In vivo potent BM635 analogue with improved drug-like properties

Article abstract of DOI:10.1016/j.ejmech.2017.12.075

BM635 is the hit compound of a promising anti-TB compound class. Herein we report systematic variations around the central pyrrole core of BM635 and we describe the design, synthesis, biological evaluation, pharmacokinetic analysis, as well as in vivo TB mouse efficacy studies of novel BM635 analogues that show improved physicochemical properties. This hit-to-lead campaign led to the identification of a new analogue, 4-((1-isopropyl-5-(4-isopropylphenyl)-2-methyl-1H-pyrrol-3-yl)methyl)morpholine (17), that shows excellent activity (MIC = 0.15 μM; SI = 133) against drug-sensitive Mycobacterium tuberculosis strains, as well as efficacy in a murine model of TB infection.

Full text of DOI:10.1016/j.ejmech.2017.12.075

Accepted Manuscript
In vivo potent BM635 analogue with improved drug-like properties
Giovanna Poce, Martina Cocozza, Salvatore Alfonso, Sara Consalvi, Giulia Venditti,
Raquel Fernandez-Menendez, Robert H. Bates, David Barros Aguirre, Lluis Ballell,
Alessandro De Logu, Giulio Vistoli, Mariangela Biava
PII:
S0223-5234(17)31106-6
10.1016/j.ejmech.2017.12.075
EJMECH 10053
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 24 July 2017
Revised Date: 20 December 2017
Accepted Date: 22 December 2017
Please cite this article as: G. Poce, M. Cocozza, S. Alfonso, S. Consalvi, G. Venditti, R. Fernandez-
Menendez, R.H. Bates, D. Barros Aguirre, L. Ballell, A. De Logu, G. Vistoli, M. Biava, In vivo potent
BM635 analogue with improved drug-like properties, European Journal of Medicinal Chemistry (2018),
doi: 10.1016/j.ejmech.2017.12.075.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
In vivo potent BM635 analogue with improved drug-like
properties
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Giovanna Pocea*, Martina Cocozzaa, Salvatore Alfonsoa,b, Sara Consalvia, Giulia Vendittia,
Raquel Fernandez-Menendezb, Robert H. Batesb, David Barros Aguirreb, Lluis Ballellb,
Alessandro De Loguc, Giulio Vistolid and Mariangela Biavaa*.
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aDepartment of Chemistry and Technology of Drugs, Sapienza University of Rome, piazzale
A. Moro 5, 00185-Rome, Italy
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b Diseases of the Developing World, Tres Cantos Medicines Development Campus, GSK,
Severo Ochoa 2, 28760-Tres Cantos, Madrid, Spain.
cDipartimento di Scienze della Vita e dell’Ambiente, Università degli Studi di Cagliari, via
Ospedale 72, 09124 - Cagliari, Italy.
dDepartment of Pharmaceutical Sciences, Università degli Studi di Milano, via Mangiagalli
25, 20133-Milan, Italy.
* To whom correspondence should be addressed: Dr. Giovanna Poce, Department of
Chemistry and Technology of Drugs, Sapienza University of Rome, Piazzale Aldo Moro 5, I-
00185 Roma, Italy, Phone: +39 06 4991 3593, Fax: +39 06 4991 3133, E-mail:
giovanna.poce@uniroma1.it
Prof. Mariangela Biava, Department of Chemistry and Technology of Drugs, Sapienza
University of Rome, Piazzale Aldo Moro 5, I-00185 Roma, Italy, Phone: +39 06 4991 3812,
Fax: +39 06 4991 3133, E-mail: mariangela.biava@uniroma1.it
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33 Abstract
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BM635 is the hit compound of a promising anti-TB compound class. Herein we report
systematic variations around the central pyrrole core of BM635 and we describe the design,
synthesis, biological evaluation, pharmacokinetic analysis, as well as in vivo TB mouse
efficacy studies of novel BM635 analogues that show improved physicochemical properties.
This hit-to-lead campaign led to the identification of a new analogue, 4-((1-isopropyl-5-(4-
isopropylphenyl)-2-methyl-1H-pyrrol-3-yl)methyl)morpholine (17), that shows excellent
activity (MIC = 0.15 ꢀM; SI = 133) against drug-sensitive Mycobacterium tuberculosis
strains, as well as efficacy in a murine model of TB infection.
43 Keywords
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Tuberculosis, pyrroles, MmpL3, drug discovery, anti-mycobacterials.
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Abbreviations: TB, tuberculosis; WHO, World Health Organization; MDR-TB, multidrug-
resistant tuberculosis; XDR-TB, extensively drug-resistant TB; Mtb, M. tuberculosis; WGS,
whole genome sequencing; MmpL3, mycobacterium membrane protein Large 3; RND,
resistance, nodulation and division; TLC, thin layer chromatography; MIC, minimum
inhibitory concentration; TFA, trifluoro acetic acid; TEA, trimethylamine; DCM,
dichloromethane; DCE, dichloroethane; TOX50, concentration of compound resulting in 50%
inhibition; HSA, human serum albumin; CLND, chemiluminescent nitrogen detection;
FaSSIF, fasted state simulated intestinal fluid; hERG, human ether-a-go-go-related gene;
SAR, structure activity relationship; Vss, volume of distribution in blood.
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1. Introduction
Despite the fact that tuberculosis (TB) mortality has fallen by 47% since 1990, TB remains
one of the world’s deadliest diseases. The World Health Organization (WHO) estimated over
10.4 million new cases and 1.8 million deaths by 2015 [1]. Drug-susceptible TB is treated
with a standard 6-months course therapy comprised of a combination of four drugs:
rifampicin, isoniazid, pyrazinamide and ethambutol. Unfortunately, standard anti-TB drugs
have been used for decades, and resistance to the medicines is widespread. Multidrug-
resistant tuberculosis (MDR-TB) is a form of TB caused by bacteria that do not respond to, at
least, isoniazid and rifampicin, the two most powerful, first-line (or standard) anti-TB drugs
[1,2]. In some cases, more severe drug resistance can develop. Extensively drug-resistant TB
(XDR-TB) is a form of multi-drug resistant tuberculosis that responds to even fewer available
medicines, including the most effective second-line anti-TB drugs. About 480 000 people
developed MDR-TB in the world in 2014, and it is estimated that about 9.7% of MDR-TB
cases had XDR-TB [1].
Over the past decades, drug discovery and development efforts have yielded a few new
interesting anti-mycobacterial agents, including the imidazo pyridine amide Q203, the
nitroimidazole PA-824, the 1,2-diamine SQ-109, and the benzothiazinone BTZ-043. Despite
that, only bedaquiline and delamanid have advanced to approval for pulmonary MDR-TB
infections when other effective treatment options are not useful [3]. Hence, there is an urgent
need to develop more effective and tolerable treatments for both drug-susceptible and drug-
resistant TB.
We have previously identified a series of 1,5-diphenyl pyrroles as a highly potent
compound class against drug-susceptible M. tuberculosis (Mtb) strains through phenotypic
screening [4]. A standard medicinal chemistry approach led to the identification of BM635
(Fig. 1), active against both replicating and non-replicating bacilli and proving to be
efficacious in a murine model of tuberculosis infection [5–10].
Using whole genome sequencing (WGS) methodology, we also determined the possible
target of this class of compounds; point mutations in the Rv0206c gene, which encodes for
Mycobacterium membrane protein Large 3 (MmpL3), were found [11]. MmpL3 is a member
of the Mycobacterial membrane protein Large (MmpL) family that belongs to the Resistance,
Nodulation and Division (RND) superfamily and is highly conserved in all the species of
mycobacteria and essential for Mtb [12–14].
Having proved the potentiality of this compound class, we started a medicinal chemistry
campaign in order to improve BM635’ liabilities: low water solubility and high proportion of
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sp2 centers. Herein we report systematic variations around the central pyrrole core of BM635
(Fig. 1) and we describe the design, synthesis, biological evaluation, pharmacokinetic
analysis, as well as in vivo TB mouse efficacy studies of novel BM635 analogues
(compounds 1-45, Figs 2 and 3) that show improved physicochemical properties. Moreover,
in order to gain some understanding on how these pyrroles interact with the MmpL3
transporter, we run a docking analysis using methods previously developed [15].
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2. Results and discussion
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2.1 Design
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The main objectives of the BM635 optimization program consisted in enhancing drug-like
properties, such as lipophilicity and solubility, as well as finding compounds with the right
potency/safety balance. For doing so, we followed different approaches like breaking
planarity, reducing aromaticity (by introducing either benzyl ring or alkyl substituents) and
introducing polarity (pyridines, polar substituents). Therefore, the new analogues were
prepared by modifying three main motifs: the N1 phenyl ring (N1Ph), the C5 phenyl ring
(C5Ph), and the C3 morpholine moiety (C3M) (Fig. 1). Both N1Ph and C5Ph modifications
(compounds 1-42) consisted in: i) removal of the phenyl ring; ii) replacement with a benzyl
ring; iii) replacement with heterocycles; iv) replacement of either the fluorine or the isopropyl
substituents; v) introduction of polar substituents; and vi) replacement with alkyl or
cycloalkyl substituents (Figs 2 and 3). C3M modifications (compounds 43-45) included
substitution of the morpholine moiety with a 3-substituted piperidine one (Fig. 3).
2.2 Chemistry
Compounds 1-35 and 38-45 were prepared following a straightforward synthetic pathway
previously optimized in our group and reported in Scheme 1 [10]. Briefly, 1,4-diketones 48a-
k were obtained by treating the suitable aldehydes 46a-k with methyl vinyl ketone 47 in a
sealed glass tube in the microwave reactor. Microwave assisted cyclization of 48a-k in the
presence of the appropriate amine gave the expected pyrroles 49a-n’ in good yields. Finally,
by treating pyrroles 49a-n’ with formaldehyde and the appropriate amine, following Mannich
reaction conditions, target compounds 1-35 and 38-45 were obtained. Compounds 36 and 37
were obtained, in turn, by deprotecting 35 with TFA and subsequent reductive methylation of
36 by means of formaldehyde and sodium triacetoxy borohydride (Na(OAc)3BH),
respectively (Scheme 2).
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2.3 Antimycobacterial activities and cytotoxicities of compounds 1-45
For each compound, we determined the minimum concentration required to inhibit (MIC)
Mtb growth in culture as well as, for some of them, the activity (expressed as IC50) against
intracellular Mtb growth in macrophages (THP-1 cells) (Table 1). Moreover, we evaluated
compound cytotoxicities by measuring the concentration of compound resulting in 50%
inhibition (TOX50) of HepG2 cell line growth, and occasionally the potential for drug-
induced human hepatotoxicity using an in vitro high-content cell-based assay (Cell Health or
CH assay) [16] (Table 1).
