Urotensin II(4-11) Azasulfuryl Peptides: Synthesis and Biological Activity

Article abstract of DOI:10.1021/acs.jmedchem.6b00108

Cyclic azasulfuryl (As) peptide analogs of the urotensin II (UII, 1, H-Glu-Thr-Pro-Asp-c[Cys-Phe-Trp-Lys-Tyr-Cys]-Val-OH) fragment 4-11 were synthesized to explore the influences of backbone structure on biological activity. N-Aminosulfamides were inserted as surrogates of the Trp⁷ and Lys⁸ residues in the biologically relevant Trp-Lys-Tyr triad. A combination of solution- and solid-phase methods were used to prepare novel UII^(4-11) analogs 6-11 by routes featuring alkylation of azasulfuryl-glycine tripeptide precursors to install various side chains. The pharmacological profiles of derivatives 6-11 were tested in vitro using a competitive binding assay and ex vivo using a rat aortic ring bioassay. Although the analogs exhibited weak affinity for the urotensin II receptor (UT) without agonistic activity, azasulfuryl-UII^(4-11) derivatives 7-9 reduced up to 50% of the effects of UII and urotensin II-related peptide (URP) without affecting their potency.

Full text of DOI:10.1021/acs.jmedchem.6b00108

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Article
(4-11)
Urotensin II Azasulfuryl Peptides: Synthesis and Biological Activity
Francesco Merlino, Ali Munaim Yousif, Etienne Billard, Julien Dufour-Gallant,
Stéphane Turcotte, Paolo Grieco, David Chatenet, and William D. Lubell
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.6b00108 • Publication Date (Web): 03 May 2016
Downloaded from http://pubs.acs.org on May 7, 2016
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(
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Urotensin II
Azasulfuryl Peptides: Synthesis and
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Biological Activity
†,‡,§
†,‡,§
⊥
†,⊥
Francesco Merlino,
Ali M. Yousif,
Étienne Billard, Julien DufourꢀGallant, Stéphane
†
‡
⊥
†,*
Turcotte, Paolo Grieco, David Chatenet and William D. Lubell
†
Département de Chimie, Université de Montréal, P.O. Box 6128, Station Centreꢀville, Montréal,
‡
QC H3C 3J7, Canada; Department of Pharmacy, University of Naples “Federico II”, via D.
⊥
Montesano 49, 80131 Naples, Italy; INRS – Institut ArmandꢀFrappier, Groupe de Recherche en
Ingénierie des Peptides et en Pharmacothérapie (GRIPP), Université du Quebéc, Ville de Laval, QC
H7V 1B7, Canada.
§
These authors equally contributed to this work.
ABSTRACT. Cyclic azasulfuryl (As) peptide analogs of the urotensin II (UII, 1, HꢀGluꢀThrꢀProꢀ
Aspꢀc[CysꢀPheꢀTrpꢀLysꢀTyrꢀCys]ꢀValꢀOH) fragment 4ꢀ11 were synthesized to explore the
influences of backbone structure on biological activity. NꢀAminosulfamides were respectively
7
8
inserted as surrogates of the Trp and Lys residues in the biologically relevant TrpꢀLysꢀTyr triad.
(
4ꢀ11)
A combination of solutionꢀ and solidꢀphase methods were used to prepare novel UII
analogs 6ꢀ
1
1 by routes featuring alkylation of azasulfurylꢀglycine tripeptide precursors to install various side
chains. The pharmacological profiles of derivatives 6ꢀ11 were tested in vitro using a competitive
binding assay and ex vivo using a rat aortic ring bioassay. Although the analogs exhibited weak
(
4ꢀ11)
affinity for the urotensin II receptor (UT) without agonistic activity, azasulfurylꢀUII
derivatives
7ꢀ9 reduced up to 50% of the effects of UII and urotensin IIꢀrelated peptide (URP) without affecting
their potency.
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INTRODUCTION
The urotensin II receptor (UT) is a G proteinꢀcoupled receptor (GPCR) that binds, in humans, two
endogenous cyclic peptide ligands: urotensin II {hUII, 1, HꢀGluꢀThrꢀProꢀAspꢀc[CysꢀPheꢀTrpꢀLysꢀ
TyrꢀCys]ꢀValꢀOH} and urotensin IIꢀrelated peptide {URP, 2, HꢀAlaꢀc[CysꢀPheꢀTrpꢀLysꢀTyrꢀCys]ꢀ
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ValꢀOH, Figure 1}. Expressed in the circulatory, excretory and central nervous systems in humans,
UT plays a determining role in the physiological regulation of various organ systems, particularly
2
the cardiovascular system. In cardiovascular and pulmonary diseases, the potent vasoactive
3
4ꢀ7
peptides UII and 2 exhibit crucial roles, which have only begun to be characterized. Notably,
multiple studies in animal models suggest that UT antagonists may have utility as potential
therapeutic agents for treating atherosclerosis, hypertension, and metabolic syndrome, as well as
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,9
cardiac and renal failure. In spite of this promise, clinical studies of UT antagonist drug
candidates {e.g., 1ꢀ[2ꢀ(4ꢀbenzylꢀ4ꢀhydroxyꢀpiperidinꢀ1ꢀyl)ꢀethyl]ꢀ3ꢀ(2ꢀmethylꢀquinolinꢀ4ꢀyl)ꢀurea
1
0
1
sulfate salt (ACTꢀ058362), a 5,6ꢀbisarylꢀ2ꢀpyridinecarboxamide derivative (SAR101099), and 4′ꢀ
[
(1R)ꢀ1ꢀ[[(6,7ꢀdichloroꢀ3ꢀoxoꢀ2,3ꢀdihydroꢀ4Hꢀ1,4ꢀbenzoxazinꢀ4ꢀyl)acetyl](methyl)amino]ꢀ2ꢀ(4ꢀ
1
1
12
morpholinyl)ethyl]ꢀ4ꢀbiphenylcarboxylicacid trifluoroacetate (GSK1440115) }, which protected
respectively against renal ischemia, diabetic nephropathy and asthma, have had limited success due
1
to lack of efficacy in humans.
Considering their critical roles in cardiovascular homeostasis, structureꢀactivity relationship
(SAR) studies have been performed on UII and 2 to identify the physicochemical and structural
determinants involved in UT recognition and activation. Sharing a conserved Cꢀterminal disulfideꢀ
1
bridged cyclic core, UII and 2 differ at their Nꢀterminal regions. Truncation of the nonꢀconserved
(
4ꢀ11)
Nꢀterminal region of UII provided the octapeptide UII
, which is the shortest sequence of the
Common to all of these cyclic peptides is
1
3,14
parent peptide that possesses full biological activity.
