Unraveling the structure-activity relationship of tomatidine, a steroid alkaloid with unique antibiotic properties against persistent forms of Staphylococcus aureus

Article abstract of DOI:10.1016/j.ejmech.2013.11.019

Staphylococcus aureus (S. aureus) is responsible for difficult-to-treat and relapsing infections and constitutes one of the most problematic pathogens due to its multiple resistances to clinically available antibiotics. Additionally, the ability of S. aureus to develop small-colony variants is associated with a reduced susceptibility to aminoglycoside antibiotics and in vivo persistence. We have recently demonstrated that tomatidine, a steroid alkaloid isolated from tomato plants, possesses anti-virulence activity against normal strains of S. aureus as well as the ability to potentiate the effect of aminoglycoside antibiotics. In addition, tomatidine has shown antibiotic activity against small-colony variants of S. aureus. We herein report the first study of the structure-activity relationship of tomatidine against S. aureus.

Full text of DOI:10.1016/j.ejmech.2013.11.019

European Journal of Medicinal Chemistry 80 (2014) 605e620
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Unraveling the structureeactivity relationship of tomatidine, a steroid
alkaloid with unique antibiotic properties against persistent forms of
Staphylococcus aureus
Félix Chagnon a, Isabelle Guay b, Marc-André Bonin a, Gabriel Mitchell b, Kamal Bouarab b,
,
François Malouin b, Éric Marsault a
*
a Institut de Pharmacologie de Sherbrooke, Département de pharmacologie, Université de Sherbrooke, 3001, 12e av nord, Sherbrooke,
Québec J1H 5N4, Canada
b Centre d’étude et de valorisation de la diversité microbienne (CEVDM), Département de biologie, Université de Sherbrooke, Sherbrooke,
Québec J1K 2R1, Canada
a r t i c l e i n f o
a b s t r a c t
Article history:
Staphylococcus aureus (S. aureus) is responsible for difficult-to-treat and relapsing infections and con-
stitutes one of the most problematic pathogens due to its multiple resistances to clinically available
antibiotics. Additionally, the ability of S. aureus to develop small-colony variants is associated with a
reduced susceptibility to aminoglycoside antibiotics and in vivo persistence. We have recently demon-
strated that tomatidine, a steroid alkaloid isolated from tomato plants, possesses anti-virulence activity
against normal strains of S. aureus as well as the ability to potentiate the effect of aminoglycoside an-
tibiotics. In addition, tomatidine has shown antibiotic activity against small-colony variants of S. aureus.
We herein report the first study of the structureeactivity relationship of tomatidine against S. aureus.
Ó 2013 Elsevier Masson SAS. All rights reserved.
Received 25 July 2013
Received in revised form
13 November 2013
Accepted 15 November 2013
Keywords:
Tomatidine
Structureeactivity relationship
Steroid alkaloid
Antibiotic
Staphylococcus aureus
Small colony variants
Aminoglycoside potentiation
1. Introduction
were discovered, including widely used classes such as the peni-
cillins, aminoglycosides and tetracyclines [3,5]. In the thirty years
Antibiotic resistance is becoming an alarming health problem
compounded by the lack of new therapeutic options to fight
resistant pathogens [1,2]. Since the first synthetic antibiotics were
introduced nearly 80 years ago, several scaffolds have become new
classes of antibiotics [3,4]. As an adaptive response to this arsenal,
pathogens have acquired efficient resistance mechanisms [5e7],
leading eventually to multi-resistant strains [3,8]. The development
of new antibacterial agents has not paralleled the development of
resistance [3,4,9], and drug approvals in the antibiotic field have
diminished by half in the past 20 years [10]. Additionally, there has
been a lack of new chemical scaffolds among new drugs. Indeed,
between 1930 and 1970, 20 different classes of antimicrobial agents
that followed, the discovery of new classes came to a standstill
[4,9]. As a result, most of the recently introduced antibiotics are still
based on scaffolds discovered or designed 60 years ago, which in-
creases the likelihood of resistance development [3,9].
Among highly resistant pathogens, Staphylococcus aureus
(S. aureus) has evolved methicillin-resistant (MRSA) and often
multi-resistant strains. Since 2002, we have witnessed the emer-
gence of strains resistant to vancomycin, which is generally
considered the last resort antibiotic for this pathogen [7,11]. MRSA
is responsible for many hospital- and community-acquired in-
fections and is often associated with recurring and difficult-to-treat
infections, which increase morbidity and mortality in both humans
and animals [5,12].
Among the mechanisms that allow S. aureus to cause resistance
and persistent infections and reduce its susceptibility to antibiotics,
this pathogen often presents itself in the form of respiratory-
deficient small-colony variants (SCVs) [13]. SCVs suffer from a
deficient electron transport chain and a weaker proton-motive
Abbreviations: S. aureus, Staphylococcus aureus; MRSA, methicillin-resistant
Staphylococcus aureus; SCV, small-colony variant; MIC, minimal inhibitory con-
centration; SAR, structureeactivity relationship.
* Corresponding author.
E-mail address: eric.marsault@usherbrooke.ca (É. Marsault).
http://dx.doi.org/10.1016/j.ejmech.2013.11.019
0223-5234/Ó 2013 Elsevier Masson SAS. All rights reserved.

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F. Chagnon et al. / European Journal of Medicinal Chemistry 80 (2014) 605e620
by hydrolysis of its glycosylated analog tomatine 2 [26]. The
structure of tomatidine was elucidated via a combination of
infrared spectroscopy, elemental analysis and X-ray crystallog-
raphy [13,27]. In the absence of an identified cellular target, our
initial objective was to elucidate SAR, based on the initial hy-
pothesis that the steroidal AeD rings act as a rigid scaffold to
orient pharmacophores defined by the 3b-hydroxyl group on ring
A and the spiroaminoketal group on rings E, F. Accordingly, our
attention focused on the two extremities of the molecule. An
important question from the outset was whether the closed or the
open form of the aminoketal is responsible for biological activity.
Indeed, the aminoketal can be opened by hydrolysis to reveal a
hydroxyl function and a tethered amino and keto functional
groups on ring D [28,29].
Analogs bearing modifications on ring A were prepared as
indicated in Scheme 1. The aminoketal moiety was first protected as
a formamide [30] by N,O-double formylation of tomatidine hy-
drochloride 3 with acetic formic anhydride, followed by chemo-
selective deprotection of the formate ester in mild basic conditions
to yield N-formyltomatidine 4 in quantitative yield. Compound 4
underwent a Mitsunobu inversion with acetic acid [31], followed by
Fig. 1. Characteristics of normal and small colony variants of S. aureus.
force, which concertedly impede bacterial growth while increasing
resistance to antibiotics such as aminoglycosides [12,14e16]. As
opposed to normal strains (Fig. 1), SCVs possess characteristics that
contribute to prevent acute infections, but allow in vivo persistence
[14,15,17].
S. aureus has the ability to switch between the normal and SCV
phenotypes. While normal strains exhibit virulence, fast growth
and dissemination and are responsible for acute infections, SCVs
are associated with colonization, biofilm production and persistent
infections [16]. They possess a deficient electron transport chain,
which modifies the proton-motive force (PMF) and affects sus-
ceptibility to aminoglycoside (AMG) antibiotics.
Tomatidine (1, Fig. 2), the aglycone version of tomatine (2), is an
important antimicrobial defense metabolite of many solanaceous
plants and possesses potential anticancer [18e21], chemosensitizer
[22], anti-Leishmania [23], anti-hyperlipidemic [24], and anti-
inflammatory properties [25]. Apart from its action on eukaryotic
organisms, our group has recently demonstrated that tomatidine
possesses anti-virulence activity against normal strains of S. aureus
without impairing growth, while also displaying potent antibiotic
activity against SCVs [12,14]. Most importantly, tomatidine has
demonstrated its ability to potentiate the bactericidal activity of
aminoglycoside antibiotics against the normal phenotype of
S. aureus [12]. An example of such potentiating effect is observed
when tomatidine is used in combination with gentamicin, which
improves the minimal inhibitory concentration (MIC) of gentamicin
protective groups acidolysis to afford 3a-hydroxyl tomatidine 6 in
high yields [32]. Palladium-catalyzed O-allylation of N-for-
myltomatidine 4 delivered O-allyltomatidine 8 in good yield after
deformylation [33]. Direct oxidation of the C3 alcohol of compound
4 using DesseMartin periodinane [34] delivered 3-ketotomatidine
9, which was subsequently deprotected to yield 10 by acidic hy-
drolysis [32]. Finally, starting from 3-ketotomatidine intermediate
9, amino analogs 12, 14, 16 and 18 were generated via reductive
amination [35] followed by deprotection in low to moderate yields.
In order to better understand the SAR of the aminoketal moiety
on rings E, F and ascertain whether a closed or open form of rings E
and F constitute the active form of the molecule, we generated
analogs modified with open rings E and/or F via two complemen-
tary approaches.
Firstly, tomatidine was used as starting materials to generate
open chain derivatives (Scheme 2). Opening of ring E was performed
via catalytic hydrogenation of the spiroaminoketal moiety on plat-
inum oxide [36] to yield piperidine derivative 19. On the other hand,
dihydrofuran 22 was obtained by acetylation of the spiroaminoketal
moiety of 3 to give intermediate 20, followed by acid-catalyzed
elimination then ester hydrolysis [37e39]. Subsequent reduction
delivered the corresponding tetrahydrofuran 23 with no control of
stereochemistry on the newly created stereogenic centers. Acety-
lation of tomatidine hydrochloride 3 also yielded triacetylated
compound 24, which was opened in acidic conditions to give in-
termediate 25 [37e39] then reduced and deprotected to afford triol
27. Unfortunately, all attempts to hydrolyze the terminal acetamide
of 27 resulted in product decomposition.
by 8-fold (0.5 mg/mL to 0.06 mg/mL in the absence or presence of
tomatidine, respectively). The antibacterial mechanism of action,
structureeactivity relationship (SAR) and target of tomatidine
remain to be elucidated.
In order to better understand the antibacterial properties of
tomatidine and explore its SAR, we undertook the synthesis of
structural analogs. Our initial goal was to firstly understand the SAR
of tomatidine, both as a potentiator of the antibiotic action of
gentamicin and as a growth inhibitor of S. aureus SCVs. The results
of these efforts are reported below.
The second approach in the synthesis of analogs modified on
rings E and F started from commercially available pregnenolone
acetate 28, which bears the same AeD ring system and stereo-
chemistry as tomatidine (Scheme 3). Hydrogenation and ester hy-
2. Chemistry
Tomatidine 1 (Fig. 2) is a steroid alkaloid structurally charac-
terized by 6 rings, 12 stereogenic centers, a 3
spiro-fused E, F rings in the form of an aminoketal. It was isolated
b-hydroxyl group and
t
drolysis [40] followed by BDMS protection of the hydroxyl group
Fig. 2. The structures of tomatidine and tomatine.

F. Chagnon et al. / European Journal of Medicinal Chemistry 80 (2014) 605e620
607
a
Tomatidine
N
N
R
hydrochloride
O
O
O
3
X
HO
Y
4
b
5: X=H, Y=OAc, R=CHO
c
6: X=H, Y=OH, R=H x HCl
8: X=OAll, Y=H, R=H x HCl
10: X, Y=O, R=H x HCl
d
7: X=OAll, Y=H, R=CHO
e
f
9: X, Y=O, R=CHO
c
N
N
R'
O
O
O
O
RHN
9
g
11: R=H, R'=CHO
e
12: R=H, R'=H x HCl
h
13: R=(CH2)2NH2, R'=CHO
e
14: R=(CH2)2NH2, R'=H x HCl
16: R=(CH2)4NH2, R'=H x HCl
18: R=(CH2)6NH2, R'=H x HCl
i
15: R=(CH2)4NH2, R'=CHO
e
j
17: R=(CH2)6NH2, R'=CHO
e
Scheme 1. Reagents and conditions: a) i) acetic formic anhydride, DIPEA, THF, rt; ii) EtOH, NaHCO3 buffer pH ¼ 9.5, rt, 79% (2 steps); b) DIAD, PPH3, AcOH, THF, rt, 86%; c) HCl, EtOH,
reflux, quant.; d) allyl methyl carbonate, Pd2(dba)3, dppp, THF, 65 ꢀC, 76%; e) HCl, MeOH, reflux, quant.; f) DesseMartin periodinane, DCM, rt, 68%; g) NH4OAc, NaBH3CN, MeOH,
pH ¼ 6, reflux, 69%; h) BocNH(CH2)2NH2, NaBH3CN, MeOH, pH ¼ 6, reflux, 89%; i) BocNH(CH2)4NH2, NaBH3CN, MeOH, pH ¼ 6, reflux, 44%; j) BocNH(CH2)6NH2, NaBH3CN, MeOH,
pH ¼ 6, reflux, 46%.
delivered keto intermediate 30 in high yields [41]. Bromination
a
to
substituted tertiary amines 32 and 33, respectively. In parallel,
reductive amination of 28 with diverse amines and monoprotected
diamines followed by acidic hydrolysis produced analogs 35, 42e
44, 46 and 48.
the keto group then yielded bromoketone 31 [42,43]. Subsequent
substitution with dimethylamine or piperidine [44] followed by
desilylation yielded dimethyl-substituted and pyrrolidinyl-
N
H2
HN
OH
a
O
x HCl
Cl
HO
HO
19
3
b
NHAc
e
NHAc
c,d
N
O
O
O
O
HO
HO
HO
AcO
22
23
20
Ac
+
N
O
OAc
NHAc
g,h
OH
OH
NHAc
f
OAc
AcO
AcO
25
24
27
Scheme 2. Reagents and conditions: a) PtO2, H2 (200 PSI), AcOH rt, 54%; b) Ac2O, pyridine, rt, 16 h, 9% 20 and 9% 24; c) AcOH, reflux, 15 min, quant.; d) NaOMe, MeOH, 16 h, rt, 46%;
e) H2 (750 PSI), Pd/C, EtOH, HCl, 16 h, rt, 12%; f) HCl, H2O, AcOH, rt, 1 h; g) LiBH4, MeOH, THF, 16 h, rt; h) HCl, MeOH, 65 ꢀC, 3.5 h, rt (31%, 2 steps).