Several compounds proved to be active against Mtb both in culture and in macrophages with
MICs ranging from 0.08 to 0.6 µM (compounds 7, 9, 13, 17-21, and 29, Table 1) and IC50s
ranging from 0.08 to 0.63 µM (compounds 9, 13, 17-21, and 29, Table 1). Some of the active
compounds showed statistically relevant toxicity against HepG2 cells (Table 1) even though
compounds 7, 13, 17, 18, 20, 21, and 29 had good selectivity indexes (Tox50/MIC) ranging
from 100 to 333. Moreover, compounds 21 and 29 exhibited initial clean profiles regarding
hepatotoxicity (Cell health > 200 µM, Table 1).
Table 1. In vitro characterization of compounds 1-45, BM635 and isoniazid.
Compound
MIC
Mtb Tox50
(µΜ)
Selectivity index
(Tox50/MIC)
Intracell
IC50 Mtb healtha
Cell
(µΜ)
(µΜ)
HepG2
>5
>100
50
ncb
ncb
ncb
>10
>10
>10
ndc
ndc
1
2
3
>5
>125
>100
>200/>20
0/>200
ndc
>5
1.25
5
80
ncb
40
ncb
>10
3.3
4
5
6
80
ndc
>100
>10
>200/>20
0/>200
ndc
0.6
>5
>100
10
167
ndc
ndc
7
8
ndc
0.6
20
33
13
ncb
0.6
2.5
ndc
50/63/80
ndc
ndc
9
1.25
>40
16
10
11
>100
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>100
20
ncb
250
ncb
ncb
13
ndc
ndc
ndc
ndc
ndc
12
13
14
15
16
17
18
19
20
21
0.08
40
0.16
ndc
>100
1.6
16
12.5
1.25
0.15
0.2
>10
1.25
0.16
0.08
0.2
40/40/50
25/32/32
ndc
20
133
100
67
20
0.3
20
40/40/50
25/25/32
>200/>20
0/>200
ndc
0.08
0.3
20
250
>267
0.08
0.63
>100
1.3
1.3
2.5
50.1
ndc
40
39
1.32
1.25
2.5
22
23
24
ncb
ncb
32/40/50
50/100/12
6
>40
>40
2.5
>100
>100
80
ncb
ncb
ncb
ncb
333
ndc
ndc
2.5
ndc
0.3
ndc
ndc
25
26
27
28
29
80/80/100
ndc
2.5
50.1
50
0.15
>200/>20
0/>200
ndc
>5
>100
32
ncb
ncb
ncb
ncb
ncb
ncb
ncb
ncb
ncb
ncb
5.28
5
30
31
32
33
34
35
36
37
38
39
5
40/50/63
ndc
ndc
ndc
ndc
ndc
ndc
ndc
ndc
>40
>5
20
ndc
>10
>10
ndc
ndc
ndc
ndc
ndc
ndc
ndc
ndc
ndc
20
ndc
>5
30
16
>80
>40
2.5
2.5
16
25
ndc
ndc
2.5
20
ncb
ndc
ndc
ndc
ndc
40
41
42
43
1.25
1.25
2.5
>100
20
>167
67
ncb
10
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2
20
ncb
ncb
ndc
ndc
0.1
ndc
ndc
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12.6
40
45
0.12
333.34
>40/>40/
>40
BM635
1.8
-
-
-
-
Isoniazid
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aNuclear morphology/membrane permeability/mitochondrial potential. ncb, not calculated.
ndc, not determined.
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2.4 SAR analysis
C5Ph modifications: modifications of this position were not well tolerated leading to
inactive compounds (1-6 and 8, Table 1) with the exception of compound 7 which showed a
remarkable activity (MIC of 0.6 µΜ). N1Ph modifications were more tolerated even if
limited to: i) the replacement of the 4-F-phenyl ring with either a 4-F-pyridin-2-yl or 4-F-
benzyl ring (compounds 9 and 13, respectively, Table 1) and ii) the replacement with alkyl,
cycloalkyl or hetero cycloalkyl substituents (compounds 17-21 and 29, Table 1). C3M
modifications, however, just led to a drastic loss of activity (compounds 43-45, Table1).
2.5 In vitro safety and DMPK profile
To identify the best hits for progression to in vivo studies, compounds with MICs <1 µΜ
were evaluated for their in vitro safety and DMPK profile. In detail, we determined the
hydrophobicity using the chromatography technique to generate Chrom log DpH7.4 values, the
artificial membrane permeability, the percentage of binding to human serum albumin (HSA),
the kinetic solubility via chemiluminescent nitrogen detection (CLND), the solubility in the
biorelevant medium to simulate the fasted states in vivo (FaSSIF), as well as human Ether-a-
go-go-related Gene (hERG) binding (Table 2).
The replacement of the isopropyl substituent with a trifluoromethyl one at the para position
of the C5 phenyl ring as well as the introduction of alkyl or cycloalkyl substituents at N1
gave 0.42-0.89 units reduction of hydrophobicity respect to BM635 (compounds 7, 17, and
19, Table 2), while the introduction of either a pyridine or a tetrahydropyrane ring led to a
1.25 unit reduction (compounds 9 and 29, Table 2). The reduction of hydrophobicity led also
to a decrease in HSA binding, with compounds 9, 17, 19, and 29 showing the best values
(Table 2). All the tested compounds proved to be highly permeable in the artificial membrane
permeability assay with values in the 10-5 range (Table 2). Compounds 9, 13, 17-20 and 29
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proved to be more soluble than BM635 with CLND values ranging from 11 to 276 µM.
Compounds 17, 19 and 29 showed grater values in the FaSSIF medium (249, 511 and 43
µg/ml, respectively). Finally, compounds 7 and 21 proved a clean profile in the hERG
activity.
Table 2. Drug-like properties and safety profile of compounds 7, 9, 13, 17-21, 29 and
BM635.
Compound
Chrom log Membrane
%HSA
CLND
FaSSIF
hERG
DpH7.4
permeability
cm/sec
binding solubility solubility
IC50 (µΜ)
(µΜ)
<1
(µg/ml)
nda
nda
nda
249
nda
511
nda
nda
43
7.67
6.85
8.2
1.5 x 10-5
6.4 x 10-5
3.0 x 10-5
7.2 x 10-5
4.3 x 10-5
nda
5.5 x 10-5
2.7 x 10-5
7.3 x 10-5
2.4 x 10-5
98.33
94.13
98.47
94.11
97.12
96.49
98.12
98.1
>50
8
7
276
11
9
5
13
7.68
8.46
7.21
8.13
9.13
6.86
8.1
199
47
16
16
8
17
18
181
71
19
8
20
<1
50
10
10
21
92.85
98.37
241
<1
29
5
BM635
178
179
nda, not determined.
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181
182
183
184
185
186
187
188
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2.6 In vivo profiling of best hits
The most promising derivatives, compounds 17 and 29 were chosen to determine their in vivo
bioavailability as well as their therapeutic efficacy.
Pharmacokinetic profiles of 17 and 29 were evaluated in female C57 mice following
intravenous and oral administration. After intravenous administration, 17 showed a moderate
mean volume of distribution in blood (Vss), which exceeded near 3-fold the total body water
in mouse, while 29 showed a greater volume of distribution (near 6-fold the total body water
in mouse). While 29 showed a very high in vivo clearance of 125 ml/min/kg, suggesting that
it would be rapidly removed from the body, 17 presented a low value of mean clearance,
compared to the hepatic blood flow in mouse, and a moderate mean half-life of 1.7 hours
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(Table 3). After oral administration, 17 showed a mean Cmax value of 177 ng/ml while 29 a
practically three-fold higher Cmax value of 548 ng/mL (Table 4). Compound 29 showed a
moderate mean bioavailability value of 23 % and compound 17 a very low mean
bioavailability of 1.2% (Table 4).
Table 3. Pharmacokinetic parameters of 17 and 29 after intravenous administration to C57
mice.
Compound
Dose
Vss
AUCinf
DNAUCinf
Clearance
t1/2 (h)
(mg/kg)
(L/kg)
(ng·h/ml)
(ng·h/ml per (ml/min/kg)
mg/kg)
4
1
1.8
3.5
4024
138
1006
138
16.9
125
1.7
0.4
17
29
197
198
199
Table 4. Pharmacokinetic parameters of 17 and 29 after oral administration to C57 mice.
Compound
Dose
(mg/kg)
50
Tmax
(h)
Cmax (ng/ml) Tlast (h)
%F
0.7
0.9
177
548
7.3
8
1.2
23
17
29
50
200
201
202
203
204
205
206
207
208
209
210
211
212
213
Compounds 17 and 29 were progressed to the in vivo acute murine model of Mtb infection.
Compounds 17 and 29 were orally administered to C57 mice once a day for eight days
starting on day 1 after infection. Although 17 showed a very poor bioavailability (Table 4), it
induced a statistically significant difference in lung bacterial counts compared to untreated
mice (Table 5). Compound 29, on the other hand, did not inhibit the growth of bacterial
burden in the lungs of infected mice (Table 5).
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Table 5. Therapeutic efficacy of 17, 29 and moxifloxacin. Differences in the lung
microorganism burden (log10 CFUs/lungs) with respect to untreated controls (Day 9 after
infection).
Compound
Dose
(mg/kg)
50
Administration
Difference in P2
logCFU/lungs
Once a day
Once a day
Once a day
1.5
0.1
3.1
p<0.05
17
29
50
p<0.05
p<0.05
Moxifloxacin 30
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220
221
222
223
224
225
226
227
228
229
230
231
232
233
234
235
236
237
238
239
240
241
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2.7 Molecular docking of compounds into MmpL3
In order to gain insight regarding the interaction between these derivatives and MmpL3, we
run a docking analysis using methods previously reported [15]. The docking analyses started
with the parent compound BM212 [4,11] to verify whether the binding site as optimized in
the previous study was also able to properly accommodate the larger pyrrole derivatives.
Figure 4 (A) shows the main interactions stabilizing the putative complex between MmpL3
and BM212 as simulated in its protonated form due to the high basicity of the piperazine
ring. Indeed, Figure 4 (A) reveals the key ionic contact between the protonated piperazine
nitrogen atom and Asp640, a contact reinforced by the charge transfer interaction with
Tyr252. Even though the relevance of Asp640 has recently been confirmed by a mutational
study showing that the D640C mutant failed to rescue an Msmg mmpL3 knockout mutant
[15], the concrete role of this ion-pair might be limited by the closeness of Arg259 which
interacts with Asp640 and which elicits an extended charge transfer interaction with the
pyrrole ring and, to minor extent, with the two phenyl rings. The non-protonated piperazine
nitrogen atom is involved in H-bonds with Ser288 and Ser325, while the carbon skeleton of
the piperazine ring is engaged in hydrophobic contacts with surrounding apolar side chains
(e.g. Ile248 and Val643). Besides the already mentioned charge transfer interaction, the N1
phenyl ring elicits electronic interactions with Phe644 and the chlorine atom stabilizes a
halogen bond with Thr280. Similarly, the chlorine atom of the phenyl ring at C5 is engaged
in a halogen bond with Thr277, while the aromatic ring contacts only alkyl side chains such
as Ile256, Val285, Leu329 and Pro330.
