (K ≥ 0.77
i
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μM). Our most potent 6-Cl containing Mcl-1 inhibitors 34 and 35 exhibit >1700-fold binding
selectivity over Bcl-xL and greater that 100-fold selectivity over Bcl-2. All structural
modifications on the series that enhance the affinity for Mcl-1 had relatively little effect on
improving binding to Bcl-xL or Bcl-2. As a result, the compounds became more selective
against Bcl-xL as they became more potent for Mcl-1. Mcl-1 inhibitors likely achieve selectivity
by filling the lower P2 pocket (Figure 2B) which is not present in Bcl-xL (PDB ID : 2YXJ) or
BCL-2 (PDB ID : 4LVT) ligand structures.
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Pulldown Experiment. To determine whether compound 34 could bind to cellular Mcl-1 and
38
inhibit the binding of a peptide derived from a pro-apoptotic protein , cell lysates from Human
chronic myelogenous leukemia K562 cells were incubated with biotin labeled MS-1, a peptide
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9
that binds specifically to Mcl-1. This peptide selectively pulls down cellular Mcl-1. As shown
in Figure 3, the addition of compound 34 blocks the ability of MS-1 to pulldown Mcl-1,
demonstrating that the compound binds to cellular Mcl-1 and blocks the interaction with biotin
MS-1 in a dose-dependent manner.
Conclusion
We have described the optimization of a novel series of small molecule Mcl-1 inhibitors
based on a hit obtained from an NMR-based fragment screen. NMR-based structural studies and
SAR exploration resulted in a binding affinity increase of 100-fold over the initial micromolar
fragment hit and further optimization, guided by structural data, resulted in an additional 30-fold
gain in affinity. The resulting compound binds to Mcl-1 with a K = 3 nM, is selective over Bcl-2
i
and Bcl-xL, and binds to cellular Mcl-1 to prevent binding of a biotin labeled peptide that binds
specifically to Mcl-1. An important result of this study is that the SAR knowledge learned from
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one fragment core could be rapidly translated to other related fragment hits. We were able to
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rapidly replace the core of our initial indole series with another fragment hit to obtain novel
compounds with high Mcl-1 potency and selectivity.
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While this manuscript was in review, Leverson et al published the characterization of a
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potent (K = 0.454 nM) small molecule inhibitor, A-1210477. This study emphasized the
i
exquisite potency (“subnanomolar and often low picomolar affinities”) needed to demonstrate
on-target cellular effects. Therefore, the low nanomolar compounds (e.g. 34, K = 3.0 nM)
i
described here are not expected to display unambiguous on-target cellular activity. However, this
tricyclic 2-indole carboxylic acid represents an excellent starting point for additional
optimization to achieve these goals.
EXPERIMENTAL SECTION
Chemistry
General. All NMR spectra were recorded at room temperature on a 400 MHz AMX Bruker
1
spectrometer. H chemical shifts are reported in δ values in ppm downfield with the deuterated
solvent as the internal standard. Data are reported as follows: chemical shift, multiplicity (s =
singlet, d = doublet, t = triplet, q = quartet, br = broad, m = multiplet), integration, coupling
constant (Hz). Low resolution mass spectra were obtained on an Agilent 1200 series 6140 mass
spectrometer with electrospray ionization. All samples were of ≥95% purity as analyzed by
LC−UV/vis-MS. Analytical HPLC was performed on an Agilent 1200 series with UV detection
at 214 and 254 nm along with ELSD detection. LC/MS parameters were as follows:
Phenomenex-C18 Kinetex column, 50 x 2.1 mm, 2 min gradient, 5% (0.1% TFA/MeCN) / 95%
(0.1% TFA/ H O) to 100% (0.1% TFA/MeCN). Preparative purification was performed on a
2
Gilson HPLC (Phenomenex-C18, 100 x 30 mm, 10 min gradient, 5→95% MeCN/H O with
2
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.1% TFA) or by automated flash column chromatography (Isco, Inc. 100sg Combiflash).
Solvents for extraction, washing, and chromatography were HPLC grade. All reagents were
purchased from chemical suppliers and used without purification.
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-Methyl-2,3-dihydro-[1,4]thiazino[2,3,4-hi]indole-5-carboxylic acid (5). General Procedure
for the Fischer Indole Reaction. To a stirred solution of 2,3-dihydro-4H-benzo[b][1,4]thiazin-
-amine (83 mg, 0.50 mmol) in absolute EtOH (2 mL) was added methyl 2-oxobutanoate (58
4
mg, 0.50 mmol) at 20 °C. The reaction mixture was stirred for 30 min at 50 °C then cooled to 0
°C. Concentrated H SO (0.2 mL) was added dropwise then the reaction mixture was warmed to
2
4
5
0 °C and stirred for an additional 4 h. The reaction mixture was cooled to 20 °C then
concentrated in vacuo. The residue was dissolved in CH Cl (5 mL), and the organic solution
2
2
was washed with saturated aqueous NaHCO solution followed by brine, dried over MgSO ,
3
4
filtered, and concentrated in vacuo. The residue was dissolved in 1:1 mixture of MeOH/THF (2
mL) then KOH (280 mg, 5.0 mmol) was added. The reaction mixture was stirred for 2 h at 50
°
C then concentrated in vacuo. The residue was dissolved in H O then acidified to pH 1 with
2
concentrated HCl. The mixture was extracted with EtOAc, dried over MgSO , filtered, and
4
concentrated in vacuo. The crude product was purified by reverse phase preparative HPLC
(
H O/CH CN gradient to 95% CH CN/0.1% TFA) to yield the title compound (103 mg, 0.44
2 3 3
1
mmol) as a white solid. HNMR(400 MHz, DMSO-d ): δ (ppm) 7.45 (d, J = 8.0 Hz, 1 H), 7.09
6
(d, J = 7.7 Hz, 1H), 7.02 (dd, J = 8.0, 7.7 Hz, 1 H), 4.73-4.71 (m, 2 H), 3.32-3.30 (m, 2 H), 2.52
+
(s, 3 H): >98% at 215 nm, MS (ESI) m/z = 234.1 [M + H] .
