Enantioselective synthesis of the ethyl analog of the marine alkaloid haliclorensin C

Article abstract of DOI:10.3390/molecules24061069

The enantioselective synthesis (3.7% overall yield in nine steps from 2) and biological screening of the ethyl analog of the macrocyclic marine alkaloid haliclorensin C (compound 5) are reported. Amino alcohol 3, generated by a LiNH2BH3-promoted reductive ring-opening/debenzylation sequence from phenylglycinol-derived lactam 2, was used as the starting chiral linear building block. Incorporation of the undecene chain via the nosyl derivative 12, methylenation of the pentanol moiety, and a ring-closing metathesis are the key steps of the synthesis.

Full text of DOI:10.3390/molecules24061069

molecules
Article
Enantioselective Synthesis of the Ethyl Analog of the
Marine Alkaloid Haliclorensin C
Guillaume Guignard 1, Núria Llor 1, David Pubill 2 , Joan Bosch 1 and Mercedes Amat 1,
*
1
Laboratory of Organic Chemistry, Department of Pharmacology, Toxicology, and Therapeutic Chemistry,
Faculty of Pharmacy and Food Sciences, and Institute of Biomedicine (IBUB), University of Barcelona,
08028 Barcelona, Spain; guignard.guillaume@gmail.com (G.G.); nllor@ub.edu (N.L.);
joanbosch@ub.edu (J.B.)
2
Pharmacology Section, Department of Pharmacology, Toxicology, and Therapeutic Chemistry, Faculty of
Pharmacy and Food Sciences, and Institute of Biomedicine (IBUB), University of Barcelona, 08028 Barcelona,
Spain; d.pubill@ub.edu
*
Correspondence: amat@ub.edu; Tel.: +34-93-402-4540
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
Academic Editors: David Díez and María Ángeles Castro
Received: 26 February 2019; Accepted: 15 March 2019; Published: 18 March 2019
ꢀꢁꢂꢃꢄꢅꢆ
Abstract: The enantioselective synthesis (3.7% overall yield in nine steps from
2) and biological
screening of the ethyl analog of the macrocyclic marine alkaloid haliclorensin C (compound
are reported. Amino alcohol , generated by a LiNH2BH3-promoted reductive ring-opening/
debenzylation sequence from phenylglycinol-derived lactam , was used as the starting chiral
5)
3
2
linear building block. Incorporation of the undecene chain via the nosyl derivative 12, methylenation
of the pentanol moiety, and a ring-closing metathesis are the key steps of the synthesis.
Keywords: alkaloids; macrocycle; amino alcohols; ring opening; reduction; enantioselective synthesis
1. Introduction
Haliclorensin C (
collected in January 2006 in Salary Bay, Madagascar [
of sponge, only 2 mg (0.002 wt.%) of haliclorensin C (
assigned as 3-methylazacyclohexadecane from its H- and 13C-NMR spectroscopic data, with the
aid of HMBC and COSY experiments, and mass spectrometry data (HREIMS). Based on biogenetic
considerations, the stereogenic center of haliclorensin C was tentatively proposed to possess the
S absolute configuration as previously determined for haliclorensin and halitulin, two related
1
) is a minor marine alkaloid isolated from the sponge Haliclona tulearensis
]. From 86 g of a frozen wet sample
) was isolated. The structure of was
1
1
1
1
azamacrocyclic alkaloids isolated from the same marine sponge [
moderate toxicity in the brine shrimp (Artemia salina) test (LD50 value 2.1 mM) [
2
,
3
]. Haliclorensin C exhibited
], whereas haliclorensin
1
and halitulin were shown to have significant cytotoxicity, the former against P-388 mouse leukemia
cells [4] and the latter against several tumor cell lines [5] (Figure 1).
Figure 1. Azamacrocyclic marine alkaloids isolated from Haliclona tulearensis.
Molecules 2019, 24, 1069; doi:10.3390/molecules24061069
www.mdpi.com/journal/molecules

Molecules 2019, 24, 1069
2 of 9
The proposed structure for haliclorensin C was confirmed in 2014 by total synthesis [
6
], although
comparison of the H- and 13C-NMR spectra of both the base and hydrochloride of the synthetic
product with the spectra reported for the natural product revealed that the isolated sample of
haliclorensin C corresponded to a protonated sample. From the stereochemical standpoint, the
synthesis relies on the use of a chiral amino alcohol building block (fragment N1-C6 of haliclorensin
C) generated from a phenylglycinol-derived oxazolopiperidone lactam that contains the required S
stereocenter at the β carbon to the amino group.
1
In recent years we have reported the preparation of enantiopure five-carbon linear building blocks
from phenylglycinol-derived bicyclic lactam scaffolds and their application to the enantioselective total
synthesis of macrocyclic natural products, such as haliclorensin marine alkaloids [
and callyspongiolide [ ]. Our approach involves the LiNH2BH3-promoted reductive opening of the
oxazolidine and lactam rings of the starting oxazolopiperidone lactam, and the subsequent reductive [
or oxidative [ ] removal of the chiral inductor to give diversely substituted 5-aminopentanols [
5-hydroxypentanoic acids or 5-hydroxypentanenitriles [ ]. Taking into account the availability
of starting lactams with a variety of substitution and stereochemical patterns [10 11], the above
6], fluvirucinin B1 [7],
8
9]
7
9],
7
,
methodology provides easy access to a range of useful functionalized chiral linear building blocks
with potential application in the enantioselective synthesis of natural product analogs.
2. Results and Discussion
We herein present the enantioselective synthesis of the ethyl analog of haliclorensin C
(compound 5) and the results of its in vitro screening in a panel of biological assays. Marine natural
products and their modified derivatives have long been recognized as one of the most important
sources of new biologically active substances and therapeutic agents [12,13].
