Identification of SQ609 as a lead compound from a library of dipiperidines

Article abstract of DOI:10.1016/j.bmcl.2011.07.015

We recently reported that compounds created around a dipiperidine scaffold demonstrated activity against Mycobacterium tuberculosis (Mtb) (Bogatcheva, E.; Hanrahan, C.; Chen, P.; Gearhart, J.; Sacksteder, K.; Einck, L.; Nacy, C.; Protopopova, M. Bioorg. Med. Chem. Lett. 2010, 20, 201). To optimize the dipiperidine compound series and to select a lead compound to advance into preclinical studies, we evaluated the structure-activity relationship (SAR) of our proprietary libraries. The (piperidin-4-ylmethyl)piperidine scaffold was an essential structural element required for antibacterial activity. Based on SAR, we synthesized a focused library of 313 new dipiperidines to delineate additional structural features responsible for antitubercular activity. Thirty new active compounds with MIC 10-20 μg/ml on Mtb were identified, but none was better than the original hits of this series, SQ609, SQ614, and SQ615. In Mtb-infected macrophages in vitro, SQ609 and SQ614 inhibited more than 90% of intracellular bacterial growth at 4 μg/ml; SQ615 was toxic to these cells. In mice infected with Mtb, weight loss was completely prevented by SQ609, but not SQ614, and SQ609 had a prolonged therapeutic effect, extended by 10-15 days, after cessation of therapy. Based on in vitro and in vivo antitubercular activity, SQ609 was identified as the best-in-class dipiperidine compound in the series.

Full text of DOI:10.1016/j.bmcl.2011.07.015

Bioorganic & Medicinal Chemistry Letters 21 (2011) 5353–5357
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Identification of SQ609 as a lead compound from a library of dipiperidines
Elena Bogatcheva a, , Colleen Hanrahan b, Boris Nikonenko a, Gladys de los Santos e, Venkata Reddy a,
⇑
Ping Chen c, Francis Barbosa d, Leo Einck a, Carol Nacy a, Marina Protopopova a
a Sequella, Inc., 9610 Medical center Drive, Suite 200, Rockville, MD 20850, USA
b John Hopkins School of Medicine & John Hopkins School of Public Health, Baltimore, MD, USA
c National Institute of Health (NIH), NIAID, 6610 Rockledge, Dr. Bethesda, MD, USA
d Sibley Hospital, 5255 Loughboro Road NW, Washington, DC 20016, USA
e Centro De Investigacion En Quimica Aplicada, Blvd. Enrique Reyna Hermosillo No.140, Saltillo, Coahuila 25250, Mexico
a r t i c l e i n f o
a b s t r a c t
Article history:
We recently reported that compounds created around a dipiperidine scaffold demonstrated activity
against Mycobacterium tuberculosis (Mtb) (Bogatcheva, E.; Hanrahan, C.; Chen, P.; Gearhart, J.; Sacksteder,
K.; Einck, L.; Nacy, C.; Protopopova, M. Bioorg. Med. Chem. Lett. 2010, 20, 201). To optimize the dipiperi-
dine compound series and to select a lead compound to advance into preclinical studies, we evaluated the
structure–activity relationship (SAR) of our proprietary libraries. The (piperidin-4-ylmethyl)piperidine
scaffold was an essential structural element required for antibacterial activity. Based on SAR, we synthe-
sized a focused library of 313 new dipiperidines to delineate additional structural features responsible for
Received 8 April 2011
Revised 29 June 2011
Accepted 6 July 2011
Available online 14 July 2011
Keywords:
Dipiperidine scaffold
(Piperidin-4-ylmethyl)piperidine
Tuberculosis
antitubercular activity. Thirty new active compounds with MIC 10–20
none was better than the original hits of this series, SQ609, SQ614, and SQ615. In Mtb-infected macro-
phages in vitro, SQ609 and SQ614 inhibited more than 90% of intracellular bacterial growth at 4 g/
lg/ml on Mtb were identified, but
l
Combinatorial library
ml; SQ615 was toxic to these cells. In mice infected with Mtb, weight loss was completely prevented
by SQ609, but not SQ614, and SQ609 had a prolonged therapeutic effect, extended by 10–15 days, after
cessation of therapy. Based on in vitro and in vivo antitubercular activity, SQ609 was identified as the
best-in-class dipiperidine compound in the series.