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the Trp ꢀLys ꢀTyr tripeptide sequence, which has been suggested to adopt a biologically active γꢀ
15ꢀ17
turn conformation that is stabilized by the disulfide bridge.
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(4ꢀ11)
Figure 1. Structures of 1, 2, 3, 4, pyrrolo[3,2ꢀe][1,4]diazepinꢀ2ꢀone (5) and azasulfurylꢀUII
analogs 6ꢀ11.
Accordingly, it has been recently demonstrated that the distinct pathophysiological roles for hUII
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and 2 most likely arise from their different interactions with their cognate receptor. In particular,
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hydrogen bonds between Glu and Asp of 1 and respectively Lys and Arg of UT were clearly
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observed in the hUII/UT complex, but were absent in the URP/UT complex. Modification and
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mimicry of the Trp ꢀLys ꢀTyr triad have recently led to peptide and small molecule UT modulators
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that differentiate UII and 2 biological activity.
For example, urocontrin, [Bip ]URP (3), and
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urocontrin A, [Pep ]URP (4, Figure 1), exhibit the ability to decrease the maximal contractile
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response of 1 without causing significant change in its potency and without noticeable effect on
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URPꢀinduced vasoconstriction.
Similar results have been also observed in vivo using urocontrin
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in anaesthetized rats. Employing a pyrrolo[3,2ꢀe][1,4]diazepinꢀ2ꢀone as a mimic of the BipꢀLysꢀ
Tyr triad of urocontrin, small molecule 5 (Figure 1) has been designed and shown to abolish the
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maximum contractile response of 1 while potentiating the contractile efficacy of 2.
In our pursuit of a better understanding of the physicochemical and structural determinants
involved in the biological activity of UT ligands, we report now the application of Nꢀ
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(4ꢀ11)
aminosulfamide surrogates to replace respectively the Trp and Lys residues in UII
. Analogs of
natural peptides (12, Figure 2), azasulfuryl (As) peptides (13) have served in proteinase inhibitors
by replicating the tetrahedral geometry of the transition state for amide bond hydrolysis, and have
22,23
adopted γꢀturn backbone geometry in azasulfuryl tripeptide crystal structures.
Although the
application of azasulfuryl peptides as ground state mimics of biologically active peptides has yet to
be explored, their potential to stabilize γꢀturn geometry and increase hydrogen bond interactions is
2
2
intriguing.
Figure 2. General structures of a peptide and an azasulfuryl peptide.
Solutionꢀphase chemistry was developed to prepare a set of azasulfuryl tripeptide building blocks,
(4ꢀ11)
which were incorporated into UII
by solidꢀphase peptide synthesis, followed by disulfide bond
formation in solution. This complementary use of solutionꢀ and solidꢀphase methods has provided
(
4ꢀ11)
six azasulfuryl peptide analogs of UII
(6ꢀ11, Figure 1), and promises to be a general approach
for introducing Nꢀaminosulfamides into various peptides. Although azasulfuryl peptides 6ꢀ11
exhibited typically very weak affinity for UT and almost no agonistic potency in an ex vivo rat
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aortic ring bioassay, certain ligands (7ꢀ9) reduced 1 and 2 contractile efficiency without influencing
their potency.
RESULTS AND DISCUSSION
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Chemistry. Azasulfurylꢀglycine (AsG) tripeptides 19 and 27 were synthesized without formation
of symmetric sulfamides by the reaction of pꢀnitrophenyl sulfamidates with amino acid hydrazides
24
(
Schemes 1 and 2). Alkylation of AsG tripeptides 19 and 27 with different alkyl halides was
subsequently performed to install various side chains on the aminosulfamide residue. Key to the
success of this strategy was the development of a functional group protection strategy for
introducing side chain diversity under the alkaline conditions of the alkylation. The pursued
strategy entailed synthesis, alkylation and saponification of two AsG containing tripeptides [Allocꢀ
PheꢀAsGꢀLys(Boc)ꢀOBn (19) and AllocꢀTrp(Boc)ꢀAsGꢀTyr(tꢀBu)ꢀODmb (27)], followed by
(4ꢀ11)
coupling their respective acids onto the resin for introduction into the core sequence of UII
by
solidꢀphase synthesis. Protecting group choice was therefore critical in order to be compatible with
base in the alkylation as well as the conditions for Fmoc/tꢀBu peptide synthesis. As expected, the
Fmoc protecting group was incompatible with the alkylation step, and the Alloc protecting group
was used to protect the backbone amine. Acid labile Boc and tertꢀbutyl protections were used for
the Trp, Lys and Tyr side chains as well as the hydrazide intermediate. Finally, orthogonal
protection of the Cꢀterminal carboxylate as a benzyl ester was required for its selective removal in
the presence of the Alloc and acid labile groups.
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Scheme 1. Synthesis and alkylation of azasulfurylꢀglycine tripeptide 19. (a) H NNHBoc, iꢀ
2
BuOCOCl, NMM, dry THF, –15 °C, 2 h; (b) pꢀO NC H OSO Cl, pꢀO NC H OH, NEt , dry DCM,
2
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–
78 °C, 2 h; (c) 1:1 TFA/DCM, 1 h; (d) ꢁW, MeCN, 60 °C, 2.5 h; (e) RBr, BTPP, dry THF, 0 °C,
3.5 h; (f) LiOH, 2:1 1,4ꢀdioxane/H O, overnight.
2
α
AllocꢀPheꢀAsGꢀLys(Boc)ꢀOBn (19) was assembled by coupling N ꢀAllocꢀLꢀphenylalanine
hydrazide (17) and pꢀnitrophenylsulfamidate benzyl ester 18. Hydrazide 17 was obtained from
α
acylation of tertꢀbutyl carbazate with N ꢀAllocꢀLꢀphenylalanine 14, and Boc group removal with
5
0% TFA in DCM (Scheme 1). Sulfamidate 18 was obtained from acylation of benzyl ester 15 with
25
4ꢀnitrophenyl chlorosulfate. The initial attempts to accomplish the synthesis of sulfamidate 18 led
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to a significant percentage (≈10%) of symmetric sulfamide. To circumvent formation of symmetric
sulfamide, three molꢀequiv of 4ꢀnitrophenol were employed in the reaction. Washings with 5%
sodium bicarbonate solution after purification by flash chromatography were used to achieve higher
purity of 18. The reaction of sulfamidate 18 with hydrazide 17 was performed under microwave
irradiation in the presence of triethylamine as base. The choice of solvent was critical to enhance
the yield of AsG tripeptide 19 during the microwave irradiation; MeCN (74% yield) and THF (72%
yield) gave better yields than 1,2ꢀdichloroethane (43% yield), which had previously been used for
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AsG tripeptide synthesis, but in which both 17 and 18 were less soluble.