The described complex affords a rather convincing validation of the capacity of the
previously optimized binding site to properly accommodate also the pyrrole derivatives and,
therefore, the MmpL3 homology model was exploited in the docking simulations involving
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the herein reported novel derivatives. When considering these novel compounds in their
neutral state, two possible binding modes can be detected. The first binding mode, as here
exemplified by the computed complex for 23 (Fig. 4 (B)), is very similar to that already seen
for BM212 with the morpholine ring which replaces the piperazine as seen in BM212. In
detail, the pyrrole stabilizes charge transfer interaction with Arg259, while the morpholine
ring elicits H-bonds with Tyr252, Ser288 and Ser321. The contacts stabilized by the phenyl
ring at C5 are largely dominated by the apolar contacts that the para-isopropyl group can
afford with the surrounding hydrophobic residues such as Ile256, Val278, Leu329, Pro330
and Leu333. Finally, the N-linked methoxy butyl chain stabilizes H-bonds with Thr280
reinforced by hydrophobic contacts with Ala281 and Ile256.
In the second observed binding mode, as exemplified by the putative complex for 13 (Fig. 4
(C)), the isopropyl phenyl moiety replaces the morpholine or the piperazine ring as seen in
the previous complexes and markedly reinforces the charge transfer interactions which the
bound molecule can stabilize with Arg259. Moreover, 13 elicits extended π-π stacking
contacts, which involve the phenyl moiety with Tyr252 as well as the benzyl group with
Phe644, while the para-isopropyl group contacts Ile248 and Val643. Finally, the morpholine
ring elicits H-bonds with Thr277 reinforced by apolar contacts with Leu329, Pro330 and
Leu333. When analyzing the best poses assumed by all simulated ligands, one may observe
that all ligands can assume both binding modes even though with different relevance in terms
of both docking scores and relative abundance. As a rule, the relevance of the second binding
mode depends on the ligand’s capacity to stabilize extended charge transfer and π-π stacking
contacts with the Arg259-Asp640 interacting dyad and the surrounding aromatic residues.
Interestingly, such a second mode appears to be the prevailing one for several potent
compounds. For example, the binding mode 2 is the preferred one for 13 out of the 42
simulated analogues when considering the PLP score function, and the compounds preferring
binding mode 2 show at most a MIC value of 2.5 µM.
Notably, the complexes computed when considering the inhibitors in their protonated state
reveal the same binding modes already seen in the neutral forms even though with different
frequency. For example, Fig. 4 (D) shows the first binding mode as assumed by 21 in its
protonated state and reveals a pattern of interactions already seen for neutral ligands further
reinforced by the clear ion pair between the protonated morpholine ring and Asp640.
Nevertheless, the capacity to yield such a salt bridge does not vastly alter the relative
abundance of the two possible binding modes. The number of compounds preferring the
binding mode 2 increases compared to what was observed for neutral inhibitors, since 18
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protonated forms out of 42 show a prevailing binding mode 2, thus confirming that the ion-
pair stabilized by the protonated morpholine ring has a very limited role. In addition, several
potent inhibitors preferentially assume the second binding mode, and indeed the compounds
with a prevailing binding mode 2 show a MIC value lower than 5 µM. A clear example of
potent inhibitor preferring the second binding mode is offered by 13 in which the protonated
morpholine ring stabilizes a reinforced H-bond with Ser325 (complex not shown).
Regardless of the ionized forms and different binding modes, the obtained docking results
allow for some general considerations. Firstly, the hydrophobic contacts appear to play a
dominant role: this result is in line with what was observed in the previous study[LIT] and is
confirmed by the following correlative study in which the best relationships are afforded by
docking scores parameterizing for apolar contacts (see Supporting Information, Table S1),
while scores encoding for ionic interactions do not yield interesting results. This finding can
be justified by considering that the explored binding site is lined by ion-pairs (i.e., Arg259-
Asp640 and Arg648-Glu263) so, that an ionized ligand cannot stabilize clear ionic
interactions without being also repelled. This can explain the limited effect of protonated
morpholine rings and can rationalize why a second ionizable group in the moiety linked to
the nitrogen atom of the pyrrolidine ring has always a detrimental role (as seen for 11 or 36).
Even though the aromatic rings of the ligand can engage in charge transfer interactions, the
N-linked aromatic ring can be conveniently replaced by an aliphatic (cyclic or acyclic)
moiety (as seen for 29), a result which is explainable considering the richness of alkyl side
chains flanking the binding site, while only one aromatic residue (Phe644) can contact this
ligand portion. The presence of some H-bonding residues can explain the positive role of the
ligand’s halogen atoms, which can be involved in halogen bonding while contributing to the
ligand apolarity.
Correlative studies (see Supporting Information) confirmed the relevance of the second
binding mode and suggested that both ionized and neutral forms can be involved in ligand
recognition with a relative weight which is related to their nucleophilicity. Overall, the
encouraging developed equations represent a mutual validation of both the MmpL3
homology model and docking results.
3. Conclusion
1,5-Diphenyl pyrroles were previously identified as a class of anti-mycobacterial
compounds endowed with in vivo efficacy within the range of commonly employed
tuberculosis drugs. Herein we presented our medicinal chemistry efforts aiming at improving
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the physicochemical properties and drug-likeness of this series while retaining their anti-
mycobacterial activity. We have designed, synthesized, and biologically evaluated a series of
45 BM635 analogues. Some of the new analogues showed improved physicochemical
properties and drug-likeness respect to the hit compound with good artificial membrane
permeability, high water solubility, no interaction with hERG, as well as excellent anti-
mycobacterial activity.
The best compound in the series was derivative 17, which showed both good intracellular
and extracellular anti-mycobacterial activities together with a good drug-like profile.
Additionally, compound 17 induced a statistically significant difference in lung bacterial
counts compared to untreated controls.
Finally, a putative homology model for the MmpL3 transporter has been used for docking
studies providing valuable further insights into the SAR of the compound series discussed.
4. Experimental section
4.1 Synthesis
Reagents and solvents were obtained from commercial sources (Fluka, Sigma-Aldrich, Alfa
Aesar). All reactions were carried out under normal atmosphere with magnetic stirring.
Microwave-assisted reactions were performed using a focused microwave reactor (Discover,
CEM Corporation, Matthews, NC, USA). Analytical thin layer chromatography (TLC) was
performed on Merck silica gel (60F254) pre-coated plates (0.25 mm). The compounds were
visualized under UV light (254 nm) and/or stained with a relevant reagent. Flash column
chromatography was performed on silica gel with pore size 60 Å, 230−400 mesh particle
size, and 40−63 ꢀm particle size, with the indicated solvents. The yields refer to purified
products, and were not optimized. All solid compounds were obtained as amorphous solids,
1
and melting points were not measured. H NMR spectra were recorded on a Bruker Avance
III NMR spectrometer and a Bruker DPX Avance 400 MHz spectrometer equipped with a
13
QNP probe and are reported in ppm using tetramethylsilane an internal standard. C NMR
spectra were recorded on a Bruker Avance III NMR spectrometer at 295 K and are reported
in ppm using solvent as an internal standard (DMSO-d6 at 39.5 ppm; CDCl3 at 77.0 ppm).
Mass spectra data and high resolution mass measurements were performed on a VG-
Analytical Autospec Q mass spectrometer. Analytical purity was ≥95% unless stated
otherwise. The purities of the final compounds were checked using a Waters ZQ2000 coupled
with LC Waters 2795 and Waters 2996 PDA detector. All mass spectra were performed using
electrospray ionization.
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4.1.1 General procedure for the preparation of pentane-1,4-diones 48a-k.
Compound 48a was commercially available. Pentanediones 48b-k were prepared as
following. In a sealed glass tube equipped with a stirring bar, aldehydes 46b-k (0.09 mol),
triethylamine (19.5 mL, 0.14mol), methyl vinyl ketone 47 (0.09 mol), and 3-ethyl-5-(2-
hydroxyethyl)-4-methylthiazolium bromide (3.53 g, 0.014 mol) were mixed together. The
flask was heated in the cavity of the microwave reactor for 15 min (150W, internal
temperature 70 °C, and internal pressure 60 psi). At the end, the obtained crude residue was
stirred with 10 ml of 2N HCl for 30 min. After extraction with ethyl acetate, the organic
layers were washed with aqueous sodium bicarbonate and brine. The organic fractions were
dried over Na2SO4, filtered, and concentrated to give a crude orange liquid. Chromatography
on aluminum oxide (activity II–III, according to Brockmann) (cyclohexane/ethyl acetate, 3:1
v/v) gave the desired 48b-k.
4.1.2 General procedure for the preparation of pyrroles 49a-n’.
The proper 1,4-pentanedione 48 (2.28 mmol) and the suitable amine (2.28 mmol) were
dissolved in ethanol (2 ml) in a sealed glass tube equipped with a stirring bar in the presence
of p-toluenesulfonic acid (30 mg, 0.17 mmol). The tube was heated in the cavity of the
microwave reactor for 30 min (150W, internal temperature 160 °C, and internal pressure 150
psi). At the end, the reaction mixture was cooled down and concentrated. The crude material
was purified by chromatography on aluminum oxide (activity II–III, according to
Brockmann) with cyclohexane to give the expected pyrroles 49a-n’ as solids in satisfactory
yields.
4.1.3 General procedure for the preparation of compounds 1-35 and 38-45.
To a stirred solution of the appropriate pyrrole 49 (5.6 mmol) in acetonitrile (20 ml), a
mixture of the appropriate amine (0.57 g, 5.6 mmol), formaldehyde (0.18 g, 5.6 mmol) (40%
in water), and 5 ml of glacial acetic acid was added drop-wise in 5 min. Following addition,
the mixture was stirred at room temperature for 1 h and then treated with a solution of sodium
hydroxide (20%, w/v) and extracted with ethyl acetate. The organic extracts were combined,
washed with brine, and dried over Na2SO4. The obtained residue after solvent evaporation
was purified by column chromatography, using silica gel and petroleum ether/ethyl acetate
(3:1 v/v) to give 1-35 and 38-45 as solids in satisfactory yields.
4.1.3.1 4-((1-(4-Fluorophenyl)-2,5-dimethyl-1H-pyrrol-3-yl)methyl)morpholine (1).
1
Yellow oil (yield 68%). H NMR (400 MHz, CDCl3): δ ppm= 7.15 (m, 4H), 5.91 (s, 1H),
3.73 (t, 4H, J= 4.8 Hz), 3.36 (s, 2H), 2.48 (m, 4H), 1.99 (s, 3H), 1.96 (s, 3H). 13C NMR (400
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MHz, CDCl3): δ ppm= 159.8, 134.8, 129.6, 126.8, 122.5, 121.9, 116.8, 111.5, 67.1, 56.2,
55.3, 12.9, 10.2. MS-ESI: m/z 289 (M + H+).