Ó 2011 Elsevier Ltd. All rights reserved.
Tuberculosis (TB), although a curable disease, remains a serious
health problem worldwide. Deadly synergy with HIV infection and
spread of multidrug-resistant (MDR) and extensively drug-resistant
(XDR) TB make it the leading cause of death due to a single etiolog-
ical agent.1 Current TB treatment takes more than 6 months and re-
quires a multidrug regimen. Moreover, existing antitubercular drugs
are not always compatiblewith common antiretrovirals and are use-
less against MDR and XDR TB. New agents are needed to more effec-
tively combat the disease in a shorter therapeutic time.2,3
We recently reported the synthesis of combinatorial libraries
containing a variety of amines, prepared on solid support or in
solution phase.4–6 Antimicrobial potency of amine derivatives has
been shown previously.7 In our research two initial criteria were
used to select active compounds: Minimal Inhibitory Concentra-
tion (MIC) against Mycobacterium tuberculosis (Mtb) H37Rv strain
and activity against a Mtb recombinant strain containing a pro-
moter fused to firefly luciferase (Luc assay). The promoter is acti-
vated and produces light in response to inhibition of cell wall
biosynthesis.8,9 The combination of these 2 assays allowed us to
identify compounds that inhibited bacterial growth and also af-
fected a process involved in Mtb cell wall biosynthesis. Several
new scaffolds were identified, including dipiperidines.5,6 Represen-
tatives of the dipiperidine series induced a strong response in the
Luc assay and demonstrated MIC in the range of 4.0–32.0 lg/ml,
acceptable cytotoxicity, and c Log P values within the range 1.91–
3.95, suggesting good absorption after oral administration. In order
to optimize this lead series, we performed extensive analyses of
the structure–activity relationship (SAR) and identified the struc-
tural requirements for the dipiperidine scaffold to maintain anti-
mycobacterial activity. Here we report the SAR evaluation of
piperidine and dipiperidine based compounds, which led to the
identification of SQ609 as a lead compound.
From our proprietary libraries, hits containing piperidine or dip-
iperidine fragments were grouped based on structural similarity
(Fig. 1) and SAR was studied within and between the groups. Series
I (9 hits) represent dipiperidines reported earlier,7 which contain
(piperidin-4-ylmethyl)piperidine, a scaffold that is represented by
two piperidine moieties connected head-to-tail through a methy-
lene bridge. To define structural features responsible for antimicro-
bial activity in this scaffold, we examined a) the size of the
heterocycles; b) replacement of one of piperidine moiety with a sec-
ondary amine bearing a variety of substituents; c) attachment of the
second amino component to the N1- or C4-position of the piperidine
⇑
Corresponding author.
E-mail address: elenabogatcheva@sequella.com (E. Bogatcheva).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.07.015

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E. Bogatcheva et al. / Bioorg. Med. Chem. Lett. 21 (2011) 5353–5357
Series I
Series II
N
NR
HO
N
N
R
HO
N
NR
n
n=0,1,2,3
8 hits
11 hits
9 hits, all active in Luc assay
no activity in Luc assay
R1
HO
no activity in Luc asay
Representatives:
SQ609: R = 1-adamantylmethyl
SQ611: R = 2-adamantyl
N
N
N
HO
SQ612: R = 2-chlorobenzyl
N
R
SQ613: R = 2-decahydronaphthyl
SQ614: R = 3-(4-hydroxyphenyl)-1-methylpropyl
SQ615: R = 2,2-diphenylethyl
SQ616: R = 3-phenylpropyl
R
17 hits
No hits
no activity in Luc assay
Series IV
Series III
N
N
R
RN
n=1,2,3,4
11 hits
no activity in Luc assay
N
R
R
N
N
N
R
n
N
HO
No hits
OH
N
No hits
R
H
No hits
N
R
N
N R
HO
1 hit
no activity in Luc assay
No hits
Figure 1. General structure of compound series containing piperidine or dipiperidine motif. R is represented by alkyl, cycloalkyl, aryl, or heteroaryl; R1 is represented by alkyl,
aryl, or heteroaryl.