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Scheme 2. Synthesis and alkylation of azasulfurylꢀglycine tripeptide 27. (a)
2 2 3 2 2 6 4 2 2 6 4
H NNHCO C(CH ) Ph, DCC, HOBt, DCM, overnight; (b) pꢀO NC H OSO Cl, pꢀO NC H OH,
NEt , dry DCM, –78 °C, 2 h; (c) 1.5% TFA:DCM, 1 h; (d) ꢁW, MeCN, 60 °C, 3 h; (e) Cl(CH ) Br,
3
2 4
BTPP, THF, 0 °C, 4 h; (f) 1% TFA:DCM, 1 h.
α
in
AllocꢀTrp(Boc)ꢀAsGꢀTyr(tꢀBu)ꢀODmb (27) was assembled by coupling N ꢀAllocꢀN ꢀBocꢀLꢀ
tryptophan hydrazide (25) and dimethoxybenzyl Oꢀtertꢀbutyl tyrosine pꢀnitrophenylsulfamate (26,
Scheme 2). Cumyl carbazate (see Supporting Information) was critical for the synthesis of
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α
in
tryptophan hydrazide 25 from N ꢀAllocꢀN ꢀBocꢀLꢀtryptophan 22 because of its stability under the
N,N'ꢀdicyclohexylcarbodiimide (DCC) with 1ꢀhydroxybenztriazole (HOBt) coupling condition to
form protected hydrazide 24 (82% yield), and orthogonal removal in 74% yield without cleavage of
the other acid labile side chain protection (e.g., Boc and tꢀBu). Tyrosine sulfamidate 26 was
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prepared from amino ester 23, which was mixed with 4ꢀnitrophenol and NEt
dropꢀwise to a solution of 4ꢀnitrophenyl chlorosulfate in DCM at –78 °C. The resulting sulfamidate
6 was obtained in 56% yield, and stored in a sealed vial at low temperature for several months
3
in DCM, then added
2
without decomposition. The coupling of hydrazide 25 and sulfamidate 26 was accomplished in 60%
yield under microwave irradiation using similar conditions as mentioned above.
A set of aryl methyl side chains was added to AsG tripeptide 19 by chemoselective alkylation
using the phosphazane base tertꢀbutyliminoꢀtri(pyrrolidino)ꢀphosphorane (BTPP) to provide
respectively azasulfuryl tripeptides 20aꢀd in 65ꢀ85% yields (Scheme 1). By employing
24
stoichiometric amounts of base and alkylating reagent, bisꢀalkylation was minimized. The position
of alkylation was assigned based on analogy with NMR experiments previously performed on the
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alkylation products from related AsG tripeptides. In the case of the azasulfurylꢀlysine (AsK )
residue, the side chain was installed by a two step process. First, alkylation of AsG tripeptide 27
was performed with 1ꢀbromoꢀ4ꢀchlorobutane to give the corresponding azasulfurylꢀ4ꢀ
chlorobutylglycine tripeptide 28 in 66% yield with minor amounts of bisꢀalkylation (Scheme 2).
Subsequently, the chloride was displaced with dimethylamine or azide (vide infra) to install the ωꢀ
26
(4ꢀ11)
nitrogen component. For introduction into the UII
sequence, the benzyl and 2,4ꢀ
dimethoxybenzyl (Dmb) esters of azasulfuryl tripeptides 20aꢀd and 28 were removed respectively
using LiOH in 1,4ꢀdioxane/THF (2:1) and 1% TFA in DCM to furnish carboxylic acids 21aꢀd and
29 in 68ꢀ86% yields.
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Scheme 3. Synthesis of azasulfurylꢀUII
peptides 6ꢀ9. (a) 21aꢀd, DIC, HOBt, 1:1 DCM/DMF,
overnight; (b) Pd(PPh ) , NDMBA, 3:2 dry DCM/DMF, 2 x 2 h; (c) Elongation; (d) 89:10:1
3
4
TFA/DMSO/anisole, 3 h.
Azasulfuryl tripeptides 21aꢀd were respectively attached to Tyr(tꢀBu)ꢀCys(Trt)ꢀVal bound to 2ꢀ
chlotrityl chloride resin 30 using DIC and HOBt as coupling reagents (Scheme 3). The Nꢀterminal
Alloc group of azasulfuryl peptides 31aꢀd was removed after two treatments with
tetrakis(triphenyphosphine)ꢀpalladium(0) and N,N’ꢀdimethylbarbituric acid (NDMBA) in
DCM/DMF (3:2) under argon. Subsequently, the resin was washed using 0.5% (w/v) sodium
(
4ꢀ11)
diethyl dithiocarbamate in DMF to remove palladium residue. The UII
sequences were
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7
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2
7
elongated using a standard Fmocꢀbased solidꢀphase peptide synthesis strategy. Azasulfurylꢀ
7
7
(4ꢀ11)
phenylalanine (AsF ) UII
peptide derivatives 6ꢀ9 were obtained following concomitant
cleavage and disulfide bond formation in a oneꢀpot reaction using a cocktail of 10% DMSO in TFA
28
in the presence of anisole as scavenger, followed by purification using HPLC on a preparative C18
column. The final isolated products were characterized by LCꢀMS analysis in two different solvent
systems and HRMS (see Table S1 in Supporting Information).
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
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7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(4ꢀ11)
Scheme 4. Synthesis of azasulfurylꢀUII
DCM/DMF, overnight; (b) HN(CH
°C, overnight; (d) Pd(PPh , NDMBA, 3:2 dry DCM/DMF, 2 x 2 h; (e) Elongation; (f) TCEP, 9:1
THF/H O, 24 h; (g) 89:10:1 TFA/DMSO/anisole, 3 h.
peptides 10 and 11. (a) 29, DIC, HOBt, 1:1
3
) , DMF, sonication, 60 °C, 4 h; (c) NaN , DMF, sonication, 60
2
3
3 4
)
2
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9
Azasulfuryl tripeptide 29 was coupled to Cys(Trt)ꢀVal bound to 2ꢀchlotrityl chloride resin 34
using similar DIC and HOBt conditions as described above (Scheme 4). Prior to elongation of the
peptide, the chloride of azasulfurylꢀ4ꢀchlorobutylglycine peptide 35 was respectively displaced with
dimethylamine (40% aqueous solution) and sodium azide in DMF using ultrasound irradiation to
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
8
26
afford AsK peptides 36 and 37. After Alloc group removal and peptide elongation as described
above, azide 41 was reduced to amine 42 using trisꢀ(2ꢀcarboxyethyl)phosphine hydrochloride
28
8
(4ꢀ11)
(
TCEP). After resin cleavage and disulfide bond formation, [AsK ]UII
peptide derivatives 10
and 11 were obtained by HPLC purification, and characterized as described above (see Table S1 in
Supporting Information).
