Discovery and early development of TMC647055, a non-nucleoside inhibitor of the hepatitis C virus NS5B polymerase

Article abstract of DOI:10.1021/jm401396p

Structure-based macrocyclization of a 6-carboxylic acid indole chemotype has yielded potent and selective finger-loop inhibitors of the hepatitis C virus (HCV) NS5B polymerase. Lead optimization in conjunction with in vivo evaluation in rats identified several compounds showing (i) nanomolar potency in HCV replicon cells, (ii) limited toxicity and off-target activities, and (iii) encouraging preclinical pharmacokinetic profiles characterized by high liver distribution. This effort culminated in the identification of TMC647055 (10a), a nonzwitterionic 17-membered-ring macrocycle characterized by high affinity, long polymerase residence time, and broad genotypic coverage. In vitro results of the combination of 10a with the HCV protease inhibitor TMC435 (simeprevir) supported an evaluation of this combination in patients with regard to virus suppression and resistance emergence. In a phase 1b trial with HCV genotype 1-infected patients, 10a was considered to be safe and well-tolerated and demonstrated potent antiviral activity, which was further enhanced in a combination study with TMC435.

Full text of DOI:10.1021/jm401396p

Article
pubs.acs.org/jmc
Discovery and Early Development of TMC647055, a Non-Nucleoside
Inhibitor of the Hepatitis C Virus NS5B Polymerase
Maxwell D. Cummings,† Tse-I Lin,‡ Lili Hu, Abdellah Tahri, David McGowan, Katie Amssoms,
Stefaan Last, Benoit Devogelaere,§ Marie-Claude Rouan, Leen Vijgen, Jan Martin Berke,
Pascale Dehertogh, Els Fransen, Erna Cleiren, Liesbet van der Helm, Gregory Fanning,
Origene Nyanguile,∥ Kenny Simmen, Pieter Van Remoortere,† Pierre Raboisson, and Sandrine Vendeville*
̀
Janssen Infectious Diseases BVBA (formerly Tibotec BVBA), Turnhoutseweg 30, 2340 Beerse, Belgium
S
* Supporting Information
ABSTRACT: Structure-based macrocyclization of a 6-carbox-
ylic acid indole chemotype has yielded potent and selective
finger-loop inhibitors of the hepatitis C virus (HCV) NS5B
polymerase. Lead optimization in conjunction with in vivo
evaluation in rats identified several compounds showing (i)
nanomolar potency in HCV replicon cells, (ii) limited toxicity
and off-target activities, and (iii) encouraging preclinical
pharmacokinetic profiles characterized by high liver distribu-
tion. This effort culminated in the identification of
TMC647055 (10a), a nonzwitterionic 17-membered-ring
macrocycle characterized by high affinity, long polymerase
residence time, and broad genotypic coverage. In vitro results
of the combination of 10a with the HCV protease inhibitor TMC435 (simeprevir) supported an evaluation of this combination
in patients with regard to virus suppression and resistance emergence. In a phase 1b trial with HCV genotype 1-infected patients,
10a was considered to be safe and well-tolerated and demonstrated potent antiviral activity, which was further enhanced in a
combination study with TMC435.
(NNIs) binding to at least four distinct allosteric sites.12−14
INTRODUCTION
■
NNI-1 inhibitors, also called thumb domain 1 or finger-loop
Hepatitis C virus (HCV) represents a major global health
inhibitors, bind in a pocket distal to the catalytic site at the
burden and is associated with increased risk of liver cirrhosis
interface of the finger and thumb domains of the polymer-
and hepatocellular carcinoma, which is the leading cause of liver
ase.15−17 A 6-carboxylic acid indole chemotype represented by
transplants in the USA.1−3 Despite the availability of new
the early lead compound 1 (Figure 1)18 was optimized by
therapies combining either of the recently approved direct-
several groups based on two distinct strategies, both of which
acting antiviral agents (DAAs) boceprevir4,5 and telaprevir,6,7
yielded clinical candidates. In the first strategy, extension from
both of which are NS3 protease inhibitors, with PEGylated
the carboxylic acid moiety and incorporation of rather large and
interferon α (as weekly injections) and ribavirin (b.i.d.), there
charged diamide moieties, combined with the more metabol-
remains a high medical need for new DAAs with comple-
ically stable cyclopentyl group rather than the cyclohexyl group,
mentary modes of action. Such regimens involving clinically
validated protease inhibitors and inhibitors of the NS5B
polymerase and the NS5A replication complex are needed in
order to (i) increase cure rates, especially in difficult-to-treat
patients; (ii) limit the development of resistant mutants; (iii)
optimize the tolerability/side-effect profile; and (iv) decrease
treatment duration. Ultimately, it is anticipated that combina-
led to the clinical candidate BILB1941 (2) (Figure 1).19 In the
second strategy, conformational rigidification by introduction of
a tether between the indole nitrogen and the C2-aryl group was
explored, leading to tetracyclic and pentacyclic structures, most
of which were zwitterions, as exemplified by MK-3281 (3)
(Figure 1), a compound that reached human phase 1 clinical
study.20
tions of several DAAs in an all-oral interferon-sparing regimen
To avoid the possible formation of acyl glucuronide
metabolites from the carboxylic acid group21 (a potential
will lead to highly effective and better-tolerated treatments.
cause of immunogenic reactions and toxicity22−24) and the
The NS5B polymerase plays an essential role in the life cycle
of HCV since it is responsible for viral replication.8,9 Two
classes of polymerase inhibitors have shown proof-of-concept in
clinical development: nucleoside analogues recognized by the
Special Issue: HCV Therapies
active site and incorporated into nascent RNA as chain (or
Received: September 10, 2013
delayed chain) terminators10,11 and non-nucleoside inhibitors
© XXXX American Chemical Society
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Figure 1. Structures of the early lead compound 1, the clinical candidates 2 (BILB1941) and 3 (MK-3281), and initial 1,6- and 2,6-indole-based
macrocycles 4−6.
presence of a zwitterionic structure (a possible cause of
exposure variability in patients25), we applied a structure-based
macrocyclization strategy centered on the introduction of a
linker in the solvent-exposed region, incorporating a carboxylic
acid bioisostere. It was anticipated that this strategy would
improve the permeability and avoid the formation of potentially
toxic glucuronide acid derivatives as phase 2 metabolites while
providing inhibitors with superior potency and pharmacokinetic
(PK) profiles due to the effect of conformational restriction.
Early crystal structures provided details on the binding mode of
this chemotype to the NNI-1 site,15,26 and we used this
information to guide the initial exploration of three macrocycle
classes. The first type of macrocycle, in which the linker is
attached to the indole nitrogen and the C6 position of the
indole via an amide, served as a proof-of-concept for this
strategy, since micromolar potency was obtained in the replicon
assay with macrocycle 4 (Figure 1) while its open precursor
(prior to the ring-closure metathesis step) was completely
inactive (not shown).27 As expected, an acyl sulfamide
replacement for the amide group proved to be a much better
bioisostere of the carboxylic acid and provided macrocycles 5a
and 5b with submicromolar EC50’s of 0.79 and 0.43 μM,
respectively. Similar findings were obtained for the second type
of macrocycle, where the linker is attached to the C2 aryl group
instead of the indole nitrogen in an effort to restrict the free
rotation of the aryl moiety and favor the bioactive
conformation. Thus, macrocycle 6 displayed an improved
potency of 0.18 μM in the replicon assay. However, while most
of our initial criteria, including cellular potency, no potential for
acyl glucuronide formation, no zwitterion, and good bioavail-
ability and distribution to the liver in rats after oral
administration, had been satisfied with this second series of
macrocycles, this series was still hampered by rather high in
vivo clearance in rats despite moderate in vitro metabolic
turnover in microsomes [clearances typically higher than 7 L
h−1 kg−1 after IV administration of 2 mg/kg compound in
70:30 PEG400/saline]. We then concentrated our efforts on a
third series of macrocycles in which the linker originates from a
bridge and forms a seven-membered ring between the indole
nitrogen and the C2 aryl group. Our subsequent optimization
efforts involved the exploration of three types of bridge:
saturated, unsaturated, and incorporating a cyclopropyl
group.28
In this article, we describe the discovery of the macrocyclic
indole TMC647055 (10a), which is currently being evaluated
in phase 2 clinical trials in combination with TMC435
(simeprevir), a once-daily protease inhibitor discovered at
Janssen during a research collaboration with Medivir. We
review the synthesis, structure−activity relationships, and
binding interactions of this new series of tetracyclic indole-
based macrocycles, and we also discuss genotypic coverage,
resistance, and mutant selection. The potential for combination
with other DAAs and preliminary results in healthy volunteers
and HCV genotype 1-infected patients, including those
obtained in combination with TMC435,29,30 are also presented.
