A gram-scale synthesis of a macrocyclic amidinourea with strong antifungal activity through a Fukuyama tri-protected polyamine intermediate

Article abstract of DOI:10.24820/ark.5550190.p010.895

Systemic fungal infections are, nowadays, of crucial importance and, thus, in the last decade, we explored the great potential of natural and synthetic guanylated compounds, a great amount of work that led to the development of new non-azole antifungal co

Full text of DOI:10.24820/ark.5550190.p010.895

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A gram-scale synthesis of a macrocyclic amidinourea with strong antifungal
activity through a Fukuyama tri-protected polyamine intermediate
Matteo Borgini,a Francesco Orofino,a Giuseppina I. Truglio,a Lorenzo Balestri,a and Maurizio Botta*a,b,c
a Department of Biotechnology, Chemistry and Pharmacy, University of Siena, I-53100 Siena, Italy
b Sbarro Institute for Cancer Research and Molecular Medicine, Center for Biotechnology, College of Science
and Technology, Temple University, Philadelphia, PA, United States of America.
c Lead Discovery Siena s.r.l, Via Vittorio Alfieri 31, I-53019 Castelnuovo Berardenga, Italy
Email: botta.maurizio@gmail.com
Dedicated to Steve, thanking him for his great ability to teach and his dedication
Received 01-30-2019
Accepted 05-04-2019
Published on line 05-16-2019
Abstract
Systemic fungal infections are, nowadays, of crucial importance and, thus, in the last decade, we explored the
great potential of natural and synthetic guanylated compounds, a great amount of work that led to the
development of new non-azole antifungal compounds bearing a macrocycle, endowed with potent antifungal
activity. We planned many biological assays to evaluate this class, implying always greater amount of
compounds needed. This triggered us to setup a convenient strategy to prepare, in an easy and affordable
way, grams of compound to be tested in excellent overall yield.
Keywords: Macrocyclic compounds, antifungals, polyamines, amidinoureas
DOI: https://doi.org/10.24820/ark.5550190.p010.895
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Introduction
The development of an effective synthetic method for amines and polyamines has always been a key interest
in organic chemistry, biochemistry and medicinal chemistry, because of the synthetic challenges these
compounds present and of the collection of interesting biological activities they possess , such as their
involvement in cancer or virus infections.1-3 The presence of dedicated polyamine transporters into cell
membranes as well as dedicated enzymatic machineries for their synthesis and metabolism stress their
importance in cell’s and organism’s life cycles.4-7
The highly polar nature of these compounds makes their handling difficult, time-consuming and sometimes
expensive. Moreover, the selection of the proper protective groups for the nitrogen atoms is usually a pivotal
moment in synthesis planning, above all if natural macromolecules preparation is involved. Hence, many
scientists devoted part of their work to the analysis of protecting group strategies, as testified by the
remarkable work done in late 1990s/early 2000s by Fukuyama and co-workers,8-12 as well as by many
others.13-23
In the last years, our research group focused its efforts on the preparation of a series of compounds bearing
interesting antifungal properties, as we reported in previous works,24-32 well represented by compound 1
(Figure 1). These compounds showed good activities against various Candida strains, most notably albicans,
guillermondii, kefyr, glabrata. Their main point of strength, though, is their ability to improve the antifungal
activity against strains presenting resistance to azoles, the most widely used antifungal drugs at the moment,
as well as a good ADMET profile.32 Being these the most desirable characteristics novel antifungals should
have, we decided to investigate them even further, so we faced the need to prepare amounts of compound 1
higher than what we usually managed. In the first described synthesis of 1,27 the commercially available
bis(8‐aminooctyl)amine was used. Two subsequent guanylations of the primary amino groups, by using N,N’-
di-Boc-S-methylisothiourea and N,N’-di-Boc-N-[(E)-but-2-en]-S-methylisothiourea respectively, followed by a
cyclization in refluxing THF in presence of TEA and a final Boc deprotection, led to the desired compound.