The conformations of human UII and 2 have previously been analyzed using molecular dynamics
16,17
with restraints based on nuclear magnetic resonance (NMR) spectroscopic data.
In the
9
7
optimized geometry, the amide NH of Tyr and the carbonyl of Trp were engaged in an
17
intramolecular hydrogen bond in an inverse γꢀturn conformation. Considering that the
aminosulfamide residue in Xꢀray crystallographic analyses of AsG tripeptides has been observed to
2
2
adopt the central residue of a γꢀturn motif, the azasulfuryl residue has been inserted respectively at
(
4ꢀ11)
positions 7 and 8 of UII
in order to explore potential for stabilizing the secondary structure
responsible for binding affinity and activity at the cognate G proteinꢀcoupled receptor. With
azasulfuryl peptides 6ꢀ11 in hand, the analogs were tested in competitive binding assays and in an
ex vivo aortic ring contraction assay.
Biological data. In the competitive binding assay, CHO cells that overꢀexpress the human
isoform of the urotensin II receptor were treated respectively with azasulfuryl peptides 6ꢀ11, and
125
their capacity to displace IꢀhUII from UT was measured. In general, 6ꢀ11 exhibited limited
–5
125
–6
ability (40ꢀ60% at 10 M) to displace IꢀhUII (Figure 3) with an IC50 greater than 10 M. The
different side chains, such as those used to mimic the aromatic moiety of the Trp residue in
azasulfuryl peptides 6ꢀ11, had limited influence on binding affinity. Among the series possessing
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8
9
1
1
1
1
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1
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1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
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4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
tryptophan surrogates, 1ꢀnaphthyl analog 8 exhibited the best affinity, perhaps due to capacity to
replicate the shape and orientation of the indole moiety of the native ligand.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
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5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
125
Figure 3. Displacement of [ I]hUII binding by nonꢀradioactive azasulfuryl peptides. Results
125
shown are the amount of bound [ I]hUII displaced when incubated in the presence of the indicated
nonꢀlabeled peptide. Data are mean (± S.E.M.) of at least three independent experiments and each
29
determination was performed in triplicate. Binding affinity for hUII have been reported elsewhere.
Azasulfuryl derivatives 6ꢀ11 were next tested for propensity to induce aortic ring contractions. In
7
agreement with their low binding affinity, AsF analogs 6ꢀ9 acted as weak partial agonists with a
8
maximum efficacy between 3% and 7% of the KClꢀinduced contraction. AsK analogs 10 and 11
7
(4ꢀ
were devoid of agonist activity (Figure 4). In contrast to the negligible activity of [AsNal(1') ]UII
11)
8
(4ꢀ11)
4
8
(4ꢀ11)
(8) and [AsK(Me
2
) ]UII
(11), the related [1ꢀNal ]URP and [Lys(Me
2
) ]UII
are potent
30,31
UT agonists.
Loss of activity may be attributed to the significant structural change, due to the
backbone modification at these particular positions.
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41
42
43
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49
50
51
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53
54
55
56
57
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60
Figure 4. Agonistic properties of 1, 2 and compounds 6ꢀ11 on the contraction of rat aortic rings.
Azasulfuryl derivatives 6ꢀ11 were subsequently tested for their ability to modulate hUII and 2
–5
vasoconstriction. Rat aortic rings were exposed to azasulfuryl peptides (10 M) and then
cumulative concentrationꢀresponses were measured using respectively increasing concentrations
–
11
–6
7
(4ꢀ11)
(
10 to 10 M) of 1 and 2 to assess a potential probeꢀdependent effect. Although [AsF ]UII
8
(4ꢀ11)
ε
8
(4ꢀ11)
(6), [AsK ]UII
(10) and [AsK(N,N ꢀMe
2
) ]UII
(11) were all unable to modulate hUIIꢀ and
URPꢀinduced contractions, azasulfuryl analogs 7ꢀ9 with increased hydrophobicity and steric bulk at
position 7 exhibited modulatory effects with varying degrees of efficacy (Figure 5 and Table 1). For
7
(4ꢀ11)
example, [AsBip ]UII
(7) reduced by >40% the contractions induced respectively by 1 and 2.
Ability to further decrease UIIꢀ and URPꢀmediated contraction (up to 65% of the total) was
7
(4ꢀ11)
7
(4ꢀ11)
exhibited by [AsNal(1’) ]UII
(8). Strikingly, [AsNal(2’) ]UII
(9) modulated differentially
19,20
UIIꢀ and URPꢀassociated contractions. Similar to urocontrin and urocontrin A,
2ꢀnaphthyl
analog 9 reduced hUIIꢀinduced contractions more significantly than those induced by 2 (Figure 5,
Table 1). Moreover, as previously observed using nonꢀpeptide pyrrolo[3,2ꢀe][1,4]diazepinꢀ2ꢀone
UT modulators, the shape and the orientation of the aromatic side chain of the Trp residue surrogate
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7
8
9
1
1
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1
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1
1
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2
2
2
2
2
2
2
2
2
3
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3
3
3
3
4
4
4
4
4
4
4
4
4
4
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5
5
5
5
5
5
5
6
mediated the effects observed on 1 and 2 contractile efficacy. The ability of 2ꢀnaphthyl analog 9 to
display different cooperativity with individual orthosteric ligands acting at the same GPCR suggests
32
an allosteric mode of action. A similar mode of action for 7 and 8 may be possible without probeꢀ
dependent effects; however, a higher UT residence time may not be excluded when accounting for
their mechanisms of action, because slow antagonist offset may delay reꢀequilibration during
experiments and noncompetitive antagonism may produce doseꢀresponse curves with depressed
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
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9
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1
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9
0
1
2
3
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5
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8
9
0
1
2
3
4
5
6
7
8
9
0
1
1
maxima. In contrast to urantide that functions as a standard, rapid, reversible, competitive
antagonist, azasulfuryl derivatives, in particular 2ꢀnaphthyl analog 9 represent novel UT antagonists
having potentially an allosteric mode of action or an enhanced UT residence time.
Figure 5. Effect of the peptidomimetics 6ꢀ11 on the hUIIꢀ (red) and URPꢀ (blue) induced
contraction’s profile of isolated rat aorta.
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Table 1. Comparison of 1 and 2 vasoactive profiles obtained in the presence or absence of
peptidomimetics 6ꢀ11.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
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34
35
36
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41
42
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a
The maximum efficacy is expressed as a percentage of the amplitude of the contraction induced by
b
KCl (40 mM). The pEC50 values (the negative logarithm of the halfꢀmaximal effective
concentration) of the ligands were determined by measuring aortic ring contraction. All values are
expressed as mean ± SEM. Statistical analysis for pEC50 and Emax were performed using unpaired
Student’s t test, * P≤0.05, ** P≤0.01, *** P≤0.001, versus values obtained for 1 or 2 alone (no
treatment). Each replicate (n) was conducted on tissue from a different animal.