The assembly of the azacyclohexadecane ring of
metathesis (RCM) reaction of an appropriate N-(hexenyl)undecenylamine
which would be prepared from amino alcohol , easily available from bicyclic lactam
5
would be accomplished by a ring-closing
(bond formed C6-C7),
, by incorporation
4
3
2
of a C11 chain bearing a terminal double bond and subsequent methylenation of the pentanol moiety
(Scheme 1).
Scheme 1. Synthetic strategy.
Lactam
was efficiently converted (75% yield) to amino diol
(LiNH2BH3) [14 15] generated in situ by deprotonation of the commercially available borane-ammonia
2, bearing the required ethyl substituent with the appropriate stereochemistry,
7 [9] by treatment with lithium amidotrihydroborate
,
complex with n-BuLi. This transformation involves an initial hydride attack on the lactam carbonyl,
followed by a Grob-type fragmentation resulting in a double ring-opening by cleavage of the C–N and
C–O bonds, with a final hydride reduction of the resulting imino aldehyde intermediate. Unfortunately,
removal of the chiral inductor of
amino alcohol , afforded the primary amine
compound , the undecene chain was introduced by reductive amination with 10-undecenal. Starting
from either the crude amino alcohol or the O-protected derivative , the corresponding secondary
amines or 10 were obtained, but also in low yield. For this reason, although the amino group of
was satisfactorily protected as the N-Boc derivative 11, this route was abandoned.
7
by hydrogenolysis, followed by silylation of the resulting crude
3
8
in low overall yield (Scheme 2). In a first approach to
5
3
8
9
9

Molecules 2019, 24, 1069
3 of 9
Scheme 2. First approach to the ethyl analog of haliclorensin C.
To overcome the above drawbacks, we then focused on an alternative approach in which the
undecene chain would be introduced by alkylation of the known [ ] nosyl-protected amine 12
with undecenyl bromide. This activated amine was satisfactorily prepared in 76% overall yield
by debenzylation of amino diol by hydrogenolysis, followed by reaction of the resulting crude amino
alcohol with o-nitrobenzenesulfonyl chloride (Scheme 3). The alkylation step was performed in the
6
7
3
presence of Cs2CO3, leading to alcohol 13 in excellent yield (84%). A subsequent Dess-Martin oxidation,
followed by a Wittig methylenation of the resulting aldehyde, afforded N-(hexenyl)undecenylamine 14
in 47% overall yield. Macrocyclization of diene 14 took place satisfactorily by ring-closing metathesis
using the second-generation Grubbs catalyst in refluxing dichloromethane under dilute solvent
conditions, providing a 88:12 diastereoisomeric mixture of E/Z cycloalkenes 15 in 70% yield. Removal
of the nosyl group by treatment with thiophenol, followed by catalytic hydrogenation of the resulting
mixture of azamacrocyclic alkenes 16, afforded the ethyl analog of haloclorensin C (5).
Scheme 3. Synthesis of the ethyl analog of haliclorensin C (5).
Compound 5 was submitted to biological screening in the context of the Lilly Open Innovation
Drug Discovery (OIDD) program, where it was subjected to a battery of assays against new
potential therapeutic targets. Among all the tests performed, the most pharmacologically relevant
results were obtained in the oncology area. Specifically, compound
5 caused 30% inhibition of
SETD8 at a concentration of 10 M, measured through a scintillation proximity assay (SPA) of
µ
enzyme inhibition [16]. SETD8 is a lysine methyltranferase that methylates histone H4 at Lys 20.
Its overactivation or overexpression has been found to play a role in the progression of certain cancers
such as neuroblastoma [17]. Accordingly, inhibition of SETD8 in neuroblastoma leads to increased p53
tumor suppressor activity and reduced tumor cell growth, resulting in prolonged survival in mouse
models of this neoplasia.

Molecules 2019, 24, 1069
4 of 9
On the other hand, compound
at a concentration of 20
5
yielded a 10.2% inhibition of cyclin-dependent kinase 2 (CDK2)
µ
M, measured through the SPA assay [18]. CDK2 is involved in cell cycle
progression, and thus has been indirectly linked to cancer through its association with cyclin E, which
activates it. Cyclin E binds to CDK2 to further phosphorylate the retinoblastoma proteins, which
repress the E2F transcription factors, thus releasing and fully activating the E2Fs. E2Fs then trigger
the transcription of S-phase proteins, including other cyclins, and promote cell cycle progression [19].
Cyclin E is frequently amplified in human tumors and is thought to promote proliferation and genome
instability in several cancers.
In conclusion, the results herein reported further demonstrate the usefulness and versatility of
chiral linear building blocks generated from phenylglycinol-derived oxazolopiperidone lactams in
the synthesis of bioactive natural products and analogs. Our synthesis also illustrates the potential of
ring-closing metathesis reactions for the efficient construction of azamacrocyclic rings [20
light of the reported structure-activity relationships and inhibitory data of some SETD8 inhibitors [23],
modifications of compound could lead to a new series of SETD8 inhibitors with therapeutic potential.
Finally, although the low inhibition of CDK2 caused by compound rules out a potential therapeutic
–22]. In the
5
5
use based on this target, the possibility of a synergistic dual effect of this compound and its derivatives
on SETD8 and CDK2 remains to be investigated.
3. Materials and Methods
3.1. General Information
All air sensitive manipulations were carried out under a dry argon or nitrogen atmosphere.
THF and CH2Cl2 were dried using a column solvent purification system. Analytical thin-layer
chromatography was performed on SiO2 (silica gel 60A 35–70 µm, Carlo Erba, Val de Reuil Cedex,
France), and the spots were located with 1% aqueous KMnO4. Chromatography refers to flash
chromatography, and was carried out on SiO2 (SDS silica gel 60 ACC, 35–75 mm, 230–240 mesh
ASTM). Hydrogenations were carried out in a Parr 4560 high-pressure reactor. NMR spectra were
recorded at 300 or 400 MHz (1H) and 100.6 MHz (13C), and chemical shifts are reported in
δ
values downfield from TMS, or relative to residual chloroform (7.26 ppm, 77.0 ppm) as an internal
standard (see Supplementary Materials). Data are reported in the following manner: chemical shift,
multiplicity, coupling constant (J) in hertz (Hz), integrated intensity, and assignment (when possible).