B
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a
Scheme 1. Synthesis of 10a
a
Reagents and conditions: (a) NaOH, THF, H2O, MeOH, RT (95%). (b) 2,2′-Oxybis(N-methylethanamine) (5 equiv), HATU, DIPEA, THF. (c)
Sulfamide, dioxane, 100 °C. (d) TFA/DCM. (e) (i) CDI, dry THF, RT; (ii) DBU, THF.
nium chloride in the presence of sodium hydride followed by
enantiomeric separation of the pair of enantiomers obtained
with the chiral auxiliary (S)-4-benzyl-2-oxazolidinone by
column chromatography, which yielded diastereoisomers 14
and 15, as depicted in Scheme 2. After removal of the chiral
auxiliary under basic conditions, the reaction sequence used for
the preparation of macrocyclic derivative 10a was applied to
carboxylic acids 16 and 17, yielding the cyclopropyl-bridged
macrocycles 18a and 19a with 100% ee. For the two pairs of
enantiomers 11a/12a and 18a/19a, assignment of stereo-
chemistry was based on antiviral data in conjunction with
conformational analysis and modeled binding modes predicated
on earlier NS5B−inhibitor complexes.28,31 The rich set of
available and relevant public structural data allowed for the
straightforward construction of what were deemed to be
relatively reliable binding models on the basis of the
assumption of common binding modes for the corresponding
features of the new inhibitors. This approach, while based on
modeled structures, seemed quite reliable, and our later results
with a macrocyclic inhibitor complex (discussed below) proved
to be fully consistent with our earlier speculations on both
stereochemical preference and macrocycle design.
The synthesis of macrocycle 25 with a “reduced” amide bond
is outlined in Scheme 3. A one-pot procedure involving the
conversion of carboxylic acid 20 to the corresponding isobutyl
carbonate followed by sodium borohydride reduction afforded
alcohol 21. Subsequent introduction of the mesylate leaving
group and its displacement by symmetrical N,N′-dimethyl-1,4-
butanediamine allowed for the synthesis of intermediate 23,
which then underwent the same sequence of reactions as
previously to yield the “reduced” macrocycle 25.
CHEMISTRY
■
Macrocyclic indole derivatives 10a−i were synthesized
following a six-step procedure starting from the orthogonally
protected bisester tetracycle 7.28 The synthetic route is
exemplified with the structure of TMC647055 (10a) in
Scheme 1. After hydrolysis of the methyl ester, a HATU-
mediated amide coupling was performed using an excess of the
symmetrical bisamine 2,2′-oxybis(N-methylethanamine) under
diluted conditions in THF to give amide 8a in 93% yield. A
monoprotected unsymmetrical bisamine could also be used
stoichiometrically and then further deprotected under acidic
conditions (i.e., TFA in DCM for the removal of the Boc
protecting group) or under basic conditions using thiophenol
in the case of the nosyl protecting group. The free amino group
was then converted to the sulfamide moiety using an excess of
sulfamide in dioxane at 100 °C to give derivative 9a in good
yield (95%). The tert-butyl ester was subsequently cleaved
under acidic conditions (TFA in DCM) in quantitative yield,
and the ring closure was performed using a tandem of reactions
involving activation of the carboxylic acid by CDI followed by
intramolecular attack of the anion of the sulfamide group,
formed by addition of DBU. This synthetic route yielded
macrocycle 10a in 49% overall yield from bisester 7 and was
also used to synthesize macrocycles 10b−i. Alternatively, ring
closure could be performed by a HATU-mediated macro-
lactamization under high dilution after successive introduction
of a sulfamide derivative containing a protected amine using the
two-step CDI/DBU procedure described above and cleavage of
the methyl ester and then the amino protecting group of the
linker. Macrocycles 10j−l were synthesized according to this
latter route (not shown).28
Upon catalytic hydrogenation, compound 10a gave a mixture
of enantiomers that could be separated by chiral chromatog-
raphy, yielding the enantiomerically pure macrocycles 11a and
12a containing a saturated bridge (Figure 2).28 The
enantiomeric excesses were 100% and 99.1%, respectively.
Derivatives with the cyclopropyl bridge were obtained by
cyclopropanation of bisester indole 7 using trimethylsulfoxo-
SAR AND PK PROPERTIES LEADING TO THE
SELECTION OF TMC647055
■
The anti-HCV potencies (EC50’s) of compounds 10a−l, 11a,
12a, 18a, 19a, and 25 were determined in the Huh-7 replicon
cell line containing the subgenomic bicistronic HCV genotype
1b replicon clone ET and luciferase readout29 (Table 1). In an
effort to better understand the observed structure−activity
relationship (SAR), and in the absence of a reliable
permeability determination for this class of macrocycles,32
binding affinities on the 1b polymerase were measured by
surface plasmon resonance (SPR)33 (Table 1). Cytotoxicity was
measured in MT4 and Huh-7 cell lines and was minimal for all
the compounds tested (CC50 > 25 μM). Studies with the
unsaturated-bridged macrocyclic series revealed several struc-
ture−activity trends. The 16- and 17-membered macrocycles
were optimal. An 18-membered analogue (10b) suffered a 2-
fold decrease in replicon potency (cf. 10a and 10c) despite
improved binding affinity (Kd = 0.8 nM). Smaller ring size
Figure 2. Structures of 11a and 12a.
C
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a
Scheme 2. Synthesis of 18a and 19a
a
Reagents and conditions: (a) (i) Trimethylsulfoxonium chloride, NaH, DMSO, RT; (ii) 7, 50 °C. (b) NaOH, H2O/THF/MeOH, RT. (c)
(COCl)2, DMF, THF, 0 °C. (d) (S)-4-Benzyl-2-oxazolidinone (1.1 equiv), n-BuLi (1.1 equiv), THF, −78 °C. (e) NaOH (1 N), MeOH/THF, RT.
(f) 2,2′-Oxybis(N-methylethanamine) (5 equiv), HATU, DIPEA,THF. (g) (i) Sulfamide, dioxane, 100 °C; (ii) TFA/DCM. (h) (i) CDI, dry THF,
RT; (ii) DBU, THF.
a
Scheme 3. Synthesis of 25
a
Reagents and conditions: (a) N-Methylmorpholine, isobutyl chloroformate, THF, −15 °C, then NaBH4, 0 °C. (b) Et3N, MeSO2Cl, THF, 0 °C. (c)
N,N′-Dimethyl-1,4-butanediamine, THF, RT. (d) Sulfamide, dioxane, 105 °C. (e) (i) TFA, DCM, RT; (ii) CDI, acetonitrile, 60 °C; (iii) DBU,
acetonitrile, RT.
(compound 10d, 15-membered ring) led to decreased binding
affinity and replicon potency. Ether and alkyl linkers were
similarly potent, while the presence of a basic nitrogen in the
linker decreased the activity (10f and the piperazine derivatives
10g and 10l), contrasting with earlier reports on non-
macrocyclic indoles.20 Substitution of the linker nitrogens had
dramatic effects. Two methyl substituents proved optimal,
while monomethyl analogues (10j and 10k) were less potent in
both binding and cellular assays, with an additional effect of
decreased permeability for macrocycle 10j. Derivatives with R1
= methoxy (10a) and fluoro (10i) showed similar potencies,
while R1 = chloro (10h) gave a 4-fold loss of potency in the
replicon assay, likely attributable to high lipophilicity in view of
the good binding affinity (Kd = 1.5 nM) observed for this
compound. While keeping the ether linker constant, the nature
and stereospecificity of the seven-membered bridge was
evaluated with the saturated analogues 11a and 12a and
cyclopropyl-bridged macrocycles 18a and 19a. For these two
subseries, only the R enantiomers (12a and 19a) showed good
antiviral activity, with 20- and 40-fold losses of potency for the
less active enantiomers 11a and 18a, respectively. Saturation of
the double bond yielded a compound with potency similar to
that of the unsaturated analogue 10a (EC50 = 79 and 77 nM for
12a and 10a, respectively), while cyclopropanation gave a 2-
fold improvement (19a, EC50 = 40 nM). Figure 3 shows
models of both enantiomers of a simple non-macrocyclic N,N-
dimethylamide, highlighting the different trajectories accessible
to this pair. In this case, the models have been adjusted to
closely mimic the bound conformation observed for 10c on
which they are overlaid; under these (idealized) conditions it is
clear that while the R amide overlays closely with that of bound
10c, such a trajectory is not possible for the S enantiomer.