CONCLUSIONS
(
4ꢀ11)
Six novel azasulfurylꢀUII
analogues have been successfully designed and synthesized by
strategies employing orthogonally protected AsG tripeptides. Chemoselective alkylation with
7
(4ꢀ11)
various alkyl halides and phosphazene base provided AsF UII
analogues 6ꢀ9 possessing
8
different aromatic side chains. Furthermore, AsK analogs 10 and 11 were synthesized by a route
featuring alkylation of protected AsG tripeptide 27 in solution with αꢀbromoꢀωꢀchlorobutane to
provide the corresponding alkyl chloride followed by displacement of the chloride with different
amines on resin. The pharmacological profiles of azasulfuryl peptides 6ꢀ11 with aminosulfamide
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9
1
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1
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1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
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5
5
5
5
5
5
5
5
6
7
8
replacements at the Trp and Lys positions were evaluated using competitive binding assays and in
an ex vivo rat aortic ring contraction bioassay. Notably, Nꢀaminosulfamide peptides 7ꢀ9 with
7
relatively large aromatic ring substituents at the Trp position reduced the maximal vasocontractile
efficiency of hUII and 2 without influencing their potency. In particular, the shape and orientation
of the aromatic sideꢀchain may be responsible for the probeꢀdependent effects observed with 1ꢀ
naphthyl analog 9. Although their relatively low potency has limited our initial efforts to assess
their mechanism of action, the ability of 7 and 8 to modulate efficiency without influencing the
potency of contraction induced by UII and 2 may represent an allosteric mode of action or slow
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
11
dissociation kinetics as previously reported. 1ꢀNaphthyl analog 9 was able to differentially
modulate hUIIꢀ and URPꢀmediated contraction, and similar to urocontrin and urocontrin A,
represents a new albeit less potent allosteric modulator of the urotensinergic system. Augmenting
the armamentarium of UT modulators, 1ꢀnaphthyl analog 9 may provide more insight into the
structural requirements for selectively modulating the effects of the endogenous UT ligands towards
understanding of the cellular mechanisms underlying receptor functions. Derivatives with improved
biological activity are being pursued to better characterize the physicochemical determinants
responsible for the observed effect and mode of action. Considering that allosteric modulation and
enhanced residence time have been linked to improved therapeutic windows and increased efficacy,
azasulfuryl derivatives 7ꢀ9 represent interesting leads towards the development of enhanced tools
for determining the pathoꢀphysiology of the urotensinergic system.
EXPERIMENTAL SECTION
2
5
Materials and general procedures. Unless specified, 4ꢀnitrophenyl chlorosulfate, as well as
α
33
α
34
N ꢀ(Alloc)ꢀLꢀPheꢀOH (14), and N ꢀ(Alloc)ꢀLꢀTrp(Boc)ꢀOH (22) were synthesized according to
ε
reported procedures. N ꢀ(Boc)ꢀLꢀlysine benzyl ester 15 was synthesized from its corresponding
35
Fmocꢀamino acid using benzyl bromide and cesium carbonate in DMF, followed by a treatment
with 20% piperidine in DMF solution. Dmb alcohol was prepared by reducing 2,4ꢀ
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3
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dimethoxybenzaldehyde with sodium borohydride in methanol for 30 min, and used in the
α
esterification of N ꢀFmocꢀLꢀTyr(tꢀBu)ꢀOH under Mitsunobu conditions employing
37
3
diisopropylazodicarboxylate (DIAD) and triphenylphosphine (PPh ) for 2 h at room temperature.
α
The N ꢀFmoc protecting group was removed using diethylamine in acetonitrile for 2 h at room
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
3
8
temperature.
Allyl chloroformate, diethylamine (DEA), diisopropylethylamine (DIEA), 1,3ꢀdimethylbarbituric
acid (NDMBA), isobutyl chloroformate (IBC), Lꢀphenylalanine, methyl benzoate,
methylmagnesium bromide (3M in diethyl ether), 4ꢀmethylmorpholine (NMM), 4ꢀnitrophenol, tertꢀ
butyl
carbazate,
triethylamine
(NEt
3
),
diisopropylazodicarboxylate
(DIAD),
and
triphenylphosphine, all were purchased from Aldrich, used as received, except the latter that was
washed with ethanol and dried under vacuum before use. Benzyl bromide was purchased from
Aldrich and filtered through a small plug of silica gel prior to use. Phosphazene base (tertꢀ
butyliminoꢀtri(pyrrolidino)phosphorane, BTPP), and 2ꢀ(bromomethyl)naphthalene were purchased
in
from Fluka and used as received. N ꢀ(Boc)ꢀLꢀTryptophan, pyridine, 4ꢀ(bromomethyl)biphenyl, 1ꢀ
(bromomethyl)naphthalene, and lithium hydroxide were purchased from Alfa Aesar, Acros, or
ChemꢀImpex. Tetrakis(triphenylphosphine)palladium(0) [Pd(PPh ) ] was purchased from Aldrich
3
4
and washed with ethanol prior to use. Methanol and trifluoroacetic acid (TFA) were respectively
purchased from Fisher Chemical and Aldrich, and used as received. Anhydrous solvents
[tetrahydrofuran (THF), N,Nꢀdimethylformamide (DMF), acetonitrile, and dichloromethane
(
DCM)] were obtained by passage through a solventꢀfiltration system (GlassContour, Irvine, CA).
Ethyl acetate and hexane were purchased from Fisher Chemical and fractionally distilled prior to
use. Amino acids, BocꢀAsp(OtꢀBu), FmocꢀAsp(OtꢀBu), FmocꢀCys(Trt), FmocꢀPhe, Fmocꢀ
Lys(Boc), FmocꢀTyr(tꢀBu), FmocꢀVal, and coupling reagents such as N,N,N′,N′ꢀtetramethylꢀOꢀ(1Hꢀ
benzotriazolꢀ1ꢀyl)uronium hexafluorophosphate (HBTU) and HOBt, all were purchased from GL
Biochem (Shangai, China), and used as received. N,N’ꢀDiisopropylcarbodiimide (DIC) was
purchased from Aldrich and used as received. 2ꢀChlorotrityl chloride resin (0.65 and 0.79 mmol/g
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6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
loading) were purchased from ChemꢀImpex, and the manufacturer’s reported loading of the resin
was used in the calculation of final product yields. Microwave irradiation was performed on a 300
MW Biotage apparatus on the highꢀabsorption level; temperature was monitored automatically.