4.1.3.2
4-((5-Cyclohexyl-1-(4-fluorophenyl)-2-methyl-1H-pyrrol-3-
yl)methyl)morpholine (2). White cristals (yield 35%). 1H NMR (400 MHz, CDCl3): δ ppm=
7.15 (m, 4H), 5.90 (s, 1H), 3.74 (t, 4H, J= 4.7 Hz), 3.38 (s, 2H), 2.48 (m, 4H), 2.19 (tt, 1H, J=
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11.6, 3.3 Hz), 1.91 (s, 3H), 1.74-1.58 (m, 6H), 1.29 (m, 2H), 1.09 (m, 2H). C NMR (400
MHz, CDCl3): δ ppm= 159.6, 134.5, 132.1, 125.7, 121.5, 120.9, 115.8, 110.8, 66.5, 55.9,
54.6, 34.3, 33.2, 25.8, 24.9, 10.2. MS-ESI: m/z 357 (M + H+).
4.1.3.3
4-((5-(5-Chloropyridin-2-yl)-1-(4-fluorophenyl)-2-methyl-1H-pyrrol-3-
1
yl)methyl)morpholine (3). White solid (yield 35%). H NMR (400 MHz, CDCl3): δ ppm=
8.26 (d, 1H, J= 2.8 Hz ), 7.37 (dd, 1H, J= 8.6, 2.8 Hz), 7.13 (m, 4H), 7.07 (d, 1H, J= 8. 6 Hz),
6.72 (s, 1H), 3.73 (t, 4H, J= 4.2 Hz), 3.43 (s, 2H), 2.51 (m, 4H), 2.05 (s, 3H). 13C NMR (400
MHz, DMSO-d6): δ ppm= 159.8, 153.9, 151.2, 146.5, 135.9, 132.7, 129.8, 125.9, 124.2,
121.9, 121.6, 116.2, 110.8, 66.8, 56.6, 55.8, 10.4. MS-ESI: m/z 386 (M + H+).
4.1.3.4
4-((5-(6-Chloropyridin-3-yl)-1-(4-fluorophenyl)-2-methyl-1H-pyrrol-3-
yl)methyl)morpholine (4). White powder (yield 41%). 1HNMR (400 MHz, CDCl3): δ ppm=
8.10 (d, 1H, J= 2.5 Hz), 7.19 (dd, 1H, J= 8.3, 2.5 Hz), 7.11 (m, 5H), 6.44 (s, 1H), 3.75 (t, 4H,
13
J= 4.3 Hz), 3.43 (s, 2H), 2.52 (m, 4H), 2.08 (s, 3H). CNMR (400 MHz, CDCl3): δ ppm=
159.5, 153.6, 150.2, 146.3, 143.5, 132.0, 129.4, 125.4, 121.8, 121.2, 120.9, 115.8, 110.3,
66.3, 56.2, 55.1, 10.0. MS-ESI: m/z 387 (M + H+).
4.1.3.5
N-(4-(1-(4-fluorophenyl)-5-methyl-4-(morpholinomethyl)-1H-pyrrol-2-
1
yl)phenyl)acetamide (5). White solid (yield 22%). H NMR (400 MHz, CDCl3): δ ppm=
7.29 (d, 2H, J= 8.4 Hz), 7.10 (m, 4H), 6.98 (d, 2H, J= 8.4 Hz), 6.33 (s, 1H), 3.75 (s broad,
4H), 3.44 (s, 2H), 2.54 (s broad, 4H), 2.14 (s, 3H), 2.07 (s, 3H). 13CNMR (400 MHz, CDCl3):
δ ppm= 168.9, 159.8, 146.7, 138.2, 135.8, 134.7, 128.2, 126.1, 122.0, 121.5, 116.3, 113.3,
66.9, 56.4, 55.8, 23.8, 10.6. MS-ESI: m/z 408 (M + H+).
4.1.3.6
4-((5-(4-Chloro-2-methylphenyl)-1-(4-fluorophenyl)-2-methyl-1H-pyrrol-3-
1
yl)methyl)morpholine (6). White solid (yield 25%). H NMR (400 MHz, CDCl3): δ ppm=
7.08 (d, 1H, J= 2.3 Hz), 6.98-6.92 (m, 6H), 6.91 (d, 1H, J= 8.6 Hz), 6.15 (s, 1H), 3.75 (m,
13
4H), 3.46 (s, 2H), 2.54 (m, 4H), 2.11 (s, 3H), 2.10 (s, 3H). C NMR (400 MHz, CDCl3): δ
ppm= 159.5, 146.3, 137.6, 134.5, 133.8, 130.1, 128.3, 127.4, 126.1, 125.4, 121.6, 121.0,
115.9, 112.9, 66.6, 56.0, 55.4, 17.8, 9.8. MS-ESI: m/z 399 (M + H+).
4.1.3.7
4-((1-(4-Fluorophenyl)-2-methyl-5-(4-(trifluoromethyl)phenyl)-1H-pyrrol-3-
1
yl)methyl)morpholine (7). White solid (yield 67%). HNMR (400 MHz, CDCl3): δ ppm=
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7.39 (d, 2H, J= 7.5 Hz), 7.11 (m, 6H), 6.47 (s, 1H), 3.75 (m, 4H), 3.44 (s, 2H), 2.53 (m, 4H),
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2.09 (s, 3H). CNMR (400 MHz, CDCl3): δ ppm= 159.9, 146.8, 143.2, 134.8, 131.3, 126.8,
126.1, 124.0, 121.9, 121.4, 116.6, 113.5, 66.8, 56.5, 55.7, 10.5. MS-ESI: m/z 419 (M + H+).
4.1.3.8
4-((5-Benzyl-1-(4-fluorophenyl)-2-methyl-1H-pyrrol-3-yl)methyl)morpholine
(8).Yellow oil (yield 46%). 1H NMR (400 MHz, DMSO-d6): δ ppm= 7.25-7.08 (m, 7H), 6.89
(m, 2H), 5.73 (s, 1H), 3.64 (s, 2H), 3.53 (m, 4H), 3.22 (s, 2H), 2.33 (m, 4H), 1.86 (s, 3H). 13C
NMR (400 MHz, DMSO-d6): δ ppm= 159.5, 146.4, 138.0, 134.2, 129.0, 128.4, 126.8, 125.3,
121.9, 121.0, 116.0, 113.0, 66.4, 56.0, 55.3, 32.1, 10.1. MS-ESI: m/z 365 (M + H+).
4.1.3.9
4-((1-(5-Fluoropyridin-2-yl)-5-(4-isopropylphenyl)-2-methyl-1H-pyrrol-3-
1
yl)methyl)morpholine (9) White solid (yield 43%). H NMR (400 MHz, CDCl3): δ ppm=
8.44 (d, 1H, J= 3.1 Hz), 7.34 (dd, 1H, J= 8.7, 3.1 Hz), 7.01 (m, 2H, J= 8.7, 3.1 Hz), 6.92 (m,
3H), 6.32 (s, 1H), 3.73 (t, 4H, J= 4.0 Hz), 3.43 (s, 2H), 2.82 (sept, 1H, J= 7.1 Hz), 2.51 (s
broad, 4H), 2.16 (s, 3H), 1.20 (d, 6H, J= 7.1 Hz). 13C NMR (400 MHz, DMSO-d6): δ ppm=
158.3, 154.9, 148.6, 137.2, 135.1, 133.8, 128.5, 126.0, 125.3, 121.6, 120.2, 115.8, 111.3,
66.9, 57.4, 56.2, 33.7, 23.5, 10.2. MS-ESI: m/z 394 (M + H+).
4.1.3.10
4-(5-(4-Isopropylphenyl)-2-methyl-3-(morpholinomethyl)-1H-pyrrol-1-yl)-
N,N-dimethylaniline (10).White solid (yield 48%). 1H NMR (400 MHz, DMSO-d6): δ ppm=
6.98 (m, 6H), 6.69 (m, 2H), 6.18 (s, 1H), 3.55 (t, 4H, J= 4.2 Hz), 3.29 (s, 2H), 2.91 (s, 6H),
13
2.76 (sept, 1H, J= 7.1 Hz), 2.37 (m, 4H), 1.93 (s, 3H), 1.11 (d, 6H, J= 7.1 Hz). C NMR
(400 MHz, DMSO-d6): δ ppm= 148.4, 147.2, 140.2, 136.8, 134.3, 128.3, 125.8, 125.2, 121.5,
117.2, 113.3, 112.1, 66.6, 56.3, 55.8, 41.5, 33.1, 23.1, 10.1. MS-ESI: m/z 418 (M + H+).
4.1.3.11
4-(5-(4-Isopropylphenyl)-2-methyl-3-(morpholinomethyl)-1H-pyrrol-1-
yl)benzoic acid (11). White solid (yield 32%). 1H NMR (400 MHz, CDCl3): δ ppm= 7.98 (d,
2H, J= 8.3 Hz), 7.11 (d, 2H, J= 8.3 Hz), 6.95 (m, 4H), 6.26 (s, 1H), 3.95 (m, 4H), 3.88 (s,
2H), 3.09 (m, 4H), 2.77 (sept, 1H, J= 6.8 Hz), 2.02 (s, 3H), 1.17 (d, 6H, J= 6.8 Hz). 13CNMR
(400 MHz, CDCl3): δ ppm= 169.6, 156.2, 148.2, 137.1, 134.2, 130.9, 129.6, 128.1, 127.2,
125.8, 125.0, 121.7, 113.5, 66.8, 56.5, 55.9, 33.6, 23.4, 10.5. MS-ESI: m/z 419 (M + H+).
4.1.3.12
3-(5-(4-Isopropylphenyl)-2-methyl-3-(morpholinomethyl)-1H-pyrrol-1-
yl)benzoic acid (12). White solid (yield 37%). 1H NMR (400 MHz, CDCl3): δ ppm= 12.50 (s
broad, 1H), 8.05 (t, 1H, 2.5 Hz), 8.00 (dt, 1H, J= 7.8 Hz, 2.5 Hz), 7.39 (t, 1H, J= 7.8 Hz),
7.22 (dt, 1H, J= 7.8 Hz, 2.5 Hz), 6.91 (d, 2H, J= 8.3 Hz), 6.82 (d, 2H, J= 8.3 Hz), 6.47(s,
1H), 4.36 (m, 2H), 4.16 (s broad, 2H), 4.02 (m, 2H), 3.58 (m, 2H), 3.00 (s broad, 2H), 2.78
(sept, 1H, J= 7.1 Hz), 2.19 (s, 3H), 1.16 (d, 6H, J= 7.1 Hz). 13C NMR (400 MHz, DMSO-d6):
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δ ppm= 166.7, 148.3, 141.7, 136.9, 134.0, 130.5, 129.0, 127.9, 126.8, 126.5, 125.4, 125.0,
121.2, 113.1, 107.2, 66.5, 56.1, 55.3, 33.0, 23.1, 10.2. MS-ESI: m/z 419 (M + H+).