Thus, we speculate that the less active S enantiomers are unable
to adopt the optimal binding conformation within the
constraint imposed by the 17-membered macrocycle. Extensive
conformational adjustment involving the core indole−phenyl
system of the inhibitor would be expected to have a profound
and likely negative effect on the binding affinity. Additional
changes in binding contacts could reasonably be expected for
the contact with Arg503 as well as the contact with His34,
D
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Table 1. Effect of the Subseries, Linker, And Phenyl
Substitution on Replicon Potency and Binding Affinity
Figure 3. Stereospecificity of the saturated bridged system. Depicted
are conformers of non-macrocyclic N,N-dimethylamide (R, green; S,
purple), showing the stereospecific difference in the trajectory from
the bridge, overlaid onto the NS5B-bound conformation of 10c
(orange).
activity relative to amide 10c (EC50 = 2.34 μM versus 0.075
μM). The antiviral potency in the presence of 40% human
serum was also measured, since this series of macrocycles
generally exhibited high binding to plasma proteins in vitro
(e.g., the free fraction of 10a in the presence of plasma is 0.3%),
which could limit the in vivo efficacy. The resulting serum shifts
were generally 10−15-fold (9-fold for 10a), with no clear SAR
for this parameter. Rather than trying to decrease the serum
shift, our strategy focused on the selection of compounds
exhibiting high distribution to the liver, since HCV replication
takes place almost exclusively in hepatocytes. We anticipated
that this parameter could be an important contributor to the
efficacy of compounds in patients34,35 and that it may
correspond to a more therapeutically relevant way of
discriminating among inhibitors with relatively good anti-
HCV replicon activities compared with the common strategy of
minimizing the serum shift. Ultimately, a concentration in the
liver at 7 h postdose that was higher than the serum-shifted
EC50 was used as a criterion to select the most promising
compounds.
A panel of analogues chosen for their good antiviral potency,
acceptable in vitro metabolic stability, and chemical diversity,
were then evaluated in vivo in Sprague−Dawley rats after IV
and oral administration at 2 and 10 mg/kg, respectively. In
addition to the classical PK parameters, concentrations in the
liver at 7 h postdose were also measured (Table 2).
The nature of the bridge had a profound impact on the PK
parameters, with the unsaturated analogue 10a exhibiting the
best overall profile, including good systemic absorption
(bioavailability higher than 66%), high distribution to the
liver (7800 ng/g, 7 h postdose), and moderate clearance. The
saturated derivative 12a showed clearance similar to that of 10a
but had low bioavailability and low distribution to the liver
(15% and 898 ng/g, respectively), while the cyclopropyl
derivative 19a showed a relatively high clearance of 5.2 L h−1
kg−1, in agreement with its low in vitro liver microsome stability
(70% of the compound metabolized after 15 min in rat liver
microsomes). The nature of the linker was also important: the
unsubstituted amide derivative 10e had good distribution to the
liver but a poor absolute bioavailability of only 6%, highlighting
the role of the amide substituent on permeability, while
a
b
Replicon potency. Dissociation constant determined by surface
plasmon resonance with NS5B con1b(Δ21).33
which has been observed for bound 10c (discussed below in
Crystallography and Modeling).
Interestingly, the carbonyl of the amide group proved to be
critical for potency, since the “reduced” macrocycle 25
exhibited a 30-fold decrease in cellular anti-HCV replicon
E
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cyclohexyl ring and the corresponding hydrophobic edge of the
relatively rigid indole−phenyl ring system sit in a hydrophobic
pocket, while the opposite and relatively flexible side of the
macrocycle (the macrocycle linker) is largely solvent-exposed.
The acyl sulfamide moiety forms two hydrogen bonds with the
guanidine of Arg503. Distinctively, the 10c complex resolves
additional N-terminal residues of the NNI-1 site in comparison
with other published NNI-1 complexes (e.g., Figure 6);15,26
interestingly, this difference may be due to a (weak) binding
contact between the amide carbonyl of the macrocycle and
His34 (Figure 4a).31
Molecular modeling analysis suggested that 10a should be
able to adopt a binding mode essentially identical to that of
10c, possibly involving slight conformational adjustments in
parts of the flexible macrocycle linker, which is one atom longer
in 10a (Figure 4b). The occupancy of the hydrophobic regions
of the binding site and the multiple contacts with Arg503 are
expected to be essentially identical to those described above for
10c, whereas the contact with His34 is less clear because of the
difference in the macrocycle linker lengths of 10c and 10a.
Binding of inhibitors at both the NNI-1 and NNI-2 sites
displaces the fingertip Λ1 loop, a change that is thought to lock
the enzyme in an inactive “open” state and thus lead to
inhibition.15,36 10c bound in the NNI-1 site closely mimics
features of the fingertip Λ1 loop, positioning hydrophobic aryl
groups similarly to the side chains of Leu30 and Leu31 of the
bound loop (Figure 5) and effectively competing with the
native intramolecular protein−protein interaction.
Since the original formulation of our macrocycle design
strategy,31 additional structures detailing indoles bound at the
NNI-1 site have been disclosed.20,37,38 These structures reveal
additional information about feasible binding interactions in the
NNI-1 site and are also consistent with previous structures.
Figure 6 shows overlays of bound 10c with two examples of
these more recent indole inhibitors, and in both cases we see a
tight overlay with the tetracyclic cyclohexyl−indole−phenyl
core of bound 10c. Similar to 10c, the new structures show
multiple hydrogen-bonding contacts with the guanidine of
Arg503. The importance of this interaction was noted
previously,15,26,31 with earlier structures incorporating a
carboxylate for this contact; one key element of our macrocycle
design strategy was the replacement of this carboxylate. In our
original report, we described the more buried pocket where the
Table 2. Pharmacokinetic Parameters of Selected
Macrocyclic Analogues (2 mg/kg IV, 10 mg/kg PO; Vehicle
70:30 PEG400/Saline IV, PEG400/2% vitE-TPGS PO; n =
3)
a
b
c
d
e
compd HLM
Cl (L h−1 kg−1
)
[liver] (ng/g)
L/P
F (%)
10a
10c
10e
10i
10l
35
56
32
38
26
63
95
3.2
2.4
4.2
3.7
9.4
3.2
5.2
7800
5000
6317
6560
299
46
23
>66
76
6
341
62
52
3
108
22
12a
19a
898
15
51
3200
38
a
Human liver microsomes stability, expressed as percentage of
compound metabolized after 15 min at a concentration of 5 μM.
b
c
Plasma clearance. Liver concentration after oral administration 7 h
d
postdose. Liver/plasma ratio after oral administration 7 h postdose.
e
Oral bioavailability.
macrocycle 10l with a basic piperazine moiety showed an
extremely poor overall profile, combining high clearance
(despite good in vitro liver microsome metabolic stability),
low absorption, and poor distribution to the liver. Finally,
replacement of the methoxy group by a fluoro substituent (10i)
led to PK properties similar to those of 10a, as did replacement
of the ether-containing linker by the alkyl linker (10c). On the
basis of the overall good in vitro and in vivo properties, 10a was
further evaluated in dogs and showed an acceptable PK profile
characterized by high oral bioavailability (F = 87%) and high
systemic exposure (Cmax = 10.2 μM and AUC0−∞ = 25.8 μM h)
after a single 10 mg/kg oral dose, combined with moderate
plasma clearance (Cl = 0.54 L h−1 kg−1) and a low volume of
distribution (Vdss = 0.32 L/kg).