Assignments are given only when they are derived from definitive two-dimensional NMR experiments
(g-HSQC-COSY). IR spectra were performed in an Avatar 320 FT-IR spectrophotometer (Thermo
Nicolet, Madison, WI, USA) and only noteworthy IR absorptions (cm−1) are listed. High resolution
mass spectra (HMRS; LC/MSD TOF, Agilent Technologies, Santa Clara, CA, USA) were performed by
Centres Científics i Tecnològics de la Universitat de Barcelona.
3.2. Experimental Procedures
(S)-4-Ethyl-5-{[(1R)-2-hydroxy-1-phenylethyl]amino}-1-pentanol (7). n-BuLi (3.90 mL of a 2.5 M solution in
hexanes, 9.67 mmol) was added to a solution of the borane–ammonia complex (300 mg, 9.67 mmol) in
anhydrous THF (6 mL) at 0 ◦C, and the resulting mixture was stirred at this temperature for 10 min
and at room temperature for 15 min. This solution was added to a solution of lactam 2 [24] (550 mg,
2.25 mmol) in anhydrous THF (5 mL), and the stirring was continued at 40 ◦C for 1 h. The reaction
mixture was quenched with H2O, and the resulting solution was extracted with Et2O. The combined
organic extracts were dried over anhydrous Na2SO4 and filtered, and the solvent was removed under
reduced pressure. Flash chromatography (from 8:2 hexane-EtOAc to 8:2 EtOAc-EtOH) of the residue
22
gave amino diol
ν
7
(425 mg, 75% yield) as a colorless oil: [
α
]
−
44.9 (c 0.16, MeOH); IR (ATR Pike)
= 0.82 (t, J = 7.6 Hz, 3H, CH3),
D
(cm−1): 3330 (NH); 1H-NMR (400 MHz, CDCl3, COSY, g-HSQC):
δ
1.23–1.40 (m, 3H, H-3, CH3CH2), 1.42–1.50 (m, 4H, H-2, H-3, H-4), 2.40 (dd, J = 11.6, 6.4 Hz, 1H, H-5),
2.46 (dd, J = 11.6, 5.0 Hz, 1H, H-5), 3.41 (br.s, 3H, OH, NH), 3.57–3.64 (m, 3H, H-1, CH2O), 3.71 (dd,

Molecules 2019, 24, 1069
5 of 9
J = 10.8, 4.0 Hz, 1H, CH2O), 3.77 (dd, J = 8.8, 4.0 Hz, 1H, CHN), 7.24–7.38 (m, 5H, ArH); 13C-NMR
(100.6 MHz, CDCl3): = 11.3 (CH3), 24.9 (CH3CH2), 27.4 (C-3), 29.2 (C-2), 38.8 (C-4), 50.2 (C-5), 60.3
δ
(C-1), 64.8 (CHN), 66.5 (CH2O), 127.3 (CH-Ar), 127.6 (CH-Ar), 128.6 (CH-Ar), 139.9 (C-i); HRMS (ESI)
calcd for [C15H25NO2 + H+]: 252.1958, found. 252.1947.
(S)-5-[(tert-Butyldiphenylsilyl)oxy]-2-ethyl-1-pentanamine (8). A solution of aminodiol 7 (241 mg,
0.96 mmol) in anhydrous MeOH (10 mL) containing 20% Pd(OH)2 (48 mg) was hydrogenated at
75 ◦C for 18 h under 10 bar of pressure. The catalyst was removed by filtration and washed with hot
MeOH. The organic solution was concentrated, and the resulting crude amino alcohol
3 was dissolved
in CH2Cl2 (3 mL). Imidazole (271 mg, 3.98 mmol) and TBDPSCl (1.09 g, 3.98 mmol) were added,
and the mixture was stirred at room temperature for 12 h and at 40 ◦C for 4 h. Saturated aqueous
NH4Cl was added, and the mixture was extracted with CH2Cl2. The combined organic extracts were
dried over anhydrous MgSO4 and filtered, and the solvent was removed under reduced pressure.
Flash chromatography (from 95:5 hexane-EtOAc to EtOAc) of the residue gave amine
8
(115 mg, 32%
5.78 (c 0.32, CHCl3); H-NMR (400 MHz, CDCl3, COSY, g-HSQC):
= 0.88 (t, J = 7.4 Hz, 3H, CH3CH2), 1.04 [s, 9H, (CH3)3], 1.32–1.44 (m, 2H, H-3, CH3CH2), 1.45–1.58 (m,
22
1
yield) as a yellow oil: [
α
]
−
D
δ
4H, H-3, H-4, CH3CH2), 1.68–1.77 (m, 1H, H-2), 2.79 (dd, J = 12.8, 7.8 Hz, 1H, H-1), 2.89 (dd, J = 12.8,
5.4 Hz, 1H, H-1), 3.64 (t, J = 7.4 Hz, 2H, H-5), 7.34–7.43 (m, 6H, ArH), 7.63–7.66 (m, 2H, ArH), 7.70–7.73
(m, 2H, HAR); 13C-NMR (100.6 MHz, CDCl3):
δ = 10.2 (CH3CH2), 19.3 [C(CH3)3], 23.6 (CH2), 26.7
(CH2), 27.0 [C(CH3)3], 29.2 (C-4), 37.5 (C-2), 42.7 (C-1), 63.9 (C-5), 127.8 (CH-Ar), 127.8 (CH-Ar), 129.7
(CH-Ar), 129.7 (CH-Ar), 134.0 (C-Ar), 134.9 (CH-Ar), 135.4 (C-Ar), 135.7 (CH-Ar).
