Synthesis of a novel tripeptidomimetic scaffold and biological evaluation for CXC chemokine receptor 4 (CXCR4) antagonism

Article abstract of DOI:10.1016/j.tet.2017.05.057

We here report the preparation of a new 2,6,8-trisubstituted bicyclic tripeptidomimetic scaffold through TFA-mediated cyclization of a linear precursor containing three side chains. The introduction of a triphenylmethyl-protected thiol into carboxylic aci

Full text of DOI:10.1016/j.tet.2017.05.057

Accepted Manuscript
Synthesis of a novel tripeptidomimetic scaffold and biological evaluation for CXC
chemokine receptor 4 (CXCR4) antagonism
Markus Baumann, Lina Marie Nome, Zack G. Zachariassen, Stefanie Karlshøj,
Torgils Fossen, Mette M. Rosenkilde, Jon Våbenø, Bengt Erik Haug
PII:
S0040-4020(17)30543-4
10.1016/j.tet.2017.05.057
TET 28723
DOI:
Reference:
To appear in: Tetrahedron
Received Date: 2 February 2017
Revised Date: 29 April 2017
Accepted Date: 15 May 2017
Please cite this article as: Baumann M, Nome LM, Zachariassen ZG, Karlshøj S, Fossen T, Rosenkilde
MM, Våbenø J, Haug BE, Synthesis of a novel tripeptidomimetic scaffold and biological evaluation for
CXC chemokine receptor 4 (CXCR4) antagonism, Tetrahedron (2017), doi: 10.1016/j.tet.2017.05.057.
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ACCEPTED MANUSCRIPT
Synthesis of a novel tripeptidomimetic scaffold and biological evaluation for CXC chemokine receptor
4 (CXCR4) antagonism
Markus Baumanna, Lina Marie Nomea, Zack G. Zachariassenb, Stefanie Karlshøjc, Torgils Fossena, Mette M.
Rosenkildec, Jon Våbenøb*, Bengt Erik Hauga*
a Department of Chemistry and Centre for Pharmacy, University of Bergen, Allégaten 41, NO-5007 Bergen,
Norway.
b Department of Pharmacy, Faculty of Health Sciences, UiT The Arctic University of Norway, Breivika, NO-
9037 Tromsø, Norway.
c
Laboratory for Molecular Pharmacology, Department of Neuroscience and Pharmacology, Faculty of
Health and Medical Sciences, University of Copenhagen, Blegdamsvej 3, DK-2200 Copenhagen, Denmark.
Abstract
We here report the preparation of a new 2,6,8-trisubstituted bicyclic tripeptidomimetic scaffold through TFA-
mediated cyclization of a linear precursor containing three side chains. The introduction of a
triphenylmethyl-protected thiol into carboxylic acid containing building blocks through sulfa Michael
additions onto α,β-unsaturated hexafluoroisopropyl esters is described. The stereoselectivity of the bicycle
formation was found to be somewhat lower than that previously reported for analogous 3,6,8-trisubstituted
scaffolds. Moreover, the configuration of the linear precursor directs the stereochemical outcome of the
cyclization differently when the R1 side chain is positioned on C2 in the bicycles (present work) instead of
C3 (previous work). Tripeptidomimetic compounds based on the new scaffold were synthesized and
evaluated for antagonistic potency toward CXCR4, and one compound (45a) displayed similar activity to
earlier reported 3,6,8-tripeptidomimetic bicycles.
Graphical abstract:
1

