Thieno[3,2-b]pyrrole-5-carboxamides as New Reversible Inhibitors of Histone Lysine Demethylase KDM1A/LSD1. Part 2: Structure-Based Drug Design and Structure-Activity Relationship

Article abstract of DOI:10.1021/acs.jmedchem.6b01019

The balance of methylation levels at histone H3 lysine 4 (H3K4) is regulated by KDM1A (LSD1). KDM1A is overexpressed in several tumor types, thus representing an emerging target for the development of novel cancer therapeutics. We have previously describe

Full text of DOI:10.1021/acs.jmedchem.6b01019

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Article
Thieno[3,2-b]pyrrole-5-carboxamides as New Reversible
Inhibitors of Histone Lysine Demethylase KDM1A/LSD1. Part 2:
Structure Based Drug Design and Structure-Activity Relationship
Paola Vianello, Luca Sartori, Federica Amigoni, Anna Cappa, Giovanni Fagá, Raimondo Fattori, Elena
Legnaghi, Giuseppe Ciossani, Andrea Mattevi, Giuseppe Meroni, Loris Moretti, Valentina Cecatiello,
Sebastiano Pasqualato, Alessia Romussi, Florian Thaler, Paolo Trifiró, Oronza A. Botrugno, Manuela
Villa, Paola Dessanti, Saverio Minucci, Stefania Vultaggio, Elisa Zagarrí, Mario Varasi, and Ciro Mercurio
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.6b01019 • Publication Date (Web): 10 Feb 2017
Downloaded from http://pubs.acs.org on February 11, 2017
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Zagarrí, Elisa; Fondazione FADOI, Centro Studi della Societá Scientifica
Varasi, Mario; IFOM - the FIRC Institute of Molecular Oncology Foundation,
Experimental Therapeutics Unit
Mercurio, Ciro; IFOM - the FIRC Institute of Molecular Oncology
Foundation, Experimental Therapeutics Unit
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Thieno[3,2ꢀb]pyrroleꢀ5ꢀcarboxamides as New
Reversible Inhibitors of Histone Lysine
Demethylase KDM1A/LSD1. Part 2: Structure
Based Drug Design and StructureꢀActivity
Relationship
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*
,†,#
*,†,§
†
†,#
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Paola Vianello,
Luca Sartori,
Federica Amigoni, Anna Cappa, Giovanni Fagá,
†,§
†
‡
‡
Raimondo Fattori,
Elena Legnaghi, Giuseppe Ciossani,
Andrea Mattevi, Giuseppe
†
,§
†,¥
Θ
†, #
∥
∥
Meroni, Loris Moretti, Valentina Cecatiello, Sebastiano Pasqualato, Alessia Romussi,
†
,§
†,§
†,§
†
†
Florian Thaler, Paolo Trifiró, Manuela Villa, Oronza A. Botrugno, Paola Dessanti,
†
,
†, #
†
†,§
†,±,#
⊥
Saverio Minucci, Stefania Vultaggio, Elisa Zagarrí, Mario Varasi, Ciro Mercurio,
† Department of Experimental Oncology, Academic Drug Discovery, European Institute of
Oncology, Via Adamello 16, 20139 Milan, Italy
‡
Department of Biology and Biotechnology, University of Pavia, Via Ferrata 1, 27100 Pavia,
Italy.
ꢀCrystallography Unit, Department of Experimental Oncology, European Institute of Oncology,
∥
Via Adamello 16, 20139 Milan, Italy
Θ IFOM, Fondazione Istituto FIRC di Oncologia Molecolare, Via Adamello 16, 20139 Milano,
Italy.
⊥ꢀDepartment of Biosciences, University of Milan, Via Celoria 26, 20133 Milan, Italy
±
Genextra Group, DAC s.r.l., Via Adamello 16, 20139 Milan, Italy
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KEYWORDS: Reversible inhibitors; LSD1; KDM1A; epigenetics; leukemia; lysineꢀspecific
demethylaseꢀ1
ABSTRACT
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The balance of methylation levels at histone H3 lysine 4 (H3K4) is regulated by KDM1A
(LSD1). KDM1A is overexpressed in several tumor types, thus representing an emerging target
for the development of novel cancer therapeutics. We have previously described (Part 1) the
identification of thieno[3,2ꢀb]pyrroleꢀ5ꢀcarboxamides, as novel reversible inhibitors of KDM1A,
whose preliminary exploration resulted in compound 2 with biochemical IC = 160 nM. We
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now report the structureꢀguided optimization of this chemical series, based on multiple
ligand/KDM1AꢀCoRest coꢀcrystal structures, which led to several extremely potent inhibitors. In
particular, compounds 46, 49 and 50 showed single digit nanomolar IC50 values for in vitro
inhibition of KDM1A, with high selectivity in secondary assays. In THPꢀ1 cells these
compounds transcriptionally affected the expression of genes regulated by KDM1A, such as
CD14, CD11b, and CD86. Moreover, 49 and 50 showed a remarkable antiꢀclonogenic cell
growth effect on MLLꢀAF9 human leukemia cells.
INTRODUCTION
Aberrant postꢀtranslational modifications of chromatin have been associated with the onset and
progression of several human diseases, including cancer. In particular, the Nꢀmethylation status
of histones is one of the crucial factors that regulate transcription and eventually control
enhancement or repression of gene expression. Histones can be methylated on lysine and
arginine residues by histone methyl transferase proteins. Lysines demethylation is specifically
catalyzed by two families of enzymes: i) Lysine specific demethylases (KDM1) which are
flavine adenine dinucleotide (FAD) dependent proteins related to monoamine oxidases (MAOs)
and catalyze methyl removal from monoꢀ and dimethylated histones H3K9 and H3K4, and ii)
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JmjCꢀdomain containing histone demethylases (KDM2; KDM3; KDM4; KDM5, JARID2;
(
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KDM6; KDM7; KDM8), which utilize alphaꢀketoglutarate (2OG) and iron (Fe ) as a cofactor,
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to catalyze demethylation of monoꢀ, diꢀ and trimethylated lysines at multiple sites. Two human
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FADꢀdependent demethylases have been identified: LSD1, also known as KDM1A and LSD2,
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also known as KDM1B . KDM1A is highly expressed in different cancer types, including acute
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myeloid leukemia, prostate cancer, ERꢀnegative breast cancer, and neuroblastoma and often
its overexpression correlates with poor prognosis.
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After the discovery of tranylcypromine as a weak irreversible KDM1A inhibitor, an extensive
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effort has been dedicated to the field.
Three irreversible tranylcypromine derivatives from
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21,22
GSK, Oryzon (see Figure 1: GSK2879552
, ORY1001
depicted as 55) and Incyte
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(INC059872 undisclosed structure) are now in clinical stages . Conversely, reports on the
preclinical development of low molecular weight reversible inhibitors have been much more
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limited.
In particular, SPꢀ2509 was reported by Salarius to be active in xenograft
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models,
although the compound itself did not demonstrate a robust correlation between
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biochemical activity against KDM1A and inhibition of cell proliferation.
Insert Figure 1
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As reported in Part 1, from an High Throughput Screening campaign (HTS) we identified a
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promising chemical series, namely 4Hꢀthieno[3,2ꢀb]pyrroleꢀ5ꢀcarboxamide (represented by
compound 1, Figure 2) with inhibitory activity against KDM1A. Initial SAR studies indicated
that substitutions at the phenyl ring of 1 could increase the inhibitory potency, resulting in
compound 2, with an IC of 160 nM for KDM1A inhibition.
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Insert Figure 2
Docking experiments of compound 2 allowed dual pose orientations (see Figure 3) directing the
positively charged piperidine tail towards two negatively charged regions. We synthesized a
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series of derivatives able to reach different portions of the protein to solve the ambiguity of
docking results and to increase inhibitory potency. The new compounds prepared gave us a clear
indication of the relevant substituents for inhibitory activity and their interactions with protein
active site of this novel class of nonꢀcovalent KDM1A inhibitors.
RESULTS AND DISCUSSION
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Chemistry
A general synthesis for the derivatives reported is depicted in Scheme 1 (Method A). Ethyl 4Hꢀ
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thieno[3,2ꢀb]pyrroleꢀ5ꢀcarboxylate (3a) was methylated at the pyrrole nitrogen and
hydrolyzed to the acid intermediate 3c. Reaction of the corresponding acyl chloride with the
appropriate aniline (3e−28e) gave final amides 3ꢀ21 and 22f−28f, in which the piperidine
nitrogen is BOCꢀprotected. Finally, compounds 22f−28f were deprotected to 22−28 by acidic
treatment, and compound 22 was treated with the appropriate aldehydes under reductive
amination conditions to give 29 or 30 or with acetyl chloride to give 31 (Scheme 1).
Insert Scheme 1
The general synthesis of the anilino derivatives is represented in Scheme 2: (bromomethyl)ꢀnitroꢀ
benzenes (15g−17g) were coupled with the appropriate biphenols to give 15iꢀ19i. Mitsunobu
reaction of tertꢀbutyl 4ꢀhydroxypiperidineꢀ1ꢀcarboxylate with the desired nitrophenols allowed
the preparation of derivatives 16j−18j, which were transformed into the Nꢀmethyl derivatives
16d−18d, by Boc removal after acidic treatment, and reductive amination with formaldehyde
and NaBH CN (15d and 19d were instead prepared by reaction of 15i and 19i with 1ꢀ
3
methylpiperidinꢀ4ꢀol). Nitro reduction originated desired anilines 15e−19e and 22e.
Insert Scheme 2
Different methods used to prepare anilino derivatives (20e, 21e, 23e−28e) are reported in
Supporting Information.
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Alternatively, a general procedure for the synthesis of the compounds is depicted in Scheme 3.
The basic moiety BOC protected was inserted at the last step followed by usual protective group
removal. The nitro group in compound 15i was reduced via catalytic hydrogenation to give the
aniline 33k, which was condensed to compound 3c previously transformed into acyl chloride.
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The thus obtained 33l through Mitsunobu reaction with alcohols R ꢀOH generated 33f−43f that
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Insert Scheme 3
Ortho, metaꢀdisubstituted compounds (Tables 5ꢀ7) were prepared according to Scheme 4 or 5.