1-Methyl-5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinoline-2-carboxylic acid (6). The general
procedure for Fischer indole reaction was followed using 3,4-dihydroquinolin-1(2H)-amine (74
mg, 0.50 mmol) and methyl 2-oxobutanoate (58 mg, 0.50 mmol) to yield the title compound (97
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mg, 0.45mmol). HNMR(400 MHz, DMSO-d ): δ (ppm) 7.45 (dd, J = 6.6, 2.6 Hz, 1 H), 7.01-
6
6
.96 (m, 2 H), 4.43 (t, J = 5.7 Hz, 2 H), 2.91 (t, J = 6.1 Hz, 2 H), 2.52 (s, 3 H), 2.15-2.09 (m, 2
+
H); >98% at 215 nm, MS (ESI) m/z = 216.1 [M + H] .
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6
-Methyl-2,3-dihydro-[1,4]oxazino[2,3,4-hi]indole-5-carboxylic acid (7). The general
procedure for Fischer indole reaction was followed using 2,3-dihydro-4H-benzo[b][1,4]oxazin-4-
amine (100 mg, 0.66 mmol) and methyl 2-oxobutanoate (85 mg, 0.73 mmol) to yield the title
1
compound (81 mg, 0.37mmol). HNMR(400 MHz, DMSO-d ): δ (ppm) 7.20 (d, J = 8.0 Hz, 1
6
H), 6.95 (dd, J = 8.0, 7.6 Hz, 1H), 6.67 (d, J = 7.6 Hz, 1 H), 4.52-4.47 (m, 4 H), 2.52 (s, 3 H).;
+
>
98% at 215 nm, MS (ESI) m/z = 218.1 [M + H] .
6
-(2-(Naphthalen-1-yloxy)ethyl)-2,3-dihydro-[1,4]thiazino[2,3,4-hi]indole-5-carboxylic acid
16). General Procedure. A solution of 2,3-dihydro-4H-benzo[b][1,4]thiazin-4-amine (336 mg,
.0 mmol) and 2-oxopentanedioic acid (325 mg, 2.2 mmol) in EtOH (5 mL) was stirred at 50 °C
for 30 min then cooled to 0 °C. To the reaction mixture was added conc. H SO (0.5 mL)
(
2
2
4
dropwise at 0 °C. The reaction mixture was stirred for 2h at 60 °C then quenched by pouring
into ice then extracted with CH Cl . The combined organic layer was washed with sat. NaHCO ,
2
2
3
water, brine, dried over MgSO , filtered and concentrated in vacuo. The residue was purified by
4
filtering through a silica gel column using Et O as an eluent. The filtrate was concentrated in
2
vacuo to give 1:1 mixture of 6-(2-ethoxy-2-oxoethyl)-2,3-dihydro-[1,4]thiazino[2,3,4-hi]indole-
5
-carboxylic acid and ethyl 6-(2-ethoxy-2-oxoethyl)-2,3-dihydro-[1,4]thiazino[2,3,4-hi]indole-5-
carboxylate as an off-white solid in 145 mg. This mixture of products was directly used for the
subsequent step without further purification.
To a solution of a mixture of products (145 mg) from the previous step in MeOH and
benzene mixture (1:10, 2.0 mL) was added TMSCHN (2M in hexane) until bubbling stopped
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and a yellow color of the reaction mixture persisted. The reaction mixture was stirred for
additional 10 min at 20 °C then concentrated in vacuo to give a mixture of methyl and ethyl 6-(2-
ethoxy-2-oxoethyl)-2,3-dihydro-[1,4]thiazino[2,3,4-hi]indole-5-carboxylate. They were directly
used for the subsequent step without further purification.
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To a solution of a mixture of methyl and ethyl 6-(2-ethoxy-2-oxoethyl)-2,3-dihydro-
[1,4]thiazino[2,3,4-hi]indole-5-carboxylate from the previous step in THF (2.0 mL) was added
BH in THF (2 mL, 2 mmol) at 20 °C. The reaction mixture was stirred for 5h at 20 °C and
3
quenched by addition of MeOH then concentrated in vacuo. The residue was purified by flash
chromatography (Combi-flash Rf Hexane/EtOAc gradient 0-100%) to give a mixture of methyl
and ethyl 6-(2-hydroxyethyl)-2,3-dihydro-[1,4]thiazino[2,3,4-hi]indole-5-carboxylate as a
colorless oil in 110 mg (0.40 mmol).
To a solution of a mixture of methyl and ethyl 6-(2-hydroxyethyl)-2,3-dihydro-
[
1,4]thiazino[2,3,4-hi]indole-5-carboxylate (50 mg, 0.18 mmol), PPh (71 mg, 0.27 mmol) and
3
naphthalen-1-ol (44 mg, 0.28mmol) in THF (1.0 mL) was added Dt-BuAD (62 mg, 0.27 mmol)
at 20 °C. The reaction mixture was stirred for 15h at 20 °C. To the reaction mixture was added
KOH (101 mg, 10 mmol) and MeOH (1.0 mL). The reaction mixture was stirred for 2h at 65 °C
then concentrated in vacuo. The crude product was purified by reverse phase preparative HPLC
(
Phenomenex Gemini C18, H O/CH CN gradient to 95% CH CN 0.1% TFA) to yield the title
2 3 3
1
compound (43 mg, 0.11 mmol) as a light yellow solid. HNMR(400 MHz, DMSO-d ): δ 8.07 (d,
6
J = 8.4 Hz, 1 H), 7.82(d, J = 8.0, Hz, 1H), 7.68 (dd, J = 7.4, 1.6 Hz, 1H), 7.49 (dd, J = 6.9, 1.6
Hz, 1H), 7.47-7.34 (comp, 3H), 7.14-7.08 (comp, 2H), 6.96 (d, J = 7.3 Hz, 1H), 4.77-4.74 (m,
2H), 4.36 (t, J = 6.6 Hz, 2H), 3.68 (t, J = 6.7 Hz, 2H), 3.35-3.10 (m, 2H); >98% at 215 nm, MS
+
(
ESI) m/z = 390.1 [M + H] .