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1. Introduction
Endogenous and exogenous peptides display a multitude of important activities related to human disease
states, and both the processing of peptide substrates and the binding of signalling peptides have been targeted
for therapeutic purposes. Thus, proteolytic enzymes (proteases) as well as peptide-binding (peptidergic)
receptors are targets for drugs. Compounds that interfere with either processing or signalling of peptides are
commonly referred to as peptidomimetics, and have been classified as either peptide backbone mimetics
(type I), functional mimetics (type II) or topographical mimetics (type III).1 Peptidomimetic structures have
found widespread use as drugs, and in a drug discovery perspective, tripeptidomimetics have been identified
as particularly interesting.2
We have previously reported the synthesis of a novel type of tripeptidomimetics based on a 3,6,8-
trisubstituted 4,7-dioxo-1-thia-5,8-diaza-bicyclo[4.4.0]decane scaffold (Figure 1A).3 These compounds were
Arg1-Arg2-2-Nal3 (2-napthylalanine, 2-Nal) mimetics and were designed as antagonists for the CXC
chemokine receptor 4 (CXCR4), which is a 7TM receptor involved in entry of T-tropic HIV strains into
CD4+ T-cells,4,5 and also has a pathological role in cancer and rheumatoid arthritis.6 The prototype
compounds 1a and 1b (Figure 1A) were shown to have EC50 values of 64 ꢀM and 80 ꢀM, respectively, in a
cell-based assay for wild-type CXCR4.3 We recently reported an array of close analogues aimed at
investigating the influence of side chain length (m = 2-4, n = 3-4, o = 1-2) on antagonistic potency, where
2a,b (Figure 1A; isolated as a mixture of diastereoisomers) showed an EC50 of 61 ꢀM.7
H
N
H
N
A
B
S
S
o
o
Ar
N
Ar
N
H
H
R3
R3
N
m
NH2
NH
N
NH2
NH
S
R1
S
O
O
N
N
m
O
O
N
n
N
n
O
R1
HN
H2N NH
HN
O
3S,6S,9aR (1a, 2a)
3R,6S,9aS (1b, 2b)
O
R2
R2
O
HN NH2
Br
Scaffold A
Scaffold B
1a and 1b: m = n = 3, o = 2, Ar =
2a,b: m = n = 3, o = 1, Ar =
Figure 1. A) Bicyclic scaffold A and tripeptidomimetic CXCR4 antagonists based on scaffold A3,7; B) Bicyclic scaffold B.
Our tripeptidomimetic compounds are inspired by a series of highly potent cyclopentapeptide CXCR4
antagonists originally reported by Fujii et al.8 The most potent cyclopentapeptides were based on the amino
acid sequence L/D-Arg1-Arg2-2-Nal3-Gly4-
D
-Tyr5, where the Arg1-position was shown to be relatively
insensitive to configurational change, as both
L- and
D
-Arg1 were tolerated.8 Later, Demmer et al. showed
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that re-positioning of the Arg1 side chain to give N-alkylated
D
-Ala1 cyclopentapeptide analogues resulted in
high antagonistic potency.9 For our approach, we envisioned that the 2,6,8-trisubstituted scaffold B (Figure
1B) would be suited to present the side chains of the Arg1-Arg2-Nal3 tripeptide fragment in a similar manner
as for the cyclopentapeptides. In this scaffold, the C2-C3 bond would correspond to the Cα-Cβ vector of the
Arg1 side chain, resulting in an additional conformational constraint. Our synthetic endeavours toward this
novel scaffold are reported herein.
2. Results and Discussion
2.1. Synthesis
In our previous syntheses of tripeptidomimetic compounds, the building blocks bearing the R1 side chain
were prepared without any attempts at controlling the configuration of the chiral centre.7,3 Consequently, two
diastereoisomers (a and b, Figure 1A) of the scaffold A bicycles were formed in the cyclization. The sulfa
Michael-type addition of the deprotected thiol onto the acyliminium ion intermediate was highly
stereoselective, as initially investigated by Grimes et al.,10 and resulted in two diastereomeric bicycles where
H6 and H9a always displayed a cis relationship.7,3
In our earlier syntheses of building blocks for scaffold A with different chain length for the R1 side chain, the
sulfur atom was introduced through a Michael addition between triphenylmethanethiol and N-Boc protected
α-methylenyl lactams of varying ring size.3,7 Subsequent hydrolysis of the N-Boc protected lactams11 gave
the required N-Boc protected ω-amino carboxylic acids. For scaffold B, we envisioned that a Michael
acceptor with an endocyclic double bond would give access to an array of building blocks with varying
methylene spacer length for the R1 side chain. Toward this end, we started out by converting N-Boc lactam 3
into α,β-unsaturated lactam
4
through reaction of the corresponding enolate with N-tert-
butylbenzenesulfinimidoyl chloride at −78 °C (Scheme 1).12 The α,β-unsaturated lactam 4 was difficult to
separate from the starting material and was isolated as a 10:1 mixture with 3; however, in the subsequent
addition step, the desired Michael adduct 5 could be isolated in pure form. Finally, hydrolysis of 5 gave
carboxylic acid 6 in high yield.
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Scheme 1. Reagents and conditions: a) i) LDA, THF, -78 °C ii) PhS(Cl)=N-t-Bu, THF, -78 °C (90%, 4/3 10:1); b) Ph3CSH,
Et3N, CH2Cl2 (68%); c) 1 M aq. LiOH, THF (82%).
For elongation of the R1 side chain, N-Boc protected lactam 7 (Scheme 2) was treated with LiHMDS in dry
THF to form the corresponding enolate, which was reacted with phenylselenyl chloride to give
phenylselenide 8.13 Oxidation with hydrogen peroxide to the selenoxide gave in turn the α,β-unsaturated
lactam 9. However, this material proved to be difficult to purify, and the crude product was therefore used
directly in the Michael addition.14 Triphenylmethanethiol was used as the Michael donor in the presence of
triethylamine as base and dry CH2Cl2 as solvent, but this reaction was unsuccessful. By changing the solvent
to anhydrous DMF, the Michael product 10 was obtained in 66% yield over two steps. Finally, hydrolysis
gave carboxylic acid 11.
Scheme 2. Reagents and conditions: a) i) LiHMDS, THF, -78 °C, ii) PhSeCl, THF, -78 °C (86%); b) H2O2, THF, 0 °C; c) Ph3CSH,
Et3N, DMF (66% over two steps); d) 1 M aq. LiOH, THF (71%).
While the Michael addition worked well both for the six- and seven-membered lactams, reaction with 12
(Scheme 3) did however not proceed according to plan (data not shown). Although the m/z for the Michael
product 13 was observed for the crude product, all attempts at isolating the product by flash column
chromatography proved to be unsuccessful. One reason for the poor outcome might be lower ring-strain for
the eight-membered α,β-unsaturated lactam 12 leading to lower reactivity than the six- and seven-membered
analogues. Since we were planning to prepare bicyclic analogues with an R1 side chain containing five
methylene groups, which would require Michael addition onto an α,β-unsaturated 9-membered lactam, we
decided to abandon the lactam strategy.
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Scheme 3. Reagents and conditions: a) Ph3CSH, Et3N, DMF.
In order to access derivatives with a longer methylene chain for the R1 group, we decided to employ α,β-
unsaturated methyl esters 19 and 20 as Michael acceptors (Scheme 4), which were prepared starting from
amino alcohols 14 and 15 through N-protection15 followed by oxidation to aldehydes and then Wittig reaction
with methyl (triphenylphosphoranylidene)acetate (Scheme 4).
Scheme 4. Reagents and conditions: a) Boc2O, 1,4-dioxane, 0 °C to rt; b) TBDMSCl, imidazole, CH2Cl2, 0 °C; c) i) n-BuLi, THF,
0 °C, ii) Boc2O, THF, 0 °C to rt; d) TBAF, THF (16: 52% over 4 steps); e) TEMPO, n-Bu4NCl, NCS, CH2Cl2 (18: 37% over 2
steps); f) Dess-Martin periodinane, CH2Cl2, 0 °C; g) MeOC(O)CHP(Ph)3, THF, 60 °C (19: 81% over 2 steps; 20: 57%).
Michael addition with triphenylmethanethiol onto α,β-unsaturated methyl esters (such as 19 and 20) proved
to be very unreliable. Complete conversion of the starting material could never be achieved, albeit a variety
of different conditions were tested.16 In addition, triphenylmethanethiol decomposed during the reactions to
give products with similar Rf values as the desired Michael addition products, which hampered purification
by flash column chromatography. As the methyl esters 19 and 20 proved to be too unreactive, we decided to
increase the electrophilicity by conversion into hexafluoroisopropyl esters 21 and 22 (Scheme 5) through
DCC mediated esterification.17 The Michael addition now gave protected thiols, which were hydrolysed to
carboxylic acids 25 and 26.
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Scheme 5. Reagents and conditions: a) 1) 1 M aq. LiOH, THF; b) 1,1,1,3,3,3-hexafluoropropan-2-ol, DMAP, DIC, CH2Cl2 (21:
62%; 22: 61%, both over 2 steps); c) Ph3CSH, Et3N, CH2Cl2 (23: 52%; 24: 91%); d) 1 M aq. LiOH, THF (25: 33%; 26: 56%).
In order to elongate the R3-side chain compared to our previously reported antagonists based on scaffold A,3
secondary amine 29 was prepared from N-Boc protected allylamine 28 through hydroboration followed by
Suzuki-Miyaura coupling and then Boc-deprotection followed by alkylation (Scheme 6).18
Scheme 6. Reagents and conditions: a) 9-BBN, THF; b) 2-Bromonapthalene, Pd(dppf)2Cl2•CH2Cl2, 1 M NaOH; c) NaOH, H2O2,
H2O, THF, 0 °C (70% over 3 steps); d) TFA, CH2Cl2, 0 °C; e) BrCH2CH(OMe)2, K2CO3, DMF, 80 °C (31% over 2 steps); f)
Fmoc-Arg(Pbf)-OH, HATU, DIPEA, CH2Cl2 (79%).
Next, the R1 side chain bearing carboxylic acids were coupled with the Fmoc-deprotected R2-R3 fragments
30 and 313 (Scheme 7). The resulting linear precursors 32-37 (inseparable mixture of diastereoisomers) were
submitted to TFA-mediated deprotection and cyclization to give bicyclic compounds 38-43, which were
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guanidinylated to give the final products 44-49 as mixtures of diastereoisomers (see Table 1 for full
structures).
Scheme 7. Reagents and conditions: a) Et2N, CH2Cl2; b) 6, 11, 25 or 26, HATU, DIPEA, CH2Cl2 (20-77% over 2 steps); c)
TFA/H2O/thioanisole (90:5:5); d) 1H-Pyrazole-1-carboxamidine hydrochloride, DIPEA, DMF (8-42% over 2 steps).
Interestingly, the outcome of the cyclisation of linear precursors for scaffold B was different than for scaffold
A (Figure 1A).10,3,7 For scaffold A, TFA-treatment of the mixture of diastereoisomeric linear precursors led
to the formation of two cyclization products, however the cyclization of 32-37 gave two major and either one
or two minor peaks as evident by HPLC analysis (see Figure S1). From this point on, the two major
guanidinylation products are denoted a and b, and the minor compounds are denoted c and d. MS analysis
revealed that all these peaks had the expected m/z of the cyclized products.
The crude guanidinylation products were purified by semi-preparative RP-HPLC, however separation proved
to be difficult due to small differences in retention time between the isomers. Pure diastereoisomers could not
be obtained for every analogue and when separation was impossible, mixtures of two isomers were collected
and analysed by NMR spectroscopy and mass spectrometry.
2.2. NMR Studies on Scaffold B Analogues
In order to assign the configuration of the newly formed chiral centres at C2 and C9a (Figure 1B) for the
1
different cyclization products, the 2D H ROESY experiment was used. As was the case for scaffold A, the
known configuration of C6 was used as a starting point. The configuration of C9a for 45a and 45b (Figure 2)
could be determined by the presence of strong cross-peaks between H9a and Hβ/Hγ of the R2 side chain in
the ROESY spectrum of 45a, while these were absent in the spectrum of 45b. Further, the ROESY spectra of
both the a and b fractions showed no cross-peaks between H2 and H9a, indicating that these are trans to each
other, which is opposite to what was observed for H3/H9a for all analogues based on scaffold A.3,7 These
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observations suggest a (2S,6S,9aS) configuration for 45a and a (2R,6S,9aR) configuration for 45b as
depicted in Figure 2.
Figure 2. Suggested configuration of major isomers a and b.
These assignments were further supported by the combination of strong cross-peaks for H9a/Hb3 (see Figure
2), a strong cross-peak for H2/Ha3, a weaker cross-peak for H2/Hb3 and the absence of a H9a/Ha3 cross-peak
in the ROESY spectrum for 45a. This suggests that H9a and Hb3 are oriented cis, while H2 is oriented trans
to Hb3 and cis to Ha3 for 45a. For 45b, the H9a/H3 and H2/H3 correlations were reversed. The NMR data for
the a and b diastereoisomers of 44 and 46-49 were consistent with those of 45a and 45b.
Isomers c and d could never be isolated in pure form, only as a mixture with varying amounts of isomer b.
For 48 and 49, the NMR signals for the two minor products could be partially assigned based on 2D NMR
1
spectra of the mixtures with the b isomer (see Table S1 for a comparison of H chemical shifts for H2, H6
1
and H9a). The 2D H ROESY spectra did however not provide a clear-cut answer with regards to the
configurational assignment of C2 and C9a, as the spectra were quite crowded and signals overlap in several
cases. Based on the 2D NMR and HRMS data, the four products are clearly all bicyclic, and all of them have
different chemical shifts for the methine protons of the bicycles. Unfortunately, our present NMR data does
not allow for an unequivocal assignment of the configuration of the chiral carbon atoms for isomers c and d.
Interestingly, Grimes et al.10 noted that in the absence of an R1 substituent, cyclization of linear precursors
gave a 90:10 mixture of diastereoisomers, which is comparable to the ratio between major and minor
cyclization products in the present work.
2.3. Biological Activity for Scaffold B Analogues
The antagonistic potency on human CXCR4 for compounds 44-49 was tested in a functional assay and 45a
showed activity with an EC50 value of 61 ꢀM, while its diastereoisomer 45b was inactive (Table 6). As none
of the other synthesised scaffold B analogues were active, this indicates that a three-carbon atom spacer in
the R1 side chain is the optimum. On the other hand, it seems detrimental to increase the R3 side chain spacer
from two to three carbon atoms in analogues based on scaffold B.
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Table 1. Structures of compounds 44 – 49 and their antagonistic potency on human CXCR4.
m
n
log EC50 ±
SEM
EC50
(ꢀM)
>100
>100
61
Purity
(220 nm)
>95%
Compd
R3
(R1) (R2)
> −4
44a
2
3
4
5
3
3
3
3
> −4
>95%
44b
−4.21 ± 0.01
> −4
>95%
45a
>100
>100
>95%
45b
> −4
>95%
46a
> −4
>100 8:2 b/c+d
46b,c,d
47a
> −4
>100
>100
>100
>100
>100
>100
>100
>100
>95%
>95%
> −4
47b
48a
> −4
>95%
3
5
3
3
> −4
8:2 b/c
2:8 b/d
>95%
48b,c
48b,d
49a
> −4
> −4
> −4
7:3 b/c
1:9 b/d
49b,c
49b,d
> −4
3. Conclusions
A new 2,6,8-trisubstituted bicyclic scaffold has been prepared for the first time, and through 2D NMR studies
we have determined the configuration for the major isomer formed. The configuration of the linear precursor
directed the stereochemical outcome of the cyclization differently whether the R1 side chain is positioned on
C2 (this work) compared to C3 (previous work) in the bicycles. All reports on cyclization to obtain
derivatives of 3,6,8-trisubstituted 4,7-dioxo-1-thia-5,8-diaza-bicyclo[4.4.0]decane gave isomers with H3 and
H9a in a cis relationship, whereas we in this work found that for derivatives of 2,6,8-trisubstituted 4,7-dioxo-
1-thia-5,8-diaza-bicyclo[4.4.0]decane, H2 and H9a were trans for the major isomers. High
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diastereoselectivity in cyclizations to give 3,6,8-trisubstituted bicycles has been reported,10,3,7 however for the
2,6,8-substitution pattern the diastereoselectivity was lower, with the major and minor isomers formed in an
approximately 9:1 ratio. With respect to antagonistic potency toward CXCR4, we found that analogue 45a
displayed comparable activity to our original hit compounds based on the 3,6,8-trisubstituted scaffold and
that a three carbon spacer seems to be optimal for the R1 side chain.
4. Experimental
4.1. Chemistry - All starting materials, reagents and solvents were purchased from Sigma-Aldrich and used
as delivered unless stated otherwise. Compounds 4,12 1619 and 313 were prepared according to literature
procedures and their analytical data were found to be in accordance with those reported. Anhydrous THF was
obtained from an anhydrous solvent delivery system (SDS-800 from mBraun) at the Department of
Chemistry, University of Bergen. CH2Cl2 was dried over molecular sieves. For analysis by thin layer
chromatography (TLC), aluminium sheets coated with Merck KGaA silica gel (60 F254) were used. The TLC
plates were visualized using either ultraviolet light or by immersion in a solution of 2% ninhydrin in ethanol
supplemented with 10 drops of concentrated sulfuric acid per 100 mL solution, followed by heating.
Purification by flash column chromatography was performed on Merck KGaA Kieselgel (230 – 400 mesh).
All final compounds were purified by semi-preparative RP-HPLC eluting with mixtures of acetonitrile and
H2O (both containing 0.1% TFA). Fractions of equal purity were pooled and lyophilized. All tested
compounds were analysed by RP-HPLC and the purity is specified for each compound below. NMR spectra
were obtained on a Bruker Biospin DPX400, a Bruker Biospin AV500, a Bruker Biospin AV600 or a Bruker
Biospin 850SB. High resolution mass spectra were obtained on a JEOL AccuTOFTM JMS T100LC which
was operated in ESI mode.
4.1.1. tert-Butyl 2-oxo-4-(tritylthio)piperidine-1-carboxylate (5)
To a stirred mixture of N-Boc-protected α,β-unsaturated lactam 4 (339 mg, 1.55 mmol) and
triphenylmethanethiol (639 mg, 2.33 mmol) in dry CH2Cl2 (9.0 mL) was added triethylamine (325 ꢀL, 2.33
mmol). The reaction mixture was stirred at room temperature for 29 h, diluted with CH2Cl2 (25 mL) and
washed with saturated NaCl (20 mL). The solvent was removed under reduced pressure to give the crude
product (1.27 g). Purification by automated column chromatography on silica gel (hexanes/EtOAc, 8:2) gave
the title compound (503 mg, 68%). Rf (hexanes/EtOAc, 8:2) = 0.21; 1H NMR (400 MHz, CDCl3): δ = 7.50 –
7.43 (m, 6H), 7.33 – 7.27 (m, 6H), 7.25 – 7.19 (m, 3H), 3.60 (dt, J = 12.9, 5.0 Hz, 1H), 3.17 – 3.08 (m, 1H),
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2.60 – 2.50 (m, 2H), 2.44 – 2.34 (m, 1H), 1.47 (s, 9H), 1.46 – 1.38 (m, 2H); 13C NMR (100.6 MHz, CDCl3):
δ = 169.1, 152.4, 144.6, 129.5, 128.2, 127.0, 83.2, 68.0, 44.9, 42.1, 37.8, 30.6, 28.0; HRMS (ESI): m/z
[M+Na]+ calcd for C29H31NO3SNa+: 496.1917; found: 496.1924.
4.1.2. 5-((tert-Butoxycarbonyl)amino)-3-(tritylthio)pentanoic acid (6)
To a stirred solution of tert-butyl 2-oxo-4-(tritylthio)piperidine-1-carboxylate (approximately 500 mg, 1.05
mmol) in THF (5.0 mL) was added LiOH (1 M, 2.0 mL, 2.0 mmol). The reaction mixture was stirred at room
temperature and monitored by TLC. After 1 h the starting material was consumed completely and the solvent
was removed under reduced pressure. The aqueous residue was acidified to pH 4 with 10% citric acid and
extracted with CH2Cl2 (20 mL, then 3 x 5 mL). The combined organic layer was washed with saturated NaCl
(40 mL), dried over anhydrous MgSO4, filtered and concentrated to give the crude product (477 mg).
Purification by flash column chromatography on silica gel (CH2Cl2/MeOH, 95:5) gave the title compound as
a colorless oil (424 mg, 82%). Rf (CH2Cl2/MeOH, 95:5) = 0.20; 1H NMR (500 MHz, CDCl3): δ = 7.55 – 7.51
(m, 6H), 7.29 (t, J = 7.7 Hz, 6H), 7.22 – 7.18 (m, 3H), 4.25 (bs, 1H), 3.16 – 2.96 (m, 2H), 2.60 (s, 1H), 2.34 –
13
2.11 (m, 2H), 1.72 – 1.62 (m, 1H), 1.56 – 1.47 (m, 1H), 1.41 (s, 9H); C NMR (150.9 MHz, CDCl3): δ =
176.5, 156.0, 144.8, 129.6, 128.1, 126.9, 79.3, 67.8, 39.7, 38.4, 37.8, 35.0, 28.5; HRMS (ESI): m/z [M+Na]+
calcd for C29H33NO4SNa+: 514.2023; found: 514.2030.
4.1.3. tert-Butyl 2-oxo-3-(phenylselanyl)azepane-1-carboxylate (8)
Lithium bis(trimethylsilyl)amide (1.0 M in THF, 1.80 mL, 1.80 mmol) was added drop-wise to a stirred
solution of N-Boc 6-aminohexanoic acid lactam (0.30 mL, 1.46 mmol) in dry THF (3 mL) at -78 °C. The
solution was stirred for 1 h before phenylselenyl chloride (0.36 g, 1.90 mmol) in dry THF (3 mL) was added
drop-wise. After 2 h of stirring at -78 °C the reaction mixture was allowed to warm to room temperature
before HCl (1.0 M, 10 mL) was added to quench the reaction. The reaction mixture was extracted with
EtOAc (3 x 10 mL) and the combined organic layers were washed with NaHCO3 (10%, 30 mL) and saturated
NaCl (30 mL) and dried over anhydrous MgSO4. The solvent was removed under reduced pressure to give
the crude product (624 mg). Purification by flash column chromatography on silica gel (hexanes/EtOAc, 9:1)
1
gave the title compound as a white solid (462 mg, 86%). Rf (hexanes/EtOAc, 9:1) = 0.16; H NMR (400
MHz, CDCl3): δ = 7.62 – 7.58 (m, 2H), 7.31 – 7.26 (m, 3H), 4.29 (dd, J = 9.4, 2.5 Hz, 1H), 4.10 – 4.02 (m,
1H), 3.75 – 3.67 (m, 1H), 2.21 – 2.11 (m, 1H), 2.09 – 1.97 (m, 1H), 1.89 – 1.79 (m, 1H), 1.76 – 1.65 (m, 2H),
1.64 – 1.55 (m, 1H), 1.52 (s, 9H); 13C NMR (100.6 MHz, CDCl3): δ = 173.7, 153.2, 134.6, 129.6, 129.3,
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128.1, 83.3, 51.1, 45.7, 31.6, 28.4, 28.3, 28.1; HRMS (ESI): m/z [M+Na]+ calcd for C17H23NO3SeNa+:
392.0735; found: 392.0743.
4.1.4. tert-Butyl 2-oxo-4-(tritylthio)azepane-1-carboxylate (10)
A stirred solution of 8 (77 mg, 0.21 mmol) in THF (0.5 mL) was cooled to 0 °C and H2O2 (30%, 50 ꢀL, 0.44
mmol) was added drop-wise. The reaction mixture was stirred at 0 °C for 17 minutes and then warmed up to
room temperature over 20 minutes. The reaction mixture was separated between CH2Cl2 (5 mL) and
saturated NaHCO3 (5 mL). The aqueous layer was extracted with CH2Cl2 (5 mL) and the combined organic
layers were washed with saturated NaCl and dried over MgSO4. The solvent was removed under reduced
pressure to give the crude N-Boc (Z) 6-aminohex-2-enoic acid lactam (9) as yellow oil (58 mg). The crude
product (58 mg) and triphenylmethanethiol (156 mg, 0.56 mmol) were dissolved in anhydrous DMF (3 mL).
Triethylamine (80 ꢀl, 0.58 mmol) was added and the reaction mixture was stirred for 18 h. The solvent was
removed under reduced pressure to give the crude product (290 mg). Purification by flash column
chromatography on silica gel (hexanes/EtOAc, 4:1) gave the title compound as white foam (68 mg, 66% over
1
two steps). Rf (hexanes/EtOAc, 4:1) = 0.19; H NMR (400 MHz, CDCl3): δ = 7.51 – 7.46 (m. 6H), 7.31 –
7.25 (m, 6H), 7.22 – 7.17 (m, 3H), 3.68 – 3.54 (m, 2H), 2.72 – 2.54 (m, 3H), 1.88 – 1.76 (m, 1H), 1.48 (s,
9H), 1.46 – 1.30 (m, 3H); 13C NMR (150.9 MHz, CDCl3): δ = 172.3, 152.6, 144.8, 129.7, 128.1, 126.9, 82.9,
68.3, 46.3, 45.3, 39.9, 35.7, 28.2, 26.2; HRMS (ESI): m/z [M+Na]+ calcd for C30H33NO3SNa+: 510.2073;
found: 510.2078; m/z [2M+Na]+ calcd for C60H66N2O6S2Na+: 997.4255; found: 997.4283.
4.1.5. 6-(tert-Butoxycarbonylamino)-3-(tritylthio)hexanoic acid (11)
To a stirred solution of 10 (118 mg, 0.24 mmol) in THF (1 mL) was added LiOH (1.0 M, 0.73 mL, 0.73
mmol). The reaction was monitored by TLC and all starting material was consumed after 1 h. The solvent
was removed under reduced pressure and the aqueous residue was acidified to pH 4 with 10% citric acid
followed by extraction with CH2Cl2 (20 mL, then 3 x 5 mL). The organic layers were combined, washed with
saturated NaCl (25 mL), dried over anhydrous MgSO4 and concentrated to give the crude product (117 mg).
Purification by flash column chromatography on silica gel (CH2Cl2/MeOH, 95:5) gave the title compound as
a colorless oil (87 mg, 71%). Rf (CH2Cl2/MeOH, 95:5) = 0.23; 1H NMR (600 MHz, CDCl3): δ = 7.55 – 7.52
(m, 6H), 7.31 – 7.27 (m, 6H), 7.23 – 7.19 (m, 3H), 4.57 (bs, 1H), 3.00 – 2.86 (m, 2H), 2.70 – 2.63 (m, 1H),
2.24 (dd, J = 16.1, 9.4 Hz, 1H), 2.04 (d, J = 15.9 Hz, 1H), 1.44 (s, 9 H), 1.41 – 1.31 (m, 4H); 13C NMR
12