However, since bis(8‐aminooctyl)amine was no longer commercially available (with the exception of custom
synthesis services), new synthetic paths had to be taken. Here follows a detailed analysis of these paths, with
their obstacles and downsides, that finally led us to setup a convenient and versatile way to prepare our lead
compound 1 in gram scale.
Figure 1: Structure of compound 1.
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Results and Discussion
Since the withdrawal from the market of the bis(8‐aminooctyl)amine, two different solutions were explored
(Figure 2). The first option was the synthesis of bis(8‐aminooctyl)amine itself in four steps starting from 1,8-
dibromooctane.33 In a second approach, the macrocyclic scaffold was obtained by reducing with DIBAL-H the
amide obtained by coupling the mono-guanilated-1,8-diaminoctane and the Cbz-protected 8-aminooctanoic
acid.25
Both the described approaches, though, are not suitable for obtaining grams of 1. The first approach, besides
using the unsafe sodium azide, implies a quite heavy workload to obtain the bis(8‐aminooctyl)amine. This is
mainly ascribed to the purification of the 1-azido-8-bromooctane, which implies long isocratic hexane
chromatography given the high similarity in terms of polarity the product and the starting material present.
The difficulties that are faced in case one of the free amines needs to be purified is another aspect that
involves overload of work. Besides, it is then used in a 3:1 ratio for the selective mono-alkylation, leading to a
waste of unrecoverable starting material (alongside the wasting of time and expensive work). The second
approach gets over this problem by anticipating the mono-guanylation in the synthetic process. DIBAL-H,
though, is the only reducing agent that can be used for the amide reduction due to the presence of other
carbonyl groups, which led to moderate to low yields, difficult purifications (including work-up with Rochelle
salt leading to a viscous mass which is hard to handle), lack of a good reproducibility (since DIBAL-H quality
tends to decay over time) and still not suitable for a scaling-up procedure. Over time, other alternatives were
explored, but they failed to solve the abovementioned problems.34
Figure 2: Summary of the synthetic strategies explored.
To overcome the described limitations, we took inspiration from Fukuyama’s work designing a novel synthetic
strategy. This allowed us to prepare grams of 1 in a single batch, in the same timeframe of the old approaches.
The keystone is to obtain intermediate bis(8‐aminooctyl)amine in the tri-protected form 6, with trityl, Cbz and
nosyl as protecting groups of choice. This intermediate offers many advantages, such as lowering of both time
and costs of production (because protecting groups make it less polar and easier to handle and purify) and
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versatility, since the three different groups are orthogonal to each other, meaning they can be selectively
removed in different conditions, allowing us to deprotect only the chosen amine; this avoids resorting to
workarounds like the use of 3:1 ratio and waste of materials by means of simple one-pot-two-steps reactions.
We prepared the diamine 4 (Scheme 1) by mono-protecting the diaminooctane with trityl chloride and,
subsequently, with nosyl chloride, at room temperature. The amino-alcohol 5 was obtained via a direct
reduction of the carboxylic acid by using borane dimethylsulfide, in high yield. The tri-amine 6, then, was
synthesized using Mitsunobu reaction conditions. The resulting product 6 was easily purified by flash
chromatography allowing the recovery of the unreacted di-protected amine 4. Another economical advantage
of this approach is that affordable diaminoctane is wasted during mono-protection in place of the more
expensive bis(8‐aminooctyl)amine. All the reaction steps occur with good yields and without difficulties.
Scheme 1: Synthesis of intermediate 6. i. Trityl chloride, DCM, 0 °C to r. t., 2 h, 96%; ii. Nosyl chloride, DIPEA,
DCM, r. t., 1 h, 95%; iii. CbzCl, NaHCO3, H2O/THF, -10 °C to r. t., o. n., 87%; iv. Me2SBH3, dry THF, r. t., o. n. 93%;
v. DIAD, PPh3, dry THF, r. t., 3 h, 76%.