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1
-(2-(Naphthalen-1-yloxy)ethyl)-5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinoline-2-carboxylic
acid (17). The title compound was prepared as a white solid according to procedures described
for preparing compound 16 by substituting 2,3-dihydro-4H-benzo[b][1,4]thiazin-4-amine with
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1
3
,4-dihydroquinolin-1(2H)-amine in 23% overall yield. H NMR (400 MHz, DMSO-d ) δ 8.10
6
(d, J = 8.3 Hz, 1H), 7.83 (d, J = 8.1 Hz, 1H), 7.67 (dd, J = 7.7, 1.5 Hz, 1H), 7.49 (ddd, J = 8.2,
.8, 1.3 Hz, 1H), 7.45 – 7.31 (m, 3H), 7.10 – 7.00 (m, 2H), 6.96 (d, J = 7.5 Hz, 1H), 4.50 – 4.42
6
(
m, 2H), 4.35 (t, J = 6.7 Hz, 2H), 3.67 (t, J = 6.7 Hz, 2H), 2.92 (t, J = 6.1 Hz, 2H), 2.18 – 2.04
+
(m, 2H); >98% at 215 nm, MS (ESI) m/z = 372.2 [M + H] .
6
-(2-(Naphthalen-1-yloxy)ethyl)-2,3-dihydro-[1,4]oxazino[2,3,4-hi]indole-5-carboxylic acid
(18). The title compound was prepared as a white solid according to procedures described for
preparing compound 16 by substituting 2,3-dihydro-4H-benzo[b][1,4]thiazin-4-amine with 2,3-
1
dihydro-4H-benzo[b][1,4]oxazin-4-amin in 18% overall yield. H NMR (400 MHz, DMSO-d ) δ
6
8.09 (d, J = 8.1 Hz, 1H), 7.83 (d, J = 8.1 Hz, 1H), 7.51 - 7.46 (m, 1H), 7.45 - 7.32 (m, 4H), 7.02
(
t, J = 7.9 Hz, 1H), 6.96 (d, J = 7.5 Hz, 1H), 6.71 (d, J = 7.3 Hz, 1H), 4.57 (t, J = 4.7 Hz, 2H),
4
.48 (t, J = 4.6 Hz, 2H), 4.37 (t, J = 6.8 Hz, 2H), 3.67 (t, J = 6.7 Hz, 2H); >98% at 215 nm, MS
+
(ESI) m/z = 374.1 [M + H] .
6
-(3-(Naphthalen-1-yloxy)propyl)-2,3-dihydro-[1,4]thiazino[2,3,4-hi]indole-5-carboxylic
acid (19). A mixture of methyl and ethyl 6-(3-hydroxypropyl)-2,3-dihydro-[1,4]thiazino[2,3,4-
hi]indole-5-carboxylate (465 mg, 1.6 mmol) was prepared as a yellow oil according to
procedures described for preparing compound 16 using 2,3-dihydro-4H-benzo[b][1,4]thiazin-4-
amine (500 mg, 3.0 mmol) and 2-oxohexanedioic acid (722 mg, 4.5 mmol) in 53% overall yield.
The title compound was prepared (25 mg, 0.062 mmol) as an off-white solid following
the Mitsunobu reaction procedure described for preparing compound 16 using a mixture of
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methyl and ethyl 6-(3-hydroxypropyl)-2,3-dihydro-[1,4]thiazino[2,3,4-hi]indole-5-carboxylate
1
(
56 mg, 0.2 mmol). H NMR (400 MHz, DMSO-d ) δ 8.21 – 8.15 (m, 1H), 7.90 – 7.82 (m, 1H),
6
7
.57 – 7.34 (m, 5H), 7.06 (d, J = 7.2 Hz, 1H), 6.97 – 6.84 (m, 2H), 4.77 – 4.67 (m, 2H), 4.17 (t, J
0
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6.0 Hz, 2H), 3.32 – 3.28 (m, 4H), 2.25 – 2.14 (m, 2H); >98% at 215 nm, MS (ESI) m/z = 404.1
+
[M + H] .
1
-(3-(Naphthalen-1-yloxy)propyl)-5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinoline-2-carboxylic
acid (20). A mixture of methyl and ethyl 1-(3-hydroxypropyl)-5,6-dihydro-4H-pyrrolo[3,2,1-
ij]quinoline-2-carboxylate (72 mg, 0.25 mmol) was prepared as a pale yellow oil according to
procedures described for preparing compound 16 using 3,4-dihydroquinolin-1(2H)-amine (74
mg, 0.50 mmol) and 2-oxohexanedioic acid (160 mg, 1.0 mmol) in 50% overall yield.
The title compound was prepared (20 mg, 0.052 mmol) as an off-white solid following
the Mitsunobu reaction procedure described for preparing compound 16 using a mixture of
methyl and ethyl 1-(3-hydroxypropyl)-5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinoline-2-carboxylate
1
(55 mg, 0.2 mmol). H NMR (400 MHz, DMSO-d ) δ 8.24 – 8.17 (m, 1H), 7.89 – 7.85 (m, 1H),
6
7
–
2
.56 – 7.41 (m, 4H), 7.38 (t, J = 7.9 Hz, 1H), 6.97 (d, J = 6.9 Hz, 1H), 6.91 – 6.84 (m, 2H), 4.48
4.40 (m, 2H), 4.16 (t, J = 6.1 Hz, 2H), 3.32 – 3.30 (m, 2H), 2.91 (t, J = 6.1 Hz, 2H), 2.25 –
+
.06 (m, 4H); >98% at 215 nm, MS (ESI) m/z = 386.2 [M + H] .
6
-(3-(Naphthalen-1-yloxy)propyl)-2,3-dihydro-[1,4]oxazino[2,3,4-hi]indole-5-carboxylic
acid (21). A mixture of methyl and ethyl 6-(3-hydroxypropyl)-2,3-dihydro-[1,4]oxazino[2,3,4-
hi]indole-5-carboxylate (375 mg, 1.3 mmol) was prepared as a pale yellow oil according to
procedures described for preparing compound 16 using 2,3-dihydro-4H-benzo[b][1,4]oxazin-4-
amin (430 mg, 2.90 mmol) and 2-oxohexanedioic acid (595 mg, 3.7 mmol) in 45% overall yield.