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F. Chagnon et al. / European Journal of Medicinal Chemistry 80 (2014) 605e620
Scheme 3. Reagents and conditions: a) H2 (200 PSI), Pd/C, rt, quant.; b) NaOH, MeOH reflux, quant.; c) tBDMSCl, imidazole, DIPEA, THF, rt, 87%; d) i) KHMDS, THF, ꢁ78 ꢀC, ii) TMSCl,
rt, iii) NBS, ꢁ78 ꢀC, 87%; e) Me2NH (32) or piperidine (33), THF, rt, 79% f) HCl, THF, rt, 75%; g) NH4OH, BocNH(CH2)2NH2 (42), BocNH(CH2)3)NH2 (43), or BocNH(CH2)4NH2 (44),
NaBH3CN, MeOH, THF, pH 6, reflux, 40e70%.
In order to further mimic the open form of tomatidine, we
synthesized analogs bearing a -hydroxyl function in position C16,
the crude mixture was converted to 16-b-hydroxybromoketone 52
[47], which was then separated from 49 by flash chromatography.
The bromide group of 52 was reduced with tributyltin hydride [47]
b
as exemplified by 55 and 57 (Scheme 4). Hydrogenation of preg-
nenolone acetate 28 on palladium yielded 49 in quantitative yield.
Ketone 49 was converted to enol acetate 50 [45], then underwent a
WohleZiegler reaction to yield enone 51 along with precursor ke-
tone 49 in a 1:1 ratio [46]. Upon reaction with N-bromoacetamide,
to generate
b-hydroxyketone 53 in 61% overall yield. Reductive
amination of 53 followed by acid-mediated deprotection gave ac-
cess to amines 55 and 57 with no control of the stereochemistry on
the newly created stereogenic center.
Scheme 4. Reagents and conditions: a) H2 (200PSI), Pd/C rt, quant.; b) pTSA, Ac2O, reflux, 99%; c) NBS, CCl4, reflux, quant.; d) N-bromoacetamide, H2O, THF, 48 h, rt, 85%; e) Bu3SnH,
Et3B, O2, DCM, rt, 73%; f) amine, NaBH3CN, MeOH, THF, pH ¼ 6, reflux, 40e60%; g) HCl, MeOH, reflux, quant.

F. Chagnon et al. / European Journal of Medicinal Chemistry 80 (2014) 605e620
609
3. Biology
The biological evaluation of tomatidine hydrochloride 3 and
newly synthesized analogs is summarized in Table 1 and expressed
according to three parameters: (1) the minimal inhibitory con-
centration (MIC) against the normal strain of S. aureus ATCC29213,
which represents the actual antibiotic activity of analogs against
S. aureus; (2) the potentiation fold, which expresses the potenti-
ating effect of analogs on the antibiotic activity of gentamicin on
normal strain of S. aureus (gentamicin alone possesses an MIC of
possess any activity on either target. This observation reinforces the
notion that specific targets are involved in the mechanism of action
of tomatidine, requiring specific orientation of heteroatoms. It also
suggests that the closed form of tomatidine is the active form.
Indeed, the open form of rings E and F of tomatidine or solasodine
would differ only by the orientation of a methyl group on a highly
flexible chain, which would be unlikely to greatly influence bio-
logical activity.
Several modifications were made for the sole purpose of testing
the importance of partial or full ring opening within the spi-
roaminoketal moiety for biological activity. While compound 19, an
analog bearing an open E ring, kept a moderate activity both as a
potentiator on normal strains and SCVs, compounds 22 and 23,
which display open F ring, as well as fully open compound 27,
showed complete loss of both activities. Moreover, compound 4,
which bears a formyl group that both traps the aminoketal in closed
configuration and masks the basic nitrogen, completely lost bio-
logical activity. Together, these observations suggest that the spi-
roaminoketal moiety is important in a closed configuration, and
that neutralizing its H-bond donating capability is detrimental for
activity. Loss of biological activity for analogs 4, 22, 23 and 27 may
also be attributed to the loss of basicity of the amine moiety. Un-
fortunately, all attempts to hydrolyze the terminal acetamide in
order to regenerate a basic amine at the extremity of the exocyclic
chain of these analogs resulted in product decomposition. All these
observations, combined with the lack of activity of dihy-
drosolasodine 58, suggest that the closed form of tomatidine is the
active form, and that a basic nitrogen is required for biological
activity.
Compounds 32, 33, 35, 42e44 (Scheme 3) were inactive against
both the normal strain and SCVs. Analogs 46 and 48 retained
noticeable potentiation activity against S. aureus compared to
tomatidine, while they lost all or partial activity against SCVs. These
results suggest that two distinct mechanisms of action might be at
play against normal strains of S. aureus and SCVs since compound
46 possesses a 64-fold decreased activity against SCVs while
retaining full gentamicin potentiation against normal strain.
Similarly to analogs 46 and 48, compound 19, which possesses
0.25e0.5
expressed as low, moderate or high (MIC ꢂ 8
g/mL, and MIC ¼ 0.06e0.25 g/mL, respectively), on S. aureus
Newbould hemB, a genetically derived strain which displays a
mg/mL on this strain), and (3) the activity of analogs
mg/mL, MIC ¼ 0.5e
4
m
m
D
stable SCV phenotype. Note that the potentiation fold (i.e., the ratio
of the MIC of gentamicin alone and the MIC of gentamicin in
combination with tomatidine analogs) is correlated to the frac-
tional inhibitory concentration (FIC index) [48]. To that effect, we
previously showed that a potentiation fold of 4 or 8 for tomatidine
is equivalent to an FIC of 0.2, which clearly demonstrates synergy
with aminoglycosides [12]. We therefore interpreted a potentiation
fold of 8, 4 and ꢃ2 as a high, moderate and low (no) synergy with
gentamicin, respectively (Table 1).
4. Results and discussion
Similarly to tomatidine, most analogs possessed no antibiotic
activity against the normal phenotype of S. aureus; as a result, these
analogs alone typically possessed MIC > 64
contrast, 8, 14 and 16 were the only analogs possessing antibiotic
activity by themselves, with MIC 8e16 g/mL.
mg/mL (Table 1). In
m
The potentiating effect of the analogs on the inhibitory activity
of gentamicin against the normal strain of S. aureus ATCC29213, and
the antibiotic properties against the SCV, varied as a function of
their structure as detailed below.
Seven compounds possessed structural variations on position 3
of ring A while keeping rings E and F of tomatidine (Scheme 1,
analogs 6e18). As reported in Table 1, all of these analogs kept a 4-
to 8-fold synergy with gentamicin against the normal strain of
S. aureus. The contrast between the moderate activity of tomatidine
derivative 6 possessing inverted stereochemistry in C3 and the high
activity of tomatidine hydrochloride 3 itself, both as aminoglyco-
side potentiator and SCV inhibitor, hints toward a specific interac-
tion at this location. Whether or not this difference is steric or due
to a hydrogen bond is undetermined. The strong activity of com-
pounds 8 and 10, both as gentamicin potentiators and against SCVs,
suggests that a hydrogen bond donor effect of the 3-hydroxyl group
is not important for activity. The moderate activity of compounds
12, 14, 16 and 18 suggests that the presence of one or two ammo-
nium groups in position C3 reduces activity against SCVs, while
being beneficial for antibiotic activity against normal strains (more
specifically as in 14 and 16). Indeed, while conserving moderate
activity both against SCVs and as aminoglycoside potentiators,
analogs 14 and 16 unexpectedly showed appreciable antibiotic
activity on their own against the normal strain of S. aureus, a
property that is absent in tomatidine or other analogs. Analog 8 also
possesses some level of antibiotic activity by itself. These results
opens the path for further optimization of tomatidine analogs as a
new class of antibiotics against normal strains of S. aureus.
both a 16b-hydroxyl functional group, a charged amino group in
close proximity to the steroidal skeleton as well as an alkyl sub-
stituent, lost some activity against SCVs. Compounds 46 and 48
both exhibit charged an amino group in close proximity to the
steroidal skeleton and alkyl substitution, but not the 16b-hydroxyl
moiety. They kept moderate to high potentiation activity against
normal strains of tomatidine, yet lost considerable activity on the
SCV phenotype (ꢂ64 fold). In order to test whether the 16
b-hy-
droxyl group rescues bacteriostatic activity, we synthesized analogs
55 and 57. However the presence of the hydroxyl group in analog
55 abrogated both potentiating and anti-SCV activity.
As a summary of SAR, we found that a hydrogen bond acceptor
in position 3 plays a role in biological activity against SCVs, and b-
configuration is preferred (e.g., 3, 8 vs 6). Alkylation of this sub-
stituent it tolerated (e.g., 8). On the other hand, potentiation of
gentamicin activity on normal strains seems to possess a broader
SAR (e.g., 14, 16, 18). Second, the stereochemistry of the spi-
roaminoketal functional group in rings E and F appears critical for
antibiotic activity against SCVs (e.g., tomatidine
1 vs dihy-
drosolasodine 58). The aminoglycoside potentiator activity, how-
ever, seems more affected by the presence of an amino group
proximal to the steroidal skeleton than by ring opening. Alkylation
of this tethered amino group seems to be preferred. Third, open
rings E, F analogs and analogs bearing a distant tethered amino
group remained inactive, as well as analogs bearing a 16b-hydroxyl
moiety. Since chemical modifications on ring A were tolerated, but
not modifications on rings E and F, the spiroaminoketal moiety
Along with analogs modified on rings E and F, we also tested
dihydrosolasodine (58, Fig. 3), a structural isomer of tomatidine
with inverted stereochemistry on the exocyclic methyl substituent
on ring F and on the spiroaminoketal rings EeF junction. It is
interesting to note that while tomatidine is highly active both as a
potentiator and SCV inhibitor, dihydrosolasodine 58 does not

610
F. Chagnon et al. / European Journal of Medicinal Chemistry 80 (2014) 605e620
Table 1
Biological activities of tomatidine analogs against S. aureus of the normal and SCV phenotypes.
Compound
MIC vs ATCC29213
g/mL)
Combination MIC vs ATCC29213
MIC vs SCVb
(mg/mL)
(
m
(potentiation fold with gentamicin)a
3
>64
>64
0.06 (8ꢄ)
0.06
High
High
4
0.25 (2ꢄ)
>8
Low
Low
6
>64
0.12 (4ꢄ)
4
Moderate
Moderate
8
16
0.06 (8ꢄ)
0.25
High
High
10
>64
0.06 (8ꢄ)
0.25
High
High
12
32
0.06 (8ꢄ)
1
High
Moderate
14
16
18
8e16
Active
0.06e0.12 (4e8ꢄ)
0.5e1
Moderate
Moderate
16
Active
0.06e0.12 (4e8ꢄ)
0.5
Moderate
Moderate
32
0.12e0.25 (2e4ꢄ)
1
Moderate
Moderate
19
22
32
0.12 (4ꢄ)
4
Moderate
Moderate
>64
0.25 (1ꢄ)
>8
Low
Low

F. Chagnon et al. / European Journal of Medicinal Chemistry 80 (2014) 605e620
611
Table 1 (continued )
Compound
MIC vs ATCC29213
g/mL)
Combination MIC vs ATCC29213
MIC vs SCVb
(mg/mL)
(
m
(potentiation fold with gentamicin)a
23
27
>64
>64
0.25 (1ꢄ)
>8
Low
Low
0.25 (1ꢄ)
>8
Low
Low
32
33
>64
>64
0.25 (2ꢄ)
>8
Low
Low
0.25 (2ꢄ)
>8
Low
Low
35
64
0.25 (2ꢄ)
>8
Low
Low
42
43
44
32
32
32
0.25 (2ꢄ)
>8
Low
Low
0.25 (2ꢄ)
>8
Low
Low
0.25 (2ꢄ)
>8
Low
Low
46
48
55
64
0.06 (8ꢄ)
4e8
Moderate
High
>64
>64
0.12 (4ꢄ)
>8
Low
Moderate
0.25 (2ꢄ)
>8
Low
Low
(continued on next page)