The three subsequent reactions have the advantage of being one-pot-two-steps. Specifically, the first one (the
deprotection of trityl group and guanylation of the primary salified amine) was carried out with TFA and Et3SiH
in DCM and subsequent treatment with N,N’-Di-Boc-1H-pyrazole-1-carboxamidine and DIPEA to afford
compound 7. In the deprotection step, the excess of Et3SiH was removed in vacuo as the remaining TFA and
DCM, leaving the salified amine and the inert triphenylmethane ready for the guanylation step. 7 is easily
purified by flash chromatography given the lack of free amine groups, with an excellent overall yield of 99%.
The following reaction, the nosyl deprotection, was carried out using thiophenol and K2CO3 in dry DMF. The
excess of thiophenol and K2CO3 were eliminated during the work-up after washing with water and 5% NaHCO3,
while the byproduct 1‐nitro‐4‐(phenylsulfanyl)benzene is left in place, since it does not interfere with the next
step. At this point, the solvent was removed and the crude product was heated at reflux with TEA in THF giving
compound 8 (52% yield), again avoiding any possible problem during the purification of the intermediate. For
this step, we exploited the tendency of di-Boc-protected guanidine to give amidinoureas when heated at
reflux in THF in presence of an amine.35,36 This ability results from the conversion of one of the Boc protecting
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group of the guanidine into isocyanate through a t-butoxide elimination and subsequent nucleophilic attack by
the amine. In our case, the reaction occurs through an intramolecular attack, which leads to the formation of a
macrocycle.
For the last guanylation, the macrocyclic amidinourea 8 was Cbz deprotected using H2 over 10% Pd/C in
presence of catalytic aqueous HCl 37%, guanylated with 9 getting compound 10 (90% yield). The latter
guanylating agent was easily prepared from N,N’-Di-Boc-1H-pyrazole-1-carboxamidine and crotyl alcohol using
Mitsunobu condition, adapting a previously described procedure.25,27
Scheme 2: Last steps of the synthesis of compound 1. i. TFA, Et3SiH, DCM, 0 °C to r. t., 1 h; ii. N,N’-Di-Boc-1H-
pyrazole-1-carboxamidine, DIPEA, THF, r. t., 16 h, 96% over two steps; iii. Thiophenol, K2CO3, dry DMF, r. t., 7
h; iv. TEA, THF, reflux, 16 h, 57% over two steps; v. H2, 10% Pd/C, cat. 37% HCl, i-PrOH, r. t., 3 h; vi. 9, TEA, THF,
r. t., 16 h, 89% over two steps; vii. HCl 4M in Dioxane, 23 h, quantitative.
Conclusions
The synthesis of compound 1 always presented drawbacks, ranging from a very low overall yield, challenging
purifications and handling of highly polar compounds, wasting of starting materials due to low molar ratios ,
and use of hazardous chemicals. During the long time we have worked on these molecules, we always
managed to prepare the lab-scale quantity required for the early development without too much hassle, but
the more advanced steps of drug discovery and development required higher amount of compounds which
were really hard to prepare following the old habits, despite the familiarity we had with them. Spending time
and efforts in the setup of an alternative synthetic strategy appeared, then, a good investment for the future
of the project. And indeed it was, since a gram-scale synthesis of 1 was successfully performed, following a
convenient, versatile and efficient route, with a great overall yield, a low cost and adequate amount of time,
despite the process still involved the use and preparation of polyamines.
Compound 1 obtained via this process proved to possess the same physico-chemical properties and
pharmacological activities of our previous batches, making this synthetic method a viable and valuable route
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to overcome all the limitations and difficulties our previous strategies implied, easing the challenging purpose
of finding always novel and more active antifungal compounds. To this aim, the synthesis goes through the
obtainment of the key intermediate 6, whose synthetic flexibility could be very important in the planning of
new derivatives. This process allowed us to perform advanced biological investigations on 1, e.g. in vivo assays,
with very little requirements in terms of costs, manpower and time, pushing our understanding of its
biological behavior even further, and allowing us to focus on the study of its mode of action.