In Scheme 4, 2ꢀ(hydroxymethyl)ꢀ6ꢀnitroꢀbenzoate 45n, obtained from 7ꢀnitroꢀ3Hꢀisobenzofuranꢀ
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ꢀone by heating in 1 M sodium hydroxide and THF, was treated with NaH and an excess of
the required alkyl halides, generating the 45v and 50v. Subsequent reduction of the ester gave the
alcohols 45w and 50w, which were converted into their bromo derivatives 45g and 50g. Reaction
with hydroquinone furnished the phenols 45l and 50l. Phenolic function was thus accessible to
Mitsunobu reaction to give 45d−50d, their nitro groups were transformed into anilines 45eꢀ50e,
useful building blocks for the benzamide preparation 45f−50f, obtained by reacting with 3c or
49c. Boc protecting groups were finally removed to give the compounds 45−50.
Insert Scheme 4
Alternatively diꢀsubstituted derivatives were prepared according to Scheme 5: 45n was alkylated
with isopropyl iodide providing 52o, which was further converted into 52p by protecting the
alcoholic group with TBDSCl. The carboxylic function was reduced to 52q and the thus formed
alcohol transformed into its mesylate 52r to facilitate the reaction with hydroquinone and the
ether formation in 52l. Free phenolic oxygen reacted with tertꢀbutyl 4ꢀhydroxypiperidineꢀ1ꢀ
carboxylate to give 52d, which, after reduction to the anilino derivative 52e, reacted with the
carboxylic acid 3c to give intermediate 52s. Removal of the protective group (52t),
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transformation of the alcoholic function into a mesylate group (52u) and subsequent reaction
with morpholine or an appropriate alcohol gave the BOC intermediates 52f−54f, which were
finally hydrolyzed to give 52−54.
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Insert Scheme 5
SAR investigation
The evidence that docking runs allowed dual placement of piperidine moiety and the lack of
unambiguous data from Xꢀray crystallography for compound 2, prompted us to perform an
electrostatic potential analysis of the protein active site (Figure 3), identifying two regions of
potential interactions for positively charged piperidine tail, both justifying such an increase in
potency of compound 2 with respect to 1. One region is located around Asp375, above the
thienopyrrole plane. The other is located around Asp555 and Asp556, which are located below
the thienopyrrole plane. The compound (green in Figure 3) would adopt an extended
conformation to interact with Asp375, whereas the interaction with Asp556 would force the
inhibitor in a uꢀshaped conformation (pink in Figure 3). The two poses of 2, obtained with
different docking programs and submitted to Molecular Dynamic, showed complexes stability
sufficient to be both considered as starting points for SAR exploration (see Supporting
Information for details).
Insert Figure 3
Taking into consideration the results obtained when meta position of the hit was modified, as
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described in Part 1, we planned a careful SAR exploration, initially directed to the ortho, meta
and para positions on benzamide ring and subsequently to the optimal residue identified.
Different spacers, hydroquinone vs resorcinol or cathecol, and different terminal basic moieties
were also investigated and to conclude the best results were combined to generate orthoꢀmeta
disubstituted derivatives (see Figure 4).
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Ortho, meta and para benzamide investigation
The investigation on the benzamide was designed with the following aims:
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_to explore the area occupied by the substituents on benzamide function,
_to modulate the substituents’ contribution to the inhibitory activity
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3_to verify whether the structural modifications of the ligand could provide Xꢀray structures.
Insert Figure 4
As reported in Table 1, the substituents were selected from those already tested in the meta
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series . The expansion was fully explored to gain a complete SAR and to direct the substituents
toward the two negatively charged regions. We used piperidine as a basic moiety to take
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advantage of activity increase showed in the meta series reported in Part 1. Whereas methoxy,
methoxymethyl, aryloxy methyl, and arylmethoxy substituents in the benzamide moiety
maintained high micromolar activity when applied to the meta position (1, 7, 10, 13), they
brought instead to almost inactive compounds when placed in ortho and para position (6, 8, 9,
11, 12, 14). When the chain bearing a piperidinoxyphenoxymethyl group was moved from meta
to ortho and para, the potency of the unsubstituted term 3 was however improved, although
while para derivative 17 resulted in the micromolar range, ortho derivatives 15 and 22 resulted
slightly more active than the corresponding meta ones, 16 and 2. We report that several attempts
to place unsubstituted piperidine in position para, failed for synthetic hurdles during BOC
deprotection of the intermediate.
Insert Table 1
This preliminary exploration resulted quite satisfactory. One of the most potent ortho derivatives
15 easily crystallized and the analysis of Xꢀray structure provided an explanation for potency
improvement (see Figure 5A). Ortho derivative 15 accommodates the benzamide moiety
favoring aromaticꢀaromatic interactions and directs the basic terminal residue towards one of the
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two negatively charged regions (Asp555). Compound 15 unambiguously follows one of the two
hypothesized positions, pointing downward with respect to the thienopyrrole plane. The ligand
occupies the catalytic site of KDM1A, the size and the shape of the active site renders the packed
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“
U shaped” conformation as the most preferred one. Main hydrophobic interactions have been
evidenced with no visible polar contacts. The thienopyrrole moiety is comprised between
residues Val333, Thr335, Leu663, Tyr765 and Thr814 into a cage that is delimitated by flavin.
The second ring system is packed between residues Val333, Phe543 and Trp699.
Compound 17 (see Figure 5B), a para substituted derivative that showed to inhibit KDM1A,
albeit with considerably less potency (2.5 µM) has also been crystallized. The benzamide moiety
of 17 is forced out of the plane altering the geometry of its aromaticꢀaromatic interaction with
flavin, a possible explanation for the lower potency, while the terminal basic moiety located in
proximity to Glu379 (2.9 A) could contribute to the residual activity.
Insert Figure 5
To conclude activity and structural comparisons between ortho, meta and para derivatives, we
speculate that meta derivative 2 could adopt a conformation similar to 15, but having interaction
with Asp556 instead of Asp555. The para derivative 17 interacts in the upward negative region
(Glu379), reachable also by obtained docking pose of 2, but the 5ꢀ5 system planarity distortion
and the lesser biochemical potency suggest that this conformation is unlikely the one adopted by
2. The possible explanation for the inactivity of shorter derivatives (4ꢀ6, 8ꢀ14) is that only meta
derivatives can reach interactions with Gln358 (docking results, see Supporting Information).
Finally compound 15 was also evaluated to define its biochemical mechanism of inhibition.
According to Figure 6A, LineweaverꢀBurk plot indicated a nearly competitive type inhibition for
compound 15, with a Ki = 2.3 µM. Accordingly, in Figure 6B we report the overlap of Xꢀray
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structures of 15 with 2V1D , clearly showing that the ortho substituent on the benzamide
moiety occupies the same region of MetꢀThrꢀArgꢀAla residues of the substrate.
Insert Figure 6
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Ortho, meta and para substitution of the hydroquinone
The influence of the ortho, meta and para position of the biꢀphenol moiety was investigated (see
Table 2), the meta being detrimental (18 resulted 3 fold less active than 15) whilst the ortho
barely active (19 IC = 68.2 µM). This trend in potency is justified by observing that the exit
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vector orientation for substituents on para biꢀphenol moiety is directed toward Asp555.
Insert Table 2
We continued our investigation by further modifying the lateral chain as depicted in Figure 4:
specifically only ortho substituted derivatives were prepared, both for the higher potency of the
analogues and for the advantage of having a support from Xꢀray crystallography.
Modifications on the spacer
Several compounds were designed and prepared to alter the hydroquinonic feature of 22 (see
Table 3). The phenyl ring has been replaced with piperidinomethyl moiety 44 resulting in a much
less potent compound. Also amide in place of ether (25) or nitrogen in place of oxygen (24 and
2
6) caused a reduction of potency. The position of oxygen seemed irrelevant being 16 (Table 1)
equipotent to 20, its removal instead barely influenced the activity (23 vs 22) and was of a much
greater impact when it occurred at the distal oxygen as in 26 or 21.
Insert Table 3
To explain these trends various aspects have been investigated:
ꢀ
ꢀ
ꢀ
Conformational analysis;
HOMOꢀLUMO energies and localization;
Partial charges on NH (NꢀMe).
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While for 44 and for 25 the conformational accessible space can quite easily explain the activity
decrease, when examining the couples 22/26 and 15/21 there are neither differences in
conformational space, nor in docking results or in nitrogen partial charges. Assuming that
electronic configuration might influence the affinity of the ligand and considering the aromaticꢀ
aromatic interaction between FAD and 5ꢀ5 thienopyrrole system, we investigated HOMOꢀ
LUMO localization and energies (see Supporting Information for details). We have noticed that
the decrease in activity is associated with the HOMO shifting toward piperidino ring, away from
the 5ꢀ5 system. The hypothesis that this shift may decrease the strength of the electronic
interaction between FAD and 5ꢀ5 system is under further investigations based on more accurate
abꢀinitio models.
10
11
12
13
14
15
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19
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21
22
23
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34
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40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Different basic moieties (ortho)
The inspection of the ring bearing the basic nitrogen was also extensively performed as reported
in Table 4: when 4ꢀpiperidinol was converted to 3ꢀpiperidinol as in 41 or shortened to 3ꢀ
pirrolidinole (42) and azetidinꢀ3ylꢀmethanol (27), or when the cycle was opened to Nꢀ
methylaminepropanol (43) or guanidinomethyl (28), the activity significantly dropped from two
to tenfold. Equipotent derivatives were obtained with 4ꢀaminocyclohexanol, where the cis isomer
36 resulted 2 fold more potent than the trans isomer (40), and (1S,5R)ꢀ8ꢀazabicyclo[3.2.1]octanꢀ
3ꢀol (37). The activity significantly improved with 4ꢀazepanole (35) and pyrrolidinꢀ3ꢀylmethanol
(34), in particular its 3R enantiomers (33) that showed an IC50 of 18 nM.
A small set of residues was prepared to alter the basicity of the terminal nitrogen and the space
occupancy of 22. Ethyl derivative 29 resulted slightly less active, while enlarging substituent as
in 30 or removing the basicity (31), as expected, resulted in complete inactivity.
34 was crystallized (see Figure 7) and showed the same orientation and interaction pattern with
protein residues as the parent 15. The length of the chain and the protein residue flexibility allow
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2
2
2
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2
2
2
2
2
2
3
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5
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5
5
5
5
6
interactions of nitrogen atoms with Asp555, where (3R)ꢀpyrrolidine group in 33 shows the best
activity among the series.
Insert Table 4
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
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9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Insert Figure 7
Orthoꢀmeta disubstituted derivatives
By observing Xꢀray crystal structure of 1 and 34 superimposed (Figure 8), it was evident that
ortho and meta substituents could occupy different pockets of KDM1A active site, thus
suggesting that their coꢀpresence could facilitate the interaction with the enzyme. With this in
mind, we approached the orthoꢀmeta double substitution, expanding separately the two positions,
according to Tables 5 and 6. We observed a further improvement in the activity: 46 in fact gave a
single digit nanomolar IC .