4.1.3.13 2-Methyl-3-[(morpholino)-methyl]-5-[4-(i-propyl)phenyl]-1-(4-fluorobenzyl)-
1
1H-pyrrole (13). White solid (yield 36%). H NMR (400 MHz, DMSO-d6): δ ppm= 7.18-
7.10 (m, 6H), 6.84 (m, 2H), 6.06 (s, 1H), 5.11 (s, 2H), 3.54 (t, 4H, J= 4.2 Hz), 3.29 (s, 2H),
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2.84 (sept, 1H, J= 7.1 Hz), 2.34 (m, 4H), 1.99 (s, 3H), 1.16 (d, 6H, J= 7.1 Hz). C NMR
(400 MHz, DMSO-d6): δ ppm= 160.2, 148.6, 136.9, 134.5, 133.2, 128.9, 128.3, 126.2, 125.7,
121.5, 115.6, 113.6, 66.8, 56.7, 55.9, 47.1, 33.5, 23.4, 10.4. MS-ESI: m/z 407 (M + H+).
4.1.3.14
4-((5-(4-Isopropylphenyl)-2-methyl-1-(pyrimidin-2-yl)-1H-pyrrol-3-
1
yl)methyl)morpholine (14). Yellow syrup (yield 13%). H NMR (400 MHz, CDCl3): δ
ppm= 8.70 (d, 2H, J= 4.9 Hz), 7.19 (t, 1H, J= 4.9 Hz), 6.92 (d, 2H, J= 8.2 Hz), 6.6.90 (d, 2H,
J= 8.2 Hz), 6.33 (s,1H), 3.71 (t, 4H, J= 4.2 Hz), 3.42 (s, 2H), 2.81 (sept, 1H, J= 6.9 Hz), 2.50
(m, 4H), 2.27 (s, 3H), 1.19 (d, 6H, J= 6.9 Hz). 13C NMR (400 MHz, CDCl3): δ ppm= 169.7,
155.8, 148.2, 136.6, 133.5, 128.0, 125.2, 124.7, 118.5, 116.9, 110.6, 66.4, 55.8, 54.9, 32.9,
23.0, 10.0. MS-ESI: m/z 376 (M + H+).
4.1.3.15
4-((5-(4-Isopropylphenyl)-2-methyl-1H-pyrrol-3-yl)methyl)morpholine
1
formate (15). White solid (yield 15%) H NMR (400 MHz, CDCl3): δ ppm= 8.44 (s, 1H),
8.34 (s broad, 1H), 7.36 (d, 2H, J= 8.3), 7.22 (d, 2H, J= 8.3 Hz), 6.35 (s, 1H), 3.91 (t, 4H, J=
4.7 Hz ), 3.87 (s, 2H), 2.97 (m, 4H), 2.90 (sept, 1H, J= 7.1 Hz), 2.32 (s, 3H), 1.26 (d, 6H, J=
7.1 Hz). 13C NMR (400 MHz, CDCl3): δ ppm= 148.4, 133.6, 130.8, 128.3, 127.8, 125.7,
118.5, 108.6, 66.7, 55.8, 55.9, 33.9, 23.6, 11.5. MS-ESI: m/z 345 (M + H+).
4.1.3.16
4-((5-(4-Isopropylphenyl)-1,2-dimethyl-1H-pyrrol-3-yl)methyl)morpholine
1
(16). Yellowish solid (yield 27%). H NMR (400 MHz, CDCl3): δ ppm= 7.29 (m, 2H), 7.24
(m, 2H), 6.10 (s, 1H), 3.73 (t, 4H, J= 4.3 Hz), 3.51 (s, 3H), 3.41 (s, 2H), 2.93 (sept, 1H, J=
6.8 Hz), 2.50 (m, 4H), 2.25 (s, 3H), 1.28 (d, 6H, J= 6.8 Hz). 13C NMR (400 MHz, CDCl3): δ
ppm= 148.2, 143.1, 130.0, 128.0, 127.3, 125.2, 119.1, 110.6, 66.7, 55.6, 55.0, 33.0, 25.6,
23.3, 10.2. MS-ESI: m/z 313 (M + H+).
4.1.3.17
4-((1-Isopropyl-5-(4-isopropylphenyl)-2-methyl-1H-pyrrol-3-
yl)methyl)morpholine (17). Yellow oil (yield 2%) 1H NMR (400 MHz, DMSO-d6): δ ppm=
7.25 (d, 2H, J= 8.3 Hz), 7.17 (d, 2H, J= 8.3 Hz), 5.80 (s, 1H), 4.42 (sept, 1H, J= 7.1 Hz), 3.52
(t, 4H, J= 4.2 Hz), 3.21 (s, 2H), 2.89 (sept, 1H, J= 7.1 Hz), 2.32 (s broad, 4H), 2.29 (s, 3H),
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1.35 (d, 6H, J= 7.1 Hz), 1.22 (d, 6H, J= 7.1 Hz). C NMR (400 MHz, DMSO-d6): δ ppm=
148.4, 143.1, 130.4, 127.9, 127.3, 125.2, 119.1, 111.0, 66.7, 55.7, 55.1, 50.9, 33.0, 23.3, 10.8.
MS-ESI: m/z 341 (M + H+).
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4-((1-Isobutyl-5-(4-isopropylphenyl)-2-methyl-1H-pyrrol-3-
1
yl)methyl)morpholine (18). Yellow oil (yield 15%). H NMR (400 MHz, DMSO- d6): δ
ppm= 7.23 (m, 4H), 5.89 (s, 1H), 3.75 (d, 2H, J= 7.8 Hz), 3.52 (t, 4H, J= 4.3 Hz), 3.25 (s,
2H), 2.88 (sept, 1H, J= 7.1 Hz ), 2.31 (m, 4H), 2.18 (s, 3H), 1.57 (sept, 1H, J= 7.8 Hz), 1.20
(d, 6H, J= 7.1 Hz), 0.54 (d, 6H, J= 7.8 Hz). 13C NMR (400 MHz, DMSO-d6): δ ppm= 148.6,
143.5, 130.7, 128.7, 127.6, 125.7, 119.6, 111.6, 66.9, 57.9, 56.8, 55.8, 33.4, 30.1, 19.7, 23.3,
10.6. MS-ESI: m/z 355 (M + H+).
4.1.3.19
4-((1-Cyclopropyl-5-(4-isopropylphenyl)-2-methyl-1H-pyrrol-3-
1
yl)methyl)morpholine (19). Yellow oil (yield 22%). H NMR (400 MHz, DMSO-d6): δ
ppm= 7.36 (d, 2H, J= 8.1 Hz), 7.20 (d, 2H, J= 8.1 Hz), 5.91 (s, 1H), 3.51 (t, 4H, J= 4.5 Hz),
3.35 (m, 1H), 3.20 (s, 2H), 2.88 (sept, 1H, J= 6.8 Hz), 2.31 (m, 4H), 2.25 (s, 3H), 1.21 (d,
13
6H, J= 6.8 Hz), 0.86 (m, 2H), 0.40 ppm (m, 2H). C NMR (400 MHz, DMSO-d6): δ ppm=
148.5, 143.3, 130.6, 128.7, 127.8, 125.9, 119.4, 111.6, 66.8, 56.8, 56.0, 33.4, 30.5, 23.4, 10.9,
4.9. MS-ESI: m/z 339 (M +H+).
4.1.3.20
4-((1-Cyclobutyl-5-(4-isopropylphenyl)-2-methyl-1H-pyrrol-3-
1
yl)methyl)morpholine (20). Yellow oil (yield 24%). H NMR (400 MHz, DMSO-d6): δ
ppm= 7.23 (d, 2H, J= 8.1 Hz), 7.17 (d, 2H, J= 8.1 Hz), 5.80 (s, 1H), 4.76 (quint, 1H, J= 8.9
Hz), 3.52 (t, 4H, J= 4.2 Hz), 3.21 (s, 2H), 2.89 (sept, 1H, J= 6.8 Hz), 2.33 (m, 9H), 2.22 (m,
2H), 1.62 (m, 2H), 1.21 (d, 6H, J= 6.8 Hz). 13C NMR (400 MHz, DMSO-d6): δ ppm= 148.4,
142.9, 130.1, 128.1, 127.2, 125.2, 119.2, 110.9, 66.7, 56.1, 55.4, 54.3, 33.0, 31.4, 23.3, 15.9,
11.0. MS-ESI: m/z 353 (M + H+).
4.1.3.21
4-((1-Cyclohexyl-5-(4-isopropylphenyl)-2-methyl-1H-pyrrol-3-
1
yl)methyl)morpholine (21). Uncolorless oil (yield 24%). H NMR (400 MHz, CDCl3): δ
ppm= 7.22 (s, 4H), 5.99 (s, 1H), 4.07 (tt, 1H, J= 12.2, 4.0 Hz), 3.71 (t, 4H, J= 4.0 Hz), 3.37
(s, 2H), 2.94 (sept, 1H, J= 7.1 Hz), 2.47 (m, 4H), 2.38 (s, 3H), 1.99-1.79 (m, 6H), 1.64 (m,
13
2H), 1.29 (d, 6H, J= 7.1 Hz), 1.20 (m, 2H). C NMR (400 MHz, CDCl3): δ ppm= 148.5,
143.2, 130.5, 128.3, 127.6, 125.7, 119.4, 111.3, 66.9, 63.8, 56.2, 55.7, 35.2, 33.4, 25.8, 25.0,
3.5, 10.9. MS-ESI: m/z 381 (M + H+).
4.1.3.22
4-((5-(4-Isopropylphenyl)-1-(2-methoxyethyl)-2-methyl-1H-pyrrol-3-
1
yl)methyl)morpholine (22). Yellow oil (yield 26%). H NMR (400 MHz, DMSO-d6): δ
ppm= 7.27 (m, 4H), 5.90 (s, 1H), 4.00 (t, 2H, J= 6.2 Hz), 3.53 (m, 4H), 3.36 (t, 2H, J= 6.2
Hz), 3.24 (s, 2H), 3.09 (s, 3H), 2.89 ( sept, 1H, J= 6.8 Hz), 2.32 (m, 4H), 2.20 (s, 3H), 1.21
(d, 6H, J= 6.8 Hz). 13C NMR (400 MHz, DMSO-d6): δ ppm= 148.2, 142.9, 130.0, 127.7,
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126.8, 125.0, 119.1, 110.8, 75.4, 66.7, 58.6, 55.9, 55.5, 47.8, 33.0, 23.3, 10.6. MS-ESI: m/z
357 (M + H+).
4.1.3.23
4-((5-(4-Isopropylphenyl)-1-(3-methoxypropyl)-2-methyl-1H-pyrrol-3-
1
yl)methyl)morpholine (23). Yellow oil (yield 32%). H NMR (400 MHz, DMSO-d6): δ
ppm= 7.24 (m, 4H), 5.90 (s, 1H), 3.92 (t, 2H, J= 7.3 Hz), 3.52 (t, 4H, J= 4.5 Hz), 3.31 (s,
2H), 3.09 (t, 2H, J= 5.8 Hz), 3.04 (s, 3H), 2.89 ( sept, 1H, J= 6.8 Hz), 2.32 (m, 4H), 2.18 (s,
3H), 1.62 (quint, 2H, J= 5.8 Hz), 1.27 (d, 6H, J= 6.8 Hz). 13C NMR (400 MHz, DMSO-d6): δ
ppm= 148.6, 143.3, 130.4, 128.1, 127.5, 125.8, 119.6, 111.3, 70.2, 66.8, 59.5, 56.7, 55.8,
45.8, 33.3, 31.7, 23.4, 10.7. MS-ESI: m/z 371 (M + H+).