Experimental Section
General. All reagents were used as purchased. CHCl3, CH3CN, DCM, TEA and DIPEA were dried by distillation
from CaH2; THF from Na/Benzophenon. Chromatographic purifications were carried out with flash columns
packaged with silica gel 60, 230-400 mesh and reactions were monitored using TLC purchased from Merck
1
with silica gel 60 F254. H-NMR and 13C-NMR data were acquired on Bruker Avance DPX 400. As internal
standard the residual signal of deuterated solvents was used and the chemical shift are reported in ppm.
Splitting patterns are described as s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet, dq = doublet
of quartet, bs = broad signal. Mass spectra data were obtained using Agilent 1100 LC/MSD VL system
(G1946C).
(8-aminooctyl)(triphenylmethyl)amine (2) To an ice-cold solution of 1,8-Diaminoctane (14.5 g, 100.4 mmol) in
DCM (85 mL), trityl chloride (8 g, 28.7 mmol) was added portionwise. The resulting solution was stirred at
room temperature for 2 h. Then, the reaction mixture was partitioned between DCM/5% NaHCO3 aq; the
organic phase was washed with brine, dried over Na2SO4, filtered and evaporated in vacuo. The crude mixture
was purified by chromatography on silica gel (DCM/MeOH 8:2) yielding compound 2 (10.6 g, 96% yield, using
1
trityl chloride as reference) as a yellow oil. Rf = 0.30 (DCM/MeOH 9:1). H NMR (400 MHz CDCl3): δ 7.47 (d, J
6.8 Hz, 6H), 7.26 (t, J 7.9 Hz, 6H), 7.17 (t, J 7.0 Hz, 3H), 2.74 (t, J 6.6 Hz, 2H), 2.11 (t, J 8.0 Hz, 2H), 1.47 (m, 3H),
1.27 (m, 7H), 0.88 (m, 2H). 13C NMR (100 MHz CDCl3): δ 146.4, 128.7, 127.6, 126.2, 70.9, 43.6, 41.7, 30.4, 30.1,
29.3, 27.8, 27.5, 27.3. LC-MS(ES) m/z: 386.9 [M+H]+.
4-nitro-N-(8-(tritylamino)octyl)benzenesulfonamide
(4)
To
a
solution
of
(8-
aminooctyl)(triphenylmethyl)amine (9.85 g, 25.49 mmol) and DIPEA (11.1 mL, 63.73 mmol) in DCM (150 mL),
nosyl chloride (5.65 g, 25.49 mmol) was added portionwise. The reaction mixture was stirred at room
temperature for 1 h. The mixture was diluted with DCM and extracted with water. The organic layer was
washed with brine, dried over Na2SO4, filtered and evaporated. The crude mixture was purified by
chromatography on silica gel (PE/AcOEt 7:3) yielding compound 4 (13.8 g, 95% yield) as a pale yellow solid. Rf =
1
0.42 (PE/AcOEt 8:2). H NMR (400 MHz CDCl3): δ 8.35 (d, J 8.2 Hz, 2H), 8.05 (d, J 8.4 Hz, 2H), 7.47 (d, J 8.4 Hz,
6H), 7.27 (t, J 7.4 Hz, 6H), 7.19 (m, 3H), 4.63 (bs, 1H), 3.00 (q, J 7.2Hz, 2H), 2.13 (m, 2H), 1.58 (m, 2H), 1.45 (m,
3H), 1.21 (m, 7H). 13C NMR (100 MHz CDCl3): δ 150.0, 146.2, 128.7, 128.3, 128.2, 127.8, 126.3, 124.4, 48.3,
43.7, 43.4, 29.6, 29.3, 28.9, 27.2, 26.7, 26.4. LC-MS(ES) m/z: 571.9 [M+H]+.