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F. Chagnon et al. / European Journal of Medicinal Chemistry 80 (2014) 605e620
Table 1 (continued )
Compound
MIC vs ATCC29213
g/mL)
Combination MIC vs ATCC29213
MIC vs SCVb
(mg/mL)
(
m
(potentiation fold with gentamicin)a
57
>64
>64
0.25 (2ꢄ)
>8
Low
Low
58
0.5 (1ꢄ)
>8
Low
Low
a
Potentiation fold of analogs is defined as the ratio of the MIC of gentamicin alone and the MIC of gentamicin in combination with the analog used at 8
g/mL).
Activity of the analogs against the SCV strain Newbould
activity).
m
g/mL (except for
compounds 14 and 16 that were used at 4
m
b
D
hemB (MIC ¼ ꢂ8
m
g/mL: low activity, MIC ¼ 0.5e4
m
g/mL: moderate activity, MIC ¼ 0.06e0.25
mg/mL: high
seems to play a critical role in this activity. Finally, analogs bearing
diamines in position 3 of ring A possess antibiotic activities of their
own against normal strains of S. aureus, although whether this new
activity is due to molecular interactions with a cellular target,
increased cellular uptake or increased solubility remains unknown.
recombination to generate a respiratory deficient strain [49], was
used as a stable small colony variant (SCV) phenotype. Both strains
were maintained on triptic soy agar (BD, Mississauga, ON, Canada).
6.1.2. Antibiotic susceptibility testing
MICs were determined by a broth microdilution technique in
cation-adjusted Mueller-Hinton broth (BD, Mississauga, ON, Can-
ada), following the recommendations of the Clinical and Laboratory
Standards Institute (CLSI) [29], except that the incubation period
was extended to 48 h (instead of 24 h) and the medium used was
brain-heart infusion (BD, Mississauga, ON, Canada) to allow
adequate growth of the SCV strain. Tomatidine hydrochloride 3
(Sigma, Oakville, ON, Canada), gentamicin sulfate (Sigma, Oakville,
ON, Canada) and the combination of gentamicin and tomatidine
were always included as comparators in all MIC tests performed for
the new chemical entities. Tomatidine and all new analogs were
solubilized at 2 mg/mL in DMSO. The gentamicin MIC for S. aureus
5. Conclusion
In conclusion, we report here the first SAR study of tomatidine
on S. aureus and SCVs, both as a synergistic agent with gentamicin,
and as an antibiotic on its own. Although no cellular target has been
identified yet, this SAR suggests a target-based rather than a
nonspecific mode of action. The segregation of biological activities
(potentiation of aminoglycoside activity and SCV growth inhibi-
tion) in some analogs also suggests the existence of two mecha-
nisms of action in S. aureus against the normal and SCV phenotypes.
Finally, these works support the hypothesis that the closed rather
than the open spiroaminoketal moiety of tomatidine is important
for activity, and that the 3-hydroxyl group can be further modified
to both refine activity and to confer to this series antibiotic activity
against normal strains of S. aureus.
ATCC29213 is 0.5e1
0.03e0.12 g/mL when gentamicin is used in combination with
tomatidine at 8 g/mL. This concentration of tomatidine is well
mg/mL when gentamicin is used alone and
m
m
above the minimal tomatidine concentration showing a potenti-
ating effect with aminoglycosides in synergy studies [12]. The MIC
of tomatidine used alone on S. aureus Newbould
DhemB is 0.03e
6. Experimental section
0.12 g/mL, whereas tomatidine has no significant activity against
m
S. aureus strains of the normal growth phenotype (MIC of >32 g/L
against ATCC29213) [12].
6.1. Biological tests
6.1.1. Bacterial strains and growth conditions
6.2. Chemistry
S. aureus strain ATCC29213 was used as the normal growth
phenotype strain. NewbouldDhemB, in which the gene hemB has
been disrupted by insertion of the ermA cassette by homologous
All moisture-sensitive reactions were performed in an inert, dry
atmosphere of argon in flame-dried glassware. Air-sensitive liquids
were transferred via syringe or cannula through rubber septa. Re-
agent grade solvents were used for extractions and flash chroma-
tography. THF was distilled from sodium/benzophenone under
argon; methanol was distilled from magnesium under argon. All
other solvents purchased from commercial sources were used
without further purification. Tomatidine hydrochloride was pur-
chased from Molekula. All other reagents were generally purchased
from Aldrich and used with no further purification.
Reactions were monitored by analytical thin-layer chromatog-
raphy (Canadian Life Science, TLC, GLASS plates SIL 60 G-25 UV
254). The plates were visualized first with UV illumination followed
by heating with ceric ammonium molybdate (CAM) [1% (w/v)
ammonium cerium sulfate, 2.5% (w/v) molybdenum trioxide,
1:9H2SO4/H2O]. Flash column chromatography was performed
manually on SiliaFlashÒ P60 silica. The solvent compositions
Fig. 3. The structure of dihydrosolasodine.

F. Chagnon et al. / European Journal of Medicinal Chemistry 80 (2014) 605e620
613
reported for all chromatographic separations are on a volume/
volume (v/v) basis.
High-pressure liquid chromatography (HPLC) was performed on
an Agilent 1100 apparatus using an ACE C18 column,
evaporated, and the resulting aqueous phase was extracted with
EtOAc (3 ꢄ 25 mL). The combined organic phases were dried on
anhydrous magnesium sulfate then evaporated under reduced
pressure.
250 ꢄ 21.2 mm, with 5
m
m silica and 15.5% carbon load.
The crude product was purified by flash chromatography (25%
EtOAc/Hexanes) to give 155 mg (79%) of the desired compound (4)
as a white solid (m.p. 184e187 ꢀC).
NMR spectra were recorded at room temperature on a Bruker
Spectrospin 300 spectrometer at 300 MHz (1H) and 75 MHz (13C),
or on a Varian Unity Inova at 600 MHz (1H) and 150 MHz (13C). 1H
1H NMR (600 MHz, CDCl3)
d (ppm) 8.41 (s, 1H), 4.29 (d, 1H,
NMR spectra are reported in parts per million (ppm) on the
relative to the residual signals of chloroform ( 7.26 ppm) or
methanol ( 3.31 ppm) as an internal standard with the following
d
scale
J ¼ 11.5 Hz), 4.13 (dd., 1H, J1 ¼ 7.3 Hz, J2 ¼ 15.5 Hz), 3.58 (quint, 1H,
J ¼ 4.7 Hz), 2.65 (t, 1H, J ¼ 11.5), 2.54 (quint, 1H, J ¼ 7.1 Hz), 1.98
(quint, 1H, J ¼ 5.28 Hz) 1.87 (d, 1H, J ¼ 13.7 Hz), 1.82e1.72 (m, 3H),
1.72e1.63 (m, 3H), 1.61e1.45 (m, 7H), 1.40 (d, 1H, J ¼ 13.0 Hz), 1.38e
1.22 (m, 8H),1.15 (dt,1H, J1 ¼12.3 Hz, J2 ¼ 3.9 Hz),1.12e1.06 (m, 2H),
1.05 (d, 3H, J ¼ 6.8 Hz), 0.95 (dt,1H, J1 ¼13.7 Hz, J2 ¼ 3.6 Hz), 0.91 (d,
3H, J ¼ 5.9 Hz), 0.89e0.84 (m, 1H), 0.82 (s, 6H), 0.64 (dt, 1H,
J1 ¼ 11.39, J2 ¼ 3.6 Hz).
d
d
multiplicity e s: singlet; d: doublet; t: triplet; q: quartet; quint:
quintet; m: multiplet. 13C NMR spectra are reported in ppm on the
d
scale relative to CDCl3 (d 77.0 ppm) or CD3OD (d 49.0 ppm). High-
resolution mass spectrometry (HRMS) was performed on a Maxis
(Bruker) Q-TOF.
13C NMR (75.5 MHz, CDCl3)
d (ppm) 158.9, 97.8, 78.5, 72.1, 62.3,
6.2.1. General procedure for synthesis of Boc-diaminoalkanes (A)
Boc-alkanediamines 36e38 were synthesized as reported by
Jensen et al. [50].
55.8, 54.3, 44.8, 40.2, 38.1, 36.9, 36.8, 35.5, 35.0, 32.2, 31.4, 30.7, 29.7,
29.2, 28.5, 27.5, 21.0, 18.9, 17.0, 15.3, 12.3.
HRMS calculated for C28H45O3NNaþ: 466.3292, found: 466.3308
(MNaþ).
6.2.2. General procedure for reductive amination of 3b-acetoxy-5-
alpha-pregnan-20-one (B)
Reductive amination with pregnanolone acetate derivatives
were synthesized as reported by Xie et al. [51].
6.2.6. 3a-Acetoxy-N-formyl-tomatidine (5)
In a 10 mL round bottom flask, N-formyltomatidine (4) (60 mg,
0.135 mmol, 1.0 eq) was dissolved in 3 mL of anhydrous THF, along
with triphenylphosphine (71 mg, 0.27 mmol, 2.0 eq) and acetic acid
6.2.3. Representative procedure for substitution of bromine by
amino compounds (C)
(22
0.202 mmol, 1.5 eq) was added, and the reaction was stirred at
room temperature. After 4 h, an additional 20 L diisopropylazo-
dicarboxylate, 30 mg triphenylphosphine and 20 L of acetic acid
mL, 0.38 mmol, 2.8 eq). Diisopropylazodicarboxylate (40 mL,
In a 20 mL vial, 53 was solubilized in THF (0.1 M). 2.0 eq of the
corresponding amine was added, and the reaction was stirred for
1 h at room temperature. THF was removed in vacuo. The resulting
solid was suspended in water then extracted three times with
EtOAc. The combined organic layers were washed with brine, dried
on anhydrous magnesium sulfate, then the solvent was removed in
vacuo. The crude compound was purified by flash chromatography.
The compound was solubilized in a mixture of THF and 1 M HCl
(4:1 solution) and stirred for 2 h while monitoring by TLC. Upon
completion, a saturated solution of aqueous sodium bicarbonate
was added until alkaline pH was achieved, and THF was removed in
vacuo. The remaining aqueous layer was extracted with EtOAc (3ꢄ),
and the combined organic layers were washed with brine, dried on
anhydrous magnesium sulfate and concentrated under reduced
pressure. The crude compound was purified by flash
chromatography.
m
m
were added, and the reaction was stirred overnight, monitored by
TLC (50% AcOEt/Hexanes, Rf: 0.5). The reaction was then concen-
trated under reduced pressure, suspended in water and extracted
with EtOAc (3ꢄ). The combined organic fractions were washed
with brine, dried on anhydrous magnesium sulfate and evaporated
under reduced pressure. The crude compound was purified by flash
chromatography (10% EtOAc/hexanes to 15% EtOAc/hexanes) to
yield 56 mg (86%) of compound 5. The compound was contami-
nated with residual diisopropylazodicarboxylate as evidenced by
1H NMR, yet was used as such for the next reaction.
1H NMR (300 MHz, CDCl3)
d (ppm) 8.43 (s, 1H), 5.00 (m, 3H,
DIAD) 4.31 (d, 1H, J ¼ 11.8 Hz), 4.99 (m, 1H), 4.16 (quad, 1H,
J ¼ 7.1 Hz), 2.68 (t, 1H, J ¼ 12.1 Hz), 2.56 (s, 4H), 2.07 (s, 3H), 2.06e
1.98 (m, 2H), 1.90 (d, 1H, J ¼ 12.4 Hz), 1.84e1.74 (m, 3H), 1.74e1.51
(m, 5H), 1.48 (s, 4H), 1.42e1.20 (m, 25H, DIAD), 1.14 (d, 2H,
J ¼ 4.7 Hz), 1.08 (d, 3H, J ¼ 7.1 Hz), 0.93 (d, 4H, J ¼ 6.0 Hz), 0.85 (s,
3H), 0.83 (s, 3H), 0.81e0.73 (dt, 1H, J1 ¼ 11.5 Hz, J2 ¼ 3.3 Hz).
6.2.4. General method for acidic deprotections (D)
In a 25 mL round bottom flash equipped with a condenser, the
starting material was solubilized in 5 mL anhydrous MeOH. To this
solution was added a solution of 0.9 mL acetyl chloride in 6 mL
anhydrous MeOH. The reaction heated to reflux for 1 h, monitored
by TLC. The reaction was allowed to cool at room temperature, then
solvent was removed in vacuo to yield the desired compound.
6.2.7. 3a-Hydroxytomatidine hydrochloride (6)
In a 25 mL round flask, 28 mg 5 (0.058 mmol) was refluxed for
3 h in 6 mL EtOH and 3 mL aqueous HCl 2.5 N (TLC: 10% MeOH/
AcOET with 0.5% NEt3, Rf: 0.5). Upon completion, the volatiles were
removed under reduced pressure and the remaining water was
removed by lyophilization.
6.2.5. N-Formyltomatidine (4)
In a 250 mL round flask, tomatidine hydrochloride (200 mg,
0.44 mmol, 1.0 eq) was suspended in dry THF (20 mL) then acetic
formic anhydride (380 mg, 4.42 mmol,10.0 eq) was added, followed
1H NMR (300 MHz, CD3OD)
d
(ppm) 4.40 (quad, 1H, J ¼ 8.8 Hz),
3.98 (s large, 1H), 3.68 (m, 2H), 3.14 (m, 1H), 2.94 (t, 1H, J ¼ 11.0 Hz),
2.23 (m, 1H), 2.13e1.99 (m, 3H), 1.91 (m, 3H), 1.80e1.45 (m, 10H),
1.40e1.15 (m, 17H), 1.12 (d, 3H, J ¼ 6.6 Hz), 1.01 (d, 3H, J ¼ 6.0 Hz),
0.93 (s, 3H), 0.86 (s, 4H).
by DIPEA (390 mL, 2.20 mmol, 5.0 eq). The reaction was stirred for
15 min, then monitored by TLC (50% EtOAc/Hexanes, CAM stain,
Rf ¼ 0.23 for mono-formylated compound, 0.49 for diformylated
compound). The volatiles were removed by evaporation under
reduced pressure. The compound was then diluted in a mixture of
125 mL EtOH and 50 mL aqueous NaHCO3 buffer (pH ¼ 9.5) and
stirred for one week, monitored by TLC until the complete disap-
pearance of the diformylated compound. EtOH was then
13C NMR (75.5 MHz, CD3OD)
d (ppm) 96.2, 81.1, 65.7, 61.7, 55.6,
54.3, 40.8, 40.7, 39.6, 38.8, 35.8, 35.2, 34.9, 32.0, 31.9, 31.4, 28.2, 28.1,
25.8, 25.3, 20.8, 20.3, 17.3, 16.0, 13.5, 10.2.
HRMS calculated for C27H46O2Nþ: 416.3523, found: 416.3534
(MHþ), 438.3350 (MNaþ).