ring; and d) length and flexibility of the linkage connecting two
nitrogen atoms (Fig. 1). We found that size of the heterocycle is
one of the key elements for maintaining antitubercular activity in
this series: substitution of one or both piperidine moieties for its
homolog pyrrolidine led to complete loss of activity. As shown in
Figure 1, compounds derived from 1-(pyrrolidin-2-ylmethyl)piper-
idine-4-ol, (Series II), [1-(piperidin-4-ylmethyl)pyrrolidin-2-yl)]
meth anol (Series III), and [1-(pyrrolidin-2-ylmethyl)pyrrolidin-2-
pound (Fig. 2). Variations in structure were made primarily to
expand SAR on the amine component and to optimize the length
of the bridge between the two piperidine rings. We replaced the 4-
hydroxypiperidine fragment that was present in original hits with
a variety of amines of different shapes and sizes. For the library syn-
thesis, we selected 16 cyclic amines bearing various substituents
and/or heteroatoms, such as 4-benzylpiperidine, 4-methylpiperi-
dine, 2- and 3-(hydroxymethyl)piperidine, morpholine, or thi-
omorpholine (Fig. 2). 4-Hydroxypiperidine was also included in a
set of amines to allow formation of the previously identified active
compounds SQ609, SQ614, SQ615, and SQ616, which were used as
an indicator of successful library synthesis and as internal standards
in biological screening. N-Boc-4-piperidinylcarboxaldehyde was
used to connect 2 piperidine rings through a methylene bridge, as
in the original compounds, and N-Boc-piperidinyl-4-acetaldehyde
was used to determine if the extension from methylene to ethylene
linkage between piperidine moieties affected activity.
The library was prepared in a 96-well format following the
scheme outlined in Figure 2. We started from the synthesis of
(piperidin-4-ylmethyl)piperidine-based compounds (Fig. 1, Series
I). First, 16 commercially available cyclic secondary amines, di-
vided into 2 groups of 8 amines, were alkylated by N-Boc-4-pipe-
ridinylcarboxaldehyde using resin bound trimethylammonium
cyanoborohydride as a reducing reagent (Fig. 2, step a). Residual
starting secondary amines in reaction mixtures were successfully
removed by commercially available polymer-supported electro-
philic scavenger macroporous (MP)-anhydride (Fig. 2, step b). To
remove unreacted residue of N-Boc-piperidinylcarboxaldehyde,
we applied a ‘catch and release’ technique using a polymer-bound
p-toluenesulphonic acid (MP-TsOH). Reaction mixtures were
loaded on a MP-TsOH resin, where the amine intermediates were
retained on polymer by forming a salt with TsOH, while the non-
basic aldehyde was washed out with dichloromethane (DCM).