(S)-4-Ethyl-5-(10-undecenylamino)-1-pentanol (
anhydrous MeOH (10 mL) containing 20% Pd(OH)2 (74 mg) was hydrogenated at 75 C for 18 h
under 10 bar of pressure. The catalyst was removed by filtration and washed with hot MeOH. The
9
). A solution of amino diol
7
(370 mg, 1.4◦7 mmol) in
organic solution was concentrated, and the resulting crude amino alcohol 3 was dissolved in CH2Cl2
(1.3 mL). 10-Undecenal (0.58 mL, 2.94 mmol), NaBH3CN (277 mg, 4.42 mmol), and AcOH (0.13 mL)
were added, and the mixture was stirred at room temperature for 12 h. Then, a saturated solution of
NaHCO3 was added, and the mixture was extracted with CH2Cl2. The combined organic extracts were
dried over anhydrous Na2SO4 and filtered, and the solvent was removed under reduced pressure.
The residue was purified by flash chromatography (from 8:2 hexane-EtOAc to 8:2 EtOAc-MeOH) to
22
afford secondary amine
Pike)
9
(104 mg, 25% yield) as a colorless oil: [
α
]
−
2.15 (c 0.3, MeOH); IR (ATR
= 0.89 (t, J = 7.4 Hz, 3H,
D
ν
(cm−1): 2926 (NH); 1H-NMR (400 MHz, CDCl3, COSY, g-HSQC):
δ
CH3CH2), 1.21–1.69 (m, 21H, H-4, 10CH2), 1.99–2.05 (m, 2H, CH2=CHCH2), 2.56–2.61 (m, 1H, H-5),
2.66–2.75 (m, 3H, H-5, CH2N), 3.62 (m, 2H, H-1), 4.43 (br.s, 1H, NH), 4.89–5.00 (m, 2H, CH2=CH), 5.79
(dddd, J = 16.9, 10.2, 10.2, 6.7 Hz, 1H, CH2=CH); 13C-NMR (100.6 MHz, CDCl3):
δ = 11.0 (CH3CH2),
24.9 (CH2), 27.1 (CH2), 27.1 (CH2), 28.3 (CH2), 28.6 (CH2), 28.9 (CH2), 29.1 (CH2), 29.3 (CH2), 29.3
(CH2), 29.5 (CH2), 33.8 (CH2=CHCH2), 37.7 (C-4), 49.5 (CH2N), 52.2 (C-5), 62.1 (C-1), 114.1 (CH2=CH),
139.1 (CH2=CH); HRMS (ESI) calcd for [C18H37NO + H+]: 284.2948, found: 284.2939.
(S)-5-[(tert-Butyldiphenylsilyl)oxy]-2-ethyl-N-(10-undecenyl)-1-pentanamine (10). 10-Undecenal (0.05 mL,
0.25 mmol) and AcOH (0.02 mL) were added to a suspension of amine
8 (74 mg, 0.20 mmol) and
NaBH3CN (25 mg, 0.40 mmol) in anhydrous CH2Cl2 (2.3 mL), and the mixture was stirred at
room temperature for 12 h. Then, water was added, and the mixture was extracted with CH2Cl2.
The combined organic extracts were dried over anhydrous Na2SO4 and filtered, and the solvent was
removed under reduced pressure. Flash chromatography (from 98:2 hexane-EtOAc to EtOAc) of the
22
residue gave secondary amine 10 (30 mg, 29% yield) as a colorless oil: [
α
]
−
12.3 (c 0.225, MeOH);
D
1H-NMR (400 MHz, CDCl3, COSY, g-HSQC):
δ
= 0.90 (t, J = 7.4 Hz, 3H, CH3CH2), 1.05 [s, 9H, (CH3)3],
1.24–1.58 (m, 18H, 9CH2), 1.65–1.77 (m, 3H, H-2, H-4), 2.01–2.06 (m, 2H, CH2=CHCH2), 2.81–2.84 (m,
2H, H-5), 2.88–2.96 (m, 2H, CH2N), 3.66–3.70 (m, 2H, H-1), 4.91–5.01 (m, 2H, CH2=CH), 5.80 (m, 1H,
CH2=CH), 7.32–7.55 (m, 6H, ArH), 7.63–7.67 (m, 4H, ArH); 13C-NMR (100.6 MHz, CDCl3):
δ
= 10.2
(CH3CH2), 19.3 [C(CH3)3], 23.1 (CH2), 23.7 (CH2), 25.8 (CH2), 26.7 (CH2), 26.8 (CH2), 27.0 [C(CH3)3],

Molecules 2019, 24, 1069
6 of 9
29.0 (CH2), 29.1 (CH2), 29.2 (CH2), 29.4 (CH2), 29.8 (CH2), 33.9 (CH2=CHCH2), 36.3 (C-4), 49.0 (CH2N),
51.6 (C-5), 63.8 (C-1), 114.3 (CH2=CH), 127.8 (CH-Ar), 129.8 (CH-Ar), 133.9 (C-Ar), 135.6 (CH-Ar), 139.3
(CH2=CH).