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The title compound was prepared (55 mg, 0.15 mmol) as an off-white solid following the
Mitsunobu reaction procedure described for preparing compound 16 using a mixture of methyl
and ethyl 6-(3-hydroxypropyl)-2,3-dihydro-[1,4]oxazino[2,3,4-hi]indole-5-carboxylate (75 mg,
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51
52
53
54
55
56
57
58
59
60
1
0
.27 mmol). H NMR (400 MHz, DMSO-d ) δ 8.18 (dd, J = 7.7, 1.8 Hz, 1H), 7.86 (dd, J = 7.4,
6
1.9 Hz, 1H), 7.56 - 7.42 (m, 3H), 7.38 (dd, J = 8.0, 7.6 Hz, 1H), 7.22 (d, J = 8.0 Hz, 1H), 6.91 -
.82 (m, 2H), 6.65 (d, J = 7.4 Hz, 1H), 4.54 (t, J = 4.6 Hz, 2H), 4.47 (t, J = 4.8 Hz, 2H), 4.17 (t, J
6
=
6.0 Hz, 2H), 3.36 - 3.31 (m, 2H), 2.26 - 2.17 (m, 2H); >98% at 215 nm, MS (ESI) m/z = 388.1
+
[M + H] .
6
-(3-(naphthalen-1-yloxy)propyl)-2,3-dihydro-[1,4]thiazino[2,3,4-hi]indole-5-carboxylic
acid 1,1-dioxide (22). To a solution of 6-(3-(naphthalen-1-yloxy)propyl)-2,3-dihydro-
[1,4]thiazino[2,3,4-hi]indole-5-carboxylic acid (20 mg, 0.050 mmol) in CH Cl (1.0 mL) and
THF (1.0 mL) was added m-CPBA (17 mg (77% pure), 1.0 mmol) at 20 °C. The reaction
mixture was stirred for 1 h at 20 °C then quenched by addition of H O, extracted with CH Cl
and concentrated in vacuo. The residue was purified by reverse-phase preparative HPLC
2
2
2
2
2
(
Phenomenex Gemini C18, H
2
O/CH
3
3
CN gradient to 25-95% CH CN 0.1% TFA) to give the title
1
compound (12 mg, 0.028 mmol) as a white solid. H NMR (400 MHz, DMSO-d ) δ 8.13 (dd, J =
6
8
7
.6, 1.8 Hz, 1H), 8.03 (d, J = 8.1 Hz, 1H), 7.89 - 7.83 (m, 1H), 7.72 (d, J = 7.4 Hz, 1H), 7.58 -
.42 (m, 3H), 7.41 - 7.33 (m, 1H), 7.21 (t, J = 7.8 Hz, 1H), 6.88 (d, J = 7.3 Hz, 1H), 5.09 - 4.93
(m, 2H), 4.17 (t, J = 6.0 Hz, 2H), 3.91 - 3.80 (m, 2H), 3.41 - 3.35 (m, 2H), 2.29 - 2.14 (m, 2H);
+
>
98% at 215 nm, MS (ESI) m/z = 436.1 [M + H] .
7
-(3-(Naphthalen-1-yloxy)propyl)-3,4-dihydro-2H-[1,4]thiazepino[2,3,4-hi]indole-6-
carboxylic acid (23). A mixture of methyl and ethyl 7-(3-hydroxypropyl)-3,4-dihydro-2H-
[
1,4]thiazepino[2,3,4-hi]indole-6-carboxylate (211 mg, 0.72 mmol) was prepared as a pale
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6
yellow oil according to procedures described for preparing compound 16 using 2,3-dihydro-4H-
benzo[b][1,4]oxazin-4-amin (261 mg, 1.45 mmol) and 2-oxohexanedioic acid (301 mg, 1.88
mmol) in 50% overall yield.
0
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The title compound was prepared (76 mg, 0.18 mmol) as a white solid following the
Mitsunobu reaction procedure described for preparing compound 16 using a mixture of methyl
and ethyl 7-(3-hydroxypropyl)-3,4-dihydro-2H-[1,4]thiazepino[2,3,4-hi]indole-6-carboxylate (63
1
6
mg, 0.20 mmol). H NMR (400 MHz, DMSO-d ) δ 8.18 (dd, J = 7.6, 1.9 Hz, 1H), 7.86 (dd, J =
7.3, 2.0 Hz, 1H), 7.61 - 7.42 (m, 4H), 7.38 (dd, J = 8.0, 7.8 Hz, 1H), 7.10 (d, J = 7.3 Hz, 1H),
6
.86 (dd, J = 8.0, 7.7 Hz, 2H), 4.86 (t, J = 5.8 Hz, 2H), 4.16 (t, J = 6.0 Hz, 2H), 3.34 (t, J = 6.6
Hz, 2H), 3.24 (t, J = 6.6 Hz, 2H), 2.34 - 2.23 (m, 2H), 2.22 - 2.13 (m, 2H); >98% at 215 nm, MS
+
(ESI) m/z = 418.1 [M + H] .