614
F. Chagnon et al. / European Journal of Medicinal Chemistry 80 (2014) 605e620
6.2.8. 3
b
-Allyloxy-N-formyltomatidine (7)
water was removed by lyophilisation to give a white solid (m.p.
In a 3 mL round bottom flask equipped with a condenser and
placed under argon atmosphere, compound 4 (30 mg, 0.068 mmol)
was dissolved in 1 mL THF. Tris(dibenzylideneacetone) dipalla-
dium(0) (3 mg, 0.003 mmol, 0.05 eq), 1,3-bis(diphenylphosphino)
propane (5 mg, 0.012 mmol, 0.18 eq) and allyl methyl carbonate
(0.2 mL,1.76 mol, 26 eq) were then added successively. The reaction
was brought to 65 ꢀC for 6 h, and monitored by TLC (50% EtOAc/
hexanes, UV/CAM, Rf: 0.20 (starting material), 0.50 (desired com-
pound)). Upon completion, the reaction was allowed to cool down
to room temperature, then the solvent was removed in vacuo. The
crude compound was purified by flash chromatography (20%
EtOAc/Hexanes) to yield 25 mg (76%) of the desired compound.
160e163 ꢀC).
1H NMR (300 MHz, CD3OD)
d (ppm) 4.60 (m, 2H), 4.37 (q, 1H,
J ¼ 9.0 Hz), 2.80 (m, 1H), 2.70 (t, 1H, J ¼ 12.0 Hz), 2.55e2.30 (m, 1H),
2.24e2.14 (m, 1H), 2.08e1.94 (m, 3H), 1.91e1.68 (m, 5H), 1.63e1.50
(m, 6H), 1.49e1.12 (m, 12H), 1.10 (s, 3H), 1.06 (d, 3H, J ¼ 7.1 Hz), 0.96
(d, 3H, J ¼ 5.5 Hz), 0.89 (s, 2H), 0.82 (s, 1H), 0.76e0.66 (m, 2H).
13C NMR (75.5 MHz, CD3OD)
d (ppm) 213.5, 100.2, 98.0, 79.2,
62.0, 55.3, 53.7, 44.0, 42.2, 41.8, 40.7, 39.7, 38.3, 35.5, 34.8, 31.9, 31.7,
28.5, 28.0, 27.4, 20.9, 18.1, 16.0, 14.3, 10.3.
HRMS calculated for C27H44O2Nþ: 414.3372, found: 414.3376
(MHþ), 436.3192 (MNaþ).
1H NMR (300 MHz, CDCl3)
d
(ppm) 8.41 (s, 1H), 5.93 (ddt, 1H,
6.2.12. N-Formyl-3-aminotomatidine (11)
J1 ¼ 17.3 Hz, J2 ¼ 10.5 Hz, J3 ¼ 5.7 Hz), 5.34 (dq, 1H, J1 ¼ 17.1 Hz,
J2 ¼ 1.4 Hz), 1.25 (dq, 1H, J1 ¼ 10.3 Hz, J2 ¼ 1.3 Hz), 4.60 (dt, 2H,
J1 ¼ 5.8 Hz, J2 ¼ 1.4 Hz), 4.53 (quint, 1H, J ¼ 5.5 Hz), 4.29 (d, 1H,
J ¼ 11.9 Hz), 4.16e4.08 (m, 1H), 2.65 (t, 1H, J ¼ 11.3 Hz), 2.54 (t, 1H,
J ¼ 7.0 Hz), 2.01e1.94 (m, 1H), 1.94e1.84 (m, 2H), 1.81e1.68 (m, 4H),
1.65 (s, 2H), 1.62e1.47 (m, 6H), 1.46e1.35 (m, 2H) 1.35e1.28 (m,
41H), 1.22e1.08 (m, 2H), 1.05 (d, 3H, J ¼ 7.0 Hz), 1.02e0.92 (m, 1H),
0.90 (d, 4H, J ¼ 5.8 Hz), 0.83 (s, 3H), 0.82 (s, 1H), 0.65 (dt, 1H,
J1 ¼ 10.7 Hz, J2 ¼ 4.3 Hz).
In a 25 mL round flask, 44 mg 4 (0.1 mmol) was dissolved in
methanol (6 mL) along with ammonium acetate (77 mg, 1.0 mmol,
10 eq). The pH was adjusted to 6 with acetic acid, sodium cyano-
borohydride (6.9 mg, 0.11 mmol, 1.1 eq) was added and the reaction
was refluxed overnight until complete as monitored by TLC (10%
MeOH/AcOEt with 0.5% NEt3, Rf: 0). Solvents were removed under
reduced pressure, and the solid was suspended in water. The pH
was adjusted to 8 with saturated aqueous NaHCO3. The mixture
was extracted with 3ꢄ EtOAc, and the combined organic fractions
were washed with brine, dried on anhydrous magnesium sulfate
and evaporated under reduced pressure. The crude compound was
purified by flash chromatography (10% MeOH/89%EtOAc/1% NEt3)
to yield 21 mg (48%) of the desired compound.
6.2.9. 3b-Allyloxytomatidine hydrochloride (8)
Following procedure D, 7 (16 mg, 0.033 mmol), was converted
into 16.2 mg (quantitative yield) of the desired compound (Rf: 0.51
in 50/50 AcOEt/Hexanes).
1H NMR (300 MHz, CDCl3)
d (ppm) 8.40 (s, 1H), 5.91 (broad s,
1H NMR (300 MHz, CD3OD)
d (ppm) 5.98e5.83 (m, 1H), 5.28 (d,
2H), 4.28 (d, 1H, J ¼ 11.5 Hz), 4.12 (m., 1H), 2.86 (quint, 1H,
J ¼ 7.1 Hz), 2.65 (t, 1H, J ¼ 11.5), 2.53 (quint, 1H, J ¼ 7.1 Hz), 2.04e
1.92 (m, 3H) 1.90e1.82 (m, 1H), 1.80e1.42 (m, 11H), 1.38e1.18 (m,
9H) 1.17e1.06 (m, 2H), 1.04 (d, 3H, J ¼ 7.1 Hz), 0.90 (d, 4H,
J ¼ 6.2 Hz), 0.81 (s, 6H), 0.69e0.57 (m, 1H).
1H, J ¼ 17.1 Hz), 5.19 (d, 1H, J ¼ 10.5 Hz), 4.53 (d, 2H, J ¼ 5.5 Hz), 4.48
(m, 1H), 4.36 (m, 1H), 3.68 (m, 1H), 3.15e3.04 (m, 1H), 2.88 (t, 1H,
J ¼ 11.87 Hz), 2.32e2.11 (m, 1H), 2.07e1.92 (m, 2H), 1.90e1.41 (m,
10H), 1.40e1.12 (m, 11H), 1.08 (d, 4H, J ¼ 6.2 Hz), 0.95 (d, 4H,
J ¼ 6.5 Hz) 0.87 (s, 3H), 0.86e0.79 (m, 4H), 0.75e0.57 (m, 2H).
13C NMR (75.5 MHz, CD3OD)
d (ppm) 154.6, 117.1, 96.1, 81.1, 77.3,
6.2.13. 3-Aminotomatidine hydrochloride (12)
Following procedure D, 21 mg (0.047 mmol) of compound 11
were converted to 23 mg (100%) of compound 12 (Rf: 0 in 10%
MeOH/AcOEt with 0.5% NEt3).
67.6, 61.7, 61.7, 55.4, 54.0, 44.4, 40.8, 40.7, 39.6, 36.3, 35.2, 34.9, 33.6,
31.9, 31.4, 28.2, 28.1, 27.0, 25.7, 25.3, 20.7, 17.2, 15.9, 13.2, 11.1.
HRMS calculated for
C
30H50O2Nþ$H2O: 474.3942, found:
1H NMR (300 MHz, CD3OD)
d
(ppm) 4.37 (q,1H, J ¼ 7.1 Hz), 3.16e
474.3590 (MHþ þ H2O).
3.00 (m, 2H), 2.88 (t, 1H, J ¼ 11.1 Hz), 2.20 (t, 1H, J ¼ 7.1 Hz), 2.09e
1.94 (m, 2H), 1.91e1.63 (m, 10H), 1.62e1.46 (m, 6H), 1.44e1.15 (m,
12H), 1.09 (d, 3H, J ¼ 7.2 Hz), 1.06e0.98 (m, 3H), 0.95 (d, 3H,
J ¼ 6.8 Hz), 0.90 (s, 3H), 0.87 (s, 5H), 0.80e0.60 (m, 3H).
6.2.10. N-Formyl-3-oxotomatidine (9)
In a 10 mL round bottom flask, N-formyltomatidine 4 (50 mg,
0.113 mmol, 1.0 eq) and DesseMartin periodinane (95 mg,
0.225 mmol, 2.0 eq) were stirred in 6.5 mL DCM. The reaction was
monitored by TLC (50% AcOEt/Hexanes, Rf: 0.24). Upon completion,
the reaction was quenched for 30 min with Na2S2O3 (0.2 M), then
extracted 3ꢄ with EtOAc. The combined organic phases were
washed with brine, dried on anhydrous magnesium sulfate then
evaporated under reduced pressure. The crude compound was
purified by flash chromatography (50% EtOAc/Hexanes) to yield
34 mg (68%) of the desired compound.
13C NMR (75.5 MHz, CD3OD)
d (ppm) 96.2, 81.1, 61.7, 55.4, 53.8,
50.3, 44.6, 40.8, 39.5, 36.2, 35.2, 34.8, 32.5, 31.7, 31.4, 29.4, 28.2,
28.0, 26.1, 25.8, 25.3, 20.6, 17.3, 15.9, 13.3, 11.1.
HRMS calculated for C27H47ONþ2 : 415.3683, found: 415.3695.
6.2.14. N-Formyl-3-(N-Boc-aminoethyl)aminotomatidine (13)
Following the procedure used for synthesis of 11, 110 mg 6
(0.25 mmol), 36 (200 mg, 1.25 mmol, 5 eq) and sodium cyanobor-
ohydride (17 mg, 0.27 mmol, 1.1 eq) were used to yield 130 mg
(85%) of compound 13 (Rf: 0.05 in 10% MeOH/AcOEt with 0.5%
NEt3). 1H NMR shows the presence of residual starting diamine,
which was removed in the next step.
1H NMR (300 MHz, CDCl3)
d (ppm) 8.46 ppm (s, 1H), 4.32 (d, 1H,
J ¼ 12.8 Hz), 4.17 (quad, 1H, J ¼ 8.9 Hz), 2.69 (t, 1H, J ¼ 12.6 Hz), 2.58
(quint, 1H, J ¼ 6.6 Hz), 2.52e2.24 (m, 3H), 2.16e2.11 (m, 1H), 2.11e
1.98 (m, 3H), 1.91 (d, 1H, J ¼ 14.0 Hz), 1.86e1.68 (m, 4H), 1.69e1.42
(m, 7H), 1.40e1.20 (m, 7H), 1.19e1.12 (m, 2H),1.09 (d, 3H. J ¼ 7.1 Hz),
1.05 (s, 2H), 0.94 (d, 4H, J ¼ 5.5 Hz), 0.88 (s, 3H), 0.77 (dt, 1H,
J1 ¼ 12.6 Hz, J2 ¼ 4.8 Hz).
1H NMR (300 MHz, CDCl3)
d (ppm) 8.39 (s, 1H), 5.02 (s large, 1H),
4.28 (d, 1H, J ¼ 11.5 Hz), 4.12 (m, 1H), 3.24e3.15 (m, 2H), 2.77e2.69
(m, 2H), 2.64 (m, 1H) 2.54 (m, 1H), 2.44 (m, 1H), 2.04e1.46 (m, 18H)
1.43 (s, 12H), 1.33e1.15 (m, 14H), 1.04 (d, 3H, J ¼ 6.9 Hz), 0.90 (d, 3H,
J ¼ 5.7 Hz), 0.87e0.82 (m, 2H), 0.81 (s, 3H), 0.78 (s, 3H), 0.69e0.57
(m, 1H).
6.2.11. 3-Oxotomatidine hydrochloride (10)
In a 25 mL round flask, 34 mg 9 (0.077 mmol) was refluxed for
2 h in 10 mL EtOH and 5 mL aqueous HCl 2.5 N (TLC: 10% MeOH/
AcOEt with 0.5% NEt3, Rf: 0.54). Upon completion, the ethanol and
HCl were removed under reduced pressure and the remaining
6.2.15. 3-(N-Aminoethyl)-aminotomatidine hydrochloride (14)
Following procedure D, 130 mg of compound 13 were depro-
tected in quantitative yield (Rf: 0 in 10% MeOH/AcOEt with 0.5%