3
9
Flash chromatography was performed on 230ꢀ400 mesh silica gel, and thin layer chromatography
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
TM
was performed on silica gel 60 F254 plates from Merck . Melting points were made on a
Gallankamp apparatus and are uncorrected. Specific rotations, [α] values, were calculated from
D
optical rotations measured at 20 °C in CHCl or THF at the specified concentration (c in g/100 mL)
3
and a 1ꢀdm cell length (l) on a PerkinElmer Polarimeter 341, using the general formula: [
(100 )/(lc). Accurate mass measurements were performed on a LCꢀMSD instrument from Agilent
technologies in positive electrospray ionisation (ESI) mode at the Université de Montréal Mass
α] =
α
+
+
Spectrometry facility. Proton and sodium adducts, [M+H] and [M+Na] , were used for empirical
1
13
formula confirmation. H NMR and C NMR spectra were recorded either on a Bruker AMX300,
or AV400, and measured in CDCl (7.26/77.16 ppm), acetoneꢀd (2.05/29.84 ppm), or pyridineꢀd
5
3
6
(7.22 ppm). Multiplicities are abbreviated: s = singlet, d = doublet, t = triplet, q = quadruplet, m =
multiplet, and br = broad. Coupling constant J values are measured in Hertz (Hz) and chemical shift
®
values in parts per million (ppm). Infrared spectra were recorded in the neat on an ATR Bruker
apparatus.
Analytical LCꢀMS analyses were performed on a Gemini reverseꢀphase column from
TM
Phenomenex (4.6 mm x 150 mm, 5 µm, C18) or a SunFire reverseꢀphase column from Waters
(2.1 mm x 50 mm, 3.5 µm, C ) with a flow rate of 0.5 mL/min using a gradient of acetonitrile
18
(0.1% formic acid) or methanol (0.1% formic acid) in water (0.1% formic acid). Purification of
azasulfuryl peptides was performed on a preparative column (Gemini C18 column, 21.2 mm x 250
mm, particle size 5 m) using specified linear gradients of acetonitrile (0.1% formic acid) in water
0.1% formic acid) with a flow rate of 10.6 mL/min and UV detection at 214 nm and 254 nm. The
ꢀ
(
purity of azasulfuryl peptides was ascertained using analytical HPLC analysis performed on a
TM
Gemini reverseꢀphase column from Phenomenex (4.6 mm x 150 mm, 5 µm, C18) or a SunFire
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7
8
9
reverseꢀphase column from Waters (2.1 mm x 50 mm, 3.5 µm, C ) with a flow rate of 0.5 mL/min
18
using gradients of acetonitrile (0.1% formic acid) or methanol (0.1% formic acid) in water (0.1%
formic acid). Azasulfuryl peptides examined for biological activity were purified to >99% (see
Supporting Information).
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
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42
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44
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α
α
N
ꢀ(Alloc)ꢀN’ꢀBocꢀLꢀPhenylalanine hydrazide (16) N ꢀ(Alloc)ꢀLꢀPhenylalanine (4.29 g, 17.21
mmol) was dissolved in dry THF (93 mL), cooled to –15 °C, treated with IBC (2.23 mL, 17.21
mmol) followed by NMM (2.37 mL, 21.51 mmol), stirred for 15 min, and treated dropꢀwise with a
solution of tertꢀbutyl carbazate in dry THF (21 mL). After stirring at –15 °C for 2 h, the reaction
mixture was treated with water (150 mL), transferred to a separatory funnel, and the phases were
separated. The aqueous layer was extracted with EtOAc (2 x 250 mL). The combined organic phase
was washed with brine, dried over MgSO , filtered and evaporated under reduced pressure. The
4
residue was purified by flash chromatography by eluting with 75:25 hexanes/EtOAc. Evaporation
of the collected fractions afforded the hydrazide 16 (4.07 g; 78% yield) as white solid: R
f
0.22
, 300 MHz)
9H, s), 3.03 (1H, dd, J = 8.0, 14.0 Hz), 3.20 (1H, dd, J = 6.0, 14.0 Hz), 4.50 (2H, dd, J = 1.4, 5.6
Hz), 4.55 (1H, d, J = 5.7 Hz), 5.18 (1H, dd, J = 1.17, 10.5 Hz), 5.23 (1H, dd, J = 1.17, 18.5 Hz),
.54 (1H, d, J = 8.0 Hz), 5.78ꢀ5.91 (1H, m), 6.64ꢀ6.75 (1H, br), 7.22ꢀ7.33 (5H, m), 8.28ꢀ8.39 (1H,
2
0
1
(75:25 hexanes/EtOAc); mp 57ꢀ60 °C; [
α]
D
–9.1 (c 0.96, THF); H NMR (CDCl
3
δ
(
5
1
3
br); C NMR (CDCl , 75 MHz)
δ
170.9, 156.2, 155.3, 136.3, 132.5, 129.5, 128.8, 127.2, 118.1,
3
–1
8
2.0, 66.2, 54.6, 38.2, 28.3; IR (neat) νmax/cm 3280, 1682, 1500, 1370, 1238, 1157, 1045; HRMS
+
(
ESI) m/z calcd for C H N NaO [M+Na] 386.1686, found 386.1691.
18 25 3 5
α
α
N
ꢀ(Alloc)ꢀLꢀPhenylalanine hydrazide (17) N ꢀ(Alloc)ꢀN’ꢀ(Boc)ꢀLꢀPhenylalanine hydrazide 16
(1.4 g, 3.85 mmol) was dissolved in TFA/DCM (1:1, 12 mL), stirred at room temperature for 1 h,
and concentrated in vacuo. The residue was partitioned between 5% aqueous NaHCO solution
100 mL) and EtOAc (100 mL), the phases were separated, and the aqueous phase was extracted
with EtOAc (100 mL x 4). The combined organic layer was dried over MgSO , filtered and
3
(
4
f
evaporated under reduced pressure to afford hydrazide 17 (905 mg; 89% yield): R 0.17 (70:30
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2
2
2
2
2
2
2
3
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3
3
3
3
3
3
3
3
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4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
2
0
1
D 5
hexanes/EtOAc); mp 131ꢀ134 °C; [α] 7.0 (c 0.95, THF); H NMR (pyridineꢀd , 400 MHz) δ 3.30
(
1H, dd, J = 7.7, 13.4 Hz), 3.52 (1H, dd, J = 7.0, 13.4 Hz), 4.60 (1H, dd, J = 5.3, 13.5 Hz), 4.68
1H, dd, J = 5.3, 13.7 Hz), 5.04 (1H, d, J = 8.0 Hz), 5.08 (1H, d, J = 10.4 Hz), 5.24 (1H, d, J = 15.9
(
13
Hz), 7.16ꢀ7.25 (5H, m), 7.36 (2H, d, J = 6.8 Hz), 8.96 (1H, d, J = 8.4 Hz); C NMR (CDCl
3
, 100
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
–1
MHz) 172.0, 156.1, 136.3, 132.5, 129.3, 128.8, 127.2, 118.1, 66.1, 55.1, 38.6; IR (neat) νmax/cm
δ
+
3
2
291, 1693, 1656, 1532, 1243, 1046; HRMS (ESI) m/z calculated for C H N O [M+H]
13 19 3 3
64.1343, found 264.1348.