Amines were later released from TsOH by washing with a 2 M solu-
tion of ammonia in methanol (Fig. 2, step c). In the next step (Fig. 2,
step d), the Boc protective group was removed with 10% TFA in
DCM. Intermediates were alkylated (Fig. 2, step e) applying the
same procedure as in Figure 2, step a. For this step we used 12
carbonyl compounds: 7 aldehydes and 5 ketones. All carbonyl
compounds were commercially available except 1-adamantane
yl]methanol (Series IV) showed MIC >64
hits. Replacement of one of the piperidine fragments with a second-
ary amine produced hits with MIC ranging from 8 to 64 g/ml. How-
lg/ml and did not produce
l
ever, they demonstrated no response in the Luc assay (Fig. 1, Series II
and Series III), suggesting that the inhibition of bacterial growth may
have been due to another pathway not associated with cell wall bio-
synthesis. The position of the second amino component in the piper-
idine ring was important but not critical. The hit rate was more
favorable for compounds bearing a second amino component at-
tached at the N1-position of the piperidine ring (Fig. 1, Series I, II),
when compared to compounds with the second amino component
attached at the C4-position of the piperidine (Fig. 1, Series III). The
majority of hits in the Series I and II were produced when 4-hydroxy-
piperidine was used as the piperidine fragment, suggesting that this
synthon may play an important role in antimicrobial activity. The
optimal linkage between 2 piperidine rings was a methylene bridge
that provides some flexibility to the scaffold. Two piperidine rings
connected to each other head-to-tail or tail-to-tail directly, without
a methylene bridge (Fig. 1, Series IV), failed to produce hits: only one
active molecule was identified. We also evaluated the R substituent
at the nitrogen atom and found that bulky lipophilic substituents
such as adamantyl, naphthyl, and benzyloxybenzyl contributed
the most often to antitubercular activity.
Thus, only hits of Series I, the dipiperidines, showed activity in
the broth microdilution assay and the luciferase luminescence
(Luc) assay.
Based on SAR, we concluded that (piperidin-4-ylmethyl)piperi-
dine is an essential structural element required for antibacterial
activity. To delineate additional structural features associated with
activity, we prepared a focused library of dipiperidines. The targeted
molecules were synthesized with 3 building blocks: the amine com-
ponent, Boc-protected piperidinealdehyde, and the carbonyl com-

E. Bogatcheva et al. / Bioorg. Med. Chem. Lett. 21 (2011) 5353–5357
5355
R1
R2
R
X
R
X
R
X
e, f
a, b, c, d
+
N
N
N
NH
BocN
CHO
m
NH
m
m
n
n
n
n=0, 1, 2
m=0, 1
amino moiety
piperidine moiety
Carbonyls used in the library preparation
Amines used in the library preparation
CHO
CHO
NH
NH
NH
NH
O
O
O
X
N
R
Ph
X=CH2,
O, NR, S
R=H, Me,CH2Ph,
OH,CH2OH
OCH3
O
O
CHO
HO
NH
HO
NH
OC2H5
CHO
N
NH
Cl
NH
n
CHO
CHO
CHO
n=0,1,2
Figure 2. Synthesis of a focused library of dipiperidines. Reagents and conditions: (a) dichloroethane (DCE), (polystyrylmethyl)trimethylammonium cyanoborohydride (2
equiv), rt, 48 h; (b) DCE, MP-anhydride resin (2 equiv), rt, 18 h; (c) DCM, MP-TsOH (2 equiv), rt, 1 h, 2 M ammonia in MeOH; (d) 10% TFA/DCM, rt, overnight; (e) DCE,
(polystyrylmethyl)trimethylammonium cyanoborohydride (2 equiv), rt, 18 h; (f) DCM, MP-TsOH (2 equiv), rt, 1 h, 2 M ammonia in MeOH.
Original compounds
HO
N
N
HO
N
N
HO
N
N
OH
SQ609, MIC 4 µg/ml
SQ614, MIC 4 µg/ml
SQ615, MIC 4 µg/ml
New hits from focused library
N
N
N
N
HO
N
N
24-017-1-A2, MIC 10 µg/ml
24-017-1-A3, MIC 5 µg/ml
24-044-6, MIC 15 µg/ml
HO
N
N
N
N
HO
N
N
24-017-1-A5, MIC 20 µg/ml
24-044-2, MIC 14 µg/ml
24-044-15, MIC 8 µg/ml
HO
N
N
N
N
N
N
O
HO
24-017-1-B9, MIC 40 µg/ml
24-017-1-A9, MIC 11 µg/ml
24-044-17, MIC 8 µg/ml
Figure 3. Representative chemical compounds from the focused library of dipiperidines.