(S)-4-Ethyl-5-[N-(tert-butoxycarbonyl)-N-(10-undecenyl)amino]-1-pentanol (11). Et3N (0.04 mL, 0.28 mmol)
was added to a solution of 9 (82 mg, 0.29 mmol) and di-tert-butyl dicarbonate (69 mg, 0.32 mmol) in
anhydrous MeOH (1.5 mL), and the resulting solution was stirred for 12 h. Water was added, and the
mixture was extracted with CH2Cl2. The combined organic extracts were dried over anhydrous MgSO4,
filtered, and concentrated under reduced pressure. Flash chromatography (from 1:1 hexane-EtOAc
22
to EtOAc) of the residue gave the N-Boc derivative 11 (95 mg, 85% yield) as a colorless oil: [
α
]
D
1
–0.82 (c 0.6, MeOH); IR (ATR Pike)
COSY, g-HSQC):
ν
(cm−1): 3453 (OH); 1694 (CO); H-NMR (400 MHz, CDCl3,
= 0.87 (t, J = 7.4 Hz, 3H, CH3CH2), 1.22–1.64 (m, 30H, C(CH3)3, H-4, 10CH2),
δ
2.00–2.05 (m, 2H, CH2=CHCH2), 3.00–3.07 (m, 1H, H-5), 3.08–3.18 (m, 3H, H-5, CH2N), 3.54–3.64 (m,
2H, H-1), 4.89–5.00 (m, 2H, CH2=CH), 5.79 (dddd, J= 16.9, 10.2, 10.2, 6.7 Hz, 1H, CH2=CH); 13C-NMR
(100.6 MHz, CDCl3):
δ = 10.8 (CH3CH2), 26.9 (CH2), 28.5 [C(CH3)3], 28.9 (CH2), 29.0 (CH2), 29.1 (CH2),
29.3 (2CH2), 29.4 (2CH2), 29.5 (CH2), 33.7 (CH2=CHCH2), 38.1 (C-4), 47.5 (CH2N), 50.3 (C-5), 63.1 (C-1),
79.1 [C(CH3)3], 114.0 (CH2=CH), 139.1 (CH2=CH), 156.0 (NCO); HRMS (ESI) calcd for [C23H45NO3 +
H+]: 384.3472, found: 384.3466.
(S)-4-Ethyl-5-[(2-nitrobenzenesulfonyl)amino]-1-pentanol (12). A solution of amino diol
7 (1.15 g,
4.58 mmol) in anhydrous MeOH (25 mL) containing 20% Pd(OH)2 (230 mg) was hydrogenated
◦
at 75 C for 18 h under 10 bar of pressure. The catalyst was removed by filtration and washed with hot
MeOH. The organic solution was concentrated, and the resulting crude amino alcohol
3 was dissolved
in anhydrous CH2Cl2 (16 mL). 2-Nitrobenzenesulfonyl chloride (1.12 g, 5.0 mmol) and Et3N (0.7 mL,
5.0 mmol) were added, and the mixture was stirred at room temperature for 18 h. The organic solvent
was removed under reduced pressure, and the residue was chromatographed (from 7:3 hexane-EtOAc
22
to EtOAc) to give N-nosyl derivative 12 (1.1 g, 76% yield) as a colorless oil: [
α
]
D +0.95 (c 0.84, MeOH);
= 0.84 (t,
1
IR (ATR Pike)
ν
(cm−1): 3348 (OH, NH); H-NMR (400 MHz, CDCl3, COSY, g-HSQC):
δ
J = 7.6 Hz, 3H, CH3), 1.30–1.40 (m, 4H, H-3, CH3CH2), 1.47–1.54 (m, 3H, H-2, H-4), 1.65 (br.s, 1H,
OH), 3.02 (dt, J = 6.1, 3.7 Hz, 2H, H-5), 3.60 (t, J = 6.4 Hz, 2H, H-1), 5.41 (t, J = 6.0 Hz, 1H, NH), 7.76
(m, 2H, H-5Ns, H-6Ns), 7.85 (m, 1H, H-4Ns), 8.13 (m, 1H, H-3Ns); 13C-NMR (100.6 MHz, CDCl3):
δ
= 10.7 (CH3), 23.9 (CH3CH2), 26.9 (C-3), 29.3 (C-2), 33.1 (C-4), 46.2 (C-5), 62.8 (C-1), 125.3, 131.1
(C-3Ns, C-6Ns), 132.7 (C-4Ns), 133.5 (C-1Ns), 133.6 (C-5Ns), 148.0 (C-2Ns); HRMS (ESI) calcd for
[C13H20N2O5S + H+]: 317.1166, found: 317.1161.
(S)-4-Ethyl-5-[N-(2-nitrobenzenesulfonyl)-N-(10-undecenyl)amino]-1-pentanol (13). 11-Bromo-1-undecene
(0.80 mL, 3.65 mmol) was added to a suspension of N-nosyl derivative 12 (1.05 g, 3.3 mmol) and
Cs2CO3 (1.3 g, 4.0 mmol) in anhydrous DMF (25 mL), and the resulting mixture was stirred at 55 ◦C
for 3 h. The mixture was cooled to room temperature, poured into brine, and extracted with Et2O.
The combined organic extracts were dried over anhydrous MgSO4, filtered, and concentrated under
reduced pressure. Flash chromatography (7:3 hexane-EtOAc) of the residue gave alkene 13 (1.30 g,
22
84% yield) as a colorless oil: [
α
]
+1.82 (c 0.7, CH2Cl2); 1H-NMR (400 MHz, CDCl3, COSY, g-HSQC):
D
δ
= 0.85 (t, J = 7.6 Hz, 3H, CH3), 1.19–1.37 (m, 16H, 8CH2), 1.40–1.65 (m, 5H, H-4, CH2, CH3CH2),
2.02 (m, 2H, CH2=CHCH2), 3.19–3.25 (m, 4H, H-5, CH2N), 3.57 (t, J = 6.8 Hz, 2H, H-1), 4.95 (m, 2H,
CH2=CH), 5.80 (m, 1H, CH2=CH), 7.62 (m, 1H, H-4Ns), 7.67 (m, 2H, C-5Ns, C-6Ns), 7.98 (m, 1H,
H-3Ns); 13C-NMR (100.6 MHz, CDCl3):
δ = 10.4 (CH3), 23.2 (CH2), 26.3 (CH2), 26.6 (CH2), 27.6 (CH2),
28.8 (CH2), 29.0 (CH2), 29.1 (CH2), 29.2 (CH2), 29.3 (CH2), 29.4 (CH2), 33.6 (CH2=CHCH2), 36.5 (C-4),
47.1 (CH2N), 50.7 (C-5), 62.8 (C-1), 114.0 (CH2=CH), 123.3 (C-3Ns), 130.6 (C-6Ns), 131.4 (C-4Ns), 133.3
(C-5Ns), 133.4 (C-1Ns), 139.0 (CH2=CH), 148.0 (C-2Ns); HRMS (ESI) calcd for [C24H40N2O5S + H+]:
469.2731, found: 469.2731.