50
Insert Figure 8
Insert Table 5
Insert Table 6
Combining best features
The last three compounds reported in Table 7 were prepared by combining all those features that
had demonstrated positive impact in terms of biochemical potency. 49 was obtained by applying
the best substitution on pyrrolic nitrogen (ethyl group identified in Part 1), compound 50 by
adding an ethyloxymethyl in meta: their IC s were more or less identical, as well as their
5
0
selectivity versus KDM1B and MAOs.
Insert Table 7
49 and 50 have been crystallized, confirming the working hypothesis. The increased potency of
diꢀsubstituted compounds can be related to additional interactions between Gln358 and
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ethyloxymethyl chain, which is also responsible of conformational restraints on the longer chain,
thus with entropic gain that further induces the Uꢀshaped conformation.
Insert Figure 9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
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29
30
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34
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37
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49
50
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54
55
56
57
58
59
60
Biology
The ability of the best compounds to inhibit KDM1A in human acute myeloid THPꢀ1 cells was
evaluated. THPꢀ1 cells, characterized by the MLLꢀAF9 translocation, were selected for the
strong experimental evidences demonstrating the relevance of KDM1A in sustaining the
3
5
oncogenic potential of MLLꢀAF9 leukemia stem cells. Moreover, the absence of evident effect
on global level of histone H3K4 methylation after genetic ablation of KDM1A both in cancer
3
6
37
cells and hematopoietc cells let us to follow KDM1A inhibition in cells looking at
transcriptionally effect on known KDM1A regulating genes (see Supporting Informations
Figure S2). To this purpose THPꢀ1 cells were incubated with the selected inhibitors at the fixed
concentration of 1 micromolar and after 24 hours CD14, CD86, CD11b mRNA levels were
measured by quantitative RTꢀPCR. As reported in Table 7 all the tested compounds, similarly to
55, were able to induce an increase of mRNA expression of the selected genes. The observed
effects being similar to what has been obtained by KDM1A down regulation by shRNA,
demonstrated their ability to block KDM1A activity in cells inducing differentiation. To
additionally confirm induction of differentiation in AML cells mediated by our compounds, we
evaluated the expression of CD86 and CD11b by flow cytometry in THPꢀ1 cells treated for 48
hours. As reported in Supporting Information (Figure S2) all the three compounds induced a
clear increase of the expression of the two differentiation markers with respect to vehicle treated
cells. The antiproliferative activity of 49, 46 and 50 was determined on three different leukemia
cell lines: THPꢀ1, NB4 and MV4ꢀ11, representative of different subtypes of AMLs. THPꢀ1 and
MV4ꢀ11 are both characterized by MLL translocation, MV4ꢀ11 by a concurrent presence of
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FLT3 mutation. As shown in Table 8, the compounds tested demonstrated to exert
antiproliferative effects on the evaluated cell lines at a micromolar level.
Insert Table 8
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
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4
5
6
7
8
9
0
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2
3
4
5
6
7
8
9
0
To verify the ability of 49 and 50 to induce apoptosis or to affect cell cycle progression Annexin
VꢀPropidium Iodide (PI) double staining was used on THPꢀ1 cells. Data (as reported in
Supporting Information, Figure S3) showed that 72 hours treatment at 1µM of 49 and 50 and 0.5
µM of 55, did not induce cell death, in agreement with the known proꢀdifferentiation effects of
3
4,46
KDM1A inhibition in AML cells.
Finally, the anticlonogenic potential of 49 and 50 on
human acute myeloid leukemia THPꢀ1 was determined. As reported in the table 9 both the
compounds strongly inhibited the colony growth of THPꢀ1 cells and similarly to 55, induced
differentiation, as demonstrated by the increased expression of CD86 marker.
Insert Table 9
CONCLUSIONS
We have reported the development of thieno[3,2ꢀb]pyrrole KDM1A inhibitors, a novel chemical
series identified by an HTS campaign, which potently and reversibly inhibit KDM1A. Several
different synthetic pathways allowed an extensive exploration of various portions of the
molecule and led to the identification of the main structural requirements for the inhibitory
activity. This work has been supported by multiple ligand/KDM1AꢀCoRest coꢀcrystal structures,
which rationalized the gain in potency due to polar interaction with 2 negatively charged areas of
the catalytic site. We identified critical molecular features for potency: ortho substitution in the
benzamide portion brought to more stable complex with the enzyme, giving optimal inhibitory
activity and allowing the solution of multiple crystal structures. 3D information thus obtained
guided subsequent optimization. Para substitution of the hydroquinone moiety was mandatory
for the potency. The lack of substituents on the terminal nitrogen facilitates the interaction with
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the enzyme and the pyrrolidinꢀ3ꢀylꢀmethanol residue 34, in particular its 3R enantiomer 33
resulted as the most efficacious basic moiety among those explored. Concurrent biꢀsubstitution
of the benzamide at positions ortho and meta brought to a clear improvement of the potency,
generating single digit nanomolar inhibitors: 46, 49 and 50. These compounds were
characterized for their cellular activity: they clearly demonstrated their ability to inhibit KDM1A
in THPꢀ1 cells and to affect the proliferation in different leukemia cell lines. Moreover, both 49
and 50 remarkably inhibit colony formation in THPꢀ1 human leukemia cells. The interesting
biological data of this new series of reversible inhibitors, prompted us to bring thienopyrroles
toward further development.
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11
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14
15
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20
21
22
23
24
25
26
27
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33
34
35
36
37
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42
43
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45
46
47
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49
50
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60
EXPERIMENTAL SECTION
General Procedures.
Melting points were determined on a BUCHI Melting point Bꢀ540 and are uncorrected. Reagents
and solvents used, unless stated otherwise, were of commercially available reagent grade quality
and were used without further purification. Reaction solvents were used anhydrous purchased in
Sure/Seal bottles stored over molecular sieves. Silica gel flash chromatography purifications
were performed on Merck silica gel 60 (0.04ꢀ0.063 mm) or Biotage Isolera One system using
1
preꢀpacked columns from Grace and Biotage. Nuclear magnetic resonance spectra ( H NMR)
were recorded on an Agilent VNMRS 500 MHz spectrometer at 300 K and are referenced in
ppm (δ) relative to TMS. Coupling constants (J) are expressed in hertz (Hz). HPLCꢀMS
experiments were performed on an Acquity UPLC apparatus, equipped with a diode array and a
Micromass SQD single quadruple (Waters). The compounds used for biological tests were
monitored at 220 and 254 nM, with two different methods and their purity was found to be at
least 95%. Semiꢀpreparative HPLC was performed on a Waters apparatus eluting the sample
through a BEH C 18 column (5µm, 19mmx100mm) using decreasingly polar mixtures of water
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(
containing 5% of MeCN and 0.1% of trifluoroacetic acid) and MeCN (containing 0.1% of
trifluoroacetic acid).
4
ꢀMethylthieno[3,2ꢀb]pyrroleꢀ5ꢀcarboxylate (3c)
0
1
2
3
4
5
6
7
8
9
0
1
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3
4
5
6
7
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9
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0
0
.92 g (38 mmol) of LiOH in 11 mL of H O was added at rt to a solution of 1.6 g (7.7 mmol) of
2
2
9
ethyl 4ꢀmethylthieno[3,2ꢀb]pyrroleꢀ5ꢀcarboxylate (3b, see compound 105a in Part 1 ) in 11 mL
of EtOH. The mixture was stirred for 30 min at reflux. The solvent was evaporated, then H O
2
was added and the solution was brought to a pH value of about 2 with 2 M HCl. The mixture was
extracted with EtOAc, the combined organic layers were dried over Na SO and the solvent was
2
4
1
evaporated to give 1.39 g of 3c (99%) as a beige solid. H NMR (CDCl ) δ (ppm): 12.45 (bs, 1
3
H), 7.54 (d, J=5.4 Hz, 1 H), 7.20 (d, J=5.4 Hz, 1 H), 7.12 (s, 1 H), 3.99 (s, 3 H); MS (ESI): m/z:
+
1
82 [M+H]
General procedure for the synthesis of compounds 3ꢀ21 and the intermediates 22fꢀ28f
0.24 g (2.0 mmol, 0.15 mL) of SOCl and 3 drops of dry DMF were added at rt to a solution of
2
0
.277 g (1.53 mmol) of 4ꢀmethylthieno[3,2ꢀb]pyrroleꢀ5ꢀcarboxylic acid (3c) in 9 mL of dry THF.
The mixture was stirred for 2 h under reflux. The reaction mixture was then cooled down to rt
and added to a solution of 1.3 mmol of aniline in 7 mL of pyridine. The mixture was stirred for
3
0 min at rt and for further 45 min at 60 °C. Then, the mixture was diluted with DCM, washed
with a saturated NaHCO solution and brine, dried over Na SO , filtered and concentrated to
3
2
4
give an oil which was purified by flash chromatography
Similarly prepared:
2
9
4ꢀMethylꢀNꢀphenylꢀthieno[3,2ꢀb]pyrroleꢀ5ꢀcarboxamide (3): see compound 23 in Part 1
Nꢀ[2ꢀ(Methoxymethyl)phenyl]ꢀ4ꢀmethylꢀthieno[3,2ꢀb]pyrroleꢀ5ꢀcarboxamide (4) (from 3c
1
and 2ꢀ(methoxymethyl)aniline): yield 57%, pale yellow solid. H NMR (DMSOꢀd ) δ (ppm):
6
9
.63 (s, 1 H), 7.69 ꢀ7.65 (m, 1 H), 7.52 (d, J = 5.4 Hz, 1 H), 7.42ꢀ7.37 (m, 1 H), 7.36ꢀ7.30 (m, 1
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H), 7.24 (d, J = 5.4 Hz, 1 H), 7.22 ꢀ 7.17 (m, 2 H), 4.51 (s, 2 H), 4.01 (s, 3 H), 3.33 (s, 3 H); MS
+
(
ESI): m/z: 301 [M+H] . Mp: 96ꢀ98 °C.
Nꢀ[4ꢀ(Methoxymethyl)phenyl]ꢀ4ꢀmethylꢀthieno[3,2ꢀb]pyrroleꢀ5ꢀcarboxamide (5) (from 3c
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
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33
34
35
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37
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47
48
49
50
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55
56
57
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60
1
and 4ꢀ(methoxymethyl)aniline): yield 45%, orange solid. H NMR (DMSOꢀd ) δ (ppm): 10.01
6
(s, 1 H) 7.73ꢀ7.69 (m, 2 H) 7.51 (d, J=5.4 Hz, 1 H) 7.32 (s, 1 H) 7.29ꢀ7.26 (m, 2 H) 7.24 (d,
+
J=5.4 Hz, 1 H) 4.36 (s, 2 H) 4.01 (s, 3 H) 3.27 (s, 3 H); MS (ESI): m/z: 301 [M+H] . Mp: 147ꢀ
150 °C.