ACCEPTED MANUSCRIPT
(125.8 MHz, CDCl3): δ = 175.5, 156.0, 144.9, 129.6, 128.1, 126.8, 79.2, 67.6, 40.6, 40.3, 39.6, 31.8, 28.5,
26.4; HRMS (ESI): m/z [M+Na]+ calcd for C30H35NO4SNa+: 528.2179; found: 528.2187.
4.1.6. tert-Butyl (6-oxohexyl)carbamate (18)
To a stirred solution of crude 17 (1.92 g) in dry CH2Cl2 (10 mL) at 0 °C was added Dess-Martin periodinane
(3.74 g, 8.81 mmol). The reaction mixture was stirred at room temperature for 25 h. Slow addition of a
mixture of saturated NaHCO3/saturated Na2S2O3 (1:1, 10 mL) and stirred for another 30 minutes. The two
layers were separated, and the aqueous layer was extracted with EtOAc (3 x 10 mL). The combined organic
layers were washed with H2O (2 x 20 mL) and saturated NaCl (20 mL), dried over anhydrous MgSO4,
filtered and concentrated to give the crude product as light yellow oil (2.03 g). Purification by flash column
chromatography on silica gel (hexanes/EtOAc, 6:4) gave the title compound as colorless liquid (661 mg, 37%
over 2 steps). Rf (hexanes/EtOAc, 6:4) = 0.38; 1H NMR (500 MHz, CDCl3): δ = 9.76 (t, J = 1.8 Hz, 1H), 4.58
(bs, 1H), 3.16 – 3.06 (m, 2H), 2.43 (td, J = 7.3, 1.6 Hz, 2H), 1.65 (p, J = 7.5 Hz, 2H), 1.54 – 1.47 (m, 2H),
13
1.44 (s, 9H), 1.39 – 1.32 (m, 2H); C NMR (125.8 MHz, CDCl3): δ = 202.6, 156.1, 79.2, 43.8, 40.4, 30.0,
28.5, 26.4, 21.8; HRMS (ESI): m/z [M + Na]+ calcd for C11H21NO3Na+: 238.1414; found: 238.1418; m/z [2M
+ Na]+ calcd for C22H42N2O6Na+: 453.2935; found: 453.2930.
4.1.7. Methyl (E)-7-((di-tertbutoxycarbonyl)amino)hept-2-enoate (19)
To a stirred solution of 16 (195 mg, 0.64 mmol) in CH2Cl2 (5 mL) at 0 °C was added Dess-Martin
periodinane (319 mg, 0.75 mmol). The reaction mixture was stirred at 0 °C for 2 h. Saturated
NaHCO3/saturated Na2S2O3 (1:1, 10 mL) was added drop wise to the reaction mixture and stirring continued
for 30 minutes. The two layers were separated and the aqueous layer was extracted with diethyl ether (4 x 10
mL). The organic layers were combined, washed with H2O (3 x 20 mL) and saturated NaCl (20 mL), dried
over anhydrous MgSO4 and concentrated to give the crude product (193 mg), which was used in the
subsequent step without purification. To a stirred solution of the crude aldehyde (193 mg) made in the
previous step in dry THF (5 mL) was added methyl (triphenylphosphoranylidene)acetate (454 mg, 1.36
mmol). The reaction mixture was stirred at 60 °C for 24 h. The solvent was removed under reduced pressure
to give the crude product as an orange solid (620 mg). Purification by flash column chromatography on silica
gel (hexanes/EtOAc, 8:2) gave the title compound (187 mg, 81%). Rf (hexanes/EtOAc, 8:2) = 0.34; 1H NMR
(500 MHz, CDCl3): δ = 6.95 (dt, J = 15.6, 7.0 Hz, 1H), 5.82 (dt, J = 15.6, 1.5 Hz, 1H), 3.72 (s, 3H), 3.57 (t,
13
7.3 Hz, 2H), 2.25 – 2.20 (m, 2H), 1.64 – 1.56 (m, 2H), 1.50 (s, 18H), 1.48 – 1.42 (m, 2H); C NMR (125.8
13

ACCEPTED MANUSCRIPT
MHz, CDCl3): δ = 167.2, 152.9, 149.2, 121.3, 82.3, 51.6, 46.2, 32.0, 28.7, 28.2, 25.4; HRMS (ESI): m/z
[M+Na]+ calcd for C18H31NO6Na+: 380.2044; found: 380.2047.
4.1.8. Methyl (E)-8-((tert-butoxycarbonyl)amino)oct-2-enoate (20)
Boc-protected aldehyde 18 (640 mg, 2.97 mmol) was dissolved in dry THF (15 mL) and
methyl(triphenylphosphoranylidene)acetate (1.40 g, 4.17 mmol) was added. The mixture was stirred at 60 °C
for 21 h under argon atmosphere. The solvent was removed under reduced pressure to give the crude product
as a yellow solid (1.95 g). Purification by flash chromatography on silica gel (hexanes/EtOAc 7:3) gave the
1
title compound as a colorless liquid (456 mg, 57%). Rf (hexanes/EtOAc, 7:3) = 0.37; H NMR (500 MHz,
CDCl3): δ = 6.95 (dt, J = 15.6, 7.0 Hz, 1H), 5.82 (dt, J = 15.7, 1.5 Hz, 1H), 4.59 (bs, 1H), 3.72 (s, 3H), 3.17 –
3.01 (m, 2H), 2.21 (qd, J = 7.2, 1.6 Hz, 2H), 1.52 – 1.46 (m, 4H), 1.44 (s, 9H), 1.38 – 1.30 (m, 2H); 13C
NMR (125.8 MHz, CDCl3): δ = 167.2, 156.1, 149.4, 121.2, 79.1, 51.5, 40.5, 32.1, 30.0, 28.5, 27.8, 26.4;
HRMS (ESI): m/z [M+Na]+ calcd for C14H25NO4Na+: 294.1676; found: 294.1683.
4.1.9. 1,1,1,3,3,3-hexafluoropropan-2-yl (E)-7-((di-tert-butoxycarbonyl)amino)hept-2-enoate (21)
LiOH (1 M, 1.6 mL, 1.6 mmol) was added to a stirred solution of unsaturated methyl ester 19 (187 mg, 0.52
mmol) in THF (2 mL). The reaction mixture was stirred for room temperature for 11.5 h. The solvent was
removed under reduced pressure and the pH of the aqueous residue was acidified to pH 4 with 10% citric
acid. The mixture was extracted with CH2Cl2 (4 x 10 mL) and the combined organic layer was washed with
saturated NaCl (25 mL), dried over anhydrous MgSO4 and concentrated to give the crude product (170 mg).
Analysis by ESI-MS showed incomplete conversion of starting material, therefore the crude product (170
mg) was dissolved in THF (2 mL) and treated with LiOH (1 M, 6 mL, 6 mmol). The reaction mixture was
stirred at room temperature for 20 h. The solvent was removed under reduced pressure and the pH of the
aqueous residue was acidified to pH 4 with 10% citric acid. The mixture was extracted with CH2Cl2 (4 x 10
mL), the combined organic layer was washed with saturated NaCl (25 mL), dried over anhydrous MgSO4
and concentrated to give the crude product (151 mg), which was used in the next step without purification.
To a stirred solution of crude carboxylic acid (151 mg) in dry CH2Cl2 (2 mL) was added DMAP (24 mg, 0.19
mmol), DIC (80 µL, 0.52 mmol) and 1,1,1,3,3,3-hexafluoropropan-2-ol (60 µL, 0.57 mmol). The reaction
mixture was stirred at room temperature for 21.5 h and then diluted with CH2Cl2 (15 mL), washed with
saturated NH4Cl (10 mL) and saturated NaCl (10 mL). The organic layer was dried over anhydrous MgSO4
and concentrated to give the crude product (408 mg). Purification by flash column chromatography on silica
14