carboxaldehyde and 1-adamantane acetaldehyde, which were syn-
thesized from corresponding alcohols by Swern oxidation.10 The
synthetic procedure is available as Supplementary data. After com-
pletion of this step, the ‘catch and release’ procedure was repeated
to remove the excess of carbonyl compounds (Fig. 2, step f) and the
products were collected. Library synthesis was monitored by posi-
tive ESI mass spectrometry. The same procedure was applied for
preparation of 1-[2-(piperidin-4-yl)ethyl]piperidine-based com-
pounds, dipiperidines with an ethylene linkage between 2 piperi-
dine rings, using N-Boc-piperidinyl-4-acetaldehyde. A complete
list of amines and carbonyl compounds used for library prepara-
tion can be found in Supplementary data. MS data confirmed the
formation of 313 1,4-methylene- and 1,4-ethylenedipiperidines
out of 384 targeted compounds.
Table 1
Activity against intracellular Mtb in J774 macrophages
Drug
Concentration (
lg/ml)
Mean RLU* on day
7
0
Experiment 1
SQ609
INH
Control
Experiment 2
SQ614
4
514
514
514
354
64
3543
0.125
0
4
255
255
255
108
4
1856
INH
Control
0.125
0
*
Relative light units.

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E. Bogatcheva et al. / Bioorg. Med. Chem. Lett. 21 (2011) 5353–5357
27
27
25
23
21
19
17
15
Non-infected
Placebo
Non-infected
25
Placebo
SQ614
23
SQ609
SQ615
21
19
17
15
0
5
10
15
20
25
0
5
10
15
20
25
Time after infection (days)
Time after infection (days)
Figure 4. Dynamics of Mtb-infected weight of mice during treatment with SQ609, SQ614, and SQ615. All drugs were administrated by gavage daily at 10 mg/kg.
The synthesized compounds were then evaluated in 2 estab-
lished biological assays used in our previous work: a broth micro-
dilution assay against Mtb strain H37Rv to determine MIC, and a
high-throughput Luc assay.5–7
Serial dilutions for the in vitro screening were made with the
assumption that compounds were formed with a 100% theoretical
yield. Two antitubercular drugs with cell-wall activity, isoniazid
(INH) and ethambutol (EMB), and internal reference standards
(original hits SQ609, SQ614, SQ615, SQ616, Fig. 1) prepared by the
100
80
60
40
20
0
Placebo
SQ609
RIF
samemethods, wereusedascontrols. Therangeof25–50
lM (which
corresponds to approximately 15–20 g/ml) was used as a cutoff for
l
Chemotherapy
hit identification in the broth microdilution assay, while in the Luc
assay a compound was considered active if its response exceeded
the background level of luminescence by P2Â. Thirty new com-
0
10
20
30
40
50
pounds with MIC 10–20 lg/ml were identified (Fig. 3). The most ac-
Time after infection (days)
tive new hits were derived when 4-benzylpiperidine (10 hits) or
3-(hydroxymethyl)piperidine (4 hits) was used as the amino com-
ponent (Fig. 2, step a). Interestingly, the 3-(hydroxymethyl)piperi-
dine derivatives showed greater response in the Luc assay,
suggesting that the presence of the hydroxyl group is important
for inducing the Rv0341 promoter. Unsubstituted or alkyl substi-
tuted piperidine, morpholine, thiomorpholine, substituted pipera-
zine, and 1,2,3,4-tetrahydroisoquinoline did not produce hits. The
following carbonyl compounds contributed most often to antituber-
cularactivity: 1-adamantanecarboxaldehyde(7 hits), its homologue
1-adamantaneacetaldehyde (11 hits), 4-(4-hydroxyphenyl)buta-
none-2 (3 hits), and diphenylacetaldehyde (2 hits). The high number
of hits containing the adamantane moiety is not surprising, as ada-
mantane is often a building block in drug discovery, likely due to
its lipophilicity and rigid structure.4–7 Since the (piperidin-4-
ylmethyl)piperidine scaffold is quite hydrophilic, we assumed that
the presence of the hydrophobic moiety increases lipophilicity and
improves bacterial cell wall permeability. The original hits SQ609,
SQ614, SQ615, and SQ616 were formed successfully, and their activ-
ity in both assays exceeded the activity of new hits. These data sug-
gest that neither structural variations in the amino component nor
extension in the methylene bridge between 2 piperidine moieties
significantly improved anti-Mtb activity in vitro. We also synthe-
sized structural isomers of SQ609, SQ614, and SQ615 where the
4-hydroxypiperidine at 1-position was connected to the 3-position
of the second piperidine ring using N-Boc-3-piperidinylcarboxalde-
hyde (1-(piperidin-3-ylmethyl)piperidine scaffold). This structural
arrangementdecreasedantibacterialactivity. Basedontheseresults,
the original hits SQ609, SQ614, and SQ615 were still considered the
most active compounds of the series.