ε
ε
4
ꢀNitrophenylꢀ
N
ꢀ(Boc)ꢀLꢀlysine benzyl ester sulfamidate (18) Under argon, a solution of N ꢀ
(Boc)ꢀLꢀlysine benzyl ester 15 (1.00 g, 2.97 mmol), 4ꢀnitrophenol (1.24 g, 8.92 mmol) and
triethylamine (2.49 mL, 17.83 mmol) in dry DCM (30 mL) was added dropꢀwise to a solution of 4ꢀ
nitrophenyl chlorosulfate (1.41 g, 5.94 mmol) in dry DCM (6 mL) at –78 °C. After stirring at –78
°
C for 2 h, the mixture was allowed to warm up to rt. The organic phase was washed with 5% citric
acid solution (100 mL x 3), dried over MgSO , filtered and evaporated. The residue was purified by
4
flash chromatography eluting with 70:30 hexanes/EtOAc to afford sulfamidate 18 as a pale yellow
solid (1.09 g; 68% yield) contaminated with <10% of 4ꢀnitrophenol, and used without further
2
0
purification in the next step: R
f
0.28 (70:30 hexanes/EtOAc); mp 93ꢀ96 °C; [
D
α] 17.2 (c 1.08,
1
THF); H NMR (CDCl , 400 MHz)
δ
 1.34ꢀ1.50 (4H, m), 1.42 (9H, s), 1.80ꢀ1.93 (2H, m), 3.07
3
(
2H, d, J = 6.3 Hz), 4.25 (1H, t, J = 5.6 Hz), 4.49ꢀ4.59 (1H, br), 5.19 (1H, d, J = 12.0 Hz), 5.23 (1H,
13
d, J = 12.0 Hz), 5.88ꢀ5.98 (1H, br), 7.32ꢀ7.40 (7H, m), 8.18ꢀ8.22 (2H, d, J = 9.2 Hz); C NMR
CDCl , 75 MHz)  171.2, 156.5, 154.5, 146.0, 134.9, 129.0, 128.9, 128.8, 125.6, 122.4, 79.6,
(
3
δ
–
1
68.1, 57.0, 39.8, 32.3, 31.7, 29.6, 28.5; IR (neat) νmax/cm 3290, 1734, 1682, 1532, 1351, 1253,
+
1
177; HRMS (ESI) m/z calculated for C24
H
31
N
3
NaO
9
S [M+Na] 560.1673; found 560.1676.
α
ε
α
N
ꢀ(Alloc)ꢀLꢀPhenylalaninylꢀazasulfurylꢀglycinylꢀ
N
ꢀ(Boc)ꢀLꢀlysine benzyl ester (19) N ꢀ
(
Alloc)ꢀLꢀPhenylalanine hydrazide 17 (29 mg, 0.11 mmol) was added to a microwave vessel
containing a solution of sulfamidate 18 (54 mg, 0.1 mmol) in MeCN (2.5 mL), and treated with
triethylamine, at which point the solution turned yellow. The vessel was sealed and heated to 60 °C
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using microwave irradiation for 2.5 h. The vessel was opened. The volatiles were evaporated under
reduced pressure. The residue was purified by flash chromatography eluting with 65:35
hexanes/EtOAc to afford azasulfurylꢀglycine tripeptide 19 as white solid (49 mg; 74% yield): R
f
20
1
0
.26 (65:35 hexanes/EtOAc); mp 119ꢀ122 °C; [
α]
3.8 (c 1.46, THF); H NMR (acetoneꢀd , 400
D 6
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
MHz) δ 1.29ꢀ1.51 (4H, m), 1.40 (9H, s), 1.76ꢀ1.84 (2H, m), 2.98 (1H, dd, J = 9.6, 13.9 Hz), 3.05
(
2H, dd, J = 6.4, 12.7 Hz), 3.20 (1H, dd, J = 5.1, 14.0 Hz), 4.19 (1H, q, J = 6.5 Hz), 4.44ꢀ4.50 (1H,
m), 4.46 (2H, d, J = 8.8 Hz), 5.11 (1H, d, J = 9.2 Hz), 5.18ꢀ5.24 (1H, m), 5.21 (2H, d, J = 3.0 Hz),
.79ꢀ5.90 (1H, m), 5.90ꢀ5.98 (1H, br), 6.54 (1H, d, J = 6.8 Hz), 6.62 (1H, d, J = 8.7 Hz), 7.19ꢀ7.46
5
13
(10H, m), 8.16ꢀ8.24 (1H, br), 9.36ꢀ9.44 (1H, br); C NMR (acetoneꢀd
6
, 100 MHz)
δ 173.1, 171.6,
1
5
1
56.8, 156.7, 138.3, 137.0, 134.2, 130.2, 129.3, 129.2, 129.1, 129.0, 127.4, 117.3, 78.4, 67.4, 65.9,
–
1
6.9, 56.0, 40.7, 40.5, 38.5, 32.9, 28.7, 22.9; IR (neat) νmax/cm 3294, 1746, 1684, 1524, 1458,
+
327, 1249, 1158; HRMS (ESI) m/z calculated for C31
H
43
N
5
NaO
9
S [M+Na] 684.2674, found
684.2678.