Nꢀ(4ꢀMethoxyphenyl)ꢀ4ꢀmethylꢀthieno[3,2ꢀb]pyrroleꢀ5ꢀcarboxamide (8) (from 3c and 4ꢀ
1
methoxyaniline): yield 73%, off white solid. H NMR (DMSOꢀd ) δ (ppm): 9.88 (s, 1 H), 7.67 ꢀ
6
7.59 (m, 2 H), 7.49 (d, J = 5.4 Hz, 1 H), 7.27 (s, 1 H), 7.23 (d, J = 5.4 Hz, 1 H), 6.95 ꢀ 6.85 (m, 2
+
H), 4.01 (s, 3 H), 3.74 (s, 3 H); MS (ESI): m/z: 287 [M+H] .
4
3
9
ꢀMethylꢀNꢀ[2ꢀ(4ꢀpyridyloxymethyl)phenyl]thieno[3,2ꢀb]pyrroleꢀ5ꢀcarboxamide (9): (from
1
c and 2ꢀ(4ꢀpyridyloxymethyl)aniline) yield 42%:off white solid. H NMR (DMSOꢀd ) δ (ppm):
6
.92 (s, 1 H), 7.59 ꢀ 7.55 (m, 2 H), 7.53 (d, J = 5.4 Hz, 1 H), 7.42 ꢀ 7.34 (m, 2 H), 7.33 ꢀ 7.28 (m,
2 H), 7.25 (d, J= 5.4 Hz, 1 H), 7.18 ꢀ 7.12 (m, 1 H), 6.10 ꢀ 5.95 (m, 2 H), 5.09 (s, 2 H), 3.99 (s, 3
+
H); MS (ESI): m/z: 364 [M+H] . Mp: 142ꢀ146 °C dec.
4
ꢀMethylꢀNꢀ[3ꢀ(4ꢀpyridyloxymethyl)phenyl]thieno[3,2ꢀb]pyrroleꢀ5ꢀcarboxamide (10): see
2
9
compound 23 in Part 1 .
4
ꢀMethylꢀNꢀ[4ꢀ(4ꢀpyridyloxymethyl)phenyl]thieno[3,2ꢀb]pyrroleꢀ5ꢀcarboxamide (11) (from
1
3c and 4ꢀ(4ꢀpyridyloxymethyl)aniline): yield 16%, off white solid. H NMR (DMSOꢀd ) δ
6
(
ppm): 10.06 (s, 1 H), 7.78 ꢀ 7.71 (m, 4 H), 7.52 (d, J = 5.4 Hz, 1 H), 7.33 ꢀ 7.28 (m, 3 H), 7.24
(
d, J= 5.4 Hz, 1 H), 6.12 ꢀ 6.04 (m, 2 H), 5.01 (s, 2 H), 4.00 (s, 3 H); MS (ESI): m/z: 364
+
[M+H] . Mp: 295 °C dec.
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ꢀMethylꢀNꢀ[2ꢀ(4ꢀpyridylmethoxy)phenyl]thieno[3,2ꢀb]pyrroleꢀ5ꢀcarboxamide (12) (from 3c
1
and 2ꢀ(4ꢀpyridylmethoxy)aniline): yield 19%, off white solid. H NMR (DMSOꢀd ) δ (ppm):
6
9.32 (s, 1 H) 8.58 ꢀ 8.49 (m, 2 H) 7.75 ꢀ 7.70 (m, 1 H) 7.54 ꢀ 7.48 (m, 3 H) 7.30 (s, 1 H) 7.24 (d,
0
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2
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6
7
8
9
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1
2
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6
7
8
9
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5
6
7
8
9
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3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
J=5.4 Hz, 1 H) 7.17 ꢀ 7.11 (m, 1 H) 7.10 ꢀ 7.06 (m, 1 H), 7.02 ꢀ 6.94 (m, 1 H) 5.27 (s, 2 H) 4.01
+
(s, 3 H); MS (ESI): m/z: 364 [M+H] . Mp: 141ꢀ145 °C.
4
ꢀMethylꢀNꢀ[3ꢀ(4ꢀpyridylmethoxy)phenyl]thieno[3,2ꢀb]pyrroleꢀ5ꢀcarboxamide (13) (from 3c
1
and 3ꢀ(4ꢀpyridylmethoxy)aniline): yield 14%, off white solid. H NMR (DMSOꢀd ) δ (ppm):
6
9.99 (s, 1 H), 8.62 ꢀ 8.54 (m, 2 H), 7.57 ꢀ 7.54 (m, 1 H), 7.52 (d, J = 5.4 Hz, 1 H), 7.47 ꢀ 7.43 (m,
2
H), 7.35 ꢀ 7.30 (m, 2 H), 7.27 ꢀ 7.21 (m, 2 H), 6.75 ꢀ 6.70 (m, 1 H), 5.18 (s, 2 H), 4.01 (s, 3 H);
+
MS (ESI): m/z: 364 [M+H] .
4
ꢀMethylꢀNꢀ[4ꢀ(4ꢀpyridylmethoxy)phenyl]thieno[3,2ꢀb]pyrroleꢀ5ꢀcarboxamide (14) (from 3c
1
and 4ꢀ(4ꢀpyridylmethoxy)aniline): yield 17%, pale yellow solid. H NMR (DMSOꢀd ) δ (ppm):
6
9.91 (s, 1 H), 8.60 ꢀ 8.55 (m, 2 H), 7.66 ꢀ 7.61 (m, 2 H), 7.50 (d, J = 5.4 Hz, 1 H), 7.46 ꢀ 7.41 (m,
2
H), 7.27 (s, 1 H), 7.23 (d, J = 5.4 Hz, 1 H), 7.03 ꢀ 6.97 (m, 2 H), 5.17 (s, 2 H), 4.00 (s, 3 H);
+
MS (ESI): m/z: 364 [M+H] . Mp: 190ꢀ193 °C.
4
ꢀMethylꢀNꢀ[2ꢀ[[4ꢀ[(1ꢀmethylꢀ4ꢀpiperidyl)oxy]phenoxy]methyl]phenyl]thieno[3,2ꢀ
b]pyrroleꢀ5ꢀcarboxamide (15 (from 3c and 2ꢀ[[4ꢀ[(1ꢀmethylꢀ4ꢀ
piperidyl)oxy]phenoxy]methyl]aniline, 15e): yield 12%, off white solid. H NMR (DMSOꢀd ) δ
)
1
6
(ppm): 9.72 (s, 1 H), 7.55 ꢀ 7.48 (m, 3 H), 7.39 ꢀ 7.33 (m, 1 H), 7.28 ꢀ 7.21 (m, 3 H), 6.91 ꢀ 6.80
(m, 4 H), 5.09 (s, 2 H), 4.22 ꢀ 4.11 (m, 1 H), 4.00 (s, 3 H), 2.61 ꢀ 2.52 (m, 2 H), 2.14 (s, 3 H),
+
2.12 ꢀ 2.05 (m, 2 H), 1.88 ꢀ 1.79 (m, 2 H), 1.61 ꢀ 1.48 (m, 2 H); MS (ESI): m/z: 476 [M+H] .
Mp: 144ꢀ147 °C.
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ꢀMethylꢀNꢀ[3ꢀ[[4ꢀ[(1ꢀmethylꢀ4ꢀpiperidyl)oxy]phenoxy]methyl]phenyl]thieno[3,2ꢀ
b]pyrroleꢀ5ꢀcarboxamide
piperidyl)oxy]phenoxy]methyl]aniline, 16e): see also 91 in Part 1 ).
ꢀMethylꢀNꢀ[4ꢀ[[4ꢀ[(1ꢀmethylꢀ4ꢀpiperidyl)oxy]phenoxy]methyl]phenyl]thieno[3,2ꢀ
b]pyrroleꢀ5ꢀcarboxamide (17 (from 3c and 4ꢀ[[4ꢀ[(1ꢀmethylꢀ4ꢀ
piperidyl)oxy]phenoxy]methyl]aniline, 17e): yield 15%, off white solid. H NMR (DMSOꢀd ) δ
(16
)
(from
3c
and
3ꢀ[[4ꢀ[(1ꢀmethylꢀ4ꢀ
2
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
4
)
1
6
(
(
(
ppm): 10.03 (s, 1 H), 7.78 ꢀ 7.70 (m, 2 H), 7.52 (d, J = 5.4 Hz, 1 H), 7.42 ꢀ 7.35 (m, 2 H), 7.32
s, 1 H), 7.24 (d, J= 5.4 Hz, 1 H), 6.94 ꢀ 6.82 (m, 4 H), 4.97 (s, 2 H), 4.25 ꢀ 4.13 (m, 1 H), 4.02
s, 3 H), 2.64 ꢀ 2.54 (m, 2 H), 2.20 ꢀ 2.04 (m, 5 H), 1.92 ꢀ 1.80 (m, 2 H), 1.63 ꢀ 1.50 (m, 2 H); MS
+
(ESI): m/z: 476 [M+H] . Mp: 164ꢀ168 °C dec.
4
ꢀMethylꢀNꢀ[2ꢀ[[3ꢀ[(1ꢀmethylꢀ4ꢀpiperidyl)oxy]phenoxy]methyl]phenyl]thieno[3,2ꢀ
b]pyrroleꢀ5ꢀcarboxamide (18 (from 3c and 2ꢀ[[3ꢀ[(1ꢀmethylꢀ4ꢀ
piperidyl)oxy]phenoxy]methyl]aniline, 18e): yield 54%, white solid. H NMR (DMSOꢀd ) δ
)
1
6
(ppm): 9.73 (s, 1 H), 7.56 ꢀ 7.47 (m, 3 H), 7.40 ꢀ 7.33 (m, 1 H), 7.29 ꢀ 7.21 (m, 3 H), 7.16 ꢀ 7.09
(m, 1 H), 6.56 ꢀ 6.46 (m, 3 H), 5.15 (s, 2 H), 4.30 ꢀ 4.21 (m, 1 H), 4.00 (s, 3 H), 2.60 ꢀ 2.51 (m, 2
H), 2.13 (s, 3 H), 2.11 ꢀ 2.03 (m, 2 H), 1.88 ꢀ 1.78 (m, 2 H), 1.61 ꢀ 1.47 (m, 2 H); MS (ESI): m/z:
+
476 [M+H] . Mp: 147ꢀ150 °C.