7
-(3-(Naphthalen-1-yloxy)propyl)-3,4-dihydro-2H-[1,4]thiazepino[2,3,4-hi]indole-6-
carboxylic acid 1-oxide (24). To a cooled (-78 °C) solution of 7-(3-(naphthalen-1-
yloxy)propyl)-3,4-dihydro-2H-[1,4]thiazepino[2,3,4-hi]indole-6-carboxylic acid (35 mg, 0.084
mmol) in THF/CH Cl (1 mL, 1:1) was added m-CPBA (19 mg, 0.084 mmol), and the mixture
2
2
was stirred for 1 h at -78 °C. The reaction was quenched by addition of saturated aqueous
NaHCO solution (0.5 mL) followed by H O (12 mL). The quenched reaction mixture was
3
2
extracted with CH Cl (2x10 mL), dried over Na SO , filtered and concentrated in vacuo. The
2
2
2
4
residue was purified by reverse-phase preparative HPLC (Phenomenex Gemini C18,
H O/CH CN gradient to 30-80% CH CN 0.1% TFA) to give the title compound (33 mg, 0.076
2
3
3
1
6
mmol) as a white amorphous solid. H NMR (400 MHz, DMSO-d ) δ 8.15 (dd, J = 7.6, 1.7 Hz,
1H), 7.91 (d, J = 8.0 Hz, 1H), 7.86 (dd, J = 7.5, 1.7 Hz, 1H), 7.61 (d, J = 7.3 Hz, 1H), 7.57 - 7.42
(m, 3H), 7.38 (dd, J = 8.0, 7.6 Hz, 1H), 7.16 (dd, J = 7.8, 7.6 Hz, 1H), 6.88 (d, J = 7.4, 1H), 4.73
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(
dt, J = 9.1, 4.2 Hz, 1H), 4.64 (dt, J = 9.1, 4.2 Hz, 1H), 4.18 (t, J = 5.9 Hz, 2H), 3.55 - 3.48 (m,
2
H), 3.39 - 3.24 (m, 2H), 3.05 (dt, J = 7.3, 6.8 Hz, 1H), 2.73 - 2.57 (m, 1H), 2.29 - 2.13 (m, 2H);
+
>
98% at 215 nm, MS (ESI) m/z = 434.1 [M + H] .
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16
17
18
19
20
21
22
23
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26
27
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32
33
34
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60
7
-(3-(Naphthalen-1-yloxy)propyl)-3,4-dihydro-2H-[1,4]thiazepino[2,3,4-hi]indole-6-
carboxylic acid 1,1-dioxide (25). To a cooled solution (0 °C) of 7-(3-(naphthalen-1-
yloxy)propyl)-3,4-dihydro-2H-[1,4]thiazepino[2,3,4-hi]indole-6-carboxylic acid (15 mg, 0.036
mmol) in CH Cl (1 mL) was added m-CPBA (18 mg, 0.072 mmol), and the mixture was stirred
2
2
for 2 h at 0 °C. The reaction was quenched by addition of saturated aqueous NaHCO solution (2
3
mL). The quenched reaction mixture was extracted with CH Cl (3x10 mL), dried over Na SO ,
2
2
2
4
filtered and concentrated in vacuo. The residue was purified by reverse-phase preparative HPLC
(Phenomenex Gemini C18, H O/CH CN gradient to 30-85% CH CN 0.1% TFA) to give the title
2
3
3
1
compound (14 mg, 0.031 mmol) as an off-white amorphous solid. H NMR (400 MHz, DMSO-
6
d ) δ 8.14 (dd, J = 7.8, 1.5 Hz, 1H), 8.06 (d, J = 8.0 Hz, 1H), 7.86 (dd, J = 7.4, 1.6 Hz, 1H), 7.82
(dd, J = 7.5, 1.0 Hz, 1H), 7.59 - 7.42 (m, 3H), 7.38 (dd, J = 8.1, 7.7 Hz, 1H), 7.22 (dd, J = 7.8,
7
.6 Hz, 1H), 6.88 (d, J = 7.2 Hz, 1H), 4.89 - 4.68 (m, 2H), 4.18 (t, J = 5.9 Hz, 2H), 3.77 (t, J =
6.8 Hz, 2H), 3.33 (t, J = 7.1 Hz, 2H), 2.42 - 2.28 (m, 2H), 2.27 - 2.12 (m, 2H); >98% at 215 nm,
+
MS (ESI) m/z = 450.1 [M + H] .
Compounds 26−33 were prepared following the general procedure outlined above in
library format. Purity of all final compounds was determined by HPLC analysis and is >95%. All
compounds were isolated as solids.
6
-(3-((5,6,7,8-Tetrahydronaphthalen-1-yl)oxy)propyl)-2,3-dihydro-[1,4]thiazino[2,3,4-
hi]indole-5-carboxylic acid (26). Coupling of a mixture of methyl and ethyl 6-(3-
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6
hydroxypropyl)-2,3-dihydro-[1,4]thiazino[2,3,4-hi]indole-5-carboxylate
and
5,6,7,8-
tetrahydronaphthalen-1-ol followed by saponification yielded 26. >98% at 215 nm, MS (ESI)
+
m/z = 408.2 [M + H] .
0
1
2
3
4
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9
0
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0
6
-(3-((4-Chloronaphthalen-1-yl)oxy)propyl)-2,3-dihydro-[1,4]thiazino[2,3,4-hi]indole-5-
carboxylic acid (27). Coupling of a mixture of methyl and ethyl 6-(3-hydroxypropyl)-2,3-
dihydro-[1,4]thiazino[2,3,4-hi]indole-5-carboxylate and 4-chloronaphthalen-1-ol followed by
+
saponification yielded 27. >98% at 215 nm, MS (ESI) m/z = 438.1 [M + H] .
6-(3-((5,6,7,8-Tetrahydronaphthalen-2-yl)oxy)propyl)-2,3-dihydro-[1,4]thiazino[2,3,4-
hi]indole-5-carboxylic acid (28). Coupling of a mixture of methyl and ethyl 6-(3-
hydroxypropyl)-2,3-dihydro-[1,4]thiazino[2,3,4-hi]indole-5-carboxylate
and
5,6,7,8-
tetrahydronaphthalen-2-ol followed by saponification yielded 28. >98% at 215 nm, MS (ESI)
+
m/z = 408.1 [M + H] .
6
-(3-(4-Chloro-3-methylphenoxy)propyl)-2,3-dihydro-[1,4]thiazino[2,3,4-hi]indole-5-
carboxylic acid (29). Coupling of a mixture of methyl and ethyl 6-(3-hydroxypropyl)-2,3-
dihydro-[1,4]thiazino[2,3,4-hi]indole-5-carboxylate and 4-chloro-3-methylphenol followed by
+
saponification yielded 29. >98% at 215 nm, MS (ESI) m/z = 402.1 [M + H] .
6-(3-(4-chloro-3,5-dimethylphenoxy)propyl)-2,3-dihydro-[1,4]thiazino[2,3,4-hi]indole-5-
carboxylic acid (30). Coupling of a mixture of methyl and ethyl 6-(3-hydroxypropyl)-2,3-
dihydro-[1,4]thiazino[2,3,4-hi]indole-5-carboxylate and 4-chloro-3,5-dimethylphenol followed
+
by saponification yielded 30. >98% at 215 nm, MS (ESI) m/z = 416.1 [M + H] .