4.1.3.24 1-(5-(4-Isopropylphenyl)-2-methyl-3-(morpholinomethyl)-1H-pyrrol-1-yl)-2-
1
methylpropan-2-ol (24). Orange oil (yield 15%). H NMR (400 MHz, DMSO-d6): δ ppm=
7.21 (m, 4H), 5.87 (s, 1H), 4.34 (s, 1H), 3.96 (s broad, 2H), 3.53 (m, 4H), 3.26 (s, 2H), 2.88
13
(sept, 1H, J= 7.1 Hz), 2.32 (m, 4H), 2.25 (s, 3H), 1.20 (d, 6H, J= 7.1 Hz), 0.68 (s, 6H). C
NMR (400 MHz, DMSO-d6): δ ppm= 148.7, 143.2, 130.4, 128.5, 127.6, 125.9, 119.7, 111.3,
71.4, 66.7, 56.2, 55.8, 33.4, 27.8, 23.5, 10.7. MS-ESI: m/z 371 (M + H+).
4.1.3.25
3-(5-(4-Isopropylphenyl)-2-methyl-3-(morpholinomethyl)-1H-pyrrol-1-
1
yl)propanoic acid (25). White solid (yield 27%). H NMR (400 MHz, DMSO-d6): δ ppm=
12.40 (s broad, 1H), 7.28 (m, 4H), 6.17 (s, 1H), 4.13 (m, 4H), 3.96 (d, 2H, J= 12.2 Hz), 3.68
(t, 2H, J= 12.2 Hz), 3.05 (m, 2H), 2.93 (sept, 1H, J= 6.8 Hz), 2.32 (s, 3H), 1.21 (d, 6H, J= 6.8
Hz). 13C NMR (400 MHz, DMSO-d6): δ ppm= 171.8, 148.2, 143.0, 130.0, 128.1, 127.3,
125.4, 119.2, 111.0, 66.7, 56.0, 55.4, 45.3, 35.1, 33.2, 23.1, 10.6. MS-ESI: m/z 371 (M + H+).
4.1.3.26
4-(5-(4-Isopropylphenyl)-2-methyl-3-(morpholinomethyl)-1H-pyrrol-1-
1
yl)butanoic acid (26). White solid (yield 17%). H NMR (400 MHz, CDCl3): δ ppm= 11.91
(s, 1H), 7.30 (m, 4H), 6.17 (s, 1H), 4.41 (t, 2H, J= 6.5 Hz), 4.03 (m, 6H), 3.66 (m, 2H), 2.96
13
(m, 3H), 2.41 (s, 3H), 2.11 (m, 2H), 1.88 (m, 2H), 1.30 (d, 6H, J= 7.1 Hz). C NMR (400
MHz, CDCl3): δ ppm= 178.7, 148.5, 143.6, 130.4, 128.4, 127.7, 125.5, 119.4, 111.1, 66.7,
56.8, 55.9, 48.2, 35.2, 33.5, 25.9, 23.3, 10.7. MS-ESI: m/z 385 (M + H+).
4.1.3.27
4-((5-(4-Isopropylphenyl)-2-methyl-1-(oxetan-3-yl)-1H-pyrrol-3-
1
yl)methyl)morpholine (27). Yellow oil (yield 28%). H NMR (400 MHz, CDCl3): δ ppm=
7.22 (d, 2H, J= 8.2 Hz), 7.14 (d, 2H, J= 8.2 Hz), 6.05 (s, 1H), 5.53 (quint,1H, J= 7.5 Hz),
4.82 (t, 2H, J= 7.5 Hz), 4.76 (t, 2H, J= 7.5 Hz), 3.73 (m, 4H), 3.40 (s broad, 2H), 2.98 (sept,
13
1H, J= 6.8 Hz), 2.51 (m, 4H), 2.38 (s, 3H), 1.27 (d, 6H, J= 6.8 Hz). C NMR (400 MHz,
CDCl3): δ ppm= 148.0, 143.1, 130.1, 127.8, 126.9, 125.2, 118.7, 110.6, 79.3, 66.6, 57.1, 55.8,
55.0, 33.0, 23.3, 10.9. MS-ESI: m/z 355 (M + H+).
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4.1.3.28 4-((5-(4-Isopropylphenyl)-2-methyl-1-(tetrahydro-2H-pyran-4-yl)-1H-pyrrol-
1
3-yl)methyl)morpholine (28). White solid (yield 43%). H NMR (400 MHz, CDCl3): δ
ppm= 7.23 (m, 4H), 6.01 (s, 1H), 4.31 (tt, 1H, J= 12.0, 4.4 Hz), 4.03 (dd, 2H, J= 12.0, 4.4
Hz), 3.73 (s broad, 4H), 3.49 (t, 1H, J= 12.0 Hz ), 3.34 (m, 4H), 2.96 (sept, 1H, J= 7.7 Hz),
2.49 (s broad, 3H), 2.41 (s, 3H), 2.32 (td, 2H, J= 12.5, 4.4 Hz), 1.76 (dd, 2H, J= 12.5, 2.0
Hz), 1.29 (d, 6H, J= 7.7 Hz), 1.21 (m, 1H). 13C NMR (400 MHz, CDCl3): δ ppm= 147.5,
142.9, 129.7, 127.5, 126.8, 124.9, 119.0, 110.5, 66.2, 64.9, 55.6, 55.0, 53.8, 32.8, 31.7, 23.0,
10.7. MS-ESI: m/z 383 (M + H+).
4.1.3.29 4-((5-(4-Isopropylphenyl)-2-methyl-1-(tetrahydro-2H-pyran-3-yl)-1H-pyrrol-
1
3-yl)methyl)morpholine (29). White solid (yield 15%). H NMR (400 MHz, DMSO-d6): δ
ppm= 7.27 (d, 2H, J= 8.1 Hz ), 7.19 (d, 2H, J= 8.1 Hz), 5.85 (s, 1H), 4.12 (m, 1H), 3.76-3.69
(m, 3H), 3.42 (m, 1H), 3.52 (t, 4H, J= 4.0 Hz), 3.25 (m, 1H), 3.21 (s, 2H), 2.91 (sept, 1H, J=
6.8 Hz), 2.31 (s, 6H), 2.15 (qd, 1H, J= 13.0, 4.0 HZ), 1.92 (d, 1H, J= 13.0 Hz), 1.68 (d, 1H,
J= 13.0 Hz), 1.48 (m, 1H), 1.22 (d, 6H, J= 6.8 Hz). 13C NMR (400 MHz, DMSO-d6): δ ppm=
148.6, 143.3, 130.5, 128.3, 127.7, 125.8, 119.5, 111.5, 75.1, 69.8, 66.7, 58.2, 56.3, 55.6, 33.0,
23.3, 20.5, 10.2. MS-ESI: m/z 383 (M + H+).
4.1.3.30
4-((5-(4-Isopropylphenyl)-2-methyl-1-((tetrahydro-2H-pyran-4-yl)methyl)-
1
1H-pyrrol-3-yl)methyl)morpholine (30). White solid (yield 28%). H NMR (400 MHz,
DMSO-d6): δ ppm= 7.24 (s, 4H), 5.88 (s, 1H), 3.84 (d, 2H, J= 7.3 Hz), 3.62 (d, 2H, J= 10.9
Hz), 3.52 (m, 4H), 3.24 (s, 2H), 3.01 (t, 2H, J= 10.9 Hz), 2.91 (sept, 1H, J= 6.8 Hz), 2.31 (s
broad, 4H), 2.20 (s, 3H), 1.56 (m, 1H), 1.21 (d, 6H, J= 6.8 Hz), 1.09 (d, 2H, J= 12.0 Hz),
0.86 (qd, 2H, J= 12.0, 4.0 Hz). 13C NMR (400 MHz, DMSO-d6): δ ppm= 148.2, 143.0, 130.0,
128.1, 127.5, 125.3, 119.2, 111.0, 69.3, 66.7, 56.3, 55.7, 52.1, 33.1, 30.8, 29.1, 23.3, 10.6.
MS-ESI: m/z 397 (M + H+).
4.1.3.31
4-((5-(4-Isopropylphenyl)-2-methyl-1-((tetrahydrofuran-2-yl)methyl)-1H-
1
pyrrol-3-yl)methyl)morpholine (31). Yellowish oil (yield 25%). H NMR (400 MHz,
DMSO-d6): δ= 7.28 (d, 2H, J= 8.6 Hz), 7.23 (d, 2H, J= 8.6 Hz), 5.89 (s, 1H), 3.93 (d, 2H, J=
5.8 Hz), 3.80 (quint, 1H, J= 5.8 Hz), 3.52 (m, 6H), 3.24 (s, 2H), 2.88 (sept, 1H, J= 6.8 Hz),
13
2.32 (s broad, 4H), 2.21 (s, 3H), 1.62 (m, 3H), 1.26 (m, 1H), 1.20 (d, 6H, J= 6.8 Hz). C
NMR (400 MHz, DMSO-d6): δ ppm= 148.8, 143.6, 130.8, 128.7, 127.9, 125.8, 119.5, 111.8,
81.7, 68.5, 66.7, 56.6, 55.9, 53.1, 28.4, 33.5, 24.9, 23.8, 10.7. MS-ESI: m/z 383 (M + H+).
4.1.3.32
3-(5-(4-Isopropylphenyl)-2-methyl-3-(morpholinomethyl)-1H-pyrrol-1-
1
yl)cyclohexan-1-ol (32). Yellow syrup (yield 9%). H NMR (400 MHz, CDCl3): δ ppm=
7.23 (m, 4H), 6.16 (s, 1H), 4.62 (m, 1H), 4.23 (m, 1H), 3.70 (s broad, 4H), 3.48 (s, 2H), 2.80-
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2.93 (m, 6H), 2.42 (s, 3H), 2.15 (m, 1H), 1.99 (m, 1H), 1.91 (m, 2H), 1.75 (m, 3H), 1.43 (m,
1H), 1.29 (d, 6H, J= 6.8 Hz). 13C NMR (400 MHz, CDCl3): δ ppm=148.5, 143.0, 130.1,
127.8, 126.9, 124.8, 118.6, 110.8, 69.0, 66.0, 59.2, 55.6, 55.0, 39.8, 35.6, 34.0, 32.9, 23.0,
16.5, 10.8. MS-ESI: m/z 397 (M + H+).