Molecules 2019, 24, 1069
7 of 9
(S)-N-[2-Ethyl-5-hexenyl)-N-(2-nitrobenzenesulfonyl)-10-undecenamine (14). Dess–Martin reagent (2.35
g, 5.54 mmol) was added to a solution of alcohol 13 (1.3 g, 2.77 mmol) in anhydrous CH2Cl2 (21
mL), and the mixture was stirred at room temperature for 1.5 h. Then, saturated aqueous Na2S2O4
(0.75 mL) and saturated aqueous NaHCO3 (0.75 mL) were added, and the resulting mixture was
stirred for 1 h. The aqueous layer was extracted with CH2Cl2, and the combined organic extracts were
washed with brine, dried over anhydrous Na2SO4, and filtered. The solvent was evaporated to give an
aldehyde, which was used in the next step without further purification. t-BuOK (13.8 mL of a 1 M
solution in THF, 13.8 mmol) was added to a solution of methyltriphenylphosphonium bromide (6.92
g, 19.4 mmol) in anhydrous THF (70 mL), and the solution was stirred at room temperature for 1 h.
Then, a solution of the above aldehyde in anhydrous THF (10 mL) was added via cannula, and the
resulting mixture was stirred at room temperature for 3 h. Saturated aqueous NH4Cl was added, and
the mixture was extracted with EtOAc. The combined organic extracts were dried over anhydrous
Na2SO4 and filtered, and the solvent was removed under reduced pressure. Flash chromatography (9:1
22
hexane-EtOAc) of the residue gave diene 14 (600 mg, 47% yield) as a colorless oil: [
α
]
D +4.09 (c 2.1,
MeOH); 1H-NMR (400 MHz, CDCl3, COSY, g-HSQC):
δ
= 0.84 (t, J = 7.5 Hz, 3H, CH3), 1.14–1.39 (m,
16H, 8CH2), 1.41–1.49 (m, 2H, CH2), 1.54–1.64 (m, 1H, CH), 1.95–2.10 (m, 4H, CH2=CHCH2), 3.14–3.26
(m,4H, 2CH2N), 4.91–5.02 (m, 4H, CH2=CHCH2), 5.74 (qt, J = 16.9, 10.0, 6.7, 6.7 Hz, 1H, CH2=CH),
5.80 (qt, J = 16.9, 10.1, 6.7, 6.7 Hz, 1H, CH2=CH), 7.59–7.62 (m, 1H, H-3Ns), 7.63–7.70 (m, 2H, H-5Ns,
H-6Ns), 7.99–8.03 (m, 1H, H-4Ns). 13C-NMR (100.6 MHz, CDCl3):
δ 10.3 (CH3), 23.2 (CH2), 26.6 (CH2),
27.5 (CH2), 28.9 (CH2), 29.0 (CH2), 29.1 (CH2), 29.3 (CH2), 29.4 (CH2), 29.5 (CH2), 30.6 (CH2=CHCH2),
33.8 (CH2=CHCH2), 36.0 (CH), 47.0 (CH2N), 50.7 (CH2N), 114.1 (CH2=CH), 114.7 (CH2=CH), 124.1
(C-3Ns), 130.9 (C-6Ns), 131.4 (C-4Ns), 133.2 (C-5Ns), 133.9 (C-1Ns), 138.5 (CH2=CH), 139.1 (CH2=CH),
148.0 (C-2Ns); HRMS (ESI) calcd for [C25H40N2O4S + H+]: 465.2782, found 465.2776.
(S)-3-Ethyl-1-(2-nitrobenzenesulfonyl)azacyclohexadec-6-ene (15). A solution of diene 14 (70 mg, 0.15
mmol) in anhydrous CH2Cl2 (15 mL) was added to a solution of second-generation Grubbs catalyst
(19 mg, 0.022 mmol) in CH2Cl2 (750 mL). The mixture was stirred at reflux temperature for 14 h.
The solvent was removed under reduced pressure, and the resulting residue was chromatographed
(95:5 hexane-EtOAc) to yield macrocycle 15 (46 mg, 70% yield) as a 88:12 mixture (calculated by
1
GC/MS) of E/Z diastereoisomers. Spectroscopic data of E diastereoisomer (from a mixture): H-NMR
(400 MHz, CDCl3, COSY, g-HSQC): δ = 0.83 (t, J = 7.5 Hz, 3H, CH3), 1.00–1.11 (m, 1H, H-4), 1.18–1.44
(m, 17H, H-4, 8CH2), 1.46–1.51 (m, 2H, CH2), 1.67–1.80 (m, 1H, H-3), 1.97–2.19 (m, 4H, H-5, H-8),
3.07 (dd, J = 13.8, 7.5 Hz, H-2), 3.11–3.15 (m, 1H, H-16), 3.21–3.26 (m, 1H, H-16), 3.28 (dd, J = 13.8,
7.5 Hz, H-2), 5.25–5.41 (m, 2H, H-6, H-7), 7.57–7.62 (m, 1H, H-3Ns), 7.63–7.73 (m, 2H, H-5Ns, H-6Ns),
7.93–8.04 (m, 1H, H-4Ns); 13C-NMR (100.6 MHz, CDCl3):
δ = 9.6 (CH3), 21.9 (CH2), 24.3 (CH2), 25.7
(CH2), 26.1 (CH2), 26.1 (CH2), 26.7 (CH2), 26.7 (CH2), 27.3 (CH2), 28.8 (CH2=CHCH2), 29.6 (CH2), 30.9
(CH2=CHCH2), 33.3 (C-3), 46.7 (C-16), 51.3 (C-2), 124.0 (C-3Ns), 130.2 (CH=), 130.7 (C-6Ns), 131.3
(C-4Ns), 131.4 (C-5Ns), 133.2 (CH=), 133.4 (C-1Ns), 148.1 (C-2Ns); HRMS (ESI) calcd for [C23H36N2O4S
+ H+]: 437.2469, found 437.2459.