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1
-(3-(4-Chloro-3,5-dimethylphenoxy)propyl)-5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinoline-2-
carboxylic acid (31). Coupling of a mixture of methyl and ethyl 1-(3-hydroxypropyl)-5,6-
dihydro-4H-pyrrolo[3,2,1-ij]quinoline-2-carboxylate and 4-chloro-3,5-dimethylphenol followed
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
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44
45
46
47
48
49
50
51
52
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57
58
59
60
1
6
by saponification yielded 31. H NMR (400 MHz, DMSO-d ) δ 7.43 (d, J = 7.6 Hz, 1H), 7.00 -
6.90 (m, 2H), 6.74 (s, 2H), 4.43 (t, J = 5.6 Hz, 2H), 3.92 (t, J = 6.4 Hz, 2H), 3.17 (t, J = 7.2 Hz,
2
H), 2.91 (t, J = 5.9 Hz, 2H), 2.27 (s, 6H), 2.17 - 2.08 (m, 2H), 2.07 - 1.96 (m, 2H); >98% at 215
+
nm, MS (ESI) m/z = 398.2 [M + H] .
6-(3-(4-Chloro-3,5-dimethylphenoxy)propyl)-2,3-dihydro-[1,4]oxazino[2,3,4-hi]indole-5-
carboxylic acid (32). Coupling of a mixture of methyl and ethyl 6-(3-hydroxypropyl)-2,3-
dihydro-[1,4]oxazino[2,3,4-hi]indole-5-carboxylate and 4-chloro-3,5-dimethylphenol followed
+
by saponification yielded 32. >98% at 215 nm, MS (ESI) m/z = 400.1 [M + H] .
7
-(3-(4-Chloro-3,5-dimethylphenoxy)propyl)-3,4-dihydro-2H-[1,4]thiazepino[2,3,4-
hi]indole-6-carboxylic acid (33). Coupling of a mixture of methyl and ethyl 7-(3-
hydroxypropyl)-3,4-dihydro-2H-[1,4]thiazepino[2,3,4-hi]indole-6-carboxylate and 4-chloro-3,5-
1
dimethylphenol followed by saponification yielded 33. H NMR (400 MHz, DMSO-d ) δ 7.46
6
(d, J = 8.0 Hz, 1H), 7.13 (d, J = 7.2 Hz, 1H), 6.91 (t, J = 7.6 Hz, 1H), 6.75 (s, 2H), 4.90 – 4.81
(
m, 2H), 3.94 (t, J = 6.3 Hz, 2H), 3.39 – 3.34 (m, 2H), 3.13 – 3.07 (m, 2H), 2.34 – 2.22 (m, 8H),
+
2
.04 – 1.95 (m, 2H); >98% at 215 nm, MS (ESI) m/z = 430.1 [M + H] .
7
-chloro-1-(3-(4-chloro-3,5-dimethylphenoxy)propyl)-5,6-dihydro-4H-pyrrolo[3,2,1-
ij]quinoline-2-carboxylic acid (34). To a stirring mixture of 2-bromo-3-chloroaniline (4.1 g, 20
mmol) in 1M HCl (25 mL) and water (5 mL) at 0 °C was added NaNO (1.38 g, 20 mmol) in
2
water (20 mL), NaCH COOH (9.23 g, 112 mmol) in water (25 mL) and ethyl 2-oxocyclopentane
3
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6
carboxylate (3.0 mL, 20 mmol) in sequence. The reaction mixture was stirred for 15 min at 0 °C
then warmed to 20 °C over 2h and extracted with CH Cl , dried over MgSO , filtered and
2
2
4
concentrated in vacuo to give 5-(2-(2-bromo-3-chlorophenyl)hydrazono)-6-ethoxy-6-
oxohexanoic acid as a red oil in 7.0 g (90% crude).
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0
To a solution of 5-(2-(2-bromo-3-chlorophenyl)hydrazono)-6-ethoxy-6-oxohexanoic acid
(
7.0 g, 18 mmol) in EtOH (30 mL) was added conc. H SO (7.5 mL), slowly. The reaction
2 4
mixture was refluxed for 1.5 h. The reaction was quenched by pouring into ice then extracted
with CH Cl . The combined organic layer was washed with saturated aqueous NaHCO solution,
2
2
3
H O, brine, dried over MgSO , filtered and concentrated in vacuo. The residue was purified by
2
4
flash chromatography (Combi-flash Rf Hex/EtOAc 0-25% gradient) to give ethyl 7-bromo-6-
chloro-3-(3-ethoxy-3-oxopropyl)-1H-indole-2-carboxylate (4.8 g, 12 mmol) as an off-white
+
solid. MS (ESI) m/z = 402.0 [M + H] .
To a solution of ethyl 7-bromo-6-chloro-3-(3-ethoxy-3-oxopropyl)-1H-indole-2-
carboxylate (2.0 g, 4.8 mmol) in THF (20 mmol) was added BH in THF (20 mL, 20 mmol) at 20
3
°C. The reaction mixture was stirred for 15h at 20 °C and quenched by addition of MeOH then
concentrated in vacuo. The residue was purified by flash chromatography (Combi-flash Rf
Hexane/EtOAc gradient 0-50%) to give ethyl 7-bromo-6-chloro-3-(3-hydroxypropyl)-1H-indole-
+
2
-carboxylate (1.44 g, 4.0 mmol) as a white solid. MS (ESI) m/z = 360.0 [M + H] .
To a solution of ethyl 7-bromo-6-chloro-3-(3-hydroxypropyl)-1H-indole-2-carboxylate
(
1.0 g, 0.28 mmol), PPh (1.1 g, 5.1 mmol) and 3,5-diMe-4-Cl-phenol (810 g, 5.2 mmol) in THF
3
(35 mL) was added Dt-BuAD (990 mg, 5.1 mmol) at 20 °C. The reaction mixture was stirred for
1
5h at 20 °C then concentrated in vacuo. The residue was purified by flash chromatography
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(
Combi-flash Rf Hexane/EtOAc gradient 0-10%) to give ethyl 7-bromo-6-chloro-3-(3-(4-chloro-
3
,5-dimethylphenoxy)propyl)-1H-indole-2-carboxylate (1.15 g, 2.3 mmol) as a white solid. MS
+
(ESI) m/z = 498.0 [M + H] .