Figure 5. Survival of mice after termination of 2 wk chemotherapy. Female C3H
mice were infected iv with 106 CFU M. tuberculosis H37Rv. From day 7 until day 20
following infection, mice were treated by gavage daily with SQ609 at 10 mg/kg, and
with RIF at 2 mg/kg. Control mice received placebo. After therapy termination,
mortality of mice was monitored.
fected with Mtb and exposed to each drug at its MIC for 4 days.
Then tissue culture medium was replaced with drug-free medium
and cultures were incubated for 3 more days. On day 0 and day 7,
MU in triplicate were lysed and the number of viable bacteria was
determined. INH was used as control. In this assay SQ609 and
SQ614 inhibited more than 90% of intracellular bacterial growth
at 4
l
g/ml (Table 1), while SQ615 at 4
aggregation.
The 3 compounds were also assessed for in vivo activity in a 20-
lg/ml was toxic and induced
MU
day Mtb-induced weight-loss mouse model that we use as a preli-
minary screen to identify lead antitubercular compounds.12 This
model predicts drug efficacy based on the ability of a compound
to prevent weight loss during Mtb infection (one of the signs of
TB severity) and correlates well with colony-forming units (CFU)
of Mtb in lungs and spleen. C3H/He mice (6 animals per group)
were inoculated intravenously (iv) with 106 CFU of virulent Mtb
H37Rv to develop rapid and progressive TB disease. Chemotherapy
was initiated 7 days after infection and continued for 2 wk. Dipi-
peridines were administered orally by gavage once daily at an
unoptimized dose of 10 mg/kg for 2 wk, and weights of mice in
all groups were monitored from day 0 through day 21. Uninfected
animals and infected untreated mice (placebo) were compared to
the test-drug treated infected animals. By day 10, the infected pla-
cebo control mice started to lose weight, and by day 20 mice in this
group lost more than 25% of their body weight (a sign of terminal
These 3 original hits were then tested for intracellular antimi-
crobial activity in Mtb-infected mouse macrophage J774A.1 cells
by methods described in detail elsewhere.11 Briefly, M
U were in-

E. Bogatcheva et al. / Bioorg. Med. Chem. Lett. 21 (2011) 5353–5357
5357
illness). SQ609 completely prevented weight loss in the Mtb-
References and notes
infected animals and prolonged the therapeutic effect following
drug withdrawal for another 10–15 days (Figs. 4 and 5). SQ615 also
demonstrated significant activity in this model (Fig. 4) by prevent-
ing rapid weight loss. SQ614 showed moderate activity in this
rapid screen (Fig. 4).
Insummary, we thoroughlyanalyzed the SAR ofdipiperidinecom-
pound series and evaluated leads in in vitro and in vivo assays. Based
on the in vivo data, SQ609 was identified as the best in class com-
pound and will be further investigated for its antitubercular activity.
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Acknowledgments
This work was supported by NIH NIAID UC1A149514 and
1R43AI060250. We would like to thank Mr. Gardner of the
Research Triangle Institute for acquiring mass spectra.
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Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2011.07.015.