CRYSTALLOGRAPHY AND MODELING
■
Macrocycle 10c, a close structural analogue of TMC647055,
was successfully cocrystallized with a ΔC21 construct of
genotype 1b strain J4 NS5B (Figures 3−6).31 The observed
binding mode for the tetracyclic core and pendant cyclohexyl
moiety of 10c bound to the NNI-1 site of NS5B is consistent
with those of the equivalent moieties of the previously
published structures (PDB IDs 2BRK and 2BRL15 and
2DXS26) that were used to guide our design strategy. The
Figure 4. Observed and modeled binding modes. (A) Crystal structure of 10c (orange) bound at the NNI-1 site, showing the pocket surface and
details of contacts with His34 and Arg503. (B) Overlay of bound 10c (orange) and the modeled low-energy conformer of 10a (purple).
F
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The mean 50% cytotoxic concentrations in Huh7 and MT4
cells in luciferase reporter assays were 42.1 μM (n = 5) and 28.9
μM (n = 16), respectively. Cytotoxicity measurements on a
panel of additional cell lines (MRC-5, HEK-293T, HepG2, and
VeroE6 cells) all resulted in CC50 values in excess of 50 μM.33
The binding kinetics of 10a were measured by SPR and
showed a high-affinity interaction with NS5B across all
genotypes (i.e., median Kd < 35 nM), except for genotype
2b, for which a more than 20-fold decrease in Kd was observed,
mainly because of a higher dissociation rate (see Table 3). For
Figure 5. Comparison of bound inhibitors with the native NNI-1 site
protein−protein interaction. Shown are bound 10c and a model of 10a
(as in Figure 4b) overlaid on the NS5B loop that occupies NNI-1 in
the absence of bound inhibitor [protein of the NNI-1 site is not
shown; the loop is from monomer B of the 10c−NS5B complex
structure (PDB ID 4DRU)].31
Table 3. Median Binding Affinities and Antiviral Activities of
10a against Different Genotypes, Using NS5B Purified
Proteins from Clinical Isolates and a Transient Replicon
Assay with Chimeric Replicons Containing the NS5B Region
Derived from Patient Isolates, Respectively
a
a
genotype
Kd (M)
n
EC50 (μM)
n
methoxyphenyl moiety of bound 10c sits, compared with the
more exposed regions seen in earlier structures. This difference
persists in the more recent structures, the two examples of
which both show the more exposed pocket in this region of the
binding site (Figure 6). The reason(s) for the observation of
the more enclosed pocket in this region of the binding site in
our structure remains unclear, but it may be due to the
methoxyphenyl group that protrudes more deeply into the
pocket and/or the weak contact between the inhibitor amide
carbonyl and His34 mentioned above. Both of these chemical
features are relatively unique to 10c in comparison with the
inhibitors bound at this site in other published structures.
wild-type 1b
4.1 × 10−9
7.3 × 10−9
3.9 × 10−9
−
0.051
0.048
0.027
12.530
>28.000
0.088
0.097
−
1a
1b
2a
2b
3a
4a
5a
6a
5
14
10
1
5
8.8 × 10−7
6.1 × 10−9
2.0 × 10−9
2.1 × 10−8
3.0 × 10−8
3
3
3
1
1
2
2
1
0.113
2
a
Number of isolates. For n = 1 or 2, the observed value or the mean of
the two observed values, respectively, is reported.
genotype 1b, the association and dissociation constants were
2.2 × 104 M−1 s−1 and 8.9 × 10−5 s−1, respectively, resulting in a
Kd of 4 nM and a half-life of 130 min. This rather long inhibitor
residence time may represent a distinctive advantage over other
indole derivatives with regard to in vivo efficacy and target
selectivity.39,40 Broad cross-genotypic coverage of 10a was also
confirmed in a transient replicon assay using chimeric replicons
containing the NS5B region derived from patient isolates
(Table 3), with 10a showing consistently high potencies against
genotypes 1a, 1b, 3a, 4a, and 6a. Similar to previous reports
describing NNI-1 inhibitors,41,42 we observed a >200-fold
reduction in potency against genotypes 2a and 2b (Table 3).
The susceptibility of 10a was tested on different genotype 1b
replicons harboring mutations in the NS5B polymerase located
in the NNI-1 pocket (L392I, V494A, and P495L), also
PRECLINICAL CHARACTERIZATION OF
TMC647055 (10A)
■
On the basis of the promising anti-HCV replicon activity and
PK profiles in preclinical species, 10a was selected as a
candidate for clinical development, and its biological profile
with respect to binding kinetics, genotypic coverage, resistance
and mutant selection, combination with other DAAs, and
preliminary safety were further characterized.33
10a elicited a mean IC50 of 34 nM in an RNA-dependent,
RNA polymerase primer-dependent transcription assay and
inhibited the replication of a genotype 1b replicon (clone ET)
in the Huh7-Luc cell line with a median EC50 of 77 nM
(luciferase readout). In Huh7-SG-1a, a stable genotype 1a cell
line, the median EC50 value was 166 nM (qRT-PCR readout).
Figure 6. Comparison with recent NS5B NNI-1 complex structures. (A) 10c with 3Q0Z (3Q0Z protein shown).38 (B) 10c with 4GMC (4GMC
protein shown).37
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identified during in vitro resistance selection experiments with
10a, and on other mutations outside of the NNI-1 pocket. The
greatest decrease in antiviral potency, a 371-fold reduction in
susceptibility relative to that of the wild-type genotype 1b
replicon, was observed for the P495L mutant, consistent with
previous reports identifying this position as a key mutation for
NNI-1 site inhibitors.16,33,42 The L392I and V494A mutations
resulted in 9- and 3-fold median decreases, respectively, in anti-
HCV replicon activity. Overall, these results mirror those of
past resistance studies. Residues 392, 494, and 495 contribute
to the NNI-1 site binding surface, making direct binding
contacts with bound 10c, 10a (model shown in Figure 7), and
Figure 8. Structure of the HCV NS3/4A protease inhibitor TMC435
(simeprevir, Sovriad).
inhibitor. Complete clearance of replicon cells was achieved
using the combination of 1.5 μM 10a and 0.10 μM TMC435,
while for each of these inhibitors alone, even at higher
concentrations (3.75 and 0.25 μM for the NNI and protease
inhibitors, respectively), incomplete clearance and rebound of
replicon RNA was observed. In addition, in the colony
formation experiments, in which Huh7-Luc replicon cells
were incubated for 2−3 weeks with either TMC435 or 10a or a
combination of the two, the combination of these two
inhibitors suppressed the formation of resistant colonies at
low concentrations (0.75 and 0.08 μM for the NNI and
protease inhibitor, respectively). At a higher concentration of
1.5 μM, 10a alone led to a similar result, while the protease
inhibitor (0.20 μM) alone did not.
The in vitro preliminary safety of 10a was found to be
acceptable in a battery of assays assessing cytotoxicity toward
six different cell lines, genotoxicity, cardiovascular toxicity,
selectivity versus human polymerases and a panel of enzymes
and receptors, and potential reactivity with regard to
glutathione adduct formation and acyl glucuronide formation.
In addition, no systemic adverse effects were observed in a 5
day tolerance study with up to 1000 mg kg−1 day−1 in rat,
except for a small decrease in body weight.
Figure 7. NS5B resistance mutations for 10a. The 10a−NS5B
complex was modeled by superimposing a low-energy conformer of
10a onto bound 10c from the 10c−NS5B complex, as shown in Figure
4b. Positions 392 (light teal), 494 (dark teal), and 495 (red) are
highlighted.
other indoles that bind at this site.15,20,26,37,38 Residue 392
contributes to the deepest part of the hydrophobic pocket
occupied by the cyclohexyl moiety, while residue 494 contacts
both the cyclohexyl ring and the face of the indole−phenyl
system. Residue 495 packs against the face of the indole and is
also near the critical acyl sulfamide moiety of the bound
inhibitor; changes at this position are also likely to impact the
positioning of Arg503, which forms multiple hydrogen bonds
with bound 10c and 10a and makes similar interactions with
other NNI-1 compounds. Thus, the resistance results seem
consistent with the observed and modeled binding modes
presented here. Additionally, the activity of 10a was not
affected by mutations associated with reduced sensitivity to
NNI-2 (M423T), NNI-3 (M414T), NNI-4 (C316Y), HCV
NS5B nucleoside/nucleotide inhibitors (S96T and S282T),
HCV NS3/4A protease inhibitors (R155K, A156V, and
D168V), and HCV NS5A inhibitors (L31V and Y93H).33
NS3/4a inhibitors (e.g., TMC435) and NS5A inhibitors
maintained full inhibitory potency against the NNI-1 mutants.