10
11
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To A solution of ethyl 7-bromo-6-chloro-3-(3-(4-chloro-3,5-dimethylphenoxy)propyl)-
1
H-indole-2-carboxylate (100 mg, 0.20 mmol) and 3-bromoprop-1-ene (0.026 mL, 0.30 mmol)
in DMF (1.5 mL) was added Cs CO (196 mg, 0.60 mmol), and the mixture was stirred for 3 h at
2
3
8
0 °C. The reaction mixture was cooled to 20 °C then concentrated in vacuo. The residue was
purified by flash chromatography (Combi-flash Rf Hexane/EtOAc gradient 0-70%) to give ethyl
1
-allyl-7-bromo-6-chloro-3-(3-(4-chloro-3,5-dimethylphenoxy)propyl)-1H-indole-2-carboxylate
+
(
105 mg 0.19 mmol) as a yellow oil. MS (ESI) m/z = 538.1 [M + H] .
To
a
solution
of
ethyl
1-allyl-7-bromo-6-chloro-3-(3-(4-chloro-3,5-
dimethylphenoxy)propyl)-1H-indole-2-carboxylate (105 mg, 0.19 mmol) and Bu SnH (0.105 ml,
3
0
.39 mmol) in toluene (1.0 mL) was added AIBN (1.6 mg, 9.7 µmol), and the mixture was
stirred for 90 min at 100 °C. The reaction mixture was cooled to 20 °C then concentrated in
vacuo. The residue was dissolved in THF (1 mL) and LiOH (0.5 mL, 2N) was added. The
reaction mixture was stirred for 24 h at 40 °C then 20 °C then concentrated in vacuo. The
residue was purified by reverse-phase preparative HPLC (Phenomenex Gemini C18,
H O/CH CN gradient to 60-95% CH CN 0.1% TFA) to give the title compound (41 mg, 0.095
2
3
3
1
mmol) as a white solid. H NMR (400 MHz, DMSO-d ) δ 7.48 (d, J = 8.0 Hz, 1 H), 7.00 (d, J =
6
8
2
.0 Hz, 1 H), 6.73 (s, 2 H), 4.42 (t, J = 6.0 Hz, 2 H), 3.91 (t, J = 6.0 Hz, 2 H), 3.15 (t, J = 6.0 Hz,
H), 2.90 (t, J = 6.0 Hz, 2 H), 2.27 (s, 6 H), 2.15 (m, 2 H), 2.00 (m, 2 H); >98% at 215 nm, MS
+
(ESI) m/z = 432.1 [M + H] .
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9
-Chloro-6-(3-(4-chloro-3,5-dimethylphenoxy)propyl)-2,3-dihydro-[1,4]oxazino[2,3,4-
hi]indole-5-carboxylic acid (35). A solution of ethyl 7-bromo-6-chloro-3-(3-(4-chloro-3,5-
dimethylphenoxy)propyl)-1H-indole-2-carboxylate (250 mg, 0.50 mmol) and
0
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bis(pinacolato)diboron (153 mg, 0.60 mmol) in DMF (2.5 mL) was degased at 20 °C then
potassium acetate (226 mg, 2.3 mmol) and 1,1'-bis(diphenylphosphino)ferrocenedichloro
palladium(ii) dichloromethane complex (18.32 mg, 0.025 mmol) was added. The reaction
mixture was stirred for 20 h at 90 °C under Ar then cooled to 20 °C and diluted with Et O (100
2
mL). The combined organic solution was washed with H O (4 x 10 mL), brine (10 mL), dried
2
over MgSO , filtered and concentrated in vacuo. The residue was purified by flash
4
chromatography (Combi-flash Rf Hexane/EtOAc gradient 0-15%) to give ethyl 6-chloro-3-(3-(4-
chloro-3,5-dimethylphenoxy)propyl)-7-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-indole-
+
2
-carboxylate (115 mg, 0.21 mmol) as a white solid. MS (ESI) m/z = 546.0 [M + H] .
To a solution of ethyl 6-chloro-3-(3-(4-chloro-3,5-dimethylphenoxy)propyl)-7-(4,4,5,5-
tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-indole-2-carboxylate (63 mg, 0.12 mmol) in THF (1.2
mL) was added 0.5 M NaOH aqueous solution (1.2 mL, 0.60 mmol) followed by 30% H O (118
2
2
µL, 1.2 mmol) at 20 °C. The reaction mixture was stirred for 15 h at 20 °C then quenched with
sat NH Cl/NH OH buffer solution. The mixture was extracted with EtOAc (2 x 10 mL), dried
4
4
over MgSO , filtered and concentrated in vacuo. The residue was purified by reverse-phase
4
preparative HPLC (Phenomenex Gemini C18, H O/CH CN gradient to 5-65% CH CN 0.1%
2
3
3
TFA) to give ethyl 6-chloro-3-(3-(4-chloro-3,5-dimethylphenoxy)propyl)-7-hydroxy-1H-indole-
+
2
-carboxylate (15 mg, 0.034 mmol). MS (ESI) m/z = 436.0 [M + H] .