ACCEPTED MANUSCRIPT
gel (hexanes/EtOAc, 9:1) gave the title compound (160 mg, 62% over 2 steps). Rf (hexanes/EtOAc, 9:1) =
0.28; 1H NMR (600 MHz, CDCl3): δ = 7.20 (dt, J = 15.6, 6.9 Hz, 1H), 5.94 (dt, J = 15.7, 1.6 Hz, 1H), 5.82
(sep, J = 6.1 Hz, 1H), 3.58 (t, J = 7.3 Hz, 2H), 2.34 – 2.29 (m, 2H), 1.64 – 1.58 (m, 2H), 1.54 – 1.45 (m,
1
20H); 13C NMR (125.8 MHz, CDCl3): δ = 162.9, 154.7, 152.8, 120.6 (q, JCF = 283.1 Hz), 118.3, 82.4, 66.4
2
(sep, JCF = 34.5 Hz), 46.0, 32.3, 28.5, 28.2, 25.0; HRMS (ESI): m/z [M+Na]+ calcd for C20H29F6NO6Na+:
516.1791; found: 516.1800.
4.1.10. 1,1,1,3,3,3-Hexafluoropropan-2-yl (E)-8-((tert-butoxycarbonyl)amino)-oct-2-enoate (22)
Methyl ester 20 (385 mg, 1.42 mmol) was dissolved in THF (10 mL) and LiOH (1 M, 4.50 mL, 4.50 mmol)
was added. The mixture was stirred at room temperature for 16.5 h. The solvent was removed under reduced
pressure upon completion and the aqueous residue was acidified to pH 4 with 10% citric acid and extracted
with CH2Cl2 (3 x 40 mL). The combined organic layers were washed with saturated NaCl (3 x 50 mL) and
dried over anhydrous MgSO4, filtered and concentrated to give the crude product as an orange solid (334
mg), which was used in the next step without purification.
To a stirred solution of crude carboxylic acid (324 mg, 1.26 mmol) in dry CH2Cl2 (3 mL) was added
1,1,1,3,3,3-hexafluoro-2-propanol (0.16 mL, 1.52 mmol), DMAP (63 mg, 0.52 mmol) and DIC (0.25 mL,
1.61 mmol). The reaction mixture was stirred for 20 h and then diluted with CH2Cl2 (25 mL), washed with
saturated NH4Cl (20 mL) and saturated NaCl (20 mL). The organic layer was dried over anhydrous MgSO4,
filtered and concentrated to yield the crude product as a red solid (622 mg). Purification by flash column
chromatography on silica gel (hexanes/EtOAc, 1:1) gave the title compound as yellow oil (315 mg, 61%). Rf
(hexanes/EtOAc, 1:1) = 0.14; 1H NMR (500 MHz, CDCl3): δ = 7.20 (dt, J = 15.7, 6.8 Hz, 1H), 5.93 (dt, J =
15.7, 1.6 Hz, 1H), 5.82 (sep, J = 6.1 Hz, 1H), 4.51 (s, 1H), 3.15 – 3.09 (m, 2H), 2.28 (dq, J = 7.1, 1.6 Hz,
2H), 1.54 – 1.47 (m, 4H), 1.44 (s, 9H), 1.39 – 1.34 (m, 2H); 13C NMR (125.8 MHz, CDCl3): δ = 163.0,
156.1, 154.9, 120.7 (q, 1JCF = 282.1 Hz), 118.2, 79.3, 66.5 (sep, 2JCF = 34.2 Hz), 40.5, 32.7, 30.1, 28.6, 27.5,
26.5; HRMS (ESI): m/z [M+Na]+ calcd for C16H23F6NO4Na+: 430.1424, found: 430.1428.
4.1.11. 1,1,1,3,3,3-Hexafluoropropan-2-yl 7-((di-tert-butoxycarbonyl)amino)-3-(tritylthio)heptanoate (23)
To a stirred solution of HFIP-ester 21 (124 mg, 0.25 mmol) in dry CH2Cl2 (5 mL) was added Et3N (0.14 mL,
1.00 mmol) and triphenylmethanethiol (339 mg, 1.23 mmol). The reaction mixture was stirred at room
temperature for 69 h and then diluted with CH2Cl2 (30 mL), washed with H2O (40 mL), dried over anhydrous
MgSO4 and concentrated to give the crude product as yellow oil (427 mg). Purification by flash column
15

ACCEPTED MANUSCRIPT
chromatography on silica gel (hexanes/EtOAc, 95:5) gave the title compound (101 mg, 52%). Rf
1
(hexanes/EtOAc, 95:5) = 0.12; H NMR (600 MHz, CDCl3): δ = 7.54 – 7.51 (m, 6H), 7.30 – 7.27 (m, 6H),
7.22 – 7.19 (m, 3H), 5.61 (sep, J = 6.1 Hz, 1H), 3.51 (t, J = 7.5 Hz, 2H), 2.70 – 2.65 (m, 1H), 2.20 (dd, J =
16.7, 9.8 Hz, 1H), 1.80 (dd, J = 16.8, 3.4 Hz, 1H), 1.59 – 1.44 (m, 4H), 1.50 (s, 18H), 1.41 – 1.25 (m, 2H);
1
13C NMR (150.9 MHz, CDCl3): δ = 168.3, 152.8, 144.7, 129.5, 128.2, 126.9, 120.4 (q, JCF = 282.2 Hz),
2
82.2, 67.7, 66.1 (sep, JCF = 34.6 Hz), 46.2, 40.7, 38.4, 34.3, 29.1, 28.2, 23.6; HRMS (ESI): m/z [M+Na]+
calcd for C39H45F6NO6SNa+: 792.2764; found: 792.2767.
4.1.12. 1,1,1,3,3,3-Hexafluoropropan-2-yl 8-((tert-butoxycarbonyl)amino)-3-(tritylthio)octanoate (24)
To a stirred solution of unsaturated HFIP ester 22 (315 mg, 0.77 mmol) in dry CH2Cl2 (5 mL) was added
triphenylmethanethiol (641 mg, 2.32 mmol) and Et3N (0.32 mL, 2.39 mmol). The reaction mixture was
stirred at room temperature for 48 h and then diluted with CH2Cl2 (30 mL) and washed with H2O (40 mL).
The organic layer was dried over anhydrous MgSO4, filtered and concentrated to give the crude product as
yellow oil (958 mg). Purification by flash column chromatography on silica gel (hexanes/EtOAc, 9:1) gave
1
the title compound as yellow foam (483 mg, 91%). Rf (hexanes/EtOAc, 9:1) = 0.19; H NMR (500 MHz,
CDCl3): δ = 7.53 – 7.51 (m, 6H), 7.30 – 7.27 (m, 6H), 7.22 – 7.19 (m, 3H), 5.61 (sep, J = 6.1 Hz, 1H), 4.47
(bs, 1H), 3.15 – 3.01 (m, 2H), 2.69 – 2.66 (m, 1H), 2.22 (dd, J = 16.6, 9.9 Hz, 1H), 1.83 (dd, J = 16.6, 3.4
Hz, 1H), 1.55 – 1.48 (m, 2H), 1.45 (s, 9H), 1.44 – 1.31 (m, 4H), 1.23 – 1.16 (m, 2H); 13C NMR (125.8 MHz,
CDCl3): δ = 168.3, 156.1, 144.7, 129.6, 128.2, 126.9, 120.4 (q, 1JCF = 282.5 Hz), 79.2, 67.7, 66.2 (sep, 2JCF =
34.8 Hz), 40.7 (2C), 38.5, 34.6, 30.0, 28.6, 26.8, 26.0; HRMS (ESI): m/z [M+Na]+ calcd for
C35H39F6NO4SNa+: 706.2396; found: 706.2402.
4.1.13. 7-((Di-tert-butoxycarbonyl)amino)-3-(tritylthio)heptanoic acid (25)
To a stirred solution of Michael product 23 (448 mg, 0.58 mmol) in THF (2 mL) was added LiOH (1 M, 4
mL, 4 mmol). The reaction mixture was stirred at room temperature for 17 h. The solvent was removed under
reduced pressure and the pH of the aqueous residue was acidified to pH 4 with 10% citric acid. The
mixture was extracted with CH2Cl2 (4 x 10 mL) and the combined organic layer was washed with saturated
NaCl (25 mL), dried over anhydrous MgSO4 and concentrated to give the crude product as yellow oil (321
mg, 33%). Purification by flash column chromatography on silica gel (CH2Cl2/MeOH, 95:5) gave the title
1
compound as a colorless oil (116 mg). Rf (CH2Cl2/MeOH, 95:5) = 0.30; H NMR (500 MHz, CDCl3): δ =
16

ACCEPTED MANUSCRIPT
7.53 – 7.50 (m, 6H), 7.29 – 7.25 (m, 6H), 7.21 – 7.17 (m, 3H), 3.48 (t, J = 7.4 Hz, 2H), 2.68 – 2.62 (m, 1H),
2.17 (dd, J = 16.4, 9.0 Hz, 1H), 1.93 (16.4, 4.2 Hz, 1H), 1.49 (s, 18H), 1.46 – 1.35 (m, 4H), 1.34 – 1.21 (m,
2H); 13C NMR (125.8 MHz, CDCl3): δ = 176.8, 152.8, 144.9, 129.7, 128.1, 126.8, 82.3, 67.7, 46.4, 41.0,
39.2, 34.5, 29.1, 28.2, 23.6; HRMS (ESI): m/z [M+Na]+ calcd for C36H45NO6SNa+: 642.2860; found:
642.2868.
4.1.14. 8-((tert-Butoxycarbonyl)amino)-3-(tritylthio)octanoic acid (26)
To a stirred solution of HFIP ester 24 (201 mg, 0.30 mmol) in THF (10 mL) was added LiOH (1 M, 2.0 mL,
2.0 mmol). The reaction mixture was stirred at room temperature for 24 h. The solvent was removed under
reduced pressure and the residue was acidified to pH 4 with 10% citric acid. The mixture was extracted with
CH2Cl2 (4 x 25 mL) and the combined organic layer was washed with saturated NaCl (50 mL), dried over
anhydrous MgSO4, filtered and concentrated to give the crude product as yellow foam (309 mg). Purification
by flash column chromatography on silica gel (CH2Cl2/MeOH, 95:5) gave the title compound as yellow foam
(197 mg, 56%). Retains CH2Cl2. Rf (CH2Cl2/MeOH, 95:5) = 0.28; 1H NMR (500 MHz, DMSO-d6): δ = 12.09
(s, 1H), 7.44 – 7.42 (m, 6H), 7.36 – 7.31 (m, 6H), 7.25 – 7.22 (m, 3H), 6.73 (t, J = 5.4 Hz, 1H), 2.84 (q, J =
6.6 Hz, 2H), 2.57 – 2.52 (m, 1H), 2.16 (dd, J = 16.3, 9.0 Hz, 1H), 1.87 (dd, J = 16.3, 4.2 Hz, 1H), 1.38 (s,
13
9H), 1.29 – 1.23 (m, 4H), 1.19 – 1.07 (m, 3H), 1.03 – 0.98 (m, 2H); C NMR (125.8 MHz, DMSO-d6): δ =
172.2, 155.5, 144.6, 129.1, 128.0, 126.6, 77.2, 66.9, 40.8, 39.8 (HSQC), 39.1 (HSQC), 34.1, 29.3, 28.3, 26.2,
25.3; HRMS (ESI): m/z [M+Na]+ calcd for C32H39NO4SNa+: 556.2492; found: 556.2499.
4.1.15. tert-Butyl (3-(naphthalene-2-yl)propyl)carbamate (28)
N-Boc allylamine 27 (1.02 g, 6.5 mmol) was dissolved in dry THF (6.0 mL) and 9-BBN (0.5 M in THF, 20.0
mL, 10.0 mmol) was added. The resulting mixture was stirred at room temperature for 2.5 h before 2-
bromonaphthalene (1.35 g, 6.5 mmol), Pd(dppf)2Cl2 CH2Cl2 (308 mg, 0.4 mmol) and NaOH (1 M, 10.0
mL, 10.0 mmol) were added. The reaction mixture was stirred at room temperature for 3 h, then degassed
and additional Pd(dppf) 2Cl2 CH2Cl2 (55 mg, 0.07 mmol) was added. The reaction mixture was stirred for
14 h at room temperature before saturated NH4Cl (40 mL) was added. The resulting mixture was extracted
with EtOAc (4 x 25 ml) and the combined organic layer was dried over MgSO4, filtered and concentrated to
yield the crude product as brown oil (3.28 g). Purification by flash column chromatography on silica gel
(hexanes/EtOAc 8.5:1.5 and second column using hexanes/EtOAc 8:2, respectively) gave a white solid (1.36
g), which still contained minor impurities (1H NMR). To a solution of the solid material (818 mg out of 1.36
17