F. Chagnon et al. / European Journal of Medicinal Chemistry 80 (2014) 605e620
615
NEt3). The crude compound was purified preparative HPLC to yield
1H NMR (300 MHz, CD3OD)
d
(ppm) 4.37 (m, 1H), 3.39 (m, 1H),
48 mg (38%) of desired compound. The HPLC elution parameters
were as follow (percentage of acetonitrile in water): 0 min, 20%;
2 min, 20%; 27 min, 95%; 32 min, 95%.
3.16e3.05 (m, 1H) 3.04e2.82 (m, 6H), 2.20 (t, 1H, J ¼ 6.7 Hz), 2.08e
1.95 (m, 3H), 1.90e1.82 (m, 3H), 1.80e1.52 (m, 15H), 1.50e1.16 (m,
18H), 1.09 (d, 3H, J ¼ 6.4 Hz), 1.06e0.99 (m, 3H), 0.96 (d, 3H,
J ¼ 6.8 Hz), 0.90 (s, 3H), 0.88 (s, 4H), 0.75e0.60 (m, 1H).
1H NMR (300 MHz, CD3OD)
d (ppm) 4.36 (m, 1H), 3.18e3.03 (m,
2H), 2.89 (t, 1H, J ¼ 10.7 Hz) 2.20 (m, 1H), 2.08e1.90 (m, 3H), 1.80e
1.50 (m, 18H), 1.45e1.14 (m, 12H) 1.09 (d, 3H, J ¼ 7.1 Hz), 0.69 (d, 3H,
J ¼ 6.7 Hz), 0.89 (s, 3H), 0.85 (s, 4H), 0.83e0.66 (m, 2H).
13C NMR (75.5 MHz, CD3OD)
d (ppm) 96.1, 81.0, 61.6, 57.0, 55.5,
53.8, 53.5, 44.5, 44.2, 40.8, 39.5, 38.8, 35.6, 34.7, 31.5, 31.3, 31.2, 30.8,
29.0, 28.2, 27.6, 26.9, 25.9, 25.5, 25.4, 22.2, 20.2, 17.2, 15.8, 13.1, 10.3.
HRMS calculated for C33H61ONþ3 þ: 257.7402, found: 257.7406.
13C NMR (75.5 MHz, CD3OD)
d (ppm) 96.1, 81.1, 61.6, 57.6, 56.3,
55.4, 53.7, 44.5, 41.1, 40.8, 40.7, 39.5, 38.6, 35.7, 35.3, 34.8, 31.7, 31.4,
30.7, 28.9, 28.0, 25.8, 25.3, 24.4, 20.6, 17.2, 15.9, 11.1, 10.4.
HRMS calculated for C29H52ONþ3 : 458.4105, found: 458.4105
(MHþ).
6.2.20. (25S)-26-Amino-3b,16b-dihydroxy-5a-cholestano-22,26-
piperidine (19)
Tomatidine hydrochloride (50 mg, 0.11 mmol) was dissolved in
20 mL glacial acetic acid. Platinum oxide (50 mg) was added and
the reaction was stirred under hydrogen atmosphere at 200 PSI
overnight. Reaction mixture was then filtered on CeliteÒ and
concentrated in vacuo (Rf: 0.67 in 10% MeOH/AcOEt with 0.5% NEt3).
The crude compound was purified by flash chromatography (50%
EtOAc/Hexanes, with 25% AcOEt step increase to 100% AcOEt, then
5% MeOH step increase to 90% EtOAc/MeOH) to yield 27 mg (54%) of
compound 19 as a white solid (m.p. 188 ꢀC with decomposition).
6.2.16. N-Formyl-3-(N-Boc-aminobutyl)aminotomatidine (15)
Following the procedure used for synthesis of 11, 27 mg 6
(0.060 mmol), 38 (57 mg, 0.30 mmol, 5 eq) and sodium cyano-
borohydride (4.1 mg, 0.066 mmol, 1.1 eq) were used to yield 16 mg
(44%) of compound 15 (Rf: 0.07 in 10% MeOH/AcOEt with 0.5%
NEt3).
1H NMR (300 MHz, CDCl3)
d (ppm) 8.40 (s, 1H), 5.02 (broad d,
1H), 4.28 (d, 1H, J ¼ 11.5 Hz), 4.12 (m, 1H), 3.14e3.04 (s large, 3H),
2.80e2.50 (m, 5H), 2.42 (m, 1H), 2.04e1.93 (m, 2H), 1.86 (d, 1H,
J ¼ 12.8 Hz), 1.79e1.62 (m, 5H), 1.60e1.46 (m, 12H), 1.42 (s, 12H),
1.35e1.10 (m, 10H), 1.04 (d, 5H, J ¼ 6.9 Hz), 0.90 (d, 4H, J ¼ 5.8 Hz),
0.81 (s, 3H), 0.78 (s, 3H), 0.69e0.57 (m, 1H).
1H NMR (300 MHz, CDCl3)
d (ppm) 4.22e4.14 (m, 1H), 3.52e3.42
(m, 1H), 3.38 (d, 1H, J ¼ 12.8 Hz), 3.14 (m, 1H), 2.65 (t, 1H,
J ¼ 12.8 Hz), 2.34e2.11 (m, 3H), 2.00e1.87 (m, 7H), 1.75e1.61 (m,
5H), 1.56e1.41 (m, 5H) 1.40e1.05 (m, 16H), 1.01 (d, 3H, J ¼ 7.2 Hz),
0.96 (d, 4H, J ¼ 6.7 Hz), 0.90 (broad s, 5H) 0.82 (s, 3H), 0.70e0.59 (m,
1H).
13C NMR (75.5 MHz, CDCl3)
d (ppm) 70.3, 70.1, 59.6, 57.0, 54.2,
6.2.17. 3-(N-Aminobutyl)-aminotomatidine hydrochloride (16)
Following procedure D, 16 mg of compound 15 were depro-
tected in quantitative yield (Rf: 0 in 10% MeOH/AcOEt with traces if
NEt3). The crude compound was purified by preparative HPLC to
yield 15 mg (100%) of desired compound. The HPLC elution pa-
rameters were as follow (percentage of acetonitrile in water):
0 min, 5%; 2 min, 5%; 27 min, 95%; 32 min, 95%.
53.9, 51.0, 44.7, 42.4, 39.9, 37.4, 36.7, 35.1, 32.7, 31.7, 31.1, 30.6, 28.9,
28.4, 22.3, 20.6, 17.3, 17.3, 12.0, 11.6, 11.3.
HRMS calculated for C27H48O2Nþ: 418.3680, found: 418.3677
(MHþ).
6.2.21. 3a-Acetoxy-N-acetyltomatidine (20)
1H NMR (300 MHz, CD3OD)
d
(ppm) 4.37 (q, 1H, J ¼ 4.8 Hz), 3.38
Compound 20 was synthesized as reported by to Sato et al. [37].
The compound was purified by flash chromatography (25% AcOEt/
Hexanes) (Rf: 0.6 in 50% AcOEt/Hexanes).
(broad s, 1H), 3.18e3.15e2.87 (m, 7H), 2.20 (t, 1H, J ¼ 6.5 Hz), 2.08e
1.82 (m, 6H) 1.82e1.63 (m, 11H), 1.61e1.50 (m, 5H), 1.44e1.14 (m,
12H), 1.08 (d, 4H, J ¼ 7.4 Hz), 0.96 (d, 3H, J ¼ 6.5 Hz), 0.89 (s, 3H),
0.87 (s, 4H), 0.79e0.69 (m, 1H).
1H NMR (300 MHz, CDCl3)
d (ppm) 4.67 (m, 1H), 4.15 (m, 1H),
4.45e3.26 (m, 2H), 3.14e3.04 (m, 1H), 2.09 (s, 3H), 2.01 (s, 3H)
2.00e1.93 (m, 1H) 1.84e1.55 (m, 8H), 1.52e1.40 (m, 4H), 1.37e1.23
(m, 5H), 1.19 (d, 3H, J ¼ 6.7 Hz), 1.10e0.94 (m, 4H), 0.86 (d, 3H,
J ¼ 6.0 Hz), 0.82 (s, 3H), 0.81 (s, 3H), 0.68e0.57 (m, 1H).
13C NMR (75.5 MHz, CD3OD)
d (ppm) 96.5, 80.7, 61.8, 57.1, 55.4,
54.9, 53.8, 45.2, 44.6, 43.6, 41.0, 39.6, 38.6, 36.2, 35.3, 34.8, 31.7, 31.5,
30.8, 29.1, 28.5, 28.0, 26.1, 25.4, 24.5, 24.3, 22.7, 22.3, 20.6, 17.4, 15.9,
13.4, 11.0.
HRMS calculated for C31H57ON23þ: 243.7245, found: 243.7253.
6.2.22. (25S)-3b-Acetoxy-27-acetylamino-5a-furost-20(22)-ene
(21)
6.2.18. N-Formyl-3-(aminohexyl)aminotomatidine (17)
Compound 21 was synthesized as reported by to Sato et al. [37]
(Rf: 0.45 in 10% MeOH/AcOEt).
Following the procedure used for the synthesis of 11, 20 mg 6
(0.045 mmol), hexamethylenediamine (30
mL, 0.225 mmol, 5 eq)
1H NMR (300 MHz, CDCl3)
d (ppm) 5.58 (broad s, 1H), 4.75e4.60
and sodium cyanoborohydride (3.2 mg, 0.050 mmol, 1.1 eq) were
used to yield 11 mg (46%) of compound 17 (Rf: 0 in 10% MeOH/
AcOEt with 0.5% NEt3).
(m, 2H), 3.12 (t., 2H, J ¼ 6.4 Hz), 2.45 (d,1H, J ¼ 10 Hz), 2.19e2.05 (m,
3H), 2.01 (s, 4H), 1.97 (s, 4H), 1.86e1.60 (m, 8H) 1.56 (s, 3H), 1.55e
1.44 (m, 5H), 1.42e1.11 (m, 12H), 1.07e0.94 (m, 4H), 0.90 (d, 3H,
J ¼ 6.8 Hz), 0.83 (s, 4H), 0.71e0.66 (m, 1H), 0.65 (s, 3H).
1H NMR (300 MHz, CDCl3)
d (ppm) 4.32e4.25 (m, 1H), 4.18e4.08
(m, 1H), 3.08 (broad s, 3H), 2.74e2.49 (m, 5H), 2.03e1.93 (m, 2H)
1.91e1.81 (m, 1H), 1.80e1.70 (m, 2H), 1.70e1.41 (m, 11H), 1.39e1.09
(m, 14H), 1.05 (d, 3H, J ¼ 7.0 Hz), 0.90 (d, 3H, J ¼ 5.4 Hz), 0.87e0.82
(m, 2H), 0.82 (s, 3H), 0.80 (s, 3H), 0.69e0.59 (m, 1H).
6.2.23. (25S)-26-Acetylamino-3b-hydroxy-5a-furost-20(22)-ene
(22)
In a 10 mL round flask, compound 21 (10 mg, 0.02 mmol, 1.0 eq)
was dissolved in 3 mL anhydrous methanol along with sodium
methoxide (1.4 mg, 0.024 mmol, 1.2 eq). The reaction was stirred at
room temperature overnight until completion as judged by TLC (Rf:
0.28 in 10% MeOH/AcOEt). Solvents were removed in vacuo, and the
crude compound was purified by flash chromatography (50%
EtOAc/Hexanes with triethylamine with 25% step increase of AcOEt
to 100% EtOAc with 0.5% triethylamine) to yield 4.3 mg (46%) of
compound 22.
6.2.19. 3-(N-Aminohexyl)-aminotomatidine hydrochloride (18)
Following procedure D,11 mg of compound 17 were deprotected
in quantitative yield (Rf: 0 in 10% MeOH/AcOEt with 0.5% NEt3). The
crude compound was purified by preparative HPLC to yield 12.5 mg
(100%) of compound 18. The HPLC elution parameters were as
follow (percentage of acetonitrile in water): 0 min, 20%; 2 min, 20%;
27 min, 95%; 32 min, 95%.