Benzyl (8-hydroxyoctyl)carbamate (5). To a solution of 3 (13 g, 44.3 mmol) in dry THF (150 mL), Me2SBH3 (12.6
mL, 132.9 mmol) was added dropwise. The reaction mixture was stirred overnight under N2 atmosphere. Once
starting material disappeared (monitored by TLC), H2O was added dropwise. Then, K2CO3 was added and the
mixture was stirred for 30’. Subsequently, AcOEt was added and the organic layer was washed with 1N NaOH
and brine, dried over Na2SO4, filtered and evaporated. The crude mixture was purified by flash
chromatography on silica gel (PE/AcOEt 6:4) yielding compound 5 as a white solid (12.4 g, 93% yield). Rf = 0.18
1
(PE/AcOEt 6:4). H NMR (400 MHz CDCl3): δ 7.33 (m, 5H), 5.09 (s, 2H), 4.72 (bs, 1H), 3.63 (t, J 6.4 Hz, 2H), 3.18
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(q, J 6.4 Hz, 2H), 1.57-1.49 (m, 6H), 1.31 (m, 6H). 13C NMR (100 MHz CDCl3): δ 156.8, 136.9, 128.7, 128.2, 128.0,
66.7, 62.9, 41.3, 32.9, 30.1, 29.5, 29.4, 26.9, 25.9. LC-MS(ES) m/z: 279.9 [M+H]+.
benzyl (8-((4-nitro-N-(8-(tritylamino)octyl)phenyl)sulfonamido)octyl)carbamate (6). 4 (5 g, 8.74 mmol) was
dissolved in dry THF (50 mL) and treated sequentially with PPh3 (2.12 g, 10.49 mmol), 5 (2.93 g, 10.49 mmol)
and DIAD (2.7 mL, 10.49 mmol) and the reaction mixture was stirred for 3 h, under N2, at room temperature.
Then, the solvent was removed and the residue was partitioned between H2O/DCM, washed with brine, dried
over Na2SO4, filtered and evaporated in vacuo. The crude was purified by column chromatography on silica gel
1
(PE/AcOEt 9:1 to 8:2) giving compound 6 as a colourless oil (5.54 g, 76% yield). Rf = 0.26 (PE/AcOEt 8:2). H
NMR (400 MHz CDCl3): δ 8.33 (d, J 8.6 Hz, 2H), 7.96 (d, J 8.0 Hz, 2H), 7.47 (d, J 7.4 Hz, 7H), 7.26 (m, 10H), 7.18
(t, J 7.6 Hz, 3H), 5.09 (s, 2H), 4.72 (bs, 1H), 3.15 (m, 6H), 2.11 (t, J 7.0 Hz, 2H), 1.57 (m, 4H), 1.49 (m, 6H), 1.26
(m, 14H). 13C NMR (100 MHz CDCl3): δ 156.2, 150.0, 146.3, 136.2, 129.1, 128.9, 128.7, 128.3, 127.6, 127.5,
127.1, 126.9, 67.4, 50.2, 45.3, 40.1, 29.6, 29.4, 29.1, 28.5, 28.3, 27.9, 27.6, 27.4, 26.9, 26.8, 26.6, 26.3. LC-
MS(ES) m/z: 833.8 [M+H]+.
benzyl
N‐[8‐(N‐{8‐[({[(tert‐butoxy)carbonyl]amino}({[(tert‐butoxy)carbonyl]imino})methyl)amino]octyl}4‐nitrobenz
enesulfonamido)octyl]carbamate (7). To a solution of 6 (3.5 g, 4.20 mmol) in DCM (38 mL), cooled at 0 °C,
Et3SiH (2.02 mL, 12.60 mmol) and TFA (1.9 mL, 5%) were added. The reaction mixture was allowed to reach
room temperature and stirred under N2 for 1 h. After that time, the mixture was concentrated in vacuo and
co-evaporated several times with DCM until a white solid formed. The crude mixture was dissolved in dry THF
(70 mL) and to this solution, dry DIPEA (1.1 mL, 6.30 mmol) was added dropwise at room temperature. Then,
N,N’-Di-Boc-1H-pyrazole-1-carboxamidine (1.70 g, 5.46 mmol) was added in one pot. The reaction mixture was
stirred at room temperature for 16 h, under nitrogen atmosphere. The mixture was partitioned between
H2O/AcOEt, the organic layer was washed with brine, dried over Na2SO4 and evaporated to dryness.