ACCEPTED MANUSCRIPT
g) in THF (20 mL), NaOH (15%, 5.0 mL) and H2O2 (30%, 10.0 mL) were added and the resulting mixture
was stirred at 0 °C for 2 h. Dilution with diethyl ether (20 mL) followed by washing with saturated NaHCO3
(20 mL) and saturated NaCl (20 mL) before the organic layer was dried over anhydrous MgSO4, filtered and
concentrated to give a yellow oil (785 mg). Purification by flash column chromatography on silica gel
1
(hexanes/EtOAc, 8.5:1.5) gave the title compound (774 mg, 70%). Rf (hexanes/EtOAc, 8.5:1.5) = 0.24; H
NMR (600 MHz, CDCl3): δ = 7.82 – 7.76 (m, 3H), 7.62 (s, 1H), 7.47 – 7.41 (m, 2H), 7.32 (dd, J = 8.4, 1.2
Hz, 1H), 4.56 (bs, 1H), 3.24 – 3.15 (m, 2H), 2.81 (t, J = 7.8 Hz, 2H), 1.90 (p, J = 7.5 Hz, 2H), 1.46 (s, 9H);
13C NMR (150.9 MHz, CDCl3): δ = 156.1, 139.2, 133.7, 132.2, 128.1, 127.7, 127.5, 127.3, 126.5, 126.1,
125.3, 79.3, 40.5, 33.4, 31.8, 28.6; HRMS (ESI): m/z [M+Na]+ calcd for C18H23NO2Na+: 308.1621; found:
308.1628.
4.1.16. N-(2,2-Dimethoxyethyl)-3-(naphthalen-2-yl)propan-1-amine (29)
To a stirred solution of 28 (321 mg, 1.12 mmol) in CH2Cl2 (5 mL) at 0 °C was added TFA (4.0 mL). The
reaction mixture was stirred at room temperature for 2.5 h, after which the solvent was removed under
reduced pressure. The residue was suspended in 10% NaOH (15 mL) and extracted with CH2Cl2 (4 x 20 mL).
The combined organic layers were dried over anhydrous MgSO4, filtered and concentrated to give the crude
product as brown oil (192 mg), which was used in the next step without purification. To a stirred solution of
crude 3-(naphthalene-2-yl)propan-1-amine (192 mg) in anhydrous DMF (5 mL) was added
bromoacetaldehyde dimethyl acetal (0.18 mL, 1.52 mmol) and K2CO3 (0.145 g, 1.05 mmol). The reaction
mixture was stirred for 27.5 h at 80 °C. The mixture was diluted with H2O (20 mL) and extracted with
EtOAc (3 x 20 mL). The combined organic layers were washed with saturated NaCl (20 mL), dried over
anhydrous MgSO4, filtered and concentrated to give the crude product as yellow oil (572 mg). Purification by
flash column chromatography on silica gel (CH2Cl2/MeOH/NH4OH, 95:4.5:0.5) gave the title compound as
pale yellow oil (96 mg, 31%). Rf (CH2Cl2/MeOH/NH4OH, 95:4.5:0.5) = 0.31; 1H NMR (400 MHz, CDCl3): δ
= 7.82 – 7.74 (m, 3H), 7.61 (s, 1H), 7.47 – 7.38 (m, 2H), 7.33 (dd, J = 8.4, 1.7 Hz, 1H), 4.47 (t, J = 5.5 Hz,
1H), 3.37 (s, 6H), 2.81 (t, J = 7.5 Hz, 2H), 2.73 (d, J = 5.5 Hz, 2H), 2.69 (t, J = 7.2 Hz, 2H), 1.92 (p, J = 7.6
13
Hz, 2H), 1.65 (bs, 1H); C NMR (100.6 MHz, CDCl3): δ = 139.6, 133.7, 132.1, 128.0, 127.7, 127.5, 127.4,
+
126.4, 126.0, 125.2, 103.9, 54.2, 51.2, 49.5, 33.8, 31.6; HRMS (ESI): m/z [M+H]+ calcd for C17H24NO2 :
274.1802; found: 274.1809.
4.1.17. (9H-Fluoren-9-yl)methyl (S)-(1-((2,2-dimethoxyethyl)(3-(naphthalen-2- yl)propyl)amino)-1-oxo-5-
18

ACCEPTED MANUSCRIPT
(3-((2,2,4,6,7-pentamethyl-2,3-dihydrobenzofuran-5-yl)sulfonyl)guanidino)pentan-2-yl)carbamate (30)
To a stirred solution of 29 (96 mg, 0.35 mmol) in dry CH2Cl2 (2 mL) was added Fmoc-Arg(Pbf)-OH (244
mg, 0.38 mmol), HATU (136 mg, 0.36 mmol) and DIPEA (0.20 mL, 1.15 mmol). The resulting mixture was
stirred for 25 h at room temperature. The solvent was removed under reduced pressure and the residue was
partitioned between H2O (10 mL) and EtOAc (10 mL). The aqueous layer was extracted with EtOAc (3 x 20
mL) and the combined organic layer was washed with 1 M KHSO4 (20 mL), H2O (20 mL), saturated
NaHCO3 (20 mL) and saturated NaCl (20 mL), dried over anhydrous MgSO4, filtered and concentrated to
give the crude product (341 mg). Purification by flash column chromatography on silica gel (EtOAc/hexanes,
8:2) gave the title compound (250 mg, 79%). Rf (EtOAc/hexanes, 8:2) = 0.10; NMR analyses did not give
useful information due to formation of rotamers; HRMS (ESI): m/z [M+H]+ calcd for C51H62N5O8S+:
904.4315; found: 904.4314; m/z [M+Na]+ calcd for C51H61N5O8SNa+: 926.4133; found: 926.4139.
4.1.18. General protocol A: Fmoc-deprotection
To a stirred solution of the Fmoc-protected amine (0.06 M in CH2Cl2) was added Et2NH (50 equiv) and the
resulting mixture was stirred at room temperature for 2-3 h (monitored by TLC). The solvent was removed
under reduced pressure and the residue was used in the next step without purification.
4.1.19. General protocol B: Formation of linear precursors
HATU (165 mg, 0.43 mmol) and DIPEA (0.19 mL, 1.1 mmol) were added to a stirred solution the carboxylic
acid (6, 11, 25 or 26) carrying the R1 side chain (0.36 mmol) in dry CH2Cl2 (2 mL) and the reaction mixture
was stirred at room temperature for 20 minutes. Fmoc-deprotected amine 30 or 31 (see general protocol A,
0.56 mmol) in dry CH2Cl2 (12 mL) was then added and stirring was continued for 24 h. The solvent was
evaporated, and the resulting red residue was partitioned between H2O (10 mL) and EtOAc (10 mL). The
aqueous layer was further extracted with EtOAc (3 x 10 mL) and the combined organic layers were washed
with 1 M KHSO4 (20 mL), H2O (20 mL), saturated NaHCO3 (20 mL) and saturated NaCl (20 mL). The
organic layer was further dried over anhydrous MgSO4, filtered and concentrated to give the crude product.
4.1.20. tert-Butyl (5-(((S)-1-((2,2-dimethoxyethyl)(2-(naphthalen-2-yl)ethyl)amino)-1-oxo-5-(3-((2,2,4,6,7-
pentamethyl-2,3-dihydrobenzofuran-5-yl)sulfonyl)-guanidino)pentan-2-yl)amino)-5-oxo-3-
(tritylthio)pentyl)carbamate (32)
19

ACCEPTED MANUSCRIPT
The title compound was prepared from carboxylic acid 6 (177 mg, 0.36 mmol) which was dissolved in DMF
(2 mL) and Fmoc-deprotected amine 31 (see general protocol B, 0.56 mmol) following general protocol A.
The crude product (red foam, 510 mg) was purified by flash column chromatography on silica gel
(EtOAc/hexane; 7.5:2.5 to 9.5:0.5 followed by EtOAc) to give an inseparable mixture of diastereoisomers of
32 (174 mg, 42%). Rf (EtOAc) = 0.7; NMR analyses did not give useful information due to formation of
+
rotamers; HRMS (ESI): m/z [M+H]+ calcd for C64H81N6O9S2 : 1141.5501; found: 1141.5502; m/z [M+Na]+
calcd for C64H80N6O9S2Na+: 1163.5320; found: 1163.5314.
4.1.21. tert-Butyl (6-(((S)-1-((2,2-dimethoxyethyl)(2-(naphthalen-2-yl)ethyl)amino)-1-oxo-5-(3-((2,2,4,6,7-
pentamethyl-2,3-dihydrobenzofuran-5-yl)sulfonyl)guanidino)pentan-2-yl)amino)-6-oxo-4-
(tritylthio)hexyl)carbamate (33)
The title compound was prepared from carboxylic acid 11 (160 mg, 0.32 mmol) and Fmoc-deprotected amine
31 (see general protocol B, 0.45 mmol) following general protocol A. The crude product (red foam, 654 mg)
was purified by flash column chromatography on silica gel (EtOAc/hexane; 8:2) to give an inseparable
mixture of diastereoisomers of 33 (216 mg, 59%). Rf (EtOAc/hexanes, 8:2) = 0.19; NMR analyses did not
+
give useful information due to formation of rotamers; HRMS (ESI): m/z [M+H]+ calcd for C65H83N6O9S2 :
1155.5658; found: 1155.5664; m/z [M+Na]+ calcd for C65H82N6O9S2Na+: 1177.5477; found: 1177.5529.
4.1.22.
Di-tert-butyl
(7-(((S)-1-((2,2-dimethoxyethyl)(2-(naphthalen-2-yl)ethyl)amino)-1-oxo-5-(3-
((2,2,4,6,7-pentamethyl-2,3-dihydrobenzofuran-5-yl)sulfonyl)-guanidino)pentan-2-yl)amino)-7-oxo-5-
(tritylthio)heptyl)carbamate (34)
The title compound was prepared from carboxylic acid 25 (116 mg, 0.19 mmol) and Fmoc-deprotected amine
31 (see general protocol B, 0.44 mmol) following general protocol A. The crude product (red oil, 444 mg)
was purified by flash column chromatography on silica gel (EtOAc/hexanes, 8:2) to give an inseparable
mixture of diastereoisomers of 34 as a colorless oil (47 mg, 20%). Rf (EtOAc/hexanes, 8:2) = 0.57; NMR
analyses did not give useful information due to formation of rotamers; HRMS (ESI): m/z [M+Na]+ calcd for
C71H92N6O11S2Na+: 1291.6158; found: 1291.6166.
4.1.23. tert-Butyl (8-(((S)-1-((2,2-dimethoxyethyl)(2-(naphthalen-2-yl)ethyl)amino)-1-oxo-5-(3-((2,2,4,6,7-
pentamethyl-2,3-dihydrobenzofuran-5-yl)sulfonyl)-guanidino)pentan-2-yl)amino)-8-oxo-6-
(tritylthio)octyl)carbamate (35)
20