616
F. Chagnon et al. / European Journal of Medicinal Chemistry 80 (2014) 605e620
1H NMR (300 MHz, CDCl3)
d
(ppm) 5.56 (broad s, 1H), 4.76e4.67
product was suspended in aqueous NaHCO3 then extracted 3ꢄ with
(m, 1H), 3.59 (sept, 1H, J ¼ 4.8 Hz), 3.12e3.09 (t, 2H, J ¼ 5.9 Hz), 2.45
(d, 1H, J ¼ 10.8 Hz), 2.20e2.06 (m, 3H), 1.98 (s, 3H), 1.84e1.63 (m,
4H) 1.56 (s, 5H), 1.54e1.43 (m, 4H), 1.42e1.33 (m, 3H), 1.32e1.21 (m,
5H) 1.15e0.94 (m, 4H), 0.90 (d, 3H, J ¼ 6.8 Hz), 0.82 (s, 4H), 0.65 (s,
4H).
EtOAc. The organic phases were dried on anhydrous magnesium
sulfate, concentrated in vacuo, and the crude product was purified
on flash chromatography (100% EtOAc) to yield 5 mg (31%) of
compound 26.
1H NMR (300 MHz, CDCl3)
d (ppm) 5.60 (s large, 1H), 5.09e5.01
13C NMR (75.5 MHz, CDCl3)
d
(ppm) 170.0,151.3, 104.1, 84.4, 71.3,
(m, 1H), 4.68 (sept, 1H, J ¼ 5.4 Hz), 3.4 (d, 1H, J1 ¼8.5 Hz), 3.28e3.05
(m, 2H), 2.41e2.30 (m, 1H), 2.23e2.06 (m, 2H), 2.03 (s, 3H), 2.01 (s,
3H), 2.00 (s, 3H),1.97e1.32 (m,18H) 1.30e0.99 (m,14H), 0.95 (d, 4H,
J ¼ 6.5 Hz), 0.92 (d, 5H, J ¼ 6.6 Hz), 0.87 (s, 3H), 0.82 (s, 4H), 0.71e
0.61 (m, 1H).
64.3, 54.8, 54.3, 45.3, 44.8, 43.6, 39.7, 38.2, 37.0, 35.6, 34.9, 34.0,
32.8, 32.4, 31.6, 31.5, 28.6, 23.4, 23.1, 21.2, 17.5, 14.2, 12.4, 11.6.
HRMS calculated for C29H47O3NNaþ: 480.3448, found: 480.3457.
6.2.24. (25S)-26-Acetylamino-3b-hydroxy-5a-furostane (23)
Compound 22 (40 mg, 0.089 mmol, 1.0 eq) was dissolved in
25 mL ethanol (95%) along with 0.5 mL HCl and 10 mg palladium on
activated charcoal (10% w/w). The reaction was stirred under
hydrogen atmosphere (750 PSI) at room temperature overnight.
TLC showed the desired compound in small proportions (Rf: 0.43 in
10% MeOH/AcOEt). The reaction mixture was filtered on CeliteÒ,
concentrated in vacuo, and the crude compound was purified by
flash chromatography (100% EtOAc) to yield 5 mg (12%) of com-
pound 23.
6.2.28. (25S)-26-Acetylamino-3b,16b,20-trihydroxy-5a-cholestane
(27)
Following procedure D, 26 (5 mg, 0.008 mmol), was converted
into 4.2 mg (quantitative yield) of compound 27 (Rf: 0.45 in 10%
MeOH/AcOEt).
1H NMR (300 MHz, CD3OD)
d
(ppm) 4.49 (q, 1H, J ¼ 6.4 Hz),
4.05e4.00 (m, 1H), 3.53e3.47 (m, 1H), 3.13e2.96 (m, 2H), 2.31e2.13
(m, 3H), 2.02e1.96 (m, 2H), 1.93 (s, 2H), 1.90 (s, 1H), 1.80e1.48 (m,
11H), 1.44e1.36 (m, 6H), 1.34e1.20 (m, 16H), 1.17e1.10 (m, 5H),
1.02e0.97 (m, 2H), 0.94e0.88 (m, 8H), 0.85 (s, 3H), 0.84 (m, 3H),
0.72e0.65 (m, 1H).
1H NMR (300 MHz, CDCl3)
d (ppm) 5.59 (broad s, 1H), 4.21e4.14
(m, 1H), 3.88 (dt, 1H, J1 ¼ 10.0 Hz, J2 ¼ 3.8 Hz), 3.58 (sept, 1H,
J ¼ 5.5 Hz), 3.23e3.03 (m, 2H), 2.75e2.61 (m, 1H), 2.04e1.99 (m,
1H), 1.97 (s, 3H) 1.95e1.75 (m, 3H), 1.74e1.62 (m, 3H), 1.56e1.32 (m,
7H), 1.31e1.22 (m, 6H), 1.16e1.11 (m, 2H) 1.08 (d, 3H, J ¼ 7.8 Hz),
0.98 (s, 3H), 0.91 (d, 3H, J ¼ 6.4 Hz), 0.81 (s, 3H), 0.65e0.54 (m, 1H).
13C NMR
d (ppm) 85.3, 82.3, 71.2, 65.2, 56.0, 55.2, 45.9, 45.6, 40.2,
38.3, 37.4, 35.4, 33.8, 33.6, 32.8, 32.5, 31.7, 31.5, 31.2, 30.1, 29.8, 29.5,
27.8, 26.7, 21.9, 21.5, 20.3, 17.3, 15.8, 14.7, 12.1.
HRMS calculated for C29H52O4NNaþ: 500.3716, found: 500.3714.
13C NMR (75.5 MHz, CDCl3)
d (ppm) 83.3, 83.1, 71.3, 61.2, 58.0,
54.3, 45.4, 44.9, 40.2, 38.2, 37.5, 37.0, 34.9, 33.4, 32.3, 32.1, 32.0, 31.5,
28.6, 28.2, 20.7, 17.8, 16.9, 12.4, 11.3.
6.2.29. 3
b
-Hydroxy-5
a
-pregnan-20-one (29)
HRMS calculated for C29H50O3Nþ: 460.3785, found: 460.3801.
In a 500 mL round bottom flask, 3
b
-acetoxy-5a-pregnan-20-one
(49) (5 g, 13.9 mmol) was dissolved in 400 mL of MeOH,150 mL H2O
and 30 mL NaOH 1 M. The mixture was refluxed for 3 h (Rf: 0.27 in
50% AcOEt/hexanes). The organic solvents were removed under
reduced pressure and a suspension could be observed in the
remaining aqueous phase. The solid was isolated by filtration,
rinsed with cold water and dried overnight at room temperature to
yield 4.34 g (98%) of desired compound.
6.2.25. (25S)-26-Amino-3b,16b-acetoxy-N-acetyl-5a-cholest-22-
eno-22,26-piperidine (24)
Compound 24 was synthesized as reported by Sato et al. [37].
The compound was purified by flash chromatography (25% AcOEt/
Hexanes) (Rf: 0.45 in 50% AcOEt/Hexanes).
1H NMR (300 MHz, CDCl3)
d
(ppm) 5.19 (t, 1H, J ¼ 3.6 Hz), 4.94 (s
1H NMR (300 MHz, CDCl3)
d
(ppm) 3.60 (m, 1H, J ¼ 4.5 Hz), 2.52
large,1H), 4.66 (sept,1H, J1 ¼4.8 Hz), 2.36e2.17 (m, 2H), 2.10 (s, 3H),
2.01 (s, 3H), 1.96 (s, 3H) 1.84e1.32 (m, 12H), 1.27 (d, 3H, J ¼ 7.2 Hz),
1.24e1.12 (m, 5H), 1.05e0.94 (m, 3H), 0.92 (d, 3H, J ¼ 6.8 Hz), 0.82
(s, 3H), 0.80 (s, 3H), 0.64 (dt, 1H, J1 ¼ 10.5 Hz, J2 ¼ 3.8 Hz).
(t, 1H, J ¼ 8.8 Hz), 2.22e2.12 (m, 1H), 2.11 (s, 1H), 2.04e1.96 (m, 1H),
1.86e1.76 (m, 1H), 1.75e1.52 (m, 7H), 1.49e1.06 (m, 12H), 1.04e0.84
(m, 3H), 0.81 (s, 3H), 0.68 (dt, 1H, J1 ¼ 10.4 Hz, J2 ¼ 3.8 Hz), 0.60 (s,
3H).
13C NMR (75.5 MHz, CDCl3)
d (ppm) 209.7, 71.2, 63.8, 56.7, 54.2,
6.2.26. (25S)-26-Acetylamino-3b,16b-diacetoxy-5a-cholest-22-one
(25)
44.8, 44.3, 39.1, 38.2, 37.0, 35.5, 32.0, 31.4, 31.0, 28.6, 24.4, 22.8, 21.3,
13.5, 12.3.
Compound 25 was synthesized as reported by Sato et al. [37] (Rf:
HRMS calculated for C21H34O2: 318.2559, found: 318.2552.
0.5 in 10% MeOH/AcOEt).
1H NMR (300 MHz, CDCl3)
d
(ppm) 5.91 (t, 1H, J ¼ 5.4 Hz), 4.97
(dt, 1H, J1 ¼8.2 Hz, J2 ¼ 3.9 Hz), 4.67 (sept, 1H, J ¼ 5.3 Hz), 3.15e3.01
(m, 2H), 2.96e2.86 (m, 1H), 2.75e2.63 (m, 1H), 2.40e2.22 (m, 2H),
2.01 (s, 3H), 1.99 (s, 3H), 1.94 (s, 3H), 1.82e1.76 (m, 3H), 1.70 (dt, 1H,
J1 ¼13.4 Hz, J2 ¼ 4.2 Hz),1.65e1.48 (m, 6H),1.47e1.31 (m, 4H),1.29e
1.17 (m, 5H), 1.12 (d, 1H, J ¼ 7.0 Hz), 1.07e0.95 (m, 4H), 0.88 (d, 3H,
J ¼ 6.5 Hz), 0.83 (s, 3H), 0.83 (s, 3H), 0.73e0.62 (m, 1H).
6.2.30. 3
b
-t-Butyldimehylsilyloxy-5a-pregnan-20-one (30)
In a 250 mL round bottom flask, 3
b
-hydroxy-5 -pregnan-20-one
a
29 (4.34 g, 13.6 mmol) was dissolved in 120 mL THF. Imidazole
(2.3 g, 34 mmol, 2.5 eq), t-butyldimethylsilyl chloride (2.56 g,
17 mmol, 1.25 eq) and diisopropylethylamine (4.7 mL, 27.2 mmol,
2.0 eq) were successively added then the reaction was stirred
overnight at room temperature (Rf: 0.39 in 50% AcOEt/hexanes).
The brown mixture was concentrated under reduced pressure then
diluted in EtOAc. The mixture was washed with water, 2ꢄ sat. so-
dium bicarbonate, 2ꢄ brine, and dried over anhydrous magnesium
sulfate. The organic solvents were removed in vacuo. The crude
product was purified by flash chromatography (25% EtOAc/Hex-
anes) to yield 5.26 g (89%) of the desired compound as a white solid
(120e123 ꢀC).
6.2.27. (25S)-26-Acetylamino-3b,16b-diacetoxy-22-hydroxy-5a-
cholestane (26)
In a 2 mL vial, 25 (16 mg, 0.028 mmol, 1.0 eq) was dissolved in
0.3 mL of a solution of THF and MeOH (30 mL THF and 0.125 mL
MeOH, for 1.1 eq of MeOH in reaction). The reaction was cooled
down to 0 ꢀC then LiBH4 (0.7 mg, 0.031 mmol, 1.1 eq) was added.
After 1 h, 1 mg of LiBH4 was added and the reaction was allowed to
stir overnight at room temperature (Rf: 0.32 in 10% MeOH/AcOEt).
The reaction was then quenched with acetone for 30 min, then with
water for 15 min. Solvents were removed in vacuo and the crude
1H NMR (300 MHz, CDCl3)
d
(ppm) 3.54 (sept, 1H, J ¼ 5.8 Hz),
2.51 (t, 1H, J ¼ 9.4 Hz), 2.21e2.11 (m, 1H), 2.10 (s, 3H), 2.05e1.96 (m,
1H), 2.72e1.54 (m, 8H), 1.49e1.01 (m, 12H), 1.00e0.90 (m, 2H), 0.88