This lack of cross-resistance suggested that combination
therapy with other DAAs may be fruitful.
EARLY CLINICAL DEVELOPMENT OF TMC647055
(10A)
■
The safety and tolerability of 10a after repeated administrations
during 5 days and a morning dose at day 6 was demonstrated in
healthy volunteers given oral b.i.d. doses of 300 mg, 1000 mg,
and 1250 mg as well as in HCV genotype 1-infected patients
given oral b.i.d. doses of 500 mg and 1000 mg. The most
commonly reported adverse events were gastrointestinal
(nausea, vomiting, loose stools, diarrhea, and abdominal
cramps/discomfort), probably related to the hydroxypropyl-β-
cyclodextrin vehicle. There were no serious adverse events
(SAEs), no grade 3 or 4 AEs, and no clinically significant effects
on laboratory parameters, vital signs, food consumption,
appetite, or ECG profile. The main pharmacokinetic observa-
tions were (i) a marginal food effect, (ii) a slightly greater than
dose-proportional increase in plasma concentrations in healthy
volunteers and a dose-proportional increase in HCV-infected
patients, (iii) an approximately 2-fold higher exposure in
infected patients relative to healthy volunteers, and (iv) a
decrease in exposure during the treatment. The plasma
concentrations of 4-β-hydroxycholesterol, the endogenous
biomarker for CYP3A4 induction, increased with increasing
To further explore the effect of a combination of an NNI and
a protease inhibitor on virus suppression and resistance
development, an in vitro clearance and rebound experiment
and a colony formation experiment were carried out with 10a
and TMC435 (Figure 8) using Huh7-Luc replicon cells.33 In
the clearance and rebound experiment, a 14 day incubation of
the cells with the two inhibitors alone and in combination was
followed by a 3 week “rebound” phase in the absence of
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TMC647055 dose in healthy volunteers, indicating that
TMC647055 induced CYP3A4 and its own metabolism.
Mean trough concentrations in patients exceeded the replicon
genotype 1a/1b EC50 values at both 500 and 1000 mg b.i.d.
The antiviral response showed dose-dependent median
maximum decreases in HCV RNA from baseline of 1.4 log10
and 2.4 log10 in GT1a patients with HCV RNA data available
up to Day 6 at 500 and 1000 mg b.i.d., respectively. In GT1b
patients, the median maximum decreases in HCV RNA were
similar at the two dose levels (3.3 log10 at 500 mg b.i.d. and 3.4
log10 at 1000 mg b.i.d.).
These results encouraged further assessment of the
combination of 10a with the protease inhibitor TMC435,
which had previously shown improved results in vitro relative
to each of these compounds alone with regard to virus
suppression and resistance development. The combination of
10a (1000 mg b.i.d.) and TMC435 (150 mg q.d.) was given to
HCV genotype 1a- and 1b-infected patients for 10 days and
found to be safe and well-tolerated. Potent antiviral activity was
measured, with a median HCV RNA decrease of 4.64 log10 IU/
mL at day 11. The decline in plasma HCV RNA was
substantially better than observed with either 10a or
TMC435 alone. Importantly, no viral breakthrough was
detected, and no emerging mutations in the NNI-1 pocket of
the polymerase associated with reduced in vitro sensitivity to
10a were seen. However, emergence of the R155K mutation in
the NS3/4A protease, which leads to a 30-fold decrease in
susceptibility to TMC435,43 was detected in six/seven
genotype 1a-infected patients during dosing or at follow-up.44
Finally, this combination was characterized by a decrease of the
mean exposures of the two compounds upon repeated dosing,
attributable to CYP3A4 induction by 10a.
ritonavir to optimize the pharmacokinetic−pharmacodynamic
parameters.
EXPERIMENTAL SECTION
General Information. NMR spectra were recorded on a Bruker
Avance 400 spectrometer operating at 400 MHz for H. Chemical
■
1
shifts are given in parts per million and J values in hertz. Multiplicities
are indicated using the following abbreviations: s for singlet, d for
doublet, t for triplet, m for multiplet, etc. Details on the different LC−
MS methods used for the characterization of the new compounds
described in this section (designated as LCMS-1 to LCMS-6 and
HPLC-chiral) can be found in the Supporting Information. Chemical
purities were >95% for all final compounds, as assessed by two LC−
MS analysis methods.
1,1-Dimethylethyl 6-[[[2-[2-[(Aminosulfonyl)methylamino]-
ethoxy]ethyl]methylamino]carbonyl]-13-cyclohexyl-3-me-
thoxy-7H-indolo[2,1-a][2]benzazepine-10-carboxylate (9a). A
solution of NaOH (6.38 g) in 25 mL of water was added to a stirred
solution of bisester 7 (2 g, 3.9 mmol, synthesized as reported in ref 28)
in THF (100 mL) and MeOH (150 mL). After 1 h, the reaction
mixture (RM) was concentrated under reduced pressure and then
diluted with ice-cold water (150 mL). The pH of the resulting solution
was adjusted to 6 with acetic acid. The resulting precipitate was
collected by filtration, washed with water, and dried under vacuum to
give 1.90 g (98% yield) of 10-(1,1-dimethylethoxycarbonyl)-13-
cyclohexyl-3-methoxy-7H-indolo[2,1-a][2]benzazepine-6-carboxylic
acid (20) as a yellowish powder. To a stirred solution of carboxylic
acid 20 (1.00 g, 2.05 mmol), DIPEA (1.07 mL, 6.15 mmol), and 2,2′-
oxybis(N-methylethanamine) (1.08 g, 8.20 mmol) in 30 mL of dry
THF was added HATU (1.17 g, 3.08 mmol) under nitrogen. After 1 h,
the RM was quenched with water (100 mL) and extracted with ethyl
acetate (75 mL). The organic layer was successively dried (Na2SO4),
filtered, and evaporated. The residue was triturated in water, filtered
off, and dried in a high-vacuum oven to give 1.15 g (93% yield) of 1,1-
dimethylethyl 13-cyclohexyl-3-methoxy-6-[[methyl[2-[2-
(methylamino)ethoxy]ethyl]amino]carbonyl]-7H-indolo[2,1-a][2]-
benzazepine-10-carboxylate (8a) as a yellowish powder, which was
used without any further purification in the next step. LCMS-1 95%
pure, tR = 3.03 min, m/z 602.4 [M + H]+; HRMS (APPI+) calcd for
C36H48N3O5 [M + H]+ 602.3594, found 602.3578. A solution of 8a
(1.15 g, 1.91 mmol) and sulfamide (1.84 g, 19.1 mmol) in dioxane (10
mL) was heated at 100 °C in a microwave oven for 20 min. The RM
was then cooled to RT and evaporated under vacuum. The residue was
triturated in water, filtered off, and washed with water. The powder
was redissolved in EtOAc, dried (Na2SO4), filtered, and evaporated to
give 1.15 g (88% yield) of the desired product 9a as a yellowish
powder. LCMS-1 100%, tR = 3.37 min, m/z 681.3 [M + H]+; HRMS
(APPI+) calcd for C36H49N4O7 [M + H]+ 681.3322, found 681.3314;
1H NMR (400 MHz, DMSO-d6) δ 1.26−1.44 (m, 4H) 1.61 (s, 9H)
Because of the good safety and tolerability combined with
potent antiviral activity, the combination of 10a and TMC435
is being evaluated in an all-oral once-daily regimen with low-
dose ritonavir to counterbalance the CYP3A4 induction, with
and without ribavirin b.i.d., in a phase 2 interferon-free study.