(S)-3-Ethylazacyclohexadec-6-ene (16). Thiophenol (0.024 mL, 0.34 mmol) and K2CO3 (82 mg, 0.59 mmol)
were added to a solution of cycloalkene 15 (86 mg, 0.20 mmol) in anhydrous DMF (4 mL), and the
mixture was stirred at room temperature for 14 h. Then, 2 M aqueous NaOH was added, and the
mixture was extracted with CH2Cl2. The combined organic extracts were dried over anhydrous MgSO4,
filtered, and concentrated under reduced pressure. Flash chromatography (from 95:5 hexane-EtOAc
to 8:2 EtOAc-Et3N) of the residue gave macrocycle 16 (23 mg, 45% yield) as a mixture of E/Z
1
diastereoisomers as a brown oil. Spectroscopic data of E diastereoisomer (from a mixture): H-NMR
(400 MHz, CDCl3, COSY, g-HSQC): δ = 0.87 (t, J = 7.5 Hz, 3H, CH3), 1.25–1.44 (m, 16H, 8CH2), 1.47–1.56
(m, 3H, CH2), 1.94–2.12 (m, 4H, H-5, H-8), 2.41 (dd, J = 12.1, 7.5 Hz, 1H, H-2), 2.51–2.57 (dd, J = 12.0,
6.4 Hz, 1H, H-16), 2.52–2.58 (dd, J = 12.1, 5.9 Hz, 1H, H-2), 2.62–2.68 (dd, J = 12.0, 5.7 Hz, 1H, H-16),
5.36–5.39 (m, 2H, H-6, H-7); 13C-NMR (100.6 MHz, CDCl3):
δ = 10.8 (CH3), 24.4 (CH2), 25.2 (CH2),

Molecules 2019, 24, 1069
8 of 9
26.2 (CH2), 26.3 (CH2), 27.2 (CH2), 27.3 (CH2), 28.0 (CH2), 28.2 (CH2), 29.1 (CH2=CHCH2), 30.8 (CH2),
31.8 (CH2=CHCH2), 36.9 (C-3), 47.6 (C-16), 52.3 (C-2), 130.8 (CH=), 130.9 (CH=); HRMS (ESI) calcd for
[C17H33N + H+]: 252.2686, found 252.2689.
(S)-3-Ethylazacyclohexadecane (5). A solution of cycloalkene 16 (19 mg, 0.076 mmol) in anhydrous MeOH
(10 mL) containing 25% Pd/C (5 mg) was hydrogenated at room temperature for 14 h under 10 bar of
pressure. The catalyst was removed by filtration and washed with hot MeOH, and the organic solution
was concentrated under reduced pressure. Flash chromatography (from 95:5 hexane-EtOAc to 8:2
22
EtOAc-Et3N) of the residue gave
1H-NMR (300 MHz, CDCl3):
(m, 3H, H-3, CH2), 2.40 (m, 2H, H-2), 2.60–2.85 (m, 2H, H-16); 13C-NMR (100.6 MHz, CDCl3):
5
(10 mg, 52% yield) as a brown oil: [
α
]
−
6.75 (c 0.15, MeOH);
D
δ
= 0.90 (t, J = 7.5 Hz, 3H, CH3), 1.25–1.41 (m, 24H, 12CH2), 1.50–1.70
= 10.8
δ
(CH3), 24.8 (CH2), 24.9 (CH2), 25.0 (CH2), 26.0 (CH2), 26.1 (CH2), 26.2 (CH2), 26.3 (CH2), 26.4 (CH2),
26.5 (CH2), 27.1 (CH2), 27.4 (CH2), 29.7 (CH2), 29.8 (CH2), 35.6 (C-3), 46.1 (C-16), 49.1 (C-2); HRMS
(ESI) calcd for [C17H35N + H+]: 254.2842, found 254.2842.
Supplementary Materials: The following are available online, Copies of 1H and 13C NMR spectra.
Author Contributions: M.A. designed and planned the research; N.L. supervised the experimental work; G.G.
performed the experimental work and characterized the compounds; D.P. analyzed the pharmacological data; J.B.
discussed the results and prepared the manuscript for publication.
Funding: This research was funded by the MINECO/FEDER, Spain (Project CTQ2015-65384-R).
Acknowledgments: Financial support from the MINECO/FEDER, Spain (Project CTQ2015-65384-R) is gratefully
acknowledged. We also acknowledge the Lilly Open Innovation Drug Discovery (OIDD) program for the
biological screening of compound 5 and the networking contribution from the COST Action CM1407.
Conflicts of Interest: The authors declare no conflict of interest.
References
1.
Sorek, H.; Rudi, A.; Aknin, M.; Gaydou, E.M.; Kashman, Y. Isohalitulin and haliclorensins B and C, three
marine alkaloids form Haliclona tulearensis. J. Nat. Prod. 2010, 73, 456–458. [CrossRef]
2.
Heinrich, M.R.; Kashman, Y.; Spiteller, P.; Steglich, W. Revision of the structure of haliclorensin to
(S)-7-methyl-1,5-diazacyclotetradecane and confirmation of the new structure by synthesis. Tetrahedron 2001
57, 9973–9978. [CrossRef]
,
3.
4.
5.
6.
Heinrich, M.R.; Steglich, W.; Banwell, M.G.; Kashman, Y. Total synthesis of the marine alkaloid halitulin.