F. Chagnon et al. / European Journal of Medicinal Chemistry 80 (2014) 605e620
617
(s, 10H), 0.79 (s, 3H), 0.66 (dt, 1H, J1 ¼ 11.6 Hz, J2 ¼ 4.8 Hz), 0.59 (s,
3H), 0.05 (s, 6H).
1H NMR (300 MHz, CDCl3)
d
(ppm) 5.28e5.11 (broad s, 1H), 3.13
(q, 2H, J ¼ 5.7 Hz), 2.76 (t, 2H, J ¼ 5.7 Hz), 2.47e2.20 (broad s, 2H),
1.38 (s, 9H).
6.2.31. 3b-t-Butyldimethylsilyloxy-21-bromo-5a-pregnan-20-one
(31)
6.2.35. N-Boc-1,3-diaminopropane (37)
In a 50 mL round bottom flask under argon atmosphere, com-
pound 30 (500 mg, 1.15 mmol) was cooled to ꢁ78 ꢀC in anhydrous
THF. Potassium bis(trimethylsilyl)amide (1 M in THF; 1.27 mL,
1.27 mmol, 1.1 eq) was added and the mixture was stirred for
Following procedure A, 970
yield 100 mg (50%) of desired product as a colorless oil.
1H NMR (300 MHz, CDCl3)
(ppm) 5.16 (broad s, 1H), 3.60
mL diaminopropane was used to
d
(broad s, 2H), 3.20 (q, 2H, J ¼ 6.1 Hz), 2.83 (t, 2H, J ¼ 6.1 Hz), 1.69
15 min. Trimethylsilyl chloride (150
mL, 1.15 mmol, 1.0 eq) was
(quint, 2H, J ¼ 6.1 Hz), 1.42 (s, 9H).
added and the mixture was stirred for 1 h at room temperature
while monitoring by TLC (50% EtOAc/Hexanes, UV/CAM, Rf: 0.35:
starting material, 0.77: silylated compound). The reaction was
cooled to ꢁ78 ꢀC before addition of N-bromosuccinimide (204 mg,
1.15 mmol, 1.0 eq). After 1 h of stirring at ꢁ78 ꢀC, monitored by TLC
(same conditions as above, Rf ¼ 0.52 for brominated compound),
the reaction was quenched with saturated aqueous sodium bicar-
bonate and THF was evaporated under reduced pressure. The crude
compound was suspended in water and the solution was extracted
with 3ꢄ EtOAc. The combined organic layers are washed with
brine, dried on anhydrous magnesium sulfate and concentrated in
vacuo. The compound was purified by flash chromatography (2%
EtOAc/Hexanes to 6% EtOAc/Hexanes) to yield 510 mg (87%) of
desired compound.
6.2.36. N-Boc-1,4-diaminobutane (38)
Following procedure A, 1.16 mL diaminobutane was used to
yield 171 mg (79%) of desired product as a colorless oil.
1H NMR (300 MHz, CDCl3)
d
(ppm) 4.94 (broad s, 1H), 3.04
(broad s, 4H), 2.69 (broad s, 2H), 1.45 (broad s, 4H), 1.36 (s, 9H).
6.2.37. 3b-Acetoxy-20-(Boc-aminoethyl)amino-5a-pregnane (39)
Following procedure B, 100 mg 36 (0.62 mmol, 1.17 eq) was used
to yield 100 mg (41%) of desired product (Rf: 0 in 25% AcOEt/
hexanes).
1H NMR (300 MHz, CDCl3)
d (ppm) 5.02 (broad s, 1H), 3.55 (m,
1H, J ¼ 4.9 Hz), 3.27e3.05 (m, 2H), 2.85e2.68 (m, 1H), 2.65e2.45
(m, 2H), 2.03e1.92 (m, 1H), 1.88e1.51 (m, 10H), 1.47e1.35 (broad s,
11H), 1.33e1.02 (m, 16H), 0.98 (d, 4H, J ¼ 9.2 Hz), 0.94e0.82 (m, 3H),
0.80 (s, 3H), 0.69 (s, 3H), 0.65e0.55 (m, 1H).
1H NMR (300 MHz, CDCl3)
d
(ppm) 3.87 (d, 2H, J ¼ 2.7 Hz), 3.51
(sept, 1H, J ¼ 5.3 Hz), 2.78 (t, 1H, J ¼ 8.9 Hz), 2.14 (q, 1H, J ¼ 9.2 Hz),
1.87 (dt, 1H, J1 ¼ 11.7 Hz, J2 ¼ 2.7 Hz), 1.73e1.52 (m, 6H), 1.46e1.11
(m, 11H), 1.08e0.88 (m, 2H), 0.85 (s, 10H), 0.76 (s, 3H), 0.63 (dt, 1H,
J1 ¼ 12.1 Hz, J2 ¼ 2.9 Hz), 0.59 (s, 3H), 0.01 (s, 6H).
6.2.38. 3b-Acetoxy-20-(Boc-aminopropyl)amino-5a-pregnane (40)
Following procedure B, 230 mg of 37 (0.605 mmol, 2.4 eq) was
used to yield 132 mg (50%) of desired product (Rf: 0 in 75% AcOEt/
hexanes).
6.2.32. 3b-Hydroxy-21-(N,N-dimethyl) amino-5a-pregnan-20-one
(32)
Following procedure C, 125 mg of 31 (0.24 mmol) were used to
give 68 mg of silylated intermediate. Upon deprotection, 20 mg of
compound 32 (38% overall yield) were obtained as a white solid
(m.p. 105e107 ꢀC) (Rf: 0.13 in 100% AcOEt with 0.5% NEt3).
1H NMR (300 MHz, CDCl3)
d (ppm) 5.60 (broad s, 0.2H) 5.07
(broad s, 0.6H), 4.65 (sept, 1H, J ¼ 5.1 Hz), 3.16 (sept, 1H, J ¼ 3.6 Hz),
1.99 (s, 3H), 1.98e1.84 (m, 2H), 1.84e1.51 (m, 7H), 1.41 (s, 9H), 1.36e
1.08 (m, 9H), 1.06 (d, 1H, J ¼ 6.1 Hz), 1.03e0.97 (m, 2H), 0.95 (d, 2H,
J ¼ 6.1 Hz), 0.79 (s, 3H), 0.66 (s, 2H), 0.63 (s, 1H), 0.62e0.57 (m, 1H).
1H NMR (300 MHz, CDCl3)
d
(ppm) 3.58 (sept, 1H, J ¼ 5.2 Hz),
3.13 (dd, 2H, J1 ¼ 21.4 Hz, J2 ¼ 21.7 Hz), 2.55 (t, 1H, J ¼ 8.8 Hz), 2.28
(s, 6H), 2.16 (d, 1H, J ¼ 9.3 Hz), 1.91e1.75 (m, 4H), 1.74e1.52 (m, 7H),
1.43e1.20 (m, 8H), 1.20e1.04 (m, 3H), 1.03e0.83 (m, 3H), 0.79 (s,
1H), 0.72e0.61 (m, 1H), 0.60 (s, 3H).
6.2.39. 3b-Acetoxy-20-(Boc-aminobutyl)amino-5a-pregnane (41)
Following procedure B, 315 mg of 38 (1.67 mmol, 3.0 eq) was
used to yield 198 mg (73%) of desired product (Rf: 0 in 50% AcOEt/
hexanes).
13C NMR (75.5 MHz, CD3OD)
d (ppm) 206.5, 70.3, 60.3, 56.6, 55.9,
54.1, 44.7, 43.8, 38.7, 38.2, 37.5, 36.8, 35.4, 31.8, 30.6, 29.5, 28.5, 24.1,
23.4, 22.4, 20.9, 12.4, 11.3.
1H NMR (300 MHz, CDCl3)
d (ppm) 4.92 (broad d), 4.65 (sept, 1H,
HRMS calculated for C23H39O2N: 362.3059, found: 362.3059.
J ¼ 6.2 Hz), 3.09 (broad s, 2H), 2.78e2.52 (m, 2H), 2.52e2.38 (m,
1H), 1.99 (s, 3H), 1.95e1.84 (m, 1H), 1.82e1.45 (m, 12H), 1.41 (s, 10H),
1.35e1.10 (m 10H), 1.10e1.05 (m, 4H) 0.97 (d, 3H, J ¼ 5.7 Hz), 0.91e
0.81 (m, 1H), 0.76 (s, 3H), 0.66 (s, 2H), 0.63 (s, 1H), 0.61e0.56
(m, 1H).
6.2.33. 3b-Hydroxy-21-piperidino-5a-pregnan-20-one (33)
Following procedure C, 100 mg of 31 (0.195 mmol) were used to
give 80 mg of silylated intermediate. Upon deprotection, 46 mg of
compound 33 (74% overall yield) were obtained (Rf: 0.16 in 100%
AcOEt with 0.5% NEt3).
6.2.40. 3b-Hydroxy-20(aminoethyl)amino-5a-pregnane
1H NMR (300 MHz, CD3OD)
d
(ppm) 3.56 (sept, 1H, J ¼ 4.8 Hz),
hydrochloride (42)
3.08 (s, 2H), 2.58 (t, 1H, J ¼ 9.8 Hz), 2.37 (s large, 4H), 2.13 (d, 1H,
J ¼ 10.3 Hz), 1.86 (dt, 2H, J1 ¼ 11.1 Hz, J2 ¼ 3.9 Hz), 1.78 (d, 1H,
J ¼ 11.1 Hz), 1.72e1.48 (m, 10H), 1.45e1.19 (m, 10H), 1.18e1.03 (m,
3H), 1.01e0.83 (m, 3H), 0.77 (s, 3H), 0.70e0.62 (m, 1H), 0.58 (s, 3H).
Following procedure D. 100 mg 39 (0.216 mmol) was used to
yield 85 mg (96%) of desired compound as a white solid (m.p.
240 ꢀC with decomposition) (Rf: 0 in 10% MeOH/AcOEt with 0.5%
NEt3).
13C NMR (75.5 MHz, CD3OD)
d (ppm) 71.3, 60.2, 56.8, 54.7, 54.2,
1H NMR (300 MHz, CD3OD)
d (ppm) 3.48 (m, 1H), 3.43e3.35 (m,
44.8, 39.0, 38.2, 37.0, 35.5, 34.5, 32.1, 31.5, 28.6, 25.7, 24.6, 23.9, 23.0,
21.3, 13.7, 12.4.
3H), 2.00e1.45 (m,10H),1.40e1.20 (m,10H),1.18e0.87 (m, 5H), 0.82
(s, 3H), 0.76 (s, 4H).
13C NMR (75.5 MHz, CD3OD)
d (ppm) 70.3, 58.6, 55.9, 55.4, 55.0,
HRMS calculated for C26H43O2N: 402.3372, found: 402.3380.
54.4, 54.0, 53.0, 52.0, 44.8, 44.7, 42.8, 42.4, 40.9, 39.1, 37.4, 36.8,
35.5, 35.2, 31.7, 30.7, 28.4, 23.5, 22.8, 20.7, 20.5, 14.9, 14.6, 11.7, 11.3.
HRMS calculated for C23H42ONþ2 : 362.3370, found: 363.3377
(MHþ).
6.2.34. N-Boc-1,2-diaminoethane (36)
Following procedure A, 770
mL diaminoethane was used to yield
76 mg (41%) of desired product as a colorless oil.