Purification by silica gel chromatography (PE/AcOEt 8:2) afforded compound 7 as a viscous oil (3.36 g, 96%
yield over two steps). Rf = 0.11 (PE/AcOEt 9:1). 1H NMR (400 MHz CDCl3): δ 11.50 (s, 1H), 8.35 (d, J 8.2 Hz, 2H),
7.98 (d, J 8.8 Hz, 2H), 7.35 (m, 5H), 5.10 (s, 2H), 4.76 (bs, 1H), 3.41 (q, J 5.8 Hz, 2H), 3.15 (m, 6H), 1.50 (m, 28H),
1.27 (m, 14H). 13C NMR (100 MHz CDCl3): δ 166.1, 157.1, 154.1, 150.1, 146.2, 136.4, 129.1, 128.7, 128.3, 127.2,
124.5, 82.1, 48.2, 43.7, 29.7, 29.2, 28.5, 28.4, 27.8, 27.4, 26.9, 26.7, 26.5, 26.3. LC-MS(ES) m/z: 834.0 [M+H]+.
benzyl (8-(4-((tert-butoxycarbonyl)imino)-2-oxo-1,3,5-triazacyclotridecan-1-yl)octyl)carbamate (8). To a
stirred solution of 7 (3.30 g, 3.96 mmol) in dry DMF (17 mL), K2CO3 (2.19 g, 15.84 mmol) and Thiophenol (0.8
mL, 7.92 mmol) were added and the reaction mixture was stirred at room temperature for 7 h. The reaction
mixture was partitioned between H2O/AcOEt, the water layer was extracted with AcOEt twice and the organic
layers were washed with brine, dried over Na2SO4, filtered and evaporated in vacuo. The crude mixture was
solubilized in dry THF (600 mL) and to this, dry TEA (0.55 mL, 3.96 mmol) was added. The resulting solution
was heated at reflux for 16 h, under N2. The solvent was evaporated and the crude purified by flash
chromatography (PE/AcOEt 85:15), affording compound 8 (1.3 g, 57% yield over two steps) as a colorless oil. Rf
1
= 0.56 (PE/AcOEt 8:2). H NMR (400 MHz CDCl3): δ 12.10 (s, 1H), 8.10 (bs, 2H), 7.35 (m, 5H), 5.09 (s, 2H), 4.71
(bs, 1H), 3.45 (m, 2H), 3.38 (q, J 7.54 Hz, 2H), 3.26-3.15 (m, 4H), 1.65 (m, 6H), 1.54 (m, 6H), 1.46 (m, 12H), 1.27
(m, 12H). 13C NMR (100 MHz CDCl3): δ 163.6, 156.0, 153.8, 153.2, 136.4, 127.7, 127.6, 81.6, 66.1, 46.7, 46.0,
40.7, 39.3, 29.5, 29.1, 28.9, 28.1, 27.5, 26.7, 26.3, 25.9, 25.5, 24.9, 23.4 ppm. LC-MS(ES) m/z: 574.0 [M+H]+,
595.9 [M+Na]+.
tert-butyl ((E)-but-2-en-1-yl)((Z)-((tert-butoxycarbonyl)imino)(1H-pyrazol-1-yl)methyl)carbamate (9). To a
stirred solution of N,N’-di-Boc-pyrazologuanidine (1,0 g, 3,22 mmol) in dry THF (20 mL) under N2 atmosphere,
PPh3 (1,27 g, 4,19 mmol) and crotyl alcohol (0,36 mL, 4,19 mmol) were added. The mixture was cooled to 0 °C
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and DIAD (0,91 mL, 4,84 mmol) was added dropwise. The mixture was stirred at 0 °C for 15 min then at room
temperature for 2 h, then it was diluted with AcOEt and washed with water. The aqueous phase was back-
extracted with AcOEt. The combined organic phases were washed with brine, dried over Na2SO4, filtered and
evaporated under reduced pressure. The crude residue was purified with flash chromatography on silica gel,
eluting with 10% AcOEt/Hex to give pure compound 9 (0.95 g, 95% yield). 1H NMR (400 MHz CDCl3) δ 7.89 (s,
1H), 7.66 (s, 1H), 6.37 (s, 1H), 5.60 (m, 2H), 4.18 (m, 2H), 1.62 (s, 3H), 1.44 (s, 9H), 1.22 (s, 9H). LC-MS(ES) m/z =
387.1 [M+Na]+, 365.1 [M+H]+.