4ꢀMethylꢀNꢀ[2ꢀ[[2ꢀ[(1ꢀmethylꢀ4ꢀpiperidyl)oxy]phenoxy]methyl]phenyl]thieno[3,2ꢀ
b]pyrroleꢀ5ꢀcarboxamide (19 (from 3c and 2ꢀ[[2ꢀ[(1ꢀmethylꢀ4ꢀ
piperidyl)oxy]phenoxy]methyl]aniline, 19e): yield 14%, off white solid. H NMR (DMSOꢀd ) δ
)
1
6
(ppm): 9.68 (s, 1 H), 7.66 ꢀ 7.62 (m, 1 H), 7.61 ꢀ 7.56 (m, 1 H), 7.52 (d, J= 5.4 Hz, 1 H), 7.39 ꢀ
7.33 (m, 1 H), 7.27 ꢀ 7.21 (m, 3 H), 7.05 ꢀ 6.98 (m, 2 H), 6.91 ꢀ 6.83 (m, 2 H), 5.18 (s, 2 H), 4.29
ꢀ 4.19 (m, 1 H), 4.01 (s, 3 H), 2.57 ꢀ 2.51 (m, 2 H), 2.09 (s, 3 H), 2.06 ꢀ 1.97 (m, 2 H), 1.83 ꢀ 1.74
+
(m, 2 H), 1.61 ꢀ 1.48 (m, 2 H); MS (ESI): m/z: 476 [M+H] .
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ꢀMethylꢀNꢀ[2ꢀ[[4ꢀ[(1ꢀmethylꢀ4ꢀpiperidyl)oxy]phenyl]methoxy]phenyl]thieno[3,2ꢀ
b]pyrroleꢀ5ꢀcarboxamide
(20
)
(from
3c
and
2ꢀ[[4ꢀ[(1ꢀmethylꢀ4ꢀ
1
piperidyl)oxy]phenyl]methoxy]aniline, 20e¸ see Supporting Info): yield 67%, white solid. H
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
NMR (DMSOꢀd ) δ (ppm): 9.08 (s, 1 H) 7.85 ꢀ 7.73 (m, 1 H) 7.56 ꢀ 7.47 (m, 1 H) 7.45 ꢀ 7.35 (m,
6
2 H) 7.28 ꢀ 7.20 (m, 1 H) 7.19 ꢀ 7.08 (m, 3 H) 7.00 ꢀ 6.84 (m, 3 H) 5.10 (s, 2 H) 4.41 ꢀ 4.27 (m, 1
H) 4.00 (s, 3 H) 2.60 (br. s., 2 H) 2.24 ꢀ 2.07 (m, 5 H) 1.98 ꢀ 1.47 (m, 4 H); MS (ESI): m/z: 476
+
[
M+H] . Mp: 137ꢀ140 °C.
4
ꢀMethylꢀNꢀ[2ꢀ[[4ꢀ[(4ꢀmethylpiperazinꢀ1ꢀyl)methyl]phenoxy]methyl]phenyl]thieno[3,2ꢀ
b]pyrroleꢀ5ꢀcarboxamide (21 (from 3c and 2ꢀ[[4ꢀ[(4ꢀmethylpiperazinꢀ1ꢀ
yl)methyl]phenoxy]methyl]aniline (21e) see Supporting Info): H NMR (DMSOꢀd ) δ (ppm):
)
1
6
9
.75 (s, 1 H), 7.56 ꢀ 7.49 (m, 3 H), 7.40 ꢀ 7.33 (m, 1 H), 7.29 ꢀ 7.21 (m, 3 H), 7.19 ꢀ 7.13 (m, 2
H), 6.94 ꢀ 6.88 (m, 2 H), 5.14 (s, 2 H), 3.99 (s, 3 H), 3.35 (s, 2 H), 2.47 ꢀ 2.06 (m, 11 H); MS
+
(ESI): m/z: 475 [M+H] . Mp: 143ꢀ147 °C.
tertꢀButyl
4ꢀ[4ꢀ[[2ꢀ[(4ꢀmethylthieno[3,2ꢀb]pyrroleꢀ5ꢀ
carbonyl)amino]phenyl]methoxy]phenoxy]piperidineꢀ1ꢀcarboxylate (22f) (from 3c and
1
aniline 22e): yield 58%, white solid. H NMR (DMSOꢀd ) δ (ppm): 9.72 (s, 1 H), 7.55ꢀ7.47 (m,
6
3
H), 7.40ꢀ7.32 (m, 1 H), 7.29ꢀ7.19 (m, 3 H), 6.94ꢀ6.83 (m, 4 H), 5.10 (s, 2 H), 4.42ꢀ4.32 (m, 1
H), 4.00 (s, 3 H), 3.68ꢀ3.55 (m, 2 H), 3.22ꢀ3.05 (m, 2 H), 1.88ꢀ1.75 (m, 2 H), 1.49ꢀ1.40 (m, 2 H),
+
1.39 (s, 9 H); MS (ESI): m/z: 584 [M+Na] .
Similarly prepared:
tertꢀButyl
4ꢀ[4ꢀ[2ꢀ[2ꢀ[(4ꢀmethylthieno[3,2ꢀb]pyrroleꢀ5ꢀ
(from 3c and tertꢀbutyl
carbonyl)amino]phenyl]ethyl]phenoxy]piperidineꢀ1ꢀcarboxylate (23f
)
4ꢀ[4ꢀ[2ꢀ(2ꢀaminophenyl)ethyl]phenoxy]piperidineꢀ1ꢀcarboxylate, 23e, see Supporting Info):
1
yield 25%, off white solid. H NMR (CDCl ) δ (ppm): 7.76 ꢀ 7.71 (m, 1 H), 7.31 (d, J = 5.4 Hz, 1
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H), 7.30 ꢀ 7.25 (m, 2 H), 7.22 ꢀ 7.17 (m, 1 H), 7.02 ꢀ 6.96 (m, 4 H), 6.84 ꢀ 6.80 (m, 2 H), 6.56 (s,
1
H), 4.44 ꢀ 4.34 (m, 1 H), 4.06 (s, 3 H), 3.74 ꢀ 3.64 (m, 2 H), 3.32 ꢀ 3.23 (m, 2 H), 2.95 ꢀ 2.86
+
(
m, 4 H), 1.94 ꢀ 1.83 (m, 2 H), 1.77 ꢀ 1.65 (m, 2 H), 1.47 (s, 9 H); MS (ESI): m/z: 560 [M+H] .
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
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60
tertꢀButyl
4ꢀ[4ꢀ[[2ꢀ[(4ꢀmethylthieno[3,2ꢀb]pyrroleꢀ5ꢀ
(from 3c and tertꢀ
carbonyl)amino]phenyl]methoxy]phenyl]piperazineꢀ1ꢀcarboxylate (24f
)
butyl 4ꢀ[4ꢀ[(2ꢀaminophenyl)methoxy]phenyl]piperazineꢀ1ꢀcarboxylate, 24e, see Supporting
1
Info): yield 83%, white solid. H NMR (CDCl ) δ (ppm): 9.05 (br. s, 1 H), 8.30 ꢀ 8.21 (m, 1 H),
3
7.46 ꢀ 7.39 (m, 1 H), 7.36 ꢀ 7.28 (m, 2 H), 7.17 ꢀ 7.11 (m, 1 H), 7.08 ꢀ 6.87 (m, 5 H), 6.81 (s, 1
H), 5.15 (s, 2 H), 4.12 (s, 3 H), 3.71 ꢀ 3.47 (m, 4 H), 3.17 ꢀ 2.95 (m, 4 H), 1.50 (s, 9 H); MS
+
(ESI): m/z: 547 [M+H] .
tertꢀButyl
4ꢀ[4ꢀ[[2ꢀ[(4ꢀmethylthieno[3,2ꢀb]pyrroleꢀ5ꢀ
(from 3c and 4ꢀ[4ꢀ
carbonyl)amino]benzoyl]amino]phenoxy]piperidineꢀ1ꢀcarboxylate (25f
)
[(2ꢀaminobenzoyl)amino]phenoxy]piperidineꢀ1ꢀcarboxylate, 25e, see Supporting Info): yield
1
57%, white solid. H NMR (DMSOꢀd ) δ (ppm): 11.74 (s, 1 H) 10.44 (s, 1 H) 8.43 ꢀ 8.56 (m, 1
6
H)7.85 ꢀ 7.99 (m, 1 H) 7.47 ꢀ 7.68 (m, 4 H) 6.90 ꢀ 7.31 (m, 5 H) 4.42 ꢀ 4.60 (m, 1 H) 4.04 (s, 3
H) 3.59 ꢀ 3.72 (m, 2 H) 3.09 ꢀ 3.27 (m, 2 H) 1.77 ꢀ 1.95 (m, 2 H) 1.45 ꢀ 1.60 (m, 2 H) 1.41 (s, 9
+
H); MS (ESI): m/z: 575 [M+H] .
tertꢀButyl
4ꢀ[Nꢀtertꢀbutoxycarbonylꢀ4ꢀ[[2ꢀ[(4ꢀmethylthieno[3,2ꢀb]pyrroleꢀ5ꢀ
(from 3c and tertꢀ
carbonyl)amino]phenyl]methoxy]anilino]piperidineꢀ1ꢀcarboxylate (26f
)
butyl 4ꢀ[4ꢀ[(2ꢀaminophenyl)methoxy]ꢀNꢀtertꢀbutoxycarbonylꢀanilino]piperidineꢀ1ꢀcarboxylate,
+
26e, see Supporting Info): yield 56%, off white solid. MS (ESI): m/z: 661 [M+H] .
tertꢀButyl
3ꢀ[[4ꢀ[[2ꢀ[(4ꢀmethylthieno[3,2ꢀb]pyrroleꢀ5ꢀ
carbonyl)amino]phenyl]methoxy]phenoxy]methyl]azetidineꢀ1ꢀcarboxylate (27f) (from 3c
and tertꢀbutyl 3ꢀ[[4ꢀ[(2ꢀaminophenyl)methoxy]phenoxy]methyl]azetidineꢀ1ꢀcarboxylate, 27e, see
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6
1
Supporting Info)): yield 31%, pale yellow solid. H NMR (CDCl ) δ (ppm): 9.05 (s, 1 H), 8.33 ꢀ
3
8.18 (m, 1 H), 7.47 ꢀ 7.40 (m, 1 H), 7.35 ꢀ 7.29 (m, 2 H), 7.16 ꢀ 7.10 (m, 1 H), 7.02 ꢀ 6.95 (m, 3
H), 6.90 ꢀ 6.85 (m, 2 H), 6.82 (s, 1 H), 5.15 (s, 2 H), 4.13 (s, 3 H), 4.12 ꢀ 4.03 (m, 4 H), 3.83 ꢀ
0
1
2
3
4
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6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
+
3
.76 (m, 2 H), 3.01 ꢀ 2.90 (m, 1 H), 1.46 (s, 9 H); MS (ESI): m/z: 548 [M+H] .
tertꢀButyl
methylthieno[3,2ꢀb]pyrroleꢀ5ꢀcarbonyl)amino]phenyl]methoxy]phenyl]methyl]carbamate
28f (from 3c and tertꢀbutyl Nꢀ[[4ꢀ[(2ꢀaminophenyl)methoxy]phenyl]methyl]ꢀNꢀ[ N,N'ꢀbis(tertꢀ
Nꢀ[(Z)ꢀN,N'ꢀbis(tertꢀbutoxycarbonyl)carbamimidoyl]ꢀNꢀ[[4ꢀ[[2ꢀ[(4ꢀ
(
)
butoxycarbonyl) carbamimidoyl]carbamate, 28e, see Supporting Info): yield 72%, white solid.