Tetrahedron 2003, 59, 9239–9247. [CrossRef]
Koren-Goldshlager, G.; Kashman, Y.; Schleyer, M. Haliclorensin, a novel diamino alkaloid from the marine
sponge Haliclona tulearensis. J. Nat. Prod. 1998, 61, 282–284. [CrossRef] [PubMed]
Kashman, Y.; Koren-Goldshlager, G.; Garcia Gravalos, M.D.; Schleyer, M. Halitulin, a new cytotoxic alkaloid
from the marine sponge Haliclona tulearensis. Tetrahedron Lett. 1999, 40, 997–1000. [CrossRef]
Amat, M.; Guignard, G.; Llor, N.; Bosch, J. Access to enantiopure 4-substituted 1,5-aminoalcohols from
phenylglycinol-derived
[CrossRef]
δ-lactams: Synthesis of Haliclona alkaloids. J. Org. Chem. 2014, 79, 2792–2802.
7.
8.
Guignard, G.; Llor, N.; Molins, E.; Bosch, J.; Amat, M. Enantioselective total synthesis of fluvirucinin B1.
Org. Lett. 2016, 18, 1788–1791. [CrossRef]
Urbina, A.; Ordeix, S.; Llor, N.; Bosch, J.; Amat, M. Studies on the enantioselective synthesis of the macrolide
callyspongiolide (OR-17). In Proceedings of the XII Spanish-Italian Symposium on Organic Chemistry
(SISOC-XII), Ferrara, Italy, 2–4 July 2018.
9.
Guignard, G.; Llor, N.; Urbina, A.; Bosch, J.; Amat, M. A general methodology for the synthesis of enantiopure
1,5-aminoalcohols. Eur. J. Org. Chem. 2016, 693–703. [CrossRef]
10. Escolano, C.; Amat, M.; Bosch, J. Chiral oxazolopiperidone lactams: Versatile intermediates for the
enantioselective synthesis of piperidine-containing natural products. Chem. Eur. J. 2006, 12, 8198–8207.
[CrossRef]
11. Amat, M.; Pérez, M.; Bosch, J. Stereoselective conjugate addition reactions to phenylglycinol-derived,
unsaturated oxazolopiperidone lactams. Chem. Eur. J. 2011, 17, 7724–7732. [CrossRef]

Molecules 2019, 24, 1069
9 of 9
12. Thakur, N.L.; Thakur, A.N.; Müller, W.E.G. Marine natural products in drug discovery. Nat. Prod. Rad. 2005
4, 471–477.
,
13. Montaser, R.; Luesch, H. Marine natural products: A new wave of drugs? Future Med. Chem. 2011, 3,
1475–1489. [CrossRef] [PubMed]
14. Pasumansky, L.; Goralski, C.T.; Singaram, B. Lithium aminoborohydrides: Powerful, selective, air-stable
reducing agents. Org. Process Res. Dev. 2006, 10, 959–970. [CrossRef]
15. Lund, A. Lithium amidotrihydroborate. In Encyclopedia of Reagents for Organic Synthesis (EROS);
Paquette, L.A., Fuchs, G.A., Molander, D.C., Eds.; Wiley: Chichester, West Sussex, UK, 2009; pp. 6082–6083.
16. Ma, A.; Yu, W.; Li, F.; Bleich, R.M.; Herold, J.M.; Butler, K.V.; Norris, J.L.; Korboukh, V.; Tripathy, A.;
Janzen, W.P.; et al. Discovery of a selective, substrate-competitive inhibitor of the lysine methyltransferase
SETD8. J. Med. Chem. 2014, 57, 6822–6833. [CrossRef] [PubMed]
17. Veschi, V.; Liu, Z.; Voss, T.C.; Ozbun, L.; Gryder, B.; Yan, C.; Hu, Y.; Ma, A.; Jin, J.; Mazur, S.J.; et al. Epigenetic
siRNA and chemical screens identify SETD8 inhibition as a therapeutic strategy for p53 activation in high-risk
neuroblastoma. Cancer Cell 2017, 31, 50–63. [CrossRef] [PubMed]
18. Byth, K.F.; Geh, C.; Forder, C.L.; Oakes, S.E.; Thomas, A.P. The cellular phenotype of AZ703, a novel selective
imidazo[1,2-a]pyridine cyclin-dependent kinase inhibitor. Mol. Cancer. Ther. 2006, 5, 655–664. [CrossRef]
19. Tadesse, S.; Caldon, E.C.; Tilley, W.; Wang, S. Cyclin-dependent kinase 2 inhibitors in cancer therapy: An
update. J. Med. Chem. 2018. [CrossRef]
20. Deiters, A.; Martin, S.F. Synthesis of oxygen- and nitrogen-containing heterocycles by ring-closing metathesis.
Chem. Rev. 2004, 104, 2199–2238. [CrossRef]
21. Nicolaou, K.C.; Bulger, P.G.; Sarlah, D. Metathesis reactions in total synthesis. Angew. Chem. Int. Ed. 2005, 44,
4490–4527. [CrossRef]
22. Gradillas, A.; Pérez-Castells, J. Macrocyclization by ring-closing metathesis in the total synthesis of natural
products: Reaction conditions and limitations. Angew. Chem. Int. Ed. 2006, 45, 6086–6101. [CrossRef]
23. Ma, A.; Yu, W.; Xiong, Y.; Butler, K.V.; Brown, P.J.; Jin, J. Structure-activity relationship studies of SETD8
inhibitors. Med. Chem. Commun. 2014, 5, 1892–1898. [CrossRef] [PubMed]
24. Amat, M.; Llor, N.; Hidalgo, J.; Bosch, J. A concise procedure for the preparation of enantiopure
3-alkylpiperidines. Tetrahedron Asymmetry 1997, 8, 2237–2240. [CrossRef]
Sample Availability: Samples of the compounds are not available from the authors.
©
2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).