tert-butyl (5-(8-(3-((E)-but-2-en-1-yl)guanidino)octyl)-4-oxo-1,3,5-triazacyclotridecan-2-ylidene)carbamate
(10). To a solution of 8 (1.30 g, 2.26 mmol) in i-PrOH (125 mL), Pd/C 10 wt.% (96 mg, 0.90 mmol) and 14 drops
of 37% HCl were added. The reaction mixture was stirred for 48 h under H2 atmosphere. Once starting
material disappeared (monitored by TLC), the reaction mixture was filtered through a pad of Celite using 85:15
DCM:MeOH as solvent. The filtrate was evaporated in vacuo to dryness and resolubilized in dry THF (38 mL).
To this solution, dry TEA (0.47 mL, 3.40 mmol) was added dropwise and then, 9 (1.07 g, 2.94 mmol) was added
in one pot. The reaction mixture was stirred at room temperature overnight, under N2. The reaction mixture
was partitioned between H2O/AcOEt, washed with brine, dried over Na2SO4 and evaporated in vacuo. The
crude mixture was purified by flash chromatography on silica gel (PE/AcOEt 85:15) yielding compound 10 (1.5
g, 89% yield over two steps) as a sticky colorless oil. Rf = 0.63 (PE/AcOEt 8:2). 1H NMR (400 MHz CDCl3): δ 12.10
(s, 1H), 8.08 (bs, 1H), 5.65 (dq, J 15, 6.2 Hz, 1H), 5.50 (m, 1H), 4.17 (s, 2H), 3.45 (t, J 8.0 Hz, 2H), 3.36 (q, J 7.0
Hz, 2H), 3.24 (m, 4H), 1.66 (m, 7H), 1.56 (m, 6H), 1.49 (s, 11H), 1.46 (s, 19H), 1.31 (m, 13H). 13C NMR (100 MHz
CDCl3): δ 164.0, 154.1, 153.6, 82.0, 47.1, 46.3, 39.6, 29.4, 29.2, 28.5, 28.2, 28.1, 27.9, 27.1, 26.8, 26.2, 25.9,
25.3, 23.7, 23.6, 17.7. LC-MS(ES) m/z: 736.1 [M+H]+.
(E)-1-(but-2-en-1-yl)-3-(8-(4-imino-2-oxo-1,3,5-triazacyclotridecan-1-yl)octyl)guanidine (1). To 10 (1.5 g, 2.04
mmol), 4N HCl in Dioxane (30 mL) was added and the resulting solution was stirred for 23 h at room
temperature. Then, the solvent was evaporated in vacuo and co-evaporated several times with DCM until a
1
white solid was obtained (1.01 g, quantitative yield). MP: 96-99°C. H NMR (400 MHz MeOD): δ 5.75 (dq, J
15.0, 8.0 Hz, 1H), 5.49 (m, 1H), 3.75 (m, 2H), 3.52 (m, 2H), 3.41-3.34 (m, 4H), 3.18 (t, J 8.0 Hz, 2H), 1.71 (m, 7H),
1.60 (m, 4H), 1.38 (m, 18H). 13C NMR (100 MHz MeOD): δ 156.0, 155.4, 153.4, 128.7, 125.1, 46.9, 46.3, 42.7,
41.3, 41.0, 28.9, 28.8, 28.6, 27.2, 26.3, 26.2, 26.1, 25.7, 23.0, 22.7, 16.6. LC-MS(ES) m/z: 436.3 [M+H]+.
Acknowledgements
This work was partially funded by Cygnet Biosciences B.V., Kaya Richard J. Beaujon Z/N, Curaçao.
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