1
H NMR (DMSOꢀd ) δ (ppm): 10.25 (br s, 1 H), 9.78 (br s, 1 H), 7.53 ꢀ 7.46 (m, 3 H), 7.38 ꢀ
6
7.32 (m, 1 H), 7.28 ꢀ 7.21 (m, 3 H), 7.20 ꢀ 7.15 (m, 1 H), 6.94 ꢀ 6.87 (m, 1 H), 5.14 (s, 2 H), 4.62
+
(
s, 2 H), 4.00 (s, 3 H), 1.49 ꢀ 1.29 (m, 27 H); MS (ESI): m/z: 734 [M+H] .
4ꢀ[(2ꢀNitrophenyl)methoxy]phenol (15i)
A mixture of 6.6 g (30 mmol) of 1ꢀ(bromomethyl)ꢀ2ꢀnitroꢀbenzene 15g 16.5 g (150 mmol)
hydroquinone (15h) and 6.2 g (45 mmol) K CO in 110 mL acetone was heated at reflux until
2
3
disappearance of the starting material. After 7 h, the mixture was cooled down to RT, K CO3
2
was filtered off and the solvent was concentrated. The crude mixture was taken up in DCM
triturated, and the filtrate was concentrated and purified on a silica pad eluting with DCM to give
1
4.87 g (66%) of 15i as a beige solid. H NMR (CDCl ) δ (ppm): 8.20ꢀ8.14 (m, 1 H), 7.95ꢀ7.88
3
(
m, 1 H), 7.72ꢀ7.66 (m, 1 H), 7.53ꢀ7.45 (m, 1 H), 6.92ꢀ6.76 (m, 4 H), 5.44 (s, 2 H), 4.44 (s, 1 H);
+
MS (ESI): m/z: 268 [M+Na] .
Similarly prepared
4ꢀ[(3ꢀNitrophenyl)methoxy]phenol (16i
)
(from 1ꢀ(bromomethyl)ꢀ3ꢀnitroꢀbenzene, 16g, and
1
benzeneꢀ1,4ꢀdiol, 15h): yield 71% (1.7 g); pale yellow solid. H NMR (DMSOꢀd ) δ (ppm): 8.99
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(
s, 1 H), 8.27 (s, 1 H), 8.21 ꢀ 8.14 (m, 1 H), 7.92 ꢀ 7.85 (m, 1 H), 7.73 ꢀ 7.65 (m, 1 H), 6.90 ꢀ 6.80
(m, 2 H), 6.72 ꢀ 6.63 (m, 2 H), 5.15 (s, 2 H); MS (ESI): m/z: 244[MꢀH]ꢀ.
4
ꢀ[(4ꢀNitrophenyl)methoxy]phenol (17i
)
(from 1ꢀ(bromomethyl)ꢀ4ꢀnitroꢀbenzene, 17g, and
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
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29
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37
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39
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45
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47
48
49
50
51
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53
54
55
56
57
58
59
60
1
benzeneꢀ1,4ꢀdiol, 15h): yield 61% (1.5g); offꢀwhite solid. H NMR (CDCl ) δ (ppm): 8.29 ꢀ 8.21
3
(m, 2 H), 7.65 ꢀ 7.56 (m, 2 H), 6.90 ꢀ 6.75 (m, 4 H), 5.13 (s, 2 H), 4.45 (s, 1 H); MS (ESI): m/z:
244[MꢀH]ꢀ.
3ꢀ[(2ꢀNitrophenyl)methoxy]phenol (18i
)
(from 1ꢀ(bromomethyl)ꢀ2ꢀnitroꢀbenzene, 15g, and
1
benzeneꢀ1,3ꢀdiol, 18h): yield 77% (4.7 g); pale yellow solid. H NMR (CDCl ) δ (ppm): 8.10
3
(
dd, J = 1.0, 8.3 Hz, 1 H), 7.81 (dd, J = 1.0, 7.8 Hz, 1 H), 7.65 ꢀ 7.59 (m, 1 H), 7.45 ꢀ 7.39 (m, 1
H), 7.1 ꢀ7.07 (m, 1 H), 6.54 ꢀ 6.49 (m, 1 H), 6.45 ꢀ6.41 (m, 1 H), 6.42 ꢀ 6.38 (m, 1 H), 5.41 (s, 2
H); MS (ESI): m/z: 244[MꢀH]ꢀ.
2ꢀ[(2ꢀNitrophenyl)methoxy]phenol (19i
)
(from 1ꢀ(bromomethyl)ꢀ2ꢀnitroꢀbenzene, 15g, and
1
benzeneꢀ1,2ꢀdiol, 19h): yield 91% (1.3g); yellow solid H NMR (DMSOꢀd ) δ (ppm): 9.12 (s, 1
6
H), 8.17 ꢀ8.12 (m, 1 H), 7.93 ꢀ7.89 (m, 1 H), 7.85 ꢀ 7.77 (m, 1 H), 7.65 ꢀ 7.57 (m, 2 H), 6.95 ꢀ
6.93 (m Hz, 2 H), 6.87 ꢀ 6.77 (m, 2 H), 6.75 ꢀ 6.65 (m, 1 H), 5.43 (s, 2 H); MS (ESI): m/z:
244[MꢀH]ꢀ.
tertꢀButyl 4ꢀ[4ꢀ[(2ꢀnitrophenyl)methoxy]phenoxy]piperidineꢀ1ꢀcarboxylate (15j)
0.85 g (4.0 mmol) of diisopropyl azodicarboxylate was added dropwise to a solution of 0.49 g
(
2.0 mmol) of 4ꢀ[(2ꢀnitrophenyl)methoxy]phenol 15i, 0.83 g (4.0 mmol) of tertꢀbutyl 4ꢀ
hydroxypiperidineꢀ1ꢀcarboxylate and 1.1 g (4.0 mmol) of triphenylphosphine in 20 mL of dry
THF cooled down to 0 °C and under a N atmosphere. The mixture was allowed to reach rt and
2
stirred overnight. The solvent was evaporated and the crude mixture purified by flash
chromatography (eluent nꢀhexane/acetone, 0% to 10% of acetone) to give 0.71 g (83%) of 15j as
1
a yellow oil. H NMR (CDCl ) δ (ppm): 8.22ꢀ8.13 (m, 1 H), 7.96ꢀ7.87 (m, 1 H), 7.74ꢀ7.65 (m, 1
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H), 7.54ꢀ7.44 (m, 1 H), 6.99ꢀ6.82 (m, 4 H), 5.45 (s, 2 H), 4.41ꢀ4.28 (m, 1 H), 3.78ꢀ3.65 (m, 2 H),
3.37ꢀ3.24 (m, 2 H), 1.96ꢀ1.85 (m, 2 H), 1.79ꢀ1.68 (m, 2 H), 1.48 (s, 9 H); MS (ESI): m/z: 373
+
[
M+Hꢀ56] .
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
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3
4
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1
2
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4
5
6
7
8
9
0
Similarly prepared
1ꢀMethylꢀ4ꢀ[4ꢀ[(2ꢀnitrophenyl)methoxy]phenoxy]piperidine (15d) (from 15i and 1ꢀmethylꢀ4ꢀ
piperidinol): yield 70% (0.72 g); purified by flash chromatography (eluent DCM: MeOH 98:2
1
until the elution of Ph PO and then DCM/MeOH/NH , 96:4:0.4 v:v:v) as a beige solid. H NMR
3
3
(DMSOꢀd6) δ (ppm): 8.15 ꢀ 8.06 (m, 1 H), 7.84 ꢀ 7.72 (m, 2 H), 7.67 ꢀ 7.55 (m, 1 H), 6.96 ꢀ 6.82
(m, 4 H), 5.37 (s, 2 H), 4.26 ꢀ 4.14 (m, 1 H), 2.60 (br s, 2 H), 2.24 ꢀ 2.05 (m, 5 H), 1.93 ꢀ 1.80 (m,
+
2
H), 1.66 ꢀ 1.50 (m, 2 H); MS (ESI): m/z: 343 [M+H] .
tertꢀButyl 4ꢀ[4ꢀ[(3ꢀnitrophenyl)methoxy]phenoxy]piperidineꢀ1ꢀcarboxylate (16j) (from 16i
1
and tertꢀbutyl 4ꢀhydroxypiperidineꢀ1ꢀcarboxylate): yield 78% (1.34 g); pale yellow solid. H
NMR (DMSOꢀd6) δ (ppm): 8.29 (s, 1 H), 8.22 ꢀ 8.15 (m, 1 H), 7.93 ꢀ 7.86 (m, 1 H), 7.75 ꢀ 7.66
(m, 1 H), 7.01 ꢀ 6.86 (m, 4 H), 5.19 (s, 2 H), 4.45 ꢀ 4.34 (m, 1 H), 3.70 ꢀ 3.57 (m, 2 H), 3.21 ꢀ
3.01 (m, 2 H), 1.89 ꢀ 1.77 (m, 2 H), 1.55 ꢀ 1.42 (m, 2 H), 1.39 (s, 9 H); MS (ESI): m/z: 373
+
[M+Hꢀ56] .