CONCLUSION
■
A structure-based macrocyclization strategy was applied to a
known 6-carboxylic acid indole chemotype, leading to the
discovery of potent and selective inhibitors of HCV NS5B
polymerase devoid of the undesirable features characterizing
non-macrocyclic analogues (potential for acyl glucuronide
formation and presence of zwitterions). Lead optimization
focused on the subseries containing the more rigid bridged-
indole macrocycles, in combination with early in vivo
evaluation in rats, culminated rapidly in the discovery of the
clinical candidate 10a. This 17-membered-ring macrocycle is
characterized by high affinity for and persistent binding to
NS5B polymerase, encouraging in vivo properties in preclinical
species (most notably high distribution to the liver in rats), and
an acceptable preliminary safety profile. Furthermore, 10a
exhibits a favorable in vitro antiviral profile with respect to
cross-genotypic coverage, virus suppression, and resistance
selection, alone or in combination with the HCV NS3 protease
inhibitor TMC435. These encouraging results translated to safe
and well-tolerated treatment for HCV genotype 1-infected
patients, demonstrating a strong antiviral effect that was more
pronounced in combination treatment with TMC435. These
results warrant further assessment of this combination in an all-
oral once-daily interferon-free regimen with a low dose of
1.75 (d, J = 13.35 Hz, 2H) 1.84 (d, J = 13.35 Hz, 2H) 2.04 (qd, J =
12.55, 3.15 Hz, 2H) 2.70 (s, 3H) 2.86 (tt, J = 12.20, 3.60 Hz, 1H),
2.95 (s, 3H) 3.13 (t, J = 5.79 Hz, 2H) 3.49−3.56 (m, 6H) 3.91 (s, 3H)
4.69 (br s, 2H) 6.31 (br s, 2H) 6.97 (s, 1H) 7.13 (d, J = 2.77 Hz, 1H)
7.16 (dd, J = 8.56, 2.77 Hz, 1H) 7.55 (d, J = 8.56 Hz, 1H) 7.58 (dd, J =
8.56, 1.51 Hz, 1H) 7.84 (d, J = 8.31 Hz, 1H) 8.05 (d, J = 1.01 Hz, 1H).
2,19-Methano-3,7:4,1-dimetheno-1H,11H-14,10,2,9,11,17-
benzoxathiatetraazacyclodocosine-8,18(9H,15H)-dione 27-Cy-
clohexyl-12,13,16,17-tetrahydro-22-methoxy-11,17-dimethyl-
10,10-dioxide (10a). TFA (3.0 g, 26.3 mmol) was added to a
solution of 9a (1.15 g, 1.70 mmol) in dichloromethane (30 mL). After
1 h, the RM was concentrated under vacuum. The residue was
triturated in ether, filtered, washed with ether, and then purified by
chromatography (gradient EtOAc to EtOAc/EtOH 9:1) to give 802
mg (76% yield) of the carboxylic acid intermediate. LCMS-1 96%, tR =
2.72 min, m/z 625 [M + H]+; HRMS (APPI+) calcd for C32H41N4O7S
1
[M + H]+ 625.2696, found 625.2683; H NMR (400 MHz, DMSO-
d6) δ 1.22−1.50 (m, 4H) 1.75 (m, 3H) 1.83 (m, 2H) 1.96−2.12 (m,
2H) 2.70 (s, 3H) 2.80−2.91 (m, 1H) 2.94 (s, 3H) 3.13 (t, J = 5.92 Hz,
2H) 3.47−3.58 (m, 6H) 3.90 (s, 3H) 4.68 (br s, 2H) 6.32 (br s, 2H)
6.96 (s, 1H) 7.12 (d, J = 2.77 Hz, 1H) 7.16 (dd, J = 8.56, 2.77 Hz, 1H)
I
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7.54 (d, J = 8.56 Hz, 1H) 7.63 (dd, J = 8.31, 1.26 Hz, 1H) 7.83 (d, J =
8.31 Hz, 1H) 8.10 (s, 1H). Carbonyldiimidazole (389 mg, 2.40 mmol)
was added to a stirred solution of the previous intermediate (500 mg,
0.80 mmol) in dry THF (8 mL). The RM was stirred at RT for 1 h
and then concentrated, and the resulting residue was purified by flash
chromatography (gradient EtOAc to CH3CN 1:0 to 0:1) to give 550
mg of the acylimidazole intermediate as a slightly yellow solid. HRMS
(APPI+) calcd for C35H43N6O6S [M + H]+ 675.2965, found 675.2950.
DBU (244 mg, 0.32 mmol) was added to a solution of the
acylimidazole (550 mg) in acetonitrile (25 mL). The RM was stirred
overnight at RT and then concentrated under reduced pressure. The
residue was dissolved in water (30 mL), and the pH of the resulting
solution was adjusted to 5 with acetic acid. The precipitate was
collected by filtration, washed with water, and dried. Recrystallization
from ethanol followed by column chromatography purification
(gradient EtOAc to EtOAc/EtOH 9:1) provided 380 mg (78%
yield) of the title product 10a as a white powder. LCMS-1 99.6%, tR =
3.81 min, m/z 607 [M + H]+, LCMS-2 100%, tR = 1.02 min; HRMS
(APPI+) calcd for C32H39N4O6S [M + H]+ 607.2590, found 607.2609;
1H NMR (DMSO-d6) δ 1.15 (m, 1H) 1.40 (m, 3H) 1.71 (m, 2H) 1.88
(4.07 mL, 47.4 mmol) was added to a solution of the previous
carboxylic acid intermediate (19.83 g, 39.5 mmol) and DMF (5 drops)
in dry tetrahydrofuran (100 mL). Upon addition of oxalyl chloride, the
immediate formation of gas was observed. The RM was stirred at 0 °C
for 1.5 h. Then an additional 0.5 equiv of oxalyl chloride was added,
and the RM was stirred for 1 h (repeated once until full conversion
was obtained). The RM was evaporated to dryness in vacuo to afford
20.5 g (97% yield) of the acid chloride as a white solid, which was used
directly in the next step. To a solution of (S)-4-benzyl-2-oxazolidinone
(7.50 g, 42.3 mmol) in dry tetrahydrofuran (60 mL) under a nitrogen
atmosphere, n-butyllithium (26.4 mL, 42.3 mmol) was added slowly at
−78 °C. The RM was stirred for 40 min at −78 °C. After 40 min, the
anion solution was added via a cannula to a solution of the acid
chloride (20 g, 38.5 mmol) in 60 mL of THF at −78 °C. The RM was
stirred for 1.5 h at −78 °C. When the reaction was finished, it was
quenched with an ammonium chloride solution at −70 °C. The RM
was then warmed to RT, extracted with EtOAc, washed with brine, and
dried over Na2SO4. The organic layer was filtered and concentrated to
afford 26.34 g of a yellow solid. The two enantiomers 14 and 15 were
separated by flash column chromatography using 5:1 heptane/EtOAc
(the faster-running spot corresponded to 15) and were obtained as
light-yellow solids with a purity of 97% in yields of 85% (11.17 g) and
81% (10.6 g), respectively; m/z 661 [M + H]+.
(4bR,5aS)-9-(tert-Butoxycarbonyl)-12-cyclohexyl-3-me-
thoxy-4b,5,5a,6-tetrahydrobenzo[3,4]cyclopropa[5,6]azepino-
[1,2-a]indole-5a-carboxylic Acid (17). Diastereoisomer 15 (11.17
g, 16.90 mmol) was first dissolved in THF (130 mL), and then
methanol (130 mL) was added. A 1 N NaOH solution (101 mL, 101
mmol) was added slowly to keep the temperature below 30 °C. The
RM was stirred at RT for 2 h. When the reaction was finished, 1 N
HCl solution was added until the pH reached 2. Water (500 mL) was
then added, and the RM was extracted with EtOAc, washed with brine,
and concentrated. Purification by flash column chromatography using
1:1 heptane/EtOAc afforded 5.24 g (60% yield) of the desired
carboxylic acid 17 with an ee of 97%. 1H NMR (400 MHz, DMSO-d6,
mixture of rotamers, 60/40 major/minor) δ 1.08−1.26 (m, 2H) 1.27−
1.48 (m, 8H) 1.51−1.62 (m, 20H) 1.66−1.79 (m, 4H) 1.81−2.11 (m,
8H) 2.65−2.92 (m, 4H) 3.41−3.44 (m, 1H major) 3.85 (s, 3H major)
3.86 (s, 3H minor) 3.95 (d, J = 15.19 Hz, 1H minor) 5.12 (d, J = 15.19
Hz, 1H minor) 5.35 (d, J = 14.74 Hz, 1H major) 6.99−7.08 (m, 2H)
7.17−7.31 (m, 4H) 7.52 (dd, J = 8.47, 1.43 Hz, 1H major) 7.60 (dd, J
= 8.58, 1.32 Hz, 1H minor) 7.79 (d, J = 8.58 Hz, 1H major) 7.85 (d, J
= 8.36 Hz, 1H minor) 7.97 (d, J = 0.88 Hz, 1H minor) 8.26 (d, J =
1.10 Hz, 1H major).
(m, 1H) 2.01 (m, 3H) 2.56 (m, 3H) 2.77 (m, 1H) 2.99 (s, 3H) 3.26
(m, 2H) 3.50−3.71 (m, 6H) 3.87 (s, 3H) 4.44 (d, J = 14.1 Hz, 1H)
5.09 (d, J = 15.0 Hz, 1H) 6.95 (s, 1H) 7.13 (s, 1H) 7.19 (d, J = 8.6 Hz,
1H) 7.47 (d, J = 8.0 Hz, 1H) 7.54 (d, J = 8.3 Hz, 1H) 7.88 (d, J = 7.8
Hz, 1H) 8.33 (s, 1H) 11.40 (s, 1H).