4.1.3.33 4-((5-(4-Isopropylphenyl)-2-methyl-1-(1-methylpiperidin-4-yl)-1H-pyrrol-3-
1
yl)methyl)morpholine (33). Yellow oil (yield 29%). H NMR (400 MHz, DMSO-d6): δ
ppm= 7.25 (d, 2H, J= 8.1 Hz), 7.17 (d, 2H, J= 8.1 Hz), 5.82 (s, 1H), 3.95 (tt, 1H, J= 12.0, 4.0
Hz), 3.52 (t, 4H, J= 4.0 Hz), 3.34 (s broad, 1H), 2.90 (sept, 1H, J= 6.8 Hz), 2.78 (d, 2H, J=
12.0 Hz), 2.30 (m, 7H), 2.11 (m, 5H), 1.76 (t, 2H, J= 11.8 Hz), 1.69 (d, 2H, J= 11.8 Hz), 1.22
(m, 7H). 13C NMR (400 MHz, DMSO-d6): δ ppm= 148.7, 143.6, 130.9, 128.5, 127.6, 125.8,
119.7, 111.6, 66.9, 56.6, 56.2, 55.8, 53.2, 47.5, 33.8, 30.1, 23.5, 11.0. MS-ESI: m/z 396 (M +
H+).
4.1.3.34
Tert-butyl-4-(5-(4-isopropylphenyl)-2-methyl-3-(morpholinomethyl)-1H-
pyrrol-1-yl)piperidine-1-carboxylate (34). Colourless oil (yield 40%). 1H NMR (400 MHz,
DMSO-d6): δ ppm= 7.24 ( d, 2H, J= 8.1 Hz), 7.18 ( d, 2H, J= 8.1 Hz), 5.82 (s, 1H), 4.16 (tt,
1H, J= 12.0, 4.0 Hz), 3.99 (m, 2H), 3.52 (m, 4H), 3.21 (s broad, 2H), 2.89 (sept, 1H, J= 7.1
Hz), 2.66 (m, 2H), 2.32 (m, 4H), 2.25 (s, 3H), 1.85 (m, 2H), 1.72 (m, 2H), 1.36 (m, 9H), 1.21
13
(d, 6H, J= 7.1 Hz). C NMR (400 MHz, CDCl3): δ ppm= 160.4,149.0, 144.2, 130.8, 128.8,
127.5, 126.1, 119.9, 111.8, 80.3, 67.2, 56.7, 56.3, 55.9, 45.4, 33.5, 30.8, 27.3, 24.1, 11.2.
MS-ESI: m/z 482 (M + H+).
4.1.3.35 Tert-butyl-(S)-3-(5-(4-isopropylphenyl)-2-methyl-3-(morpholinomethyl)-1H-
1
pyrrol-1-yl)piperidine-1-carboxylate (35). Yellow oil (yield 72%). H NMR (400 MHz,
CDCl3): δ ppm= 7.23 (m, 4H), 6.02 (s, 1H), 4.11 (m, 3H), 3.71 (t, 4H, J= 4.2 Hz), 3.36 (s,
2H), 3.18 (m, 1H), 2.93 (sept, 1H, J= 7.0 Hz), 2.56 (t, 1H, J= 12.0 Hz), 2.47 (s broad, 4H),
2.37 (s, 3H), 2.07-1.93 (m, 3H), 1.71 (d, 1H, J= 12.0 Hz), 1.40 (s, 9H), 1.27 (d, 6H, J= 7.0
Hz). 13C NMR (400 MHz, CDCl3): δ ppm= 154.8, 148.8, 143.7, 130.5, 128.6, 127.9, 125.4,
119.8, 110.9, 80.1, 66.7, 58.5, 56.4, 55.5, 52.8, 49.3, 33.4, 31.5, 29.6, 23.5, 22.0, 10.9. MS-
ESI: m/z 482 (M + H+).
4.1.3.36
4-((5-(4-isopropylphenyl)-2-methyl-1-(piperidin-3-yl)-1H-pyrrol-3-
yl)methyl)morpholine (36) Yellow solid (yield 28%). 1H NMR (400 MHz, CDCl3): δ ppm=
7.22 (m, 4H), 5.99 (s, 1H), 4.18 (m, 1H), 3.73 (m, 4H), 3.41 (s, 2H), 3.10 (m, 2H), 2.96 (m
3H), 2.52 (s broad, 4H), 2.39 (s, 3H), 2.09-20.2 (m, 2H), 1.68 (m broad, 1H), 1.47 (m, 2H),
13
1.28 (d, 6H, J= 7.1 Hz). C NMR (400 MHz, CDCl3): δ ppm= 148.5, 143.12, 132.6, 128.7,
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127.8, 126.6, 118.5, 111.7, 66.9, 61.2, 55.9, 54.8, 49.8, 48.7, 33.8, 30.12, 23.5, 22.8, 10.8.
MS-ESI: m/z 382 (M + H+).
4.1.3.37 4-((5-(4-isopropylphenyl)-2-methyl-1-(1-methylpiperidin-3-yl)-1H-pyrrol-3-
1
yl)methyl)morpholine (37) White solid (yield 42%). H NMR (400 MHz, CDCl3): δ ppm=
7.20 (m, 4H), 5.99 (s, 1H), 4.35 (m, 1H), 3.71 (m, 4H), 3.36 (s, 2H), 2.88 (m, 2H), 2.79 (m
1H), 2.47 (s broad, 4H), 2.39 (s, 3H), 2.26 (s, 3H), 1.91-1.62 (m, 6H), 1.28 (d, 6H, J= 7.1
Hz). 13C NMR (400 MHz, CDCl3): δ ppm= 148.5, 142.12, 132.6, 128.7, 126.8, 125.6, 118.5,
111.7, 65.9, 61.2, 55.9, 54.8, 49.8, 48.7, 33.8, 30.12, 22.96, 23.5, 22.8, 10.8. MS-ESI: m/z
396 (M + H+).
4.1.3.38
4-((5-(4-Chlorophenyl)-1-isopropyl-2-methyl-1H-pyrrol-3-
yl)methyl)morpholine (38) Yellow solid (yield 12%). 1H NMR (400 MHz, CDCl3): δ ppm=
7.34 (m, 2H), 7.24 (m, 2H), 6.00 (s, 1H), 4.47 (sept, 1H, J= 7.1 Hz), 3.73 (m, 4H), 3.38 (s
broad, 2H), 2.49 (m, 4H), 2.37 (s, 3H), 1.43 (d, 6H, J= 7.1 Hz). 13C NMR (400 MHz,
CDCl3): δ ppm= 143.0, 133.6, 129.8, 127.8, 127.5, 126.6, 118.5, 110.7, 66.0, 55.8, 54.9, 49.7,
22.8, 10.8. MS-ESI: m/z 333 (M + H+).
4.1.3.39
4-((1-Isopropyl-5-(4-methoxyphenyl)-2-methyl-1H-pyrrol-3-
1
yl)methyl)morpholine (39) White solid (yield 75%). H NMR (400 MHz, DMSO-d6): δ
ppm= 7.17 (d, 2H, J= 8.6 Hz), 6.94 (d, 2H, J= 8.6 Hz), 5.75 (s, 1H), 4.36 (sept, 1H, J= 7.1
Hz), 3.76 (s, 3H), 3.52 (t, 4H, J= 4.0 Hz), 3.20 (s, 2H), 2.31 (s broad, 4H), 2.28 (s, 3H), 1.32
(d, 6H, J= 7.1 Hz). 13C NMR (400 MHz, DMSO-d6): δ ppm= 161.2, 144.5, 128.7, 127.8,
126.0, 120.1, 115.3, 112.0, 67.1, 56.8, 56.0, 55.3, 51.6, 23.5, 10.9. MS-ESI: m/z 329 (M +
H+).
4.1.3.40
4-((1-Isopropyl-2-methyl-5-(4-(trifluoromethyl)phenyl)-1H-pyrrol-3-
1
yl)methyl)morpholine (40) Colourless oil (yield 62%). H NMR (400 MHz, CDCl3): δ
ppm= 7.61 (d, 2H, J= 7.5 Hz), 7.41 (d, 2H, J= 7.5 Hz), 6.08 (s, 1H), 4.50 (sept, 1H, J= 6.8
Hz), 3.72 (s broad, 4H), 3.36 (s, 2H), 2.47 (s broad, 4H), 2.38 (s, 3H), 1.45 (d, 6H, J= 6.8
13
Hz). C NMR (400 MHz, CDCl3): δ ppm= 142.6, 135.9, 129.2, 126.6, 125.4, 124.8, 123.2,
119.3, 111.5, 65.9, 55.8, 54.7, 49.6, 22.8, 10.7. MS-ESI: m/z 367 (M + H+).
4.1.3.41
4-((1-Cyclohexyl-2-methyl-5-(4-(trifluoromethyl)phenyl)-1H-pyrrol-3-
1
yl)methyl)morpholine (41) Colourless oil (yield 56%). H NMR (400 MHz, CDCl3): δ
ppm= 7.60 (d, 2H, J= 8.1 Hz), 7.39 (d, 2H, J= 8.1 Hz), 6.08 (s, 1H), 4.02 (tt, 1H, J= 12.0 Hz,
3.8 Hz), 3.72 (t, 4H, J= 3.8 Hz), 3.37 (s broad, 2H), 2.47 (m, 4H), 2.40 (s, 3H), 1.98-1.82 (m,
6H), 1.68 (d, 1H, J= 11.4 Hz) 1.29-1.17 (m, 3H). 13C NMR (400 MHz, CDCl3): δ ppm=
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138.2, 131.6, 127.9, 127.0, 126.3, 124.1, 120.5, 112.4, 66.9, 64.6, 56.6, 55.8, 35.2, 26.1, 25.3,
11.0. MS-ESI: m/z 407 (M + H+).
4.1.3.42
4-((1-Cyclobutyl-2-methyl-5-(4-(trifluoromethyl)phenyl)-1H-pyrrol-3-
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yl)methyl)morpholine (42) Colourless oil (yield 26%). H NMR (400 MHz, CDCl3): δ
ppm= 7.60 (d, 2H, J= 8.5 Hz), 7.40 (d, 2H, J= 8.5 Hz), 6.06 (s, 1H), 4.79 (quint, 1H, J= 8.7
Hz ), 3.71 (t, 4H, J= 4.0 Hz), 3.37 (s, 2H), 2.48 (m, 4H), 2.41 (s, 3H), 2.38-2.28 (m, 4H), 1.73
(m, 2H). 13C NMR (400 MHz, CDCl3): δ ppm= 145.0, 137.4, 132.3, 128.2, 126.8, 125.7,
124.3, 120.6, 112.5, 66.9, 56.8, 55.4, 54.0, 31.8, 16.3, 10.9. MS-ESI: m/z 379 (M + H+).
4.1.3.43
1-((1-(4-Fluorophenyl)-5-(4-isopropylphenyl)-2-methyl-1H-pyrrol-3-
1
yl)methyl)piperidin-3-ol (43) Colourless oil (yield 15%). H NMR (400 MHz, CDCl3): δ
ppm= 7.14 (m, 2H), 7.05 (m, 2H), 6.99 (d, 2H, J= 7.1 Hz), 6.95 (d, 2H, J= 7.1 Hz), 6.28 (s,
1H), 3.85 (s broad, 1H), 3.45 (s, 2H), 2.80 (sept, 1H, J= 6.8 Hz), 2.62 (m, 2H), 2.49 (s broad,
1H), 2.28 (m, 1H), 2.05 (s, 3H), 1.83-1.57 (m, 5H), 1.18 (d, 6H, J= 6.8 Hz). 13C NMR (400
MHz, CDCl3): δ ppm= 158.7, 147.2, 145.9, 136.3, 133.1, 127.8, 124.6, 124.2, 121.4, 120.7,
115.4, 112.8, 66.4, 63.8, 55.9, 54.8, 33.0, 29.3, 20.2, 10.1. MS-ESI: m/z 407 (M + H+).