ACCEPTED MANUSCRIPT
The title compound was prepared from carboxylic acid 26 (105 mg, 0.20 mmol) and Fmoc-deprotected amine
31 (see general protocol B, 0.10 mmol) following general protocol A. The crude product (205 mg) was
purified by flash column chromatography on silica gel (EtOAc/hexanes, 9:1) to give an inseparable mixture
of diastereoisomers of 35 as a colorless oil (80 mg, 68%). Rf (EtOAc/hexanes, 9:1) = 0.25; NMR analyses did
not give useful information due to formation of rotamers; HRMS (ESI): m/z [M+Na]+ calcd for
C67H86N6O9S2Na+: 1205.5790; found: 1205.5796.
4.1.24. tert-Butyl (6-(((S)-1-((2,2-dimethoxyethyl)(3-(naphthalen-2-yl)propyl)amino)-1-oxo-5-(3-((2,2,4,6,7-
pentamethyl-2,3-dihydrobenzofuran-5-yl)sulfonyl)-guanidino)pentan-2-yl)amino)-6-oxo-4-
(tritylthio)hexyl)carbamate (36)
The title compound was prepared from carboxylic acid 11 (166 mg, 0.33 mmol) and Fmoc-deprotected amine
30 (see general protocol B, 0.10 mmol) following general protocol A. The crude product (273 mg) was
purified by flash column chromatography on silica gel (EtOAc/hexane, 9:1) to give an inseparable mixture of
diastereoisomers of 36 as a colorless oil (90 mg, 73%). Rf (EtOAc/hexanes, 9:1) = 0.35; NMR analyses did
not give useful information due to formation of rotamers; HRMS (ESI): m/z [M+Na]+ calcd for
C66H84N6O9S2Na+: 1191.5633; found: 1191.5640.
4.1.25. tert-Butyl (8-(((S)-1-((2,2-dimethoxyethyl)(3-(naphthalen-2-yl)propyl)amino)-1-oxo-5-(3-((2,2,4,6,7-
pentamethyl-2,3-dihydrobenzofuran-5-yl)sulfonyl)-guanidino)pentan-2-yl)amino)-8-oxo-6-
(tritylthio)octyl)carbamate (37)
The title compound was prepared from carboxylic acid 27 (93 mg, 0.17 mmol) and Fmoc-deprotected amine
30 (see general protocol B, 0.13 mmol) following general protocol A. The crude product (238 mg) was
purified by flash column chromatography on silica gel (EtOAc/hexane, 8:2) to give an inseparable mixture of
diastereoisomers of 36 as a colorless oil (117 mg, 77%). Rf (EtOAc/hexanes, 8:2) = 0.22; NMR analyses did
not give useful information due to formation of rotamers; HRMS (ESI): m/z [M+H]+ calcd for
C68H89N6O9S2 : 1197.6127; found: 1197.6134; m/z [M+Na]+ calcd for C68H88N6O9S2Na+: 1219.5946; found:
+
1219.6000.
4.1.26. General procedure C: Global deprotection and cyclization
The linear precursor was dissolved in a mixture of TFA, thioanisole and H2O (90:5:5, 100 mL per mmol) and
the resulting mixture was stirred at room temperature for 2-4 h. The TFA was removed under reduced
21

ACCEPTED MANUSCRIPT
pressure and the crude product was precipitated by addition of cold diethyl ether. The diethyl ether was
removed under reduced pressure and the residue was dried under high vacuum to give the crude product as a
white solid.
4.1.27.
1-(3-((2S,6S,9aS)-2-(2-Guanidinoethyl)-8-(2-(naphthalen-2-yl)ethyl)-4,7-dioxohexahydro-2H,6H-
pyrazino[2,1-b][1,3]thiazin-6-yl)propyl)guanidine (44a)
Linear precursor 32 (174 mg, 0.15 mmol) was deprotected and cyclized following general procedure C. The
crude product (approx. 200 mg) was dissolved in dry DMF (5 mL) and 1H-pyrazole-1-carboxamidine
hydrochloride (305 mg, 2.06 mmol) and DIPEA (0.58 mL, 3.33 mmol) were added and the mixture was
stirred under inert atmosphere for 70 h during which the reaction was monitored by RP-HPLC. Diethyl ether
(20 mL) was then added and the mixture was cooled at 4 °C and stirred for an additional hour, resulting in
the precipitation of a white solid. The crude product was purified by semi-preparative RP-HPLC to give the
title compound as a fluffy white solid (24 mg, 42%). 1H NMR (600 MHz, CD3OD): δ = 7.86 – 7.83 (m, 2H),
7.80 (d, J = 8.3 Hz, 1H), 7.64 (s, 1H), 7.50 – 7.44 (m, 2H), 7.42 (dd, J = 8.5, 1.5 Hz, 1H), 5.07 (dd, J = 10.0,
5.4 Hz, 1H), 5.01 (dd, J = 8.8, 4.2 Hz, 1H), 3.72 – 3.69 (m, 2H), 3.43 (dd, J = 12.8, 4.3 Hz, 1H), 3.29 – 3.25
(m, 1H), 3.22 – 3.16 (m, 1H), 3.15 – 3.03 (m, 5H), 2.86 – 2.80 (m, 1H), 2.59 (dd, J = 16.4, 3.6 Hz, 1H), 2.49
(dd, J = 16.4, 11.9 Hz, 1H), 1.87 – 1.72 (m, 3H), 1.70 – 1.64 (m, 1H), 1.52 – 1.44 (m, 2H); 13C NMR (150.9
MHz, CD3OD): δ = 170.8, 169.2, 158.6, 158.5, 137.5, 135.0, 133.8, 129.4, 128.7, 128.64, 128.59, 128.5,
127.3, 126.8, 56.6, 53.1, 52.1, 49.7, 41.7, 41.6, 39.8, 35.3, 35.2, 34.1, 29.3 26.2; HRMS (ESI): m/z [M+H]+
calcd for C26H37N8O2S+: 525.2755; found: 525.2766. Purity = 99.8% (UV 220 nm).
4.1.28.
1-(3-((2R,6S,9aR)-2-(2-guanidinoethyl)-8-(2-(naphthalen-2-yl)ethyl)-4,7-dioxohexahydro-2H,6H-
pyrazino[2,1-b][1,3]thiazin-6-yl)propyl)guanidine (44b)
Bicycle 44b was prepared as described for 44a above. The crude product was purified by semi-preparative
RP-HPLC to give the title compound as a fluffy white solid (22 mg, 38%). 1H NMR (600 MHz, CD3OD): δ =
7.84 – 7.78 (m 3H), 7.71 (s, 1H), 7.48 – 7.42 (m, 3H), 5.38 (dd, J = 4.0, 2.8 Hz, 1H), 4.65 (app t, J = 7.8 Hz,
1H), 4.20 (dt, J = 13.4, 7.7 Hz, 1H), 3.98 (dd, J = 15.0, 4.4 Hz, 1H), 3.69 – 3.65 (m, 1H), 3.54 (dd, J = 15.0,
2.5 Hz, 1H), 3.49 – 3.44 (m ,1H), 3.17 (app t, J = 7.0 Hz, 2H), 3.13 – 3.04 (m, 3H), 2.99 – 2.90 (m, 2H), 2.61
13
(dd, J = 14.8, 3.9 Hz, 1H), 1.84 – 1.77 (m, 1H), 1.60 – 1.45 (m, 3H), 1.35 – 1.23 (m, 2H); C NMR (150.9
MHz, CD3OD); δ = 172.3, 169.4, 158.6, 158.4, 137.2, 135.0, 133.9, 129.3, 128.7, 128.54, 128.45 (2C),
22

ACCEPTED MANUSCRIPT
127.2, 126.7, 56.1, 55.1, 49.2 (HSQC), 48.5 (HSQC), 41.4, 40.02, 39.98, 38.1, 37.8, 34.6, 30.6, 26.1; HRMS
(ESI): m/z [M+H]+ calcd for C26H37N8O2S+: 525.2755; found: 525.2764. Purity = 97.4 % (UV 220 nm).
4.1.29.
1,1'-(((2S,6S,9aS)-8-(2-(naphthalen-2-yl)ethyl)-4,7-dioxohexahydro-2H,6H-pyrazino[2,1-
b][1,3]thiazine-2,6-diyl)bis(propane-3,1-diyl))diguanidine (45a)
Linear precursor 39 (128 mg, 111 mmol) was deprotected and cyclized following general procedure C. The
crude product (58 mg) was dissolved in anhydrous DMF (4 mL) and 1H-pyrazole-1-carboxamidine
hydrochloride (27 mg, 0.18 mmol) and DIPEA (30 ꢀL, 0.17 mmol) were added. The reaction mixture was
stirred under inert atmosphere for 144 h during which the reaction was monitored by analytical RP-HPLC.
Additional 1H-pyrazole-1-carboxamidine hydrochloride and DIPEA was added during this time. Diethyl
ether (25 mL) was added and the mixture was cooled to 0 °C and stirred for an additional hour, resulting in
the precipitation of a yellow solid (203 mg). The crude product was purified by semi-preparative RP-HPLC
1
to give the title compound as a fluffy white solid (6.5 mg, 15%). H NMR (600 MHz, CD3OD): δ = 7.86 –
7.82 (m, 2H), 7.81 – 7.77 (m, 1H), 7.62 (s, 1H), 7.51 – 7.44 (m, 2H), 7.44 – 7.40 (m, 1H), 5.15 (dd, J = 10.1,
5.1 Hz, 1H), 4.89 (HSQC, 1H), 3.88 – 3.81 (m, 1H), 3.58 – 3.50 (m, 1H), 3.31 (HSQC, 1H), 3.22 – 3.08 (m,
5H), 3.07 – 2.99 (m, 2H), 2.53 – 2.44 (m, 2H), 2.41 – 2.33 (m, 1H), 1.94 – 1.86 (m, 1H), 1.81 – 1.73 (m, 1H),
1.57 – 1.27 (m, 6H); 13C NMR (150.9 MHz, CD3OD): δ = 170.7, 169.2, 158.62, 158.57, 137.7, 135.0, 133.8,
129.4, 128.71, 128.68, 128.67, 128.63, 127.4, 126.8, 56.6, 54.1, 51.7, 49.9, 42.2, 42.0, 41.6, 37.0, 34.1, 33.0,
29.1, 27.2, 26.2; HRMS (ESI): m/z [M+2H]2+ calcd for C27H40N8O2S2+: 270.1492; found 270.1499. Purity =
100.0% (UV 220 nm).
4.1.30.
1,1'-(((2R,6S,9aR)-8-(2-(naphthalen-2-yl)ethyl)-4,7-dioxohexahydro-2H,6H-pyrazino[2,1-
b][1,3]thiazine-2,6-diyl)bis(propane-3,1-diyl))diguanidine (45b)
Bicycle 45b was prepared as described for 45a. The crude product was purified by semi-preparative RP-
HPLC gave the title compound as a fluffy white solid (6.6 mg, 16%). 1H NMR (600 MHz, CD3OD): δ = 7.83
– 7.78 (m, 3H), 7.71 (s, 1H), 7.49 – 7.42 (m, 3H), 5.35 – 5.33 (m, 1H), 4.66 (t, J = 7.8 Hz, 1H), 4.20 – 4.14
(m, 1H), 3.95 (dd, J = 14.8, 4.3 Hz, 1H), 3.64 – 3.58 (m, 1H), 3.53 (dd, J = 14.9, 2.8 Hz, 1H), 3.51 – 3.46 (m,
1H), 3.13 – 2.92 (m, 7H), 2.59 (dd, J = 14.9, 3.8 Hz, 1H), 1.62 – 1.47 (m, 5H), 1.40 – 1.28 (m, 3H); 13C
NMR (150.9 MHz, CD3OD): δ = 172.6, 169.5, 158.6, 158.5, 137.2, 135.0, 133.9, 129.3, 128.7, 128.51,
128.45, 128.4, 127.2, 126.7, 56.1, 55.0, 49.2 (HSQC), 48.8 (HSQC), 42.0, 41.5, 40.4, 40.2, 36.2, 34.6, 30.6,
23