(19S)-2,19-Methano-3,7:4,1-dimetheno-1H,11H-
14,10,2,9,11,17-benzoxathiatetraazacyclodocosine-8,18-
(9H,15H)-dione 27-Cyclohexyl-12,13,16,17,19,20-hexahydro-
22-methoxy-11,17-dimethyl-10,10-dioxide (11a) and (19R)-
2,19-Methano-3,7:4,1-dimetheno-1H,11H-14,10,2,9,11,17-ben-
zoxathiatetraazacyclodocosine-8,18(9H,15H)-dione 27-Cyclo-
hexyl-12,13,16,17,19,20-hexahydro-22-methoxy-11,17-di-
methyl-10,10-dioxide (12a). A solution of 10a (56 mg, 0.092
mmol) in MeOH (15 mL) and THF (5 mL) was hydrogenated in an
H-cube apparatus using a 10% Pd on carbon cartridge. Then the
solvent was evaporated, and the residue was purified by column
chromatography (CH2Cl2/CH3CN 9:1) to give 23 mg (41% yield) of
the desired saturated product as a racemic mixture, which was further
separated by SFC using a 6.5 min run on a chiral CHIRALCEL OD-H
column (250 × 10 mm, coated on 5 μm silica gel) with 55%
methanol/45% CO2 as the mobile phase at a flow rate of 10 mL/min
to give the two pure enantiomers 11a and 12a. Retention times under
these conditions were observed at 4.25 and 5.54 min, respectively.
Compound 11a: HPLC-chiral 99.57%, tR = 2.80 min, ee = 99.1%;
LCMS-3 99.7%, tR = 5.12 min, m/z 609 [M + H]+. Compound 12a:
HPLC-chiral 100%, tR = 2.27 min, ee = 100%; LCMS-3 99.7%, tR =
(1aR,12bS)-8-Cyclohexyl-11-methoxy-16,22-dimethyl-
1,12b-dihydro-5,1a-(methanoiminothioiminoethanooxy-
ethanoiminomethano)cyclopropa[d]indolo[2,1-a][2]benz-
azepine-13,23(2H)-dione 15,15-Dioxide (19a). This macrocycle
was synthesized following the same sequence of reactions as described
for the synthesis of the unsaturated macrocyclic analogue 10a, starting
from carboxylic acid 17 instead of 20, and was obtained as a white
solid. LCMS-5 100%, tR = 6.57 min; LCMS-6 100%, tR = 1.90 min;
1
5.12 min, m/z 609 [M + H]+; H NMR (400 MHz, DMSO-d6) δ
1.15−1.30 (m, 1H) 1.32−1.46 (m, 2H) 1.55−1.81 (m, 3H) 1.82−2.09
(m, 4H) 2.54−2.59 (m, 1H) 2.79−2.91 (m, 1H) 2.92−3.09 (m, 5H)
3.34−3.49 (m, 6H) 3.51−3.60 (m, 1H) 3.64−3.74 (m, 2H) 3.76−3.96
(m, 6H) 4.53 (d, J = 14.81 Hz, 1H) 6.98−7.11 (m, 2H) 7.32 (m, 2H)
7.75−7.88 (m, 2H) 11.30 (br s, 1H).
1
HPLC-chiral 100%, tR = 5.00 min, ee = 100%; H NMR (400 MHz,
(4bS,5aR)-tert-Butyl 5a-((S)-4-Benzyl-2-oxooxazolidine-3-
c a r b o n y l ) - 1 2 - c y c l o h e x y l - 3 - m e t h o x y - 4 b , 5 , 5 a , 6 -
tetrahydrobenzo[3,4]cyclopropa[5,6]azepino[1,2-a]indole-9-
carboxylate (14) and (4bR,5aS)-tert-Butyl 5a-((S)-4-Benzyl-2-
oxooxazolidine-3-carbonyl)-12-cyclohexyl-3-methoxy-
4b,5,5a,6-tetrahydrobenzo[3,4]cyclopropa[5,6]azepino[1,2-a]-
indole-9-carboxylate (15). Sodium hydride (1.54 g, 64 mmol) was
added to a stirred suspension of trimethylsulfoxonium chloride (9.07 g,
2.2 equiv) in anhydrous DMSO (100 mL) under nitrogen. After 30
min at RT, 16 g (32 mmol, 1 equiv) of neat bisester 7 was added
portionwise. After 2 h at RT, TLC showed completion of the reaction,
and the RM was quenched with water. The resulting precipitate was
filtered off, washed with water, and dried in the vacuum oven.
Purification by flash chromatography on silica gel (gradient of MeOH
in DCM) provided 9.5 g (57% yield) of the desired cyclopropyl
intermediate 13 as a racemic mixture. Subsequent cleavage of the
methyl ester using the same procedure as described for the synthesis of
20 gave the corresponding carboxylic acid in 95% yield as a light-
yellow solid. At 0 °C under a protective atmosphere, oxalyl chloride
DMSO-d6, 100 °C) δ 1.21−2.08 (m, 12H) 2.48−2.51 (m, 1H) 2.82−
3.90 (m, 16H) 5.13 (d, J = 15.34 Hz, 1H) 7.01 (dd, J = 8.48, 2.83 Hz,
1H) 7.08−7.17 (m, 1H) 7.28 (d, J = 8.48 Hz, 1H) 7.38 (dd, J = 8.48,
1.21 Hz, 1H) 7.77 (d, J = 8.48 Hz, 1H) 8.04 (d, J = 1.21 Hz, 1H)
10.93 (br s, 1H).
(1aS,12bR)-8-Cyclohexyl-11-methoxy-16,22-dimethyl-
1,12b-dihydro-5,1a-(methanoiminothioiminoethanooxy-
ethanoiminomethano)cyclopropa[d]indolo [2,1-a][2]benz-
azepine-13,23(2H)-dione 15,15-Dioxide (18a). This macrocycle
was synthesized following the same sequence of reactions as described
for the synthesis of the other enantiomer 19a, starting from
diastereoisomer 14 instead of 15, and was obtained as a white solid.
LCMS-5 100%, tR = 6.56 min; LCMS-6 100%, tR = 1.90 min; HPLC-
chiral 100%, tR = 4.62 min, ee = 100%; 1H NMR (400 MHz, DMSO-
d6) δ 1.13−2.14 (m, 12H) 2.45 (t, J = 7.26 Hz, 1H) 2.75−3.90 (m,
16H) 5.11 (d, J = 15.19 Hz, 1H) 7.05 (dd, J = 8.47, 2.53 Hz, 1H) 7.13
(d, J = 2.20 Hz, 1H) 7.26 (d, J = 8.36 Hz, 1H) 7.28−7.35 (m, 1H)
7.79 (d, J = 8.36 Hz, 1H) 8.01−8.22 (m, 1H) 11.34 (s, 1H).
J
dx.doi.org/10.1021/jm401396p | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
1,1-Dimethylethyl 13-Cyclohexyl-6-hydroxymethyl-3-me-
thoxy-7H-indolo[2,1-a][2]benzazepine-10-carboxylate (21).