4.1.3.44
1-((1-Isopropyl-5-(4-isopropylphenyl)-2-methyl-1H-pyrrol-3-
1
yl)methyl)piperidin-3-ol (44). Colourless oil (yield 15%). H NMR (400 MHz, CDCl3): δ
ppm= 7.23 (s, 4H), 5.95 (s, 1H), 4.54 (sept, 1H, J= 6.8 Hz), 3.91 (s broad, 1H), 3.52 (s, 2H),
2.94 (sept, 1H, J= 6.8 Hz), 2.73 (m, 2H), 2.43 (s, 3H), 2.37 (s, 3H), 1.93 (s broad, 1H) 1.60
(s broad, 3H), 1.43 (d, 6H, J= 6.8 Hz), 1.28 (d, 6H, J= 6.8 Hz). 13C NMR (400 MHz, CDCl3):
δ ppm= 147.9, 142.5, 129.3, 127.4, 126.8, 124.7, 118.5, 110.6, 65.7, 63.6, 56.3, 55.8, 49.4,
32.5, 31.9, 23.3, 23.0, 19.9, 10.7. MS-ESI: m/z 355 (M + H+).
4.1.3.45
1-((1-Isopropyl-5-(4-isopropylphenyl)-2-methyl-1H-pyrrol-3-yl)methyl)-3-
1
methoxypiperidine (45). Colourless oil (yield 18%). H NMR (400 MHz, CDCl3): δ ppm=
7.23 (m, 4H), 5.98 (s, 1H), 4.54 (sept, 1H, J= 7.1 Hz), 3.43 (s, 2H), 3.35 (s, 3H), 3.32 (m,
1H), 3.05 (m, 1H), 2.93 (sept, 1H, J= 7.1 Hz), 2.74 (m, 1H), 2.36 (s, 3H), 1.98-1.74 (m, 3H),
13
1.71 (m, 2H), 1.50 (m, 1H), 1.44 (dd, 6H, J= 7.1, 1.8 Hz Hz), 1.28 (d, 6H, J= 7.1 Hz). C
NMR (400 MHz, CDCl3): δ ppm= 148.1, 142.9, 129.8, 128.0, 127.2, 125.3, 119.0, 110.9,
76.4, 61.2, 57.2, 56.3, 55.8, 50.1, 33.0, 28.9, 23.0, 22.8, 21.3, 10.7. MS-ESI: m/z 369 (M +
H+).
4.1.4 Preparation of compound 36.
To a stirred solution of 35 in dichloromethane (2.5 ml), TFA (2.5 ml) was added and the
reaction mixture was stirred at room temperature for 1.5 h. After solvent evaporation, sat.
NaHCO3 was added (2.5 ml), the mixture was extracted with dichloromethane, washed with
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brine and dried under MgSO4. After solvent evaporation, 36 was obtained in satisfactory
yield without further purification.
4.1.4.1
(S)-4-((5-(4-isopropylphenyl)-2-methyl-1-(piperidin-3-yl)-1H-pyrrol-3-
1
yl)methyl)morpholine (36). Yellow syrup (yield 72%). H NMR (400 MHz, CDCl3): δ
ppm= 7.22 (m, 4H), 5.99 (s, 1H), 4.18 (tt, 1H, J= 12.0, 4.0 Hz), 3.73 (t, 4H, J= 4.0 Hz), 3.41
(s broad, 2H), 2.97-3.17 (m, 5H), 2.52 (s broad, 4H), 2.39 (s, 3H), 2.06 (m, 2H), 1.70 (s
13
broad, 1H), 1.47 (m, 2H), 1.28 (d, 6H, J= 6.8 Hz). C NMR (400 MHz, CDCl3): δ ppm=
147.8, 142.5, 129.6, 127.7, 126.8, 124.9, 118.1, 108.6, 66.0, 60.1, 55.4, 54.0, 50.4, 46.8, 32.8,
3.7, 23.3, 20.9, 13.2. MS-ESI: m/z 382 (M + H+).
4.1.5 Preparation of compound 37.
To a stirred solution of 36 (0.038 mmol) in dichloroethane (5ml), formaldehyde (0.042
mmol) and a drop of acetic acid, sodium (triacetoxy)borohydride was added in one portion at
0° C. The mixture was stirred at room temperature for 15 hours. The reaction was quenched
with NaOH 2N (10 ml), extracted with ethyl acetate, washed with brine and dried over
MgSO4. After solvent evaporation, 37 was obtained in satisfactory yield without further
purification.
4.1.5.1 (S)-4-((5-(4-Isopropylphenyl)-2-methyl-1-(1-methylpiperidin-3-yl)-1H-pyrrol-
1
3-yl)methyl)morpholine (37). Yellow syrup (yield 67%). H NMR (400 MHz, CDCl3): δ
ppm= 7.20 (m, 4H), 5.99 (s, 1H), 4.35 (m, 1H), 3.71 (m, 4H), 3.36 (s, 2H), 2.91 (m, 3H), 2.88
(m, 1H), 2.47 (s broad, 4H), 2.38 (s, 3H), 2.26 (s, 3H), 1.91-1.73 (m, 5H), 1.29 (d, 6H, J= 6.8
Hz ). 13C NMR (400 MHz, CDCl3): δ ppm= 148.0, 142.7, 129.8, 127.6, 126.9, 125.0, 118.9,
110.7, 66.7, 63.2, 6.4, 57.2, 55.8, 55.0, 48.3, 33.2, 30.8, 23.3, 20.9, 10.7. MS-ESI: m/z 396
(M + H+).
4.1.6 Declaration of Purity. All assayed final compounds (1-45) were more than 95% pure
by UPLC-MS analysis (see Supporting Information).
4.2 Biological assays
4.2.1 Materials and methods
The human biological samples were sourced ethically and their research use was according
to the terms of the informed consent. All animal studies were ethically reviewed and carried
out in accordance with European Directive 2010/63/EU and the GSK Policy on the Care,
Welfare, and Treatment of Animals. This research was approved by the Comité Etico de
Experimentacion Animal de GSK R&D, protocolos number: PROEX 63/14 and PROEX
71/14
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4.2.2 MIC determination
The measurement of the minimum inhibitory concentration (MIC) against M. tuberculosis
strains for each tested compound was performed in 96-well flat-bottom, polystyrene
microtiter plates in a final volume of 100 ml. Ten two-fold drug dilutions in neat DMSO
starting at 50 mM were performed. Drug solutions were added to Middlebrook 7H9 medium
(Difco) and isoniazid (Sigma Aldrich) was used as a positive control with two-fold dilutions
of isoniazid starting at 160 mg/ml. The inoculum was standardized to approximately 16e7
cfu/ml and diluted 1 in 100 in Middlebrook 7H9 broth (Difco). This inoculum (100 ml) was
added to the entire plate but G-12 and H-12 wells were used as blank controls. All plates
were placed in a sealed box to prevent drying out of the peripheral wells and incubated at 37
°C without shaking for six days. A Resazurin solution was prepared by dissolving one tablet
of resazurin (Resazurin Tablets for Milk Testing; Ref 330884Y’ VWR International Ltd) in
30 ml of sterile PBS (phosphate buffered saline). Of this solution, 25 ml were added to each
well. Fluorescence was measured (Spectramax M5 Molecular Devices, Excitation 530 nm,
Emission 590 nm) after 48 hours to determine the MIC value.
4.2.3 In vivo assay.
Specific pathogen-free, 8-10 week-old female C57BL/6 mice were purchased from Harlan
Laboratories and were allowed to acclimate for one week. In brief, mice were intratracheally
infected with 100.000 CFU/mouse (M. tuberculosis H37Rv strain). Products were
administered for 4 consecutive days starting on day five after infection. Lungs were harvested
on day nine (24 hours after the last administration). All lung lobes were aseptically removed,
homogenized and frozen. Homogenates were plated in 10% OADC-7H11 medium for 14
days at 37 ºC.
4.2.4 Docking.
The proposed docking simulations are based on the homology model for the MmpL3 channel
previously generated [15]. Briefly, the homology model was developed by an iterative
approach which analyzed different suitable templates to finally select the model based on the
Multidrug Exporter MEXB (PDB ID 2V50) as the template. The model was then refined and
validated by combining molecular docking simulations as reported elsewhere [15]. The
considered compounds were simulated in both neutral and ionized forms. Indeed, the weak
basicity of the morpholine ring should favor the neutral form at physiological pH although
the protonated state cannot be completely excluded depending on the micro-environment of
the MmpL3 binding site. In detail, the conformational profile of the simulated compounds
was explored by MonteCarlo simulations, which generated 1000 conformers by randomly
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rotating the rotors. The so obtained lowest energy geometry was then optimized by PM7
semi-empirical calculations and underwent docking simulations by using PLANTS [17]. In
detail, docking simulations were focused on a 10 Å radius sphere around the barycenter of the
two interacting residues Agr259-Asp640. For each docking run, 10 poses were generated and
scored by using the ChemPLP function with speed equal to 1. All computed complexes were
then minimized by keeping fixed all atoms outside a 10 Å radius sphere around a bound
inhibitor and finally used to recalculate the scoring functions by using the ReScore+ tool as
implemented in the VEGA suite of programs [18]. The so minimized and rescored complexes
were then visually inspected in order to extract the best complexes for the two possible
binding modes as above detailed. All mentioned minimizations were performed using the
conjugate gradient algorithm until the r.m.s. gradient was smaller than 0.01 kcal mol-1 Å-1.
All calculations were carried out by Namd2.10 with the force-field CHARMm v22 and the
Gasteiger’s atomic charges.
Supplementary data
Additional protocols, synthesis, and experimental properties of intermediate compounds.
Acknowledgment
We gratefully acknowledge María Teresa Fraile for formulation, Ana Álvarez for physical
chemical evaluations, Sophie Huss and Ángel Santos for in vitro DMPK studies.
This work was supported by the Tres Cantos Open Lab Foundation [grant number TC028].
References
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Fig. 1. Chemical structure of BM635 and design of compounds 1-45.
Fig. 2. Chemical structures of compounds 1-37.
Fig. 3. Chemical structures of compounds 38-45.
Fig. 4. (A) Key interactions stabilizing the computed complexes between the MmpL3
homology model (based on PDB ID 2V50 as reported in ref. 15) and BM212 in its protonated
state as a reference ligand; (B) 23 in its neutral form assuming the proposed binding mode 1;
(C) 13 in its neutral form assuming the proposed binding mode 2; (D) 21 in its protonated
state assuming the proposed binding mode 1. Notice that usually the alkyl side-chains are not
shown for clarity.
Scheme 1. Synthetic pathway for compounds 1-35, 38-45.
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Scheme 2. Synthetic pathway for compounds 36 and 37.
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