ACCEPTED MANUSCRIPT
27.5, 26.2; HRMS (ESI): m/z [M + 2H]2+ calcd for C27H40N8O2S2+: 270.1492; found 270.1498. Purity = 95.2
% (UV 220 nm).
4.1.31.
1-(3-((2S,6S,9aS)-2-(4-guanidinobutyl)-8-(2-(naphthalen-2-yl)ethyl)-4,7-dioxohexahydro-2H,6H-
pyrazino[2,1-b][1,3]thiazin-6-yl)propyl)guanidine (46a)
Linear precursor 40 (47 mg, 0.04 mmol) was deprotected and cyclized following general procedure C. The
crude product (29 mg) was dissolved in anhydrous DMF (2 mL) and 1H-pyrazole-1-carboxamidine
hydrochloride (85 mg, 0.58 mmol) and DIPEA (0.1 mL, 0.57 mmol) were added. The reaction mixture was
stirred under inert atmosphere for 94 h during which the reaction was monitored by analytical RP-HPLC.
Cold diethyl ether (10 mL) was then added and the mixture was cooled at 0 °C and stirred for an additional
hour, resulting in the precipitation of a white solid. The crude product was purified by semi-preparative RP-
HPLC to give the title compound as fluffy white solid (3.3 mg, 22%). 1H NMR (850 MHz, CD3OD): δ = 7.84
(t, J = 8.4 Hz, 2H), 7.79 (d, J = 8.0 Hz, 1H), 7.61 (s, 1H), 7.50 – 7.45 (m, 2H), 7.41 (dd, J = 8.3, 1.8 Hz, 1H),
5.16 (dd, J = 10.4, 5.0 Hz, 1H), 4.88 (HSQC, 1H), 3.88 – 3.84 (m, 1H), 3.52 – 3.47 (m, 1H), 3.27 (dd, J =
12.6, 4.3 Hz, 1H), 3.21 – 3.11 (m, 5H), 3.06 – 3.02 (m, 1H), 2.98 (dd, J = 12.6, 9.9 Hz, 1H), 2.48 (dd, J =
16.3, 3.1 Hz, 1H), 2.45 – 2.41 (m, 1H), 2.35 (dd, J = 16.3, 11.9 Hz, 1H), 1.93 – 1.88 (m, 1H), 1.81 – 1.75 (m,
13
1H), 1.56 – 1.45 (m, 4H), 1.44 – 1.38 (m, 1H), 1.37 – 1.28 (m, 2H), 1.20 – 1.14 (m, 1H); C NMR (213.8
MHz, CD3OD): δ = 170.8, 169.3, 158.7, 158.6, 137.7, 135.0, 133.8, 129.4, 128.70, 128.69 (2C), 128.63,
127.4, 126.8, 56.6, 54.3, 51.6, 50.0, 42.3, 42.2, 41.6, 37.0, 35.5, 34.0, 29.5, 29.0, 26.2, 24.8; HRMS (ESI):
m/z [M+H]+ calcd for C28H41N8O2S: 553.3068; found: 553.3076. Purity = 99.7% (UV 220 nm).
4.1.32.
1-(3-((2R,6S,9aR)-2-(4-guanidinobutyl)-8-(2-(naphthalen-2-yl)ethyl)-4,7-dioxohexahydro-2H,6H-
pyrazino[2,1-b][1,3]thiazin-6-yl)propyl)guanidine (46b)
Bicycles 46b was prepared as described for 46a above. The crude product was purified by semi-preparative
RP-HPLC to give the title compounds as a fluffy white solid (3.6 mg, 2:8 mixture between 46c+d and 46b).
1H NMR (850 MHz, CD3OD): 7.83 – 7.78 (m, 3H), 7.71 (s, 1H), 7.48 – 7.42 (m, 3H), 5.32 (t, J = 3.6 Hz,
1H), 4.67 (t, J = 7.8 Hz, 1H), 4.16 (dt, J = 13.7, 7.7 Hz, 1H), 3.93 (dd, J = 14.8, 4.3 Hz, 1H), 3.60 – 3.56 (m,
1H), 3.53 – 3.45 (m, 2H), 3.14 – 3.04 (m, 4H), 3.00 – 2.93 (m, 3H), 2.58 (dd, J = 14.9, 3.9 Hz, 1H), 1.68 –
13
1.48 (m, 5H), 1.42 – 1.29 (m, 5H); C NMR (213.8 MHz, CD3OD): δ = 172.7, 169.6, 158.6, 158.5, 137.2,
135.0, 133.9, 129.3, 128.7, 128.51, 128.46, 128.43, 127.2, 126.7, 56.1, 54.9, 49.0 (2C, HSQC), 42.2, 41.5,
24

ACCEPTED MANUSCRIPT
40.6, 40.3, 38.7, 34.6, 30.6, 29.5, 26.2, 25.1; HRMS (ESI) m/z [M+H]+ calcd for C28H41N8O2S+: 553.3068;
found: 553.3075.
Due to signal overlap, all chemical shifts could not be unambiguously identified for 46c. See supporting
information for key chemical shifts for 46c.
4.1.33.
1-(3-((2S,6S,9aS)-2-(5-guanidinopentyl)-8-(2-(naphthalen-2-yl)ethyl)-4,7-dioxohexahydro-2H,6H-
pyrazino[2,1-b][1,3]thiazin-6-yl)propyl)guanidine (47a)
Linear precursor 41 (80 mg, 0.07 mmol) was deprotected and cyclized following general procedure C. The
crude product (108 mg) was dissolved in anhydrous DMF (5 mL) and 1H-pyrazole-1-carboxamidine
hydrochloride (118 mg, 0.80 mmol) and DIPEA (0.11 mL, 0.63 mmol) were added. The reaction mixture
was stirred under inert atmosphere for 99 h during which the reaction was monitored by analytical RP-HPLC.
Cold diethyl ether (20 mL) was then added and the mixture was cooled at 4 °C and stirred for an additional
hour, resulting in the precipitation of a white solid. The crude product was purified by semi-preparative RP-
HPLC to give the title compound as fluffy white solid (3.5 mg, 13%). 1H NMR (850 MHz, CD3OD): δ = 7.84
(t, J = 7.7 Hz, 2H), 7.79 (d, J = 7.9 Hz, 1H), 7.62 (s, 1H), 7.50 – 7.45 (m, 2H), 7.41 (dd, J = 8.4, 1.7 Hz, 1H),
5.16 (dd, J = 10.3, 5.1 Hz, 1H), 4.86 (HSQC, 1H), 3.89 – 3.85 (m, 1H), 3.51 – 3.47 (m, 1H), 3.27 (dd, J =
12.6, 4.4 Hz, 1H), 3.21 – 3.11 (m, 5H), 3.05 – 3.02 (m, 1H), 2.97 (dd, J = 12.6, 10.0 Hz, 1H), 2.48 (dd, J =
16.4, 3.1 Hz, 1H), 2.43 – 2.38 (m, 1H), 2.33 (dd, J = 16.3, 11.9 Hz, 1H), 1.94 – 1.88 (m, 1H), 1.81 – 1.75 (m,
13
1H), 1.59 – 1.48 (m, 4H), 1.41 – 1.36 (m, 1H), 1.34 – 1.25 (m, 4H), 1.17 – 1.10 (m, 1H); C NMR (213.8
MHz, CD3OD): δ = 170.8, 169.3, 158.7, 158.6, 137.7, 135.0, 133.8, 129.4, 128.71, 128.692, 128.688, 128.64
, 127.4, 126.8, 56.6, 54.4, 51.6, 50.0, 42.4, 42.3, 41.6, 37.1, 35.8, 34.0, 29.7, 29.0, 27.37, 27.36, 26.2; HRMS
(ESI): m/z [M+H]+ calcd for C29H43N8O2S+: 567.3224; found: 567.3226. Purity = 99.9% (UV 220 nm).
4.1.34. 1-(3-((2R,6S,9aR)-2-(5-guanidinopentyl)-8-(2-(naphthalen-2-yl)ethyl)-4,7-dioxohexahydro-2H,6H-
pyrazino[2,1-b][1,3]thiazin-6-yl)propyl)guanidine (47b)
Bicycles 47b and was prepared as described for 47a above. The crude product was purified by semi-
preparative RP-HPLC to give the title compounds as a fluffy white solid (3.5 mg, 13%). 1H NMR (850 MHz,
CD3OD): δ = 7.84 – 7.78 (m, 3H), 7.71 (s, 1H), 7.48 – 7.42 (m, 3H), 5.31 (t, J = 3.7 Hz, 1H), 4.67 (t, J = 7.8
Hz, 1H), 4.18 – 4.13 (m, 1H), 3.92 (dd, J = 14.8, 4.3 Hz, 1H), 3.59 – 3.55 (m, 1H), 3.52 – 3.48 (m, 2H), 3.17
– 3.11 (m, 2H), 3.11 – 3.05 (m, 2H), 3.01 – 2.94 (m, 3H), 2.57 (dd, J = 14.9, 4.0 Hz, 1H), 1.61 – 1.49 (m,
5H), 1.39 – 1.29 (m, 7H); 13C NMR (213.8 MHz, CD3OD): δ = 172.7, 169.5, 158.6, 158.5, 137.2, 135.0,
25

ACCEPTED MANUSCRIPT
133.9, 129.3, 128.7, 128.51, 128.46, 128.43, 127.2, 126.7, 56.0, 54.8, 49.2 (HSQC), 49.1 (HSQC), 42.3,
41.5, 40.6, 40.4, 39.1, 34.6, 30.6, 29.7, 27.6, 27.3, 26.2; HRMS (ESI): m/z [M + H]+ calcd for C29H43N8O2S+:
567.3224; found: 567.3225. Purity = 98.9% (UV 220 nm).
4.1.35.
1,1'-(((2S,6S,9aS)-8-(3-(naphthalen-2-yl)propyl)-4,7-dioxohexahydro-2H,6H-pyrazino[2,1-
b][1,3]thiazine-2,6-diyl)bis(propane-3,1-diyl))diguanidine (48a)
Linear precursor 42 (90 mg, 0.08 mmol) was deprotected and cyclized following general procedure C. The
crude product (97 mg) was dissolved in anhydrous DMF (2 mL) and 1H-pyrazole-1-carboxamidine
hydrochloride (85 mg, 0.58 mmol) and DIPEA (0.10 mL, 0.57 mmol) were added. The reaction mixture was
stirred under inert atmosphere for 78 h during which the reaction was monitored by analytical RP-HPLC.
Cold diethyl ether (10 mL) was then added and the mixture was cooled at 4 °C and stirred for an additional
hour, resulting in the precipitation of a white solid. The crude product was purified by semi-preparative RP-
HPLC to give the title compound as fluffy white solid (3.3 mg, 11%). 1H NMR (850 MHz, CD3OD): δ = 7.82
– 7.78 (m, 2H), 7.76 (d, J = 8.3 Hz, 1H), 7.69 (s, 1H), 7.46 – 7.41 (m, 2H), 7.37 (dd, J = 8.4, 1.8 Hz, 1H),
5.01 (dd, J = 10.1, 5.0 Hz, 1H), 4.95 (dd, J = 8.4, 4.7 Hz, 1H), 3.68 – 3.63 (m, 1H), 3.54 – 3.48 (m, 2H), 3.41
– 3.37 (m, 1H), 3.27 – 3.23 (m, 1H), 3.22 – 3.16 (m, 3H), 3.02 – 2.98 (m, 1H), 2.90 – 2.86 (m, 1H), 2.84 –
2.80 (m, 1H), 2.58 (dd, J = 16.6, 3.4 Hz, 1H), 2.42 (dd, J = 16.5, 12.0 Hz, 1H), 2.11 – 2.03 (m, 2H), 1.97 –
1.92 (m, 1H), 1.80 – 1.74 (m, 1H), 1.71 – 1.65 (m, 1H), 1.64 – 1.57 (m, 4H), 1.52 – 1.47 (m, 1H); 13C NMR
(213.8 MHz, CD3OD): δ = 170.9, 169.2, 158.7, 158.6, 140.5, 135.1, 133.6, 129.1, 128.7, 128.5, 128.1, 127.4,
127.1, 126.4, 56.7, 52.6, 52.3, 48.3, 42.1, 42.0, 41.7, 37.1, 34.5, 33.3, 29.7, 28.9, 27.2, 26.4; HRMS (ESI):
m/z [M+H]+ calcd for C28H41N8O2S+: 553.3068; found: 553.3077. Purity = 100.0% (UV 220 nm).
4.1.36.
1,1'-(((2R,6S,9aR)-8-(3-(naphthalen-2-yl)propyl)-4,7-dioxohexahydro-2H,6H-pyrazino[2,1-
b][1,3]thiazine-2,6-diyl)bis(propane-3,1-diyl))diguanidine (48b)
Bicycle 48b was prepared as described for 48a above. The crude product was purified by semi-preparative
RP-HPLC to give the title compound as a fluffy white solid (4.6 mg, 2:8 mixture between 48c and 48b). 1H
NMR (850 MHz, CD3OD): δ = 7.82 – 7.77 (m, 3H), 7.68 (s, 1H), 7.46 – 7.40 (m, 2H), 7.40 – 7.37 (m, 1H),
5.41 – 5.40 (m, 1H), 4.79 – 4.76 (m, 1H), 4.11 (dd, J = 14.9, 4.3 Hz, 1H), 3.67 – 3.63 (m, 1H), 3.59 – 3.55
(m, 1H), 3.53 (dd, J = 15.0, 2.7 Hz, 1H), 3.52 – 3.48 (m, 1H), 3.27 – 3.18 (m, 3H), 3.12 – 3.09 (m, 2H), 3.05
(dd, J = 14.8, 6.0 Hz, 1H), 2.91 – 2.86 (m, 1H), 2.83 – 2.79 (m, 1H), 2.63 (dd, J = 14.9, 3.8 Hz, 1H), 2.09 –
1.97 (m, 2H), 1.83 – 1.75 (m, 2H), 1.69 – 1.65 (m, 2H), 1.64 – 1.57 (m, 3H), 1.46 – 1.39 (m, 1H); 13C NMR
26