To a stirred solution of acid 20 (1300 mg, 2.67 mmol) and N-
methylmorpholine (270 mg, 2.67 mmol) in dry THF (10 mL) was
added dropwise at −15 °C isobutyl chloroformate (364 mg, 2.67
mmol), and the mixture was stirred at −15 °C for 15 min. The RM
turned from yellow to orange. The suspension was filtered, and NaBH4
(202 mg, 5.33 mmol) was added at 0 °C to the filtrate, followed by 2
drops of water. The RM was stirred in an ice bath for 2 h and then
diluted with water and extracted with EtOAc. The organic phase was
washed with water until neutral, dried (MgSO4), filtered, and
evaporated under vacuum. Purification by flash chromatography
(heptane/EtOAc 9:1 to 1:1) gave 1 g (78% yield) of the desired
alcohol 21 as a slightly yellow solid. LCMS-1 99.17%, tR = 3.58 min,
10,10-dioxide (25). To a solution of tert-butyl ester 24 (310 mg,
0.476 mmol) in DCM was added TFA (3.2 g, 60 equiv) at RT. The
RM immediately turned yellow upon addition of TFA. After 2 h, the
RM was concentrated to dryness, and the residue was triturated in
diisopropyl ether. The resulting cream solid was filtered off and dried
under high vacuum to give 350 mg (100% yield) of the corresponding
carboxylic acid, which was used directly in the next step as a TFA salt.
LCMS-1 98.5%, tR = 2.39 min, m/z 595 [M + H]+. To the carboxylic
acid intermediate (TFA salt) dissolved in acetonitrile (20 mL) was
added CDI (88 mg, 1.1 equiv) at RT. The RM was then heated at 50
°C for 2 h, at which time LC−MS showed complete conversion of the
carboxylic acid to the acylimidazole intermediate. The RM was then
diluted with ACN (50 mL), and DBU (225 mg, 3 equiv) was added at
RT. After 16 h, the RM was quenched with diluted HCl (pH 2),
concentrated in vacuum, and extracted with DCM. The organic layer
was washed with water (until the water layer reached neutral pH),
dried over MgSO4, filtered, and concentrated under vacuum. The
residue was triturated in DCM, and the resulting cream solid was
filtered off to give 146 mg (46% yield) of compound 25. LCMS-2 98%,
tR = 1.28 min, m/z 577 [M + H]+; LCMS-4 100%, tR = 1.51 min; 1H
NMR (400 MHz, DMSO-d6) δ 1.06−1.21 (m, 1H) 1.24−1.51 (m,
8H) 1.60−2.09 (m, 8H) 2.13−2.28 (m, 1H) 2.55−2.68 (m, 1H)
2.70−2.84 (m, 2H) 3.02 (s, 3H) 3.10−3.22 (m, 1H) 3.36−3.49 (m,
2H) 3.85 (s, 3H) 4.08 (d, J = 13.80 Hz, 1H) 5.11 (d, J = 13.80 Hz,
1H) 6.65 (s, 1H) 7.02 (d, J = 2.51 Hz, 1H) 7.10 (dd, J = 8.78, 2.76 Hz,
1H) 7.35 (d, J = 8.28 Hz, 1H) 7.49 (d, J = 8.53 Hz, 1H) 7.82 (d, J =
8.53 Hz, 1H) 8.14 (s, 1H) 11.50 (br s, 1H).
1
m/z 474 [M + H]+; H NMR (400 MHz, CDCl3) δ 1.24−1.29 (m,
1H) 1.32−1.46 (m, 2H) 1.63 (s, 9H) 1.66−1.72 (m, 1H) 1.73−1.82
(m, 2H) 1.87−2.17 (m, 4H) 2.76−2.93 (m, 1H) 3.89 (s, 3H) 4.10−
4.28 (m, 1H) 4.35 (br s, 2H) 4.74−4.99 (m, 1H) 6.68 (s, 1H) 6.88 (d,
J = 2.35 Hz, 1H) 6.99 (dd, J = 8.61, 2.54 Hz, 1H) 7.48 (d, J = 8.61 Hz,
1H) 7.68 (d, J = 8.41 Hz, 1H) 7.83 (d, J = 8.41 Hz, 1H) 8.12 (s, 1H).
1,1-Dimethylethyl 13-Cyclohexyl-3-methoxy-6-
[[(methylsulfonyl)oxy]methyl]-7H-indolo[2,1-a][2]-
benzazepine-10-carboxylate (22). To a solution of alcohol 21
(500 mg, 1.056 mmol, 1.0 equiv) in THF (10 mL) were added Et3N
(160 mg, 1.5 equiv) and methanesulfonyl chloride (181 mg, 1.5 equiv)
at 0 °C. The resulting solution was stirred at 0 °C for 2 h. The RM was
then quenched with water and extracted with EtOAc. The organic
layer was washed with water two times, dried over MgSO4, filtered,
and concentrated to give 555 mg (90% yield) of mesyl alcohol 22 as a
yellow solid, which was used without further purification in the next
step. LCMS-3 99%, tR = 6.46 min, m/z 552 [M + H]+; 1H NMR (400
MHz, CDCl3) δ 1.22−1.29 (m, 1H) 1.31−1.47 (m, 2H) 1.53−1.67
(m, 10H) 1.70−1.82 (m, 2H) 1.90−2.15 (m, 4H) 2.72−2.90 (m, 4H)
3.89 (br s, 3H) 4.19−4.41 (m, 1H) 4.77−4.87 (m, 1H) 4.92 (br s, 2H)
6.84 (br s, 1H) 6.89 (br s, 1H) 6.99−7.10 (m, 1H) 7.49 (d, J = 8.61
Hz, 1H) 7.70 (d, J = 8.41 Hz, 1H) 7.80−7.87 (m, 1H) 8.15 (br s, 1H).
1,1-Dimethylethyl 13-Cyclohexyl-6-[methyl[4-
(methylamino)butyl]amino]methyl-3-methoxy-7H-indolo[2,1-
a][2]benzazepine-10-carboxylate (23). To N,N′-dimethyl-1,4-
butanediamine (105 mg, 5 equiv) dissolved in THF (5 mL) was
added mesyl alcohol 22 (100 mg, 0.181 mmol) at RT. The RM was
stirred at RT for 48 h and then diluted with water and extracted with
EtOAc. The organic layer was dried (MgSO4), filtered, and
concentrated to give 450 mg (96% yield) of compound 23 as a
slightly yellow solid, which was used without further purification in the
next step. LCMS-1 99%, tR = 3.64 min (broad peak), m/z 572 [M +
ASSOCIATED CONTENT
* Supporting Information
Details of LCMS and chiral HPLC methods. This material is
available free of charge via the Internet at http://pubs.acs.org.
■
S
AUTHOR INFORMATION
Corresponding Author
*Phone: + 32 14 64 17 84. E-mail: svendev1@its.jnj.com.
■
Present Addresses
†Janssen Research & Development, Welsh and McKean Roads,
Spring House, PA 19477, USA.
‡Roche R&D China Ltd., 720 Ca iLun Rd., Building 5, Pudong,
201203 Shanghai, P. R. China.
§Cartagenia, Technologie laan 3, 3001 Leuven, Belgium.
∥University of Applied Sciences and Arts Western Switzerland,
Institute of Life Technologies, tRe du Rawyl 47, 1950 Sion 2,
Switzerland.
1
H]+; H NMR (400 MHz, CDCl3) δ 1.20−1.26 (m, 1H) 1.36−1.40
(m, 2H) 1.59 (s, 12H) 1.68−1.80 (m, 2H) 1.81−1.97 (m, 2H) 1.97−
2.14 (m, 7H) 2.18−2.33 (m, 2H) 2.41 (s, 3H) 2.44−2.58 (m, 2H)
2.74−2.93 (m, 1H) 3.10 (br s, 2H) 3.90 (s, 3H) 3.99−4.11 (m, 1H)
4.94−5.19 (m, 1H) 6.55 (s, 1H) 6.81−6.90 (m, 1H) 6.91−7.02 (m,
1H) 7.48 (d, J = 8.41 Hz, 1H) 7.67 (d, J = 8.61 Hz, 1H) 7.76−7.85
(m, 1H) 8.16 (s, 1H).
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
We thank the analytical chemistry team for their technical
assistance.
■
1,1-Dimethylethyl 13-Cyclohexyl-6-[[methyl[4-
[(aminosulfonyl)methylamino]butyl]amino]methyl]-3-me-
thoxy-7H-indolo[2,1-a][2]benzazepine-10-carboxylate (24). A
mixture of compound 23 (400 mg, 0.7 mmol) and sulfamide (336 mg,
5 equiv) in dioxane (20 mL) was heated at 105 °C. After 3 h, the RM
was cooled to RT, concentrated in vacuum, redissolved in DCM,
extracted with water, dried over MgSO4, filtered, and concentrated.
Purification by flash chromatography (DCM to EtOAc) gave 350 mg
(63%) of compound 24 as a white foam. LCMS-1 100%, tR = 3.18 min,
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