618
F. Chagnon et al. / European Journal of Medicinal Chemistry 80 (2014) 605e620
6.2.41. 3
b-Hydroxy-20(aminopropyl)amino-5a-pregrane
1H NMR (300 MHz, CDCl3)
d (ppm) 5.62 (s large, 2H), 4.65 (sept,
hydrochloride (43)
1H, J ¼ 6.2 Hz), 3.26e2.57 (m, 2H), 1.99 (s, 5H), 1.85e1.37 (m, 11H),
1.35e1.15 (m, 8H),1.12 (d, 2H, J ¼ 6.3 Hz),1.09e0.96 (m, 3H), 0.92 (d,
1H, J ¼ 6.3 Hz), 0.89 (d, 6H, J ¼ 6.4 Hz), 0.790 (s 3H), 0.69e0.55 (m,
4H).
Following procedure D, 132 mg 40 (0.277 mmol) was used to
yield 125 mg (100%) of desired compound as a white solid (m.p.
205 ꢀC with decomposition) (Rf: 0 in 10% MeOH/AcOEt with 0.5%
NEt3).
1H NMR (300 MHz, CD3OD)
d (ppm) 3.45e3.34 (m, 2H), 3.13 (t,
6.2.46. 3b-Hydroxy-20-isopentylamino-5a-pregnane
2H, J ¼ 6.9 Hz), 3.05 (t, 2H, J ¼ 7.9 Hz) 2.09 (m, 2H), 1.97e1.79 (m,
1H), 1.79e1.62 (m, 5H), 1.56e1.45 (m, 2H), 1.43 (m, 4H), 1.40e1.33
(m, 3H), 1.33e1.21 (m, 7H), 1.17e1.04 (m, 3H), 1.03e1.86 (m, 2H),
0.82 (s, 3H), 0.73 (d, 3H, J ¼ 6.1 Hz), 0.71e0.60 (m, 1H).
hydrochloride (48)
Following procedure D, 46 mg of 47 (0.11 mmol), were con-
verted to 48 mg (100%) of desired product (Rf: 0.07 in 10% MeOH/
AcOEt with 0.5% NEt3).
13C NMR (75.5 MHz, CD3OD)
d (ppm) 70.3, 57.8, 55.9, 55.4, 54.4,
1H NMR (300 MHz, CD3OD)
d (ppm) 3.49 (m, 1H), 2.98 (m, 1H),
54.0, 52.7, 51.8, 44.8, 42.7, 42.3, 40.9, 40.1, 39.1, 37.4, 36.8, 35.5, 35.2,
31.7, 30.7, 28.4, 25.9, 23.9, 23.5, 23.0, 20.7, 20.5, 15.1, 14.2, 11.7, 11.3.
HRMS calculated for C24H45ONþ2 : 377.3526, found: 376.3535
(MHþ).
1.89e1.45 (m, 12H), 1.45e1.00 (m, 17H), 0.96 (d, 6H, J ¼ 6.4 Hz),
0.91e0.86 (m, 2H), 0.82 (s, 3H), 0.72 (s, 3H), 0.70e0.63 (m, 2H).
13C NMR (75.5 MHz, CD3OD)
d (ppm) 70.4, 55.6, 54.4, 54.0, 52.0,
44.8, 42.3, 37.5, 37.4, 36.8, 35.2, 34.4, 31.7, 30.7, 28.4, 25.9, 23.5, 23.4,
21.4, 21.2, 20.6, 14.3, 11.5, 11.3.
HRMS calculated for C26H48ONþ: 390.3730, found: 390.3733
(MHþ).
6.2.42. 3
b-Hydroxy-20(aminobutyl)amino-5a-pregrane
hydrochloride (44)
Following procedure D,198 mg 41 (0.40 mmol) was used to yield
186 mg (98%) of desired compound as a white solid (m.p. 255 ꢀC
with decomposition) (Rf: 0 in 10% MeOH/AcOEt with 0.5% NEt3).
6.2.47. 3
b
,21-Diacetoxy-5
a
-pregn-17,20-diene (50)
In a 250 mL round bottom flask 3
b
-acetoxypregnan-20-one 49
1H NMR (300 MHz, CD3OD)
d (ppm) 3.48 (m, 1H), 3.09e2.91 (m,
(4 g, 11 mmol) was dissolved in acetic anhydride (150 mL) along
with 4 g (21 mmol, 1.9 eq) p-toluenesulfonic acid. The mixture was
brought to 150 ꢀC for 4 h (Rf: 0.53 in 50% AcOEt/hexanes). After-
ward, the reaction was concentrated in vacuo then dissolved in
DCM. The resulting mixture was neutralized with 1 M NaOH, then
washed subsequently with water and brine. The solution was dried
on anhydrous magnesium sulfate and solvent was removed in
vacuo to yield 4.42 g (100%) of crude compound as a brown oil.
4H), 2.01e1.63 (m, 11H), 1.61e1.46 (m, 3H), 1.36 (d, 3H, J ¼ 6.2 Hz),
1.32e1.21 (m, 6H), 1.20e1.03 (m, 3H), 1.03e0.86 (m, 2H), 0.82 (d,
3H, J ¼ 3.4 Hz), 0.73 (s, 4H).
13C NMR (75.5 MHz, CD3OD)
d (ppm) 70.3, 57.6, 55.9, 55.5, 54.4,
54.2, 52.8, 51.9, 44.8, 43.2, 42.3, 39.1, 38.6, 37.4, 36.8, 35.2, 31.7, 30.7,
28.4, 25.9, 24.3, 23.5, 23.3, 22.8, 20.7, 20.5, 15.1, 14.2, 11.7, 11.3.
HRMS calculated for C25H47ONþ2 : 391.3683, found: 391.3689
(MHþ).
1H NMR (300 MHz, CDCl3)
d (ppm) 4.73e4.41 (m, 1H), 2.37e2.16
(m, 2H), 2.09 (s, 3H), 2.02 (s, 3H), 1.87 (t, 1H, J ¼ 1.8 Hz), 1.77 (s, 3H),
1.75e1.59 (m, 4H), 1.57 (s, 3H, H2O), 1.54e1.09 (m, 11H), 1.07e0.86
(m 3H), 0.82 (s, 3H), 0.81 (s, 3H), 0.74e0.58 (m, 1H).
6.2.43. 3
Following procedure B, 50 mg of 3
one 49 (0.14 mmol), 137 L (1.39 mmol, 10 eq) of butylamine and
b
-Acetoxy-20-butylamino-5
a
-pregnane (45)
b-acetoxy-5
a-pregnan-20-
m
10 mg of sodium cyanoborohydride (0.15 mmol, 1.1 eq) were con-
verted to 24.2 mg (41%) of desired product (Rf: 0.33 in 10% MeOH/
AcOEt with 0.5% NEt3).
6.2.48. 3
Compound 51 was synthesized as reported by Djerassi et al.
[46].
The compound was obtained as a mixture of desired enone 51
(Rf: 0.69 in 100% AcOEt) and 3 -acetoxy-5 -pregnan-20-one 49 in a
b-Acetoxy-5a-pregn-16-en-20-one (51)
1H NMR (300 MHz, CDCl3)
d
(ppm) 4.67 (sept, 1H, J ¼ 5.9 Hz),
2.75e2.65 (m, 1H), 2.60e2.52 (m, 1H), 2.50e2.41 (m, 1H), 2.01 (s,
3H), 2.00e1.84 (m, 5H), 1.82e1.44 (m, 11H), 1.40e1.10 (m 12H), 1.09
(d, 5H, J ¼ 6.5 Hz), 1.07e0.95 (m, 3H), 0.91 (t, 3H, J ¼ 7.4 Hz), 0.81 (s,
3H), 0.65 (s, 4H).
b
a
1:1 ratio (determined by NMR). The mixture was used as such for
the subsequent reactions.
1H NMR (300 MHz, CDCl3)
d (ppm) 6.68e6.64 (m, 0.5H), 4.65
(sept, 1H, J ¼ 6.2 Hz), 2.35e2.24 (m, 1H), 2.22 (s, 3H), 2.17e2.07 (m,
2H), 1.98 (s, 3H), 1.95e1.58 (m, 6H), 1.52e1.28 (m, 5H), 1.20e1.10 (m,
1H), 1.07e0.93 (m, 2H), 0.87 (s, 3H), 0.85e0.81 (m, 4H), 0.78e0.58
(m, 1H).
6.2.44. 3b-Hydroxy-20-butylamino-5a-pregnane hydrochloride
(46)
Following procedure D, 15 mg of 45 (0.036 mmol), were con-
verted to 13.5 mg (100%) of desired product (Rf: 0.05 in 10% MeOH/
AcOEt with 0.5% NEt3).
6.2.49. 17.3b-Acetoxy-16b-hydroxy-17a-bromo-5a-pregn-16-ene
(52)
1H NMR (300 MHz, CD3OD)
d (ppm) 3.48 (m, 1H), 3.20 (m, 1H),
2.97 (m, 2H), 1.97e1.84 (m, 2H), 1.79e1.07 (m, 23H), 1.01e0.85 (m,
5H), 0.81 (s, 3H), 0.72 (s, 3H), 0.69e0.61 (m, 1H).
Compound 52 was synthesized as reported by Ravindar et al.
[47] (Rf: 0.63 in 100% AcOEt).
13C NMR (75.5 MHz, CD3OD)
d (ppm) 70.4, 57.4, 56.0, 54.1, 52.7,
1H NMR (300 MHz, CDCl3)
d (ppm) 4.88e4.82 (m,1H), 4.61 (sept,
44.7, 42.7, 42.6, 39.1, 37.4, 36.8, 35.2, 35.2, 31.7, 30.7, 28.4, 27.9, 25.8,
23.8, 20.8, 19.5, 15.1, 12.5, 11.3, 10.9.
1H, J ¼ 6.2 Hz), 2.34 (s, 3H), 2.32e2.03 (m, 1H), 1.94 (s, 3H), 1.86e
1.48 (m, 10H), 1.47e1.34 (m, 3H), 1.33e1.10 (m 10H), 1.08 (s, 3H),
1.03e0.80 (m, 4H), 0.78 (s, 3H), 0.73e0.62 (m, 1H).
HRMS calculated for C25H46ONþ: 376.3574, found: 376.3578
(MHþ).
6.2.50. 3b-Acetoxy-16b-hydroxy-5a-pregn-16-ene (53)
6.2.45. 3
Following procedure B, 100 mg of 3
one 49 (0.28 mmol), 200 L (1.72 mmol, 6.2 eq) of isopentyl-
amine and 18 mg of sodium cyanoborohydride (0.29 mmol, 1.05 eq)
were converted to 46 mg (41%) of desired product (Rf: 0.36 in 10%
MeOH/AcOEt with 0.5% NEt3).
b
-Acetoxy-20-isopentylamino-5
a
-pregnane (47)
Compound 53 was synthesized as reported by Ravindar et al.
[47] (Rf: 0.69 in 100% AcOEt).
b
-acetoxy-5 -pregnan-20-
a
m
1H NMR (300 MHz, CDCl3)
d
(ppm) 4.68 (sept, 1H, J ¼ 6.2 Hz),
4.58e4.51 (m, 1H), 2.32e2.22 (m, 2H), 2.20 (s, 3H), 2.02 (s, 3H),
2.00e1.96 (m, 1H), 1.85e0.50 (m, 9H), 1.50e1.10 (m, 8H), 1.08e0.95
(m, 3H), 0.94 (s, 3H), 0.84 (s, 3H), 0.76e0.54 (m, 1H).

F. Chagnon et al. / European Journal of Medicinal Chemistry 80 (2014) 605e620
619
6.2.51. 3
amino-5
Following procedure B, 20 mg of 16-hydroxypregnanolone ac-
etate 53 (0.053 mmol), 40 L (0.32 mmol, 6.0 eq) of dimethylami-
b
-Acetoxy-16
b
-hydroxy-20 (N,N-dimethylaminopropyl)
the Centre de Recherche Cliniques Étienne-Le Bel and the FQRNT-
funded Proteo Network.
a
-pregnane (54)
m
Appendix A. Supplementary data
nopropylamine and 3.6 mg of sodium cyanoborohydride
(0.058 mmol, 1.1 eq) were converted to 10.3 mg (42%) of desired
product (Rf: 0.49 in 10% MeOH/AcOEt with 0.5% NEt3).
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2013.11.019.
1H NMR (300 MHz, CDCl3)
d
(ppm) 4.66 (sept, 1H, J ¼ 4.7 Hz),
4.37 (m, 1H), 3.12 (d quint, 1H, J1 ¼ 47.7 Hz, J2 ¼ 6.8 Hz), 2.58e2.45
(m, 1H), 2.38 (t, 1H, J ¼ 6.3 Hz), 2.21 (s, 6H), 2.00 (s, 3H), 1.97e1.34
(m, 12H), 1.33e1.02 (m, 12H), 0.97 (s, 3H), 0.96e0.85 (m, 3H), 0.82
(s, 0.69e0.57 (m, 2H)).
References
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bugs need drugs: an update on the development pipeline from the Antimi-
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6.2.52. 3b,16b-Dihydroxy-20 (N,N-dimethylaminopropyl)amino-
5
a-pregnane (55)
Following procedure D, 10.3 mg of 54 (0.022 mmol), were con-
verted to 9.4 mg (100%) of desired product as a white solid (m.p.
225 ꢀC with decomposition) (Rf: 0 in 10% MeOH/AcOEt with 0.5%
NEt3).
1H NMR (300 MHz, CD3OD)
d (ppm) 4.38 (m, 1H), 3.73 (m, 1H),
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3.47 (m, 3H), 3.11 (m, 1H), 2.90 (s, 6H), 2.28e2.08 (m, 2H),1.98e1.90
(m, 1H), 1.77e1.53 (m, 6H), 1.48 (d, 4H, J ¼ 6.56 Hz) 1.45e1.15 (m,
11H), 1.06 (s, 3H), 1.04e0.85 (m, 3H), 0.82 (d, 3H, J ¼ 6.8 Hz), 0.75e
0.63 (m, 1H).
13C NMR (75.5 MHz, CD3OD)
d (ppm) 72.2, 70.3, 56.8, 54.7, 54.3,
54.1, 53.9, 44.7, 42.2, 42.1, 42.0, 41.5, 38.8, 37.4, 36.7, 36.4, 35.2, 34.7,
31.6, 30.7, 28.4, 21.1, 20.5, 14.8, 13.1, 11.3.
HRMS calculated for C26H46O2Nþ2 : 421.3789, found: 421.3793.
6.2.53. 3b-Acetoxy-16b-hydroxy-20-butylkamino-5a-pregnane
(56)
Following procedure B, 20 mg of 16-hydroxypregnanolone ac-
etate 55 (0.053 mmol), 31 L (0.32 mmol, 6.0 eq) of butylamine and
m
3.6 mg of sodium cyanoborohydride (0.058 mmol, 1.1 eq) were
converted to 15.6 mg (68%) of desired product (Rf: 0.55 in 10%
MeOH/AcOEt with 0.5% NEt3).
1H NMR (300 MHz, CDCl3)
d
(ppm) 4.68 (sept, 1H, J ¼ 4.7 Hz),
4.35e4.15 (m, 1H), 2.32e2.19 (m 1H), 2.10e2.03 (m, 1H), 2.01 (s,
3H), 1.85e1.59 (m, 4H), 1.56 (H2O), 1.53e1.39 (m, 5H), 1.33 (d, 3H,
J ¼ 6.3 Hz),1.32e1.08 (m, 8H), 1.01 (s, 3H), 0.0.98e0.86 (m, 3H), 0.84
(s, 3H), 0.71e0.59 (m, 1H).
[15] S.S. Grant, D.T. Hung, Persistent bacterial infections, antibiotic tolerance, and
the oxidative stress response, Virulence 4 (2013).
6.2.54. 3
hydrochloride (57)
b,16b-Dihydroxy-20-butylamino-5a-pregnane
[16] G. Mitchell, D.L. Seguin, A.E. Asselin, E. Deziel, A.M. Cantin, E.H. Frost,
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d (ppm) 4.23e4.00 (dm, 1H), 3.55e
3.45 (m, 1H), 3.02e2.88 (m, 1H), 2.22e2.08 (m, 2H), 1.77e1.64 (m,
4H), 1.56e1.40 (m, 6H), 1.35e1.25 (m, 8H), 1.22e1.05 (m, 4H), 0.97
(s, 3H), 0.92e0.86 (m, 4H), 0.85 (S, 3H), 0.71e0.60 (m, 1H).
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27.7, 20.4, 19.5, 14.9, 12.5, 11.3.
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Acknowledgments
This study was supported by a team grant from the Fonds
Québécois de Recherche sur la Nature et les Technologies (FQRNT)
to Éric Marsault, Kamal Bouarab and François Malouin. Gabriel
Mitchell was a recipient of doctoral research studentship from
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