ACCEPTED MANUSCRIPT
(850 MHz, CD3OD): δ = 172.7, 169.9, 158.61, 158.55, 140.3, 135.2, 133.6, 129.1, 128.6, 128.4, 128.1,
127.4, 127.0, 126.3, 56.1, 55.1, 48.4, 47.8, 42.0, 41.7, 40.4, 40.3, 36.3, 34.3, 30.7, 29.8, 27.5, 26.5; HRMS
(ESI): m/z [M+H]+ calcd for C28H41N8O2S+: 553.3068; found: 553.3077. Purity = 97.2% (sum of
diastereoisomers 48b and 48c, UV 220 nm)
Due to signal overlap, all chemical shifts could not be unambiguously identified for 48c. See supporting
information for key chemical shifts for 48c.
4.1.37.
1,1'-(((2RS,6S,9aRS)-8-(3-(naphthalen-2-yl)propyl)-4,7-dioxohexahydro-2H,6H-pyrazino[2,1-
b][1,3]thiazine-2,6-diyl)bis(propane-3,1-diyl))diguanidine (48d)
Bicycle 48d was prepared as described for 48a above. The crude product was purified by semi-preparative
1
RP-HPLC to give the title compound as a fluffy white solid (0.9 mg, 2:8 mixture of 48b and 48d). H (850
MHz, CD3OD): δ = 7.82 – 7.77 (m, 3H), 7.68 (s, 1H), 7.45 – 7.41 (m, 2H), 7.38 (dd, J = 8.3, 1.7 Hz, 1H),
4.66 (dd, J = 9.2, 6.0 Hz, 1H), 4.50 (dd, J = 11.4, 4.4 Hz, 1H), 3.72 – 3.68 (m, 1H), 3.67 – 3.63 (m, 1H), 3.59
(dd, J = 13.7, 4.3 Hz, 1H), 3.49 – 3.44 (m, 1H), 3.26 – 3.19 (m, 3H), 3.16 (t, J = 6.8 Hz, 2H), 2.91 – 2.87 (m,
1H), 2.83 – 2.79 (m, 1H), 2.62 (dd, J = 16.1, 5.3 Hz, 1H), 2.43 (dd, J = 16.2, 7.1 Hz, 1H), 2.08 – 2.00 (m,
2H), 1.85 – 1.80 (m, 1H), 1.78 – 1.73 (m, 1H), 1.70 – 1.65 (m, 3H), 1.65 – 1.59 (m, 3H), 1.51 – 1.46 (m,
13
1H); C (213.8 MHz, CD3OD): δ = 171.3, 170.2, 158.64, 158.59, 140.6, 135.1, 133.6, 129.0, 128.6, 128.5,
128.3, 127.4, 127.2, 126.5, 57.4, 56.5, 48.2, 47.8, 41.9, 41.6, 41.0, 40.8, 35.1, 34.5, 31.2, 29.6, 27.3, 26.5;
HRMS (ESI): m/z [M+H]+ calcd for C28H41N8O2S+: 553.3068; found: 553.3076. Purity = 82.7% (sum of
diastereoisomers 48b and 48d, UV 220 nm)
4.1.38. 1-(3-((2S,6S,9aS)-2-(5-guanidinopentyl)-8-(3-(naphthalen-2-yl)propyl)-4,7-dioxohexahydro-2H,6H-
pyrazino[2,1-b][1,3]thiazin-6-yl)propyl)guanidine (49a)
Linear precursor 43 (117 mg, 0.10 mmol) was deprotected and cyclized following general procedure C. The
crude product (87 mg) was dissolved in anhydrous DMF (5 mL), 1H-pyrazole-1-carboxamidine
hydrochloride (100 mg, 0.69 mmol) and DIPEA (0.12 mL, 0.69 mmol) were added. The reaction mixture
was stirred at room temperature for 93 h at room temperature during which the reaction was monitored by
analytical RP HPLC. The reaction was cooled to 0 °C, cold diethyl ether (20 mL) was added and stirring
continued for an additional hour, resulting in the precipitation of a brown oil (152 mg). The crude product
was purified by semi-preparative RP-HPLC to give the title compound as a white fluffy solid (3.2 mg, 8%).
1H NMR (850 MHz, CD3OD): δ = 7.80 (dd, J = 17.7, 8.0 Hz, 2H), 7.76 (d, J = 8.3 Hz, 1H), 7.69 (s, 1H),
27

ACCEPTED MANUSCRIPT
7.46 – 7.41 (m, 2H), 7.36 (dd, J = 8.4, 1.7 Hz, 1H), 5.01 (dd, J = 10.1, 4.9 Hz, 1H), 4.91 (dd, J = 8.8, 4.5 Hz,
1H), 3.67 (dt, J = 13.6, 7.4 Hz, 1H), 3.52 – 3.45 (m, 2H), 3.37 (ddd, J = 13.4, 7.4, 6.0 Hz, 1H), 3.27 – 3.23
(m, 1H), 3.21 – 3.15 (m, 3H), 2.97 – 2.93 (m, 1H), 2.90 – 2.86 (m, 1H), 2.84 – 2.79 (m, 1H), 2.55 (dd, J =
16.6, 3.4 Hz, 1H), 2.39 (dd, J = 16.6, 12.0 Hz, 1H), 2.11 – 2.03 (m, 2H), 1.97 – 1.92 (m, 1H), 1.80 – 1.74 (m,
13
1H), 1.63 – 1.57 (m, 4H), 1.57 – 1.53 (m, 1H), 1.48 – 1.41 (m, 2H), 1.41 – 1.34 (m, 3H); C NMR (213.8
MHz, CD3OD): δ = 171.0, 169.3, 158.65, 158.60, 140.5, 135.1, 133.5, 129.1, 128.7, 128.5, 128.1, 127.4,
127.1, 126.3, 56.7, 52.8, 52.1, 48.3, 42.4, 42.2, 41.7, 37.3, 36.2, 34.5, 29.73, 29.67, 28.9, 27.43, 27.38, 26.3;
HRMS (ESI): m/z [M+H]+ calcd for C30H45N8O2S+: 581.3381; found: 581.3389. Purity = 99.0% (UV 220
nm).
4.1.39. 1-(3-((2R,6S,9aR)-2-(5-guanidinopentyl)-8-(3-(naphthalen-2-yl)propyl)-4,7-dioxohexahydro-2H,6H-
pyrazino[2,1-b][1,3]thiazin-6-yl)propyl)guanidine (49b)
Bicycles 49b was prepared as described for 49a above. The crude product was purified by semi-preparative
RP-HPLC to give the title compound as a fluffy white solid (2.2 mg, 3:7 mixture between 49c and 49b). 1H
NMR (850 MHz, CD3OD): δ = 7.81 – 7.77 (m, 3H), 7.69 (s, 1H), 7.46 – 7.37 (m, 3H), 5.40 – 5.39 (m, 1H),
4.80 – 4.77 (m, 1H), 4.10 (dd, J = 14.9, 4.3 Hz, 1H), 3.67 – 3.63 (m, 1H), 3.63 – 3.59 (m, 1H), 3.53 (dd, J =
15.0, 2.7 Hz, 1H), 3.47 – 3.43 (m, 1H), 3.27 – 3.20 (m, 2H), 3.17 (t, J = 7.2 Hz, 1H), 3.05 – 3.01 (m, 2H),
2.92 – 2.87 (m, 1H), 2.83 – 2.78 (m, 1H), 2.62 (dd, J = 14.9, 3.8 Hz, 1H), 2.10 – 2.02 (m, 1H), 2.01 – 1.96
(m, 1H), 1.84 – 1.75 (m, 2H), 1.70 – 1.64 (m, 2H), 1.62 – 1.56 (m, 2H), 1.47 – 1.42 (m, 1H), 1.41 – 1.24 (m,
5H); 13C (213.8 MHz, CD3OD): δ = 172.9, 169.9, 158.6, 158.5, 140.4, 135.2, 133.6, 129.0, 128.6, 128.4,
128.1, 127.4, 127.0, 126.3, 56.1, 55.0, 48.4, 47.7, 42.3, 41.7, 40.8, 40.5, 39.3, 34.3, 30.7, 29.9, 29.6, 27.7,
27.3, 26.5; HRMS (ESI): m/z [M+H]+ calcd for C30H45N8O2S+: 581.3381; found: 581.3382; Purity = 96.6%
(sum of diastereoisomers 49b and 49c, UV 220 nm).
Due to signal overlap, all chemical shifts could not be unambiguously identified for 49c. See supporting
information for key chemical shifts for 49c.
4.1.40. 1-(3-(2-(5-guanidinopentyl)-8-(3-(naphthalen-2-yl)propyl)-4,7-dioxohexahydro-2H,6H-pyrazino[2,1-
b][1,3]thiazin-6-yl)propyl)guanidine (49d)
Bicycle 49d was prepared as described for 49a above. The crude product was purified by semi-preparative
RP-HPLC to give the title compound as a fluffy white solid (1.3 mg, 1:9 mixture of 49b and 49d). 1H NMR
(850 MHz, CD3OD): δ = 7.82 – 7.77 (m, 3H), 7.68 (s, 1H), 7.46 – 7.41 (m, 2H), 7.39 (dd, J = 8.3, 1.8 Hz,
28

ACCEPTED MANUSCRIPT
1H), 4.65 (dd, J = 9.0, 6.1 Hz, 1H), 4.49 (dd, J = 11.4, 4.5 Hz, 1H), 3.72 – 3.68 (m, 1H), 3.66 (dd, J = 13.6,
11.4 Hz, 1H), 3.58 (dd, J = 13.7, 4.4 Hz, 1H), 3.49 – 3.45 (m, 1H), 3.26 – 3.19 (m, 3H), 3.16 (t, J = 7.2 Hz,
2H), 2.91 – 2.87 (m, 1H), 2.83 – 2.79 (m, 1H), 2.59 (dd, J = 16.0, 5.5 Hz, 1H), 2.41 (dd, J = 16.1, 6.9 Hz,
1H), 2.09 – 1.99 (m, 2H), 1.85 – 1.80 (m, 1H), 1.78 – 1.72 (m, 1H), 1.70 – 1.65 (m, 2H), 1.61 – 1.56 (m, 3H),
1.48 – 1.34 (m, 5H); 13C NMR (213.8 MHz, CD3OD): δ = 171.6, 170.2, 158.63, 158.59, 140.6, 135.1, 133.6,
129.0, 128.6, 128.5, 128.3, 127.4, 127.2, 126.5, 57.4, 56.4, 48.2, 47.8, 42.4, 41.6, 41.1, 41.0, 38.2, 34.5, 31.2,
29.7, 29.6, 27.5, 27.3, 26.5: HRMS (ESI): m/z [M + H]+ calcd for C30H45N8O2S+: 581.3381; found 581.3389.
Purity = 90.9% (UV 220 nm).
4.2. Biology – The final compounds were tested for antagonistic activity against the human CXCR4 in a
scintillation proximity-based inositol-phosphate accumulation assay (SPA-IP) following the same protocol as
recently described.7
Acknowledgements
Financial support for this project was obtained from the University of Bergen (M.B. and B.E.H.), UiT The
Arctic University of Norway (Z.G.Z and J.V.), and from the University of Copenhagen and the Danish
Medical Research Council, the Hørslev Foundation, the AP-Møller Foundation and the Danish Research
Council | Health and Disease (S.K. and M.M.R.). The authors would like to thank associate professor Nils
Åge Frøystein and associate professor Jarl Underhaug at the University of Bergen for excellent technical
support with NMR experiments, and Dr. Bjarte Holmelid at the University of Bergen and Maibritt S.
Baggesen at the University of Copenhagen for excellent technical support for MS analyses and biological
analyses respectively. The Research Council of Norway is acknowledged for support through the Norwegian
NMR Platform, NNP (226244 / F50)
Supplementary Data
Supplementary data (HPLC traces for key cyclization reactions and key NMR data for bicycles) associated
with this article can be found, in the online version, at http://
References and Notes
1.
2.
Ripka, AS; Rich, DH. Curr. Opin. Chem. Biol. 1998, 2, 441-452.
Ung, P; Winkler, DA. J. Med. Chem. 2011, 54, 1111-1125.
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