tertꢀButyl 4ꢀ[4ꢀ[(4ꢀnitrophenyl)methoxy]phenoxy]piperidineꢀ1ꢀcarboxylate (17j
)
(from 17i
1
and tertꢀbutyl 4ꢀhydroxypiperidineꢀ1ꢀcarboxylate): yield 62% (0.53 g); white solid. H NMR
(CDCl ) δ (ppm): 8.29 ꢀ 8.22 (m, 2 H), 7.65 ꢀ 7.57 (m, 2 H), 6.93 ꢀ 6.82 (m, 4 H), 5.14 (s, 2 H),
3
4.39 ꢀ 4.29 (m, 1 H), 3.77 ꢀ 3.65 (m, 2 H), 3.37 ꢀ 3.25 (m, 2 H), 1.96 ꢀ 1.84 (m, 2 H), 1.79 ꢀ 1.66
+
(
m, 2 H), 1.48 (s, 9 H); MS (ESI): m/z: 373 [M+Hꢀ56] .
tertꢀButyl 4ꢀ[3ꢀ[(2ꢀnitrophenyl)methoxy]phenoxy]piperidineꢀ1ꢀcarboxylate (18j
)
(from 18i
1
and tertꢀbutyl 4ꢀhydroxypiperidineꢀ1ꢀcarboxylate): yield 70% (0.6 g); yellow solid. H NMR
(
CDCl ) δ (ppm): 8.21ꢀ8.16 (m, 1 H), 7.93 ꢀ 7.88 (m, 1 H), 7.72 ꢀ 7.67 (m, 1 H), 7.53 ꢀ 7.47 (m, 1
3
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H), 7.24 ꢀ 7.17 (m, 1 H), 6.61 ꢀ 6.55 (m, 3 H), 5.48 (s, 2 H), 4.50 ꢀ 4.41 (m, 1 H), 3.76 ꢀ 3.67 (m,
2
H), 3.39 ꢀ 3.28 (m, 2 H), 1.97 ꢀ 1.86 (m, 2 H), 1.80 ꢀ 1.70 (m, 2 H), 1.48 (s, 9 H) MS (ESI):
+
m/z: 451 [M+Na] .
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ꢀ Methyl ꢀ4ꢀ[2ꢀ[(2ꢀnitrophenyl)methoxy]phenoxy]piperidine (19d) (from 19i): yield 71%
1
(1.29 g); yellow oil. H NMR (DMSOꢀd6) δ (ppm): 8.15ꢀ8.11 (m,1 H), 7.91ꢀ7.87 (m, 1 H), 7.85 ꢀ
7
.77 (m, 2 H), 7.68 ꢀ 7.60 (m, 2 H), 7.09 ꢀ 7.01 (m, 2 H), 6.95 ꢀ 6.85 (m, 2 H), 5.44 (s, 2 H), 4.28
(br s, 1 H), 2.60 (br. s, 2 H), 2.16 (br s, 5 H), 1.86 (br s, 2 H), 1.71 ꢀ 1.58 (m, 2 H); MS (ESI):
+
m/z: 343 [M+H] .
1
ꢀMethylꢀ4ꢀ[4ꢀ[(3ꢀnitrophenyl)methoxy]phenoxy]piperidine (16d)
5
mL of 4 M HCl in 1,4ꢀdioxane was added to a solution of 0.655 g (1.53 mmol) tertꢀbutyl 4ꢀ[4ꢀ
[(3ꢀnitrophenyl)methoxy]phenoxy]piperidineꢀ1ꢀcarboxylate (16j) in 4 mL of dry 1,4ꢀdioxane and
the mixture was stirred at rt for 90 min. The reaction mixture was then concentrated, the residue
was taken up in saturated NaHCO solution and extracted with EtOAc. The combined organic
3
phases were dried over Na SO and concentrated to afford 0.49 g (98%) of 4ꢀ[4ꢀ[(3ꢀ
2
4
nitrophenyl)methoxy]phenoxy]piperidine as a yellow oil.
1
H NMR (DMSOꢀd ) δ (ppm): 8.29 (s, 1 H), 8.22ꢀ8.14 (m, 1 H), 7.92ꢀ7.83 (m, 1 H), 7.73ꢀ7.66
6
(m, 1 H), 6.99ꢀ6.82 (m, 4 H), 5.19 (s, 2 H), 4.29ꢀ4.16 (m, 1 H), 2.97ꢀ2.84 (m, 2 H), 2.57ꢀ2.50 (m,
+
2
0
H), 1.90ꢀ1.79 (m, 2 H), 1.47ꢀ1.33 (m, 2 H); MS (ESI): m/z: 329 [M+H] .
.13 mL (1.788 mmol) of formaldehyde (37% in H O), 0.085 mL (1.5 mmol) of glacial acetic
2
acid and 0.118 g (1.788 mmol) of NaBH CN were added to 0.49 g (1.492 mmol) of 4ꢀ[4ꢀ[(3ꢀ
3
nitrophenyl)methoxy]phenoxy]piperidine in 15 mL of MeOH. The reaction mixture was stirred
at rt until disappearance of the starting material. Then, the solvent was removed and the crude
mixture was partitioned between H O and DCM. The organic phase was dried over Na SO ,
2
2
4
filtered off and concentrated to afford a yellow oil. The crude product was purified by flash
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chromatography (eluent DCM/MeOH/NH , 96:4:0.4 v:v:v) to afford 0.407 g (80%) of 16d as a
3
1
yellow oil. H NMR (DMSOꢀd ) δ (ppm): 8.29 (s, 1 H), 8.22ꢀ8.13 (m, 1 H), 7.94ꢀ7.83 (m, 1 H),
6
7.74ꢀ7.65 (m, 1 H), 7.03ꢀ6.80 (m, 4 H), 5.20 (s, 2 H), 4.38 (br s, 1 H), 3.11ꢀ2.64 (m, 4 H), 2.55
0
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9
0
1
2
3
4
5
6
7
8
9
0
+
(
br s, 3 H), 2.04ꢀ1.65 (m, 4 H); MS (ESI): m/z: 343 [M+H] .
Similarly prepared
ꢀMethylꢀ4ꢀ[4ꢀ[(4ꢀnitrophenyl)methoxy]phenoxy]piperidine (17d) (from 17j): according to
1
the procedure for 16d, except for nitrogen deprotection, which was carried out with TFA in
1
DCM; yield 79% (0.073 g); pale yellow solid. H NMR (CDCl3) δ (ppm): 8.34 ꢀ 8.19 (m, 2 H),
7.68 ꢀ 7.52 (m, 2 H), 6.98 ꢀ 6.79 (m, 4 H), 5.14 (s, 2 H), 4.42 ꢀ 4.17 (m, 1 H), 2.93 ꢀ 2.76 (m, 2
+
H), 2.58 ꢀ 2.35 (m, 5 H), 2.13 ꢀ 2.02 (m, 2 H), 1.97 ꢀ 1.83 (m, 2 H). MS (ESI): m/z: 343 [M+H] .
1ꢀMethylꢀ4ꢀ[3ꢀ[(2ꢀnitrophenyl)methoxy]phenoxy]piperidine (18d) (from 18j): yield 75% (117
1
mg); yellow oil. H NMR (DMSOꢀd6) δ (ppm): 8.16 ꢀ 8.06 (m, 1 H), 7.81 ꢀ 7.75 (m, 2 H), 7.67 ꢀ
7.56 (m, 1 H), 7.23 ꢀ 7.10 (m, 1 H), 6.62 ꢀ 6.51 (m, 3 H), 5.42 (s, 2 H), 4.46 ꢀ 4.30 (m, 1 H), 2.82
ꢀ
2.64 (m, 2 H), 2.38 ꢀ 2.18 (m, 5 H), 1.97 ꢀ 1.83 (m, 2 H), 1.72 ꢀ 1.54 (m, 2 H); MS (ESI): m/z:
+
3
43 [M+H] .
General procedure to reduce Nitro group to Aniline
.50 mmol of nitro derivative was dissolved in 60 mL of EtOH. The solution was hydrogenated
1
in an HꢀCube apparatus (PtO cartridge, atmospheric pressure, 70°C, flow 0.5 mL/min). The
2
solvent was evaporated to give the desired aniline.
Similarly prepared:
2ꢀ[[4ꢀ[(1ꢀ Methyl ꢀ4ꢀpiperidyl)oxy]phenoxy]methyl]aniline (15e) (from 15d): yield 83% (218
1
mg); off white solid. H NMR (DMSOꢀd ) δ (ppm): 7.19 ꢀ 7.10 (m, 1 H), 7.06 ꢀ 6.98 (m, 1 H),
6
6.95 ꢀ 6.82 (m, 4 H), 6.71 ꢀ 6.63 (m, 1 H), 6.58 ꢀ 6.48 (m, 1 H), 4.88 (s, 2 H), 4.25 ꢀ 4.09 (m, 1
H), 2.62 ꢀ 2.52 (m, 2 H), 2.21 ꢀ 2.05 (m, 5 H), 1.92 ꢀ 1.80 (m, 2 H), 1.64 ꢀ 1.48 (m, 2 H); MS
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+
(
ESI): m/z: 313 [M+H] .
3
ꢀ[[4ꢀ[(1ꢀ Methyl ꢀ4ꢀpiperidyl)oxy]phenoxy]methyl]aniline (16e) (from 16d): yield 63% (147
1
mg); pale yellow solid. H NMR (DMSOꢀd ) δ (ppm): 7.03ꢀ6.95 (m, 1 H), 6.94ꢀ6.83 (m, 4 H),
6
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
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31
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44
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47
48
49
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59
60
6
.60 (s, 1 H), 6.55ꢀ6.44 (m, 2 H), 5.09 (br. s, 2 H), 4.86 (s, 2 H), 4.31 (br s, 1 H), 2.91 (br s, 2 H),
+
2.62 (br.s, 2 H), 2.45 (br s, 3 H), 1.92 (br s, 2 H), 1.71 (br s, 2 H); MS (ESI): m/z: 313 [M+H] .
4ꢀ[[4ꢀ[(1ꢀ Methyl ꢀ4ꢀpiperidyl)oxy]phenoxy]methyl]aniline (17e) (from 17d): yield 90% (58
1
mg); pale yellow solid. H NMR (CDCl3) δ (ppm): 7.25ꢀ7.18 (m, 2 H), 6.93ꢀ6.82 (m, 4 H), 6.74ꢀ
6
.66 (m, 2 H), 4.89 (s, 2 H), 4.22 (br s, 1 H), 3.72 (br s, 2 H), 2.81ꢀ2.71 (m, 2 H), 2.43ꢀ2.27 (m, 5
+
H), 2.10ꢀ1.96 (m, 2 H), 1.92ꢀ1.79 (m, 2 H); MS (ESI): m/z: 313 [M+H] .