A mixture of ethyl 6-chloro-3-(3-(4-chloro-3,5-dimethylphenoxy)propyl)-7-hydroxy-1H-
indole-2-carboxylate (10 mg, 0.023 mmol), cesium carbonate (37 mg, 0.12 mmol) and
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dibromoethane (4.0 µL, 0.046 mmol) in DMF (460 µL) was stirred at 100 °C for 15 h. The
mixture was cooled to 20 °C, filtered through a hydrophobic frit and concentrated in vacuo. The
residue was dissolved in THF (230 µL) and 2N aqueous LiOH solution (115 µL, 0.23 mmol) was
added. The reaction mixture was stirred for 15 h 20 °C, acidified with TFA and concentrated in
vacuo. The residue was purified by reverse-phase preparative HPLC (Phenomenex Gemini C18,
H O/CH CN gradient to 50-95% CH CN 0.1% TFA) to give the title compound (7.5 mg, 0.017
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mmol) as a white solid. H NMR (400 MHz, DMSO-d ) δ 7.23 (d, J = 8.6 Hz, 1H), 6.98 (d, J =
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8.6 Hz, 1H), 6.71 (s, 2H), 4.57 (m, 4H), 3.93 (t, J = 6.2 Hz, 2H), 3.31 (s, 3H), 3.15 (t, J = 7.6 Hz,
+
2
H), 2.25 (s, 6H), 2.00 (t, J = 7.1 Hz, 2H); >98% at 215 nm, MS (ESI) m/z = 434.1 [M + H] .
Protein expression and purification. A codon-optimized gene sequence encoding residues 172-
327 of human Mcl-1 (Uniprot: Q07820) was purchased (Genscript) and cloned into a Gateway
entry vector (pDONR-221, Invitrogen) using the protocols provided. This construct was further
sub-cloned into an expression vector (pDEST-HisMBP) containing a maltose binding protein
(MBP) tag for increased solubility, a tobacco etch virus (TEV) protease recognition site for tag-
removal and an N-terminal His-tag to facilitate purification. The integrity of all plasmids was
checked by sequencing. Soluble Mcl-1 protein was expressed in Escherichia coli BL21
CodonPlus (DE3) RIL (Stratagene) using ampicillin and chloramphenicol for selection.
In brief, a colony from a fresh transformation plate was picked to inoculate 100 mL of LB
medium (37°C). The overnight culture was used to start a 10 L fermentation (BioFlo 415, New
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Brunswick Scientific) grown at 37°C. For NMR studies, uniformly N and N/ C isotopically
1
5
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labeled protein samples were produced in minimal M9 media, where NH Cl and [U- C]-D-
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glucose were used as sole nitrogen and carbon sources (Cambridge Isotope Laboratories). When
the cell density corresponded to OD =2, the temperature was lowered to 20°C. After one hour,
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protein expression was induced with 0.5 mM IPTG. Cells were harvested after 16 h by
centrifugation. Pellets were frozen and re-dissolved in lysis buffer (20 mM TRIS pH 7.5, 300
mM NaCl, 20 mM imidazole, 5 mM BME), approximately 100 mL / 10 g pellet, before the cells
were broken by homogenization (APV-2000, APV). Prior to application to an affinity column
(140 mL, ProBond, Invitrogen), lysate was cleared by centrifugation (18,000 rpm) and filtration
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(0.44 µm). Bound protein was washed on the column and then eluted by a gradient (20 mM
TRIS pH 7.5, 300 mM NaCl, 500 mM imidazole, 5 mM BME). To enhance TEV protease
cleavage, samples were buffer exchanged (50 mM TRIS pH 7.5, 100 mM NaCl, 5 mM BME) on
three serially connected columns (HiPrep 26/10 Desalting, GE Healthcare). TEV protease was
added to a molar ratio of 1:10 (TEV:Mcl-1) and incubated at room temperature until cleavage
was complete. After adding 20 mM imidazole to the samples, they were passed over a
subtractive second nickel-column (120 mL, Ni-NTA Superflow, Qiagen) to remove the MBP-
tag, non-cleaved protein, and TEV protease. Mcl-1 protein for NMR screening was buffer
exchanged into an optimized NMR buffer (25 mM sodium phosphate pH 6.3, 25 mM NaCl, 1
mM DTT, 0.01% NaN ). To achieve highly pure samples (e.g. for crystal screening), a
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supplementary step of size-exclusion chromatography (HiLoad 26/60, Superdex 75, GE
Healthcare) was implemented. The running buffer also acted as the Mcl-1 storage and
crystallography buffer (20 mM HEPES pH 6.8, 50 mM NaCl, 3 mM DTT, 0.01% NaN3).
Purifications were done at 4°C, and concentration steps were performed in stirred ultrafiltration
cells (Amicon, Millipore).
To improve protein sample quality and to increase crystal diffraction, protein mutants
were created by site-directed mutagenesis (QuikChange, Agilent Technologies). Primers for a C-
terminal deletion (Δ5) were designed using their online tool and ordered from Eurofins MWG
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Operon. Mutations were made on the entry vector above, analyzed by in-house sequencing, and
subsequently transferred into the pDEST-HisMBP expression vector. Mutant proteins were
purified in the same way as wild-type (WT) proteins.
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NMR experiments NOE-guided fragment docking. NOE-derived distance restraints were
acquired to enable NMR-based docking of fragments into a previously determined X-ray
structure of a Mcl-1/ligand complex (PDB: 4HW2). Spectra were recorded on a Bruker 800-
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MHz spectrometer equipped with a cryo-probe and pulsed field gradients. 300 µM N/ C-
labeled samples of Mcl-1 was prepared in a D O-based NMR buffer and mixed with fragment 2
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at a concentration of 1 mM. Side-chain H and C NMR signals were assigned from C-edited
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NOE and HCCH-TOCSY experiments.
NOE distance restraints were obtained from three-
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dimensional C-edited NOESY spectra, as well as three-dimensional N- and C-filter/edited
NOESY spectra acquired with a mixing time of 80 ms. Compounds were docked into a X-ray
structure using the NMR-derived restraints and a simulated annealing protocol using the program
4
3
-1
Xplor-NIH. A square-well potential (F
= 50 kcal mol ) was employed to constrain NOE-
NOE
derived distances. Five low energy models from this process were energy minimized using MOE
011.10 (Chemical Computing Group Inc., Montreal). The lowest energy structures obtained by
2
this method were consistent with the observed NOEs.
Protein crystallization, data collection and structure refinement. Fresh batches of Mcl-1
proteins, WT and Δ5, were concentrated to 600 µM (10.7 mg/mL) and 1 mM (17.4 mg/mL),
respectively, and screened for crystallization conditions with a 1.2x excess of ligand. Crystals
were obtained by mixing 1 µL protein with 1 µL reservoir solution (25-30% PEG 3350, 0.1 M
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