2ꢀ[[3ꢀ[(1ꢀ Methyl ꢀ4ꢀpiperidyl)oxy]phenoxy]methyl]aniline (18e) (from 18d): yield 91% (118
1
mg); pale yellow oil. H NMR (DMSOꢀd ) δ (ppm): 7.20 ꢀ 7.11 (m, 2 H), 7.05 ꢀ 6.97 (m, 1 H),
6
6.70 ꢀ 6.64 (m, 1 H), 6.61 ꢀ 6.48 (m, 4 H), 5.04 (br. s, 2 H), 4.92 (s, 2 H), 4.37 ꢀ 4.26 (m, 1 H),
2
.67 ꢀ 2.55 (m, 2 H), 2.21 ꢀ 2.09 (m, 5 H), 1.94 ꢀ 1.84 (m, 2 H), 1.67 ꢀ 1.51 (m, 2 H); MS (ESI):
+
m/z: 313 [M+H] .
2ꢀ[[2ꢀ[(1ꢀmethylꢀ4ꢀpiperidyl)oxy]phenoxy]methyl]aniline (19e) (from 19d): yield 75% (175
1
mg); pale yellow oil. H NMR (DMSOꢀd ) δ (ppm): 7.28ꢀ7.24 (m, 1 H), 7.22 ꢀ 7.17 (m, 1 H),
6
7.12 ꢀ 7.06 (m, 2 H), 7.01 ꢀ 6.93 (m, 2 H), 6.77ꢀ6.74 (m, 1 H), 6.64 ꢀ 6.58 (m, 1 H), 5.23 (br. s, 2
H), 5.00 (s, 2 H), 4.41 ꢀ 4.30 (m, 1 H), 2.78 ꢀ 2.65 (m, 2 H), 2.25 (br s, 5 H), 2.01 ꢀ 1.89 (m, 2 H),
+
1.78 ꢀ 1.61 (m, 2 H); MS (ESI): m/z: 313 [M+H] .
tertꢀButyl 4ꢀ[4ꢀ[(2ꢀaminophenyl)methoxy]phenoxy]piperidineꢀ1ꢀcarboxylate (22e) (from
1
1
5j): yield 98% (0.59 g); yellow oil. H NMR (CDCl ) δ (ppm): 7.22ꢀ7.15 (m, 2 H), 6.97ꢀ6.83
3
(m, 4 H), 6.82ꢀ6.75 (m, 2 H), 5.00 (s, 2 H), 4.39ꢀ4.26 (m, 1 H), 3.78ꢀ3.62 (m, 2 H), 3.36ꢀ3.21 (m,
+
2
H), 1.97ꢀ1.82 (m, 2 H), 1.79ꢀ1.67 (m, 2 H), 1.48 (s, 9 H); MS (ESI): m/z: 399 [M+H] .
1
4
ꢀ[(2ꢀAminophenyl)methoxy]phenol (33k) (from 15i): yield 98% (1.1 g); pale yellow oil. H
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NMR (CDCl ) δ (ppm): 7.22ꢀ7.14 (m, 2 H), 6.93ꢀ6.85 (m, 2 H), 6.82ꢀ6.72 (m, 4 H), 4.99 (s, 2
3
+
H), 4.32 (bs, 3 H); MS (ESI): m/z: 238 [M+Na] .
General procedure for the synthesis of 22ꢀ28 (BOCꢀdeprotection)
0
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ꢀMethylꢀNꢀ[2ꢀ[[4ꢀ(4ꢀpiperidyloxy)phenoxy]methyl]phenyl]thieno[3,2ꢀb]pyrroleꢀ5ꢀ
carboxamide tertꢀbutyl 4ꢀ[4ꢀ[[2ꢀ[(4ꢀMethylthieno[3,2ꢀb]pyrroleꢀ5ꢀ
carbonyl)amino]phenyl]methoxy]phenꢀoxy]piperidineꢀ1ꢀcarboxylate (22)
.407 (0.725 mmol) of tertꢀbutyl 4ꢀ[4ꢀ[[2ꢀ[(4ꢀmethylthieno[3,2ꢀb]pyrroleꢀ5ꢀ
0
g
carbonyl)amino]phenyl]methoxy]phenoxy]piperidineꢀ1ꢀcarboxylate (22f) was dissolved in a
mixture of 3 mL of dry 1,4ꢀdioxane and 3 mL of dry MeOH. 4.17 mL of 4 M HCl in 1,4ꢀdioxane
was added and the mixture was stirred at rt for 3 h. The reaction mixture was then concentrated
and the residue was taken up in a saturated NaHCO solution and extracted with EtOAc. The
3
combined organic phases were dried over Na SO , concentrated and triturated in Et O give 0.309
2
4
2
1
g (92%) of 22 as a white solid. H NMR (DMSOꢀd ) δ (ppm): 9.72 (s, 1 H), 7.57ꢀ7.46 (m, 3 H),
6
7.40ꢀ7.31 (m, 1 H), 7.29ꢀ7.17 (m, 3 H), 6.93ꢀ6.79 (m, 4 H), 5.09 (s, 2 H), 4.27ꢀ4.13 (m, 1 H),
4.00 (s, 3 H), 2.94ꢀ2.84 (m, 2 H), 2.49ꢀ2.42 (m, 2 H), 1.89ꢀ1.73 (m, 2 H), 1.43ꢀ1.30 (m, 2 H); MS
+
(
ESI): m/z: 462 [M+H] .
Similarly prepared:
4ꢀMethylꢀNꢀ[2ꢀ[2ꢀ[4ꢀ(4ꢀpiperidyloxy)phenyl]ethyl]phenyl]thieno[3,2ꢀb]pyrroleꢀ5ꢀ
carboxamide, hydrochloride (23
)
(from 23f): The alkaline workꢀup reported for compound 22
1
was not performed. Yield 77% (26 mg); beige solid. H NMR (DMSOꢀd6) δ (ppm): 9.67 (s, 1
H), 8.51 (br. s, 2 H), 7.51 (d, J = 5.4 Hz, 1 H), 7.33 ꢀ 7.27 (m, 3 H), 7.26 ꢀ 7.17 (m, 3 H), 7.10 ꢀ
7.05 (m, 2 H), 6.87 ꢀ 6.82 (m, 2 H), 4.59 ꢀ 4.49 (m, 1 H), 4.00 (s, 3 H), 3.24 ꢀ 3.15 (m, 2 H), 3.10
ꢀ
2.99 (m, 2 H), 2.89 ꢀ 2.72 (m, 4 H), 2.07 ꢀ 1.96 (m, 2 H), 1.81 ꢀ 1.70 (m, 2 H). MS (ESI): m/z:
+
4
60 [M+H] .
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ꢀMethylꢀNꢀ[2ꢀ[(4ꢀpiperazinꢀ1ꢀylphenoxy)methyl]phenyl]thieno[3,2ꢀb]pyrroleꢀ5ꢀ
carboxamide (24
)
(from 24f): the compound was purified as a free base with silica gel flash
1
chromatograpy obtaining 36 mg (yield 47%) pale orange solid. H NMR (CDCl ) δ (ppm): 9.06
3
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
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(
s, 1 H), 8.29 ꢀ 8.25 (m, 1 H), 7.45 ꢀ 7.39 (m, 1 H), 7.35 ꢀ 7.28 (m, 2 H), 7.16 ꢀ 7.11 (m, 1 H),
7.03 ꢀ 6.90 (m, 5 H), 6.81 (s, 1 H), 5.14 (s, 2 H), 4.13 (s, 3 H), 3.19 ꢀ 3.10 (m, 8 H); MS (ESI):
+
m/z: 447 [M+H] . Mp: 195ꢀ200 °C.
4
ꢀMethylꢀNꢀ[2ꢀ[[4ꢀ(4ꢀpiperidyloxy)phenyl]carbamoyl]phenyl]thieno[3,2ꢀb]pyrroleꢀ5ꢀ
carboxamide (25
)
(from 25f): the compound was purified as a free base with silica gel flash
1
chromatograpy yielding 20 mg (48%) of a beige solid. H NMR (DMSOꢀd6) δ (ppm): 11.76 (br.
s., 1 H), 10.43 (br. s., 1 H), 8.60 ꢀ 8.38 (m, 1 H), 8.01 ꢀ 7.80 (m, 1 H), 7.71 ꢀ 7.51 (m, 4 H), 7.31 ꢀ
6
.87 (m, 5 H), 4.36 (br. s., 1 H), 4.04 (s, 3 H), 3.02 ꢀ 2.88 (m, 2 H), 2.64 ꢀ 2.52 (m, 3 H), 1.98 ꢀ
+
1
.82 (m, 2 H), 1.53 ꢀ 1.36 (m, 2 H); MS (ESI): m/z: 475 [M+H] .
4ꢀMethylꢀNꢀ[2ꢀ[[4ꢀ(4ꢀpiperidylamino)phenoxy]methyl]phenyl]thieno[3,2ꢀb]pyrroleꢀ5ꢀ
carboxamide trifluoroacetate (26 (from 26f): the alkaline workꢀup reported for compound 22
)
was not performed, the compound was purified as trifluoroacetate preparative HPLC, yielding 7
1
mg (35%) of brownish oil. H NMR (DMSOꢀd6) δ (ppm): 10.36 (s, 1 H), 9.28 ꢀ 8.79 (m, 2 H),
8
7
2
.53 (d, J = 9.8 Hz, 1 H), 8.15 ꢀ 7.95 (m, 1 H), 7.80 ꢀ 7.68 (m, 1 H), 7.54 (d, J = 5.4 Hz, 1 H),
.32 ꢀ 7.15 (m, 4 H), 7.07 ꢀ 7.00 (m, 1 H), 6.90 ꢀ 6.76 (m, 2 H), 6.64 ꢀ 6.53 (m, 2 H), 4.33 (br. s.,
H), 4.03 (s, 3 H), 3.65 ꢀ 3.54 (m, 1 H), 3.32 ꢀ 3.22 (m, 2 H), 2.98 ꢀ 2.85 (m, 2 H), 2.01 ꢀ 1.88
+
(m, 2 H), 1.61 ꢀ 1.46 (m, 2 H); MS (ESI): m/z: 461 [M+H] .
Nꢀ[2ꢀ[[4ꢀ(Azetidinꢀ3ꢀylmethoxy)phenoxy]methyl]phenyl]ꢀ4ꢀmethylꢀthieno[3,2ꢀb]pyrroleꢀ5ꢀ
carboxamide trifluoroacetate (27) (from 27f): The alkaline workꢀup reported for compound 22
was not performed. The compound was purified as trifluoroacetate preparative HPLC, yielding
1
53 mg (65%) of beige solid. H NMR (DMSOꢀd6) δ (ppm): 9.74 (s, 1 H), 8.52 (br. s., 2 H), 7.56
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