α
ε
N
ꢀ(Alloc)ꢀLꢀPhenylalaninylꢀazasulfurylꢀphenylalaninylꢀN ꢀ(Boc)ꢀLꢀlysine benzyl ester (20a)
A 0 °C solution of azasulfurylꢀglycine tripeptide 19 (271 mg, 0.41 mmol) in THF (8 mL) was
treated with BTPP (138 µL, 0.45 mmol), stirred for 30 min, treated with benzyl bromide (55 µL,
0.45 mmol) and stirred at 0 °C for 3.5 h. The volatiles were evaporated. The residue was purified by
flash chromatography eluting with 65:35 hexanes/EtOAc to afford azasulfurylꢀphenylalanine
tripeptide 20a as a white solid (209 mg; 68% yield): R
f
0.37 (65:35 hexanes/EtOAc); mp 125ꢀ128
20
1
°C; [
α]
D
–4.3 (c 1.77, THF); H NMR (acetoneꢀd
6
, 400 MHz) 1.33ꢀ1.51 (4H, m), 1.40 (9H, s),
δ
1.72ꢀ1.87 (2H, m), 2.78 (1H, dd, J = 10.1, 13.9 Hz), 2.94ꢀ2.98 (1H, br), 3.05 (2H, dd, J = 6.7, 12.7
Hz), 4.30ꢀ4.38 (2H, m), 4.42 (2H, d, J = 3.8 Hz), 4.49 (1H, d, J = 14.6 Hz), 4.58 (1H, d, J = 14.3
Hz), 5.10 (1H, d, J = 10.0 Hz), 5.21 (1H, d, J = 17.0 Hz), 5.21 (2H, s), 5.78ꢀ5.88 (1H, m), 5.89ꢀ5.97
1
3
(
1H, br), 6.48ꢀ6.59 (1H, br), 6.81 (1H, d, J = 7.3 Hz), 7.16ꢀ7.49 (15H, m), 9.13ꢀ9.18 (1H, br); C
NMR (acetoneꢀd , 75 MHz) 172.9, 171.8, 156.8, 156.6, 141.1, 138.3, 137.1, 136.6, 134.2, 130.2,
30.0, 129.3, 129.1, 129.1, 129.0, 128.6, 127.4, 117.3, 78.4, 67.4, 65.9, 57.2, 57.1, 55.9, 55.2, 40.7,
6
δ
1
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5
6
–
1
38.3, 33.1, 28.7, 23.0; IR (neat) νmax/cm 3320, 1685, 1601, 1506, 1436, 1392, 1238, 1163; HRMS
+
(
ESI) m/z calculated for C H N NaO S [M+Na] 774.3143, found 774.3156.
38
49
5
9
α
ε
N
ꢀ(Alloc)ꢀLꢀPhenylalaninylꢀazasulfurylꢀpꢀphenylphenylalaninylꢀN ꢀ(Boc)ꢀLꢀlysine benzyl
ester (20b) Employing the protocol described for the synthesis of azasulfurylꢀphenylalanine
tripeptide 20a, 4ꢀ(bromomethyl)biphenyl (86 mg, 0.35 mmol) was reacted with azasulfurylꢀglycine
tripeptide 19 (210 mg, 0.32 mmol). The residue was purified by flash chromatography eluting with
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
8
0:20 hexanes/EtOAc to afford azasulfurylꢀpꢀphenylphenylalanine tripeptide 20b as white solid
20
(
225 mg; 85% yield): R
f
0.27 (70:30 hexanes/EtOAc); mp 121ꢀ126 °C; [
, 400 MHz)
1H, br), 2.95ꢀ3.02 (1H, br), 3.04 (2H, t, J = 5.7 Hz), 4.34ꢀ4.39 (2H, m), 4.41 (2H, d, J = 2.2 Hz),
α
D
4.7 (c 1.50, THF);
1
H NMR (acetoneꢀd
6
δ 1.35ꢀ1.55 (4H, m), 1.40 (9H, s), 1.74ꢀ1.88 (2H, m), 2.76ꢀ2.87
(
4
5
.54 (1H, d, J = 14.0 Hz), 4.62 (1H, d, J = 14.3 Hz), 5.08 (1H, d J = 10.8 Hz), 5.13ꢀ5.30 (1H, br),
.22 (2H, s), 5.77ꢀ5.87 (1H, m), 5.88ꢀ5.97 (1H, br), 6.50ꢀ6.60 (1H, br), 6.77ꢀ6.87 (1H, br), 7.16ꢀ
13
7.66 (19H, m), 9.12ꢀ9.25 (1H, br); C NMR (acetoneꢀd , 100 MHz)
δ
172.9, 171.8, 156.8, 156.7,
41.4, 141.2, 138.2, 137.0, 135.7, 134.2, 130.6, 130.2, 129.7, 129.3, 129.1, 129.1, 129.0, 128.2,
27.7, 127.6, 127.3, 117.3, 78.4, 67.4, 65.9, 57.2, 55.9, 55.8, 55.0, 40.7, 38.3, 33.0, 28.7, 23.0; IR
6
1
1
–1
(
neat) νmax/cm 3163, 1601, 1506, 1435, 1393, 1222, 1163; HRMS (ESI) m/z calculated for
+
C
44
H
53
N
5
Na0
9
S [M+Na] 850.3456, found 850.3448.
α
ε
N
ꢀ(Alloc)ꢀLꢀPhenylalaninylꢀazasulfurylꢀ1ꢀnaphthylalaninylꢀN ꢀ(Boc)ꢀLꢀlysine benzyl ester
(
20c) Employing the protocol described for the synthesis of azasulfurylꢀphenylalanine tripeptide
2
0a, 1ꢀ(bromomethyl)naphthalene (24 mg, 0.11 mmol) was reacted with azasulfurylꢀglycine
tripeptide 19 (66 mg, 0.1 mmol). The residue was purified by flash chromatography eluting with
75:25 hexanes/EtOAc to afford azasulfurylꢀ1ꢀnaphthylalanine tripeptide 20c as white solid (52 mg;
20
1
6
5% yield): R
f
0.28 (75:25 hexanes/EtOAc); mp 145ꢀ147 °C; [
D
α] –11.7 (c 1.12, THF); H NMR
(
acetoneꢀd , 400 MHz) δ
6
1.34ꢀ1.47 (4H, m), 1.39 (9H, s), 1.72ꢀ1.87 (2H, br), 2.69 (1H, dd, J = 10.5,
13.5 Hz), 2.81ꢀ2.95 (1H, m), 2.97ꢀ3.09 (2H, br), 4.30 (1H, m), 4.37 (1H, d, J = 6.4 Hz), 4.40 (2H, d,
J = 4.6 Hz), 4.95 (1H, d, J = 13.0 Hz), 5.09 (1H, d, J = 10.5 Hz), 5.15ꢀ5.28 (2H, m), 5.20 (2H, d, J
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= 2.0 Hz), 5.77ꢀ5.85 (1H, m), 5.86ꢀ5.96 (1H, br), 6.38ꢀ6.52 (1H, br), 6.81ꢀ6.92 (1H, br), 7.14ꢀ7.52
13
(
14H, m), 7.72ꢀ7.98 (2H, m), 8.24ꢀ8.38 (1H, m), 9.12ꢀ9.33 (1H, br); C NMR (acetoneꢀd , 100
6
MHz)
δ 172.9, 171.9, 156.7, 156.7, 138.3, 137.0, 134.8, 134.2, 133.0, 131.8, 130.1, 129.7, 129.5,
1
29.3 (3C), 129.1, 129.0, 129.0, 127.3, 127.2, 126.6, 126.0, 125.1, 117.3, 78.4, 67.4, 65.8, 57.3,
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
–
1
55.8, 52.6, 40.7, 38.3, 33.0, 30.3, 28.7, 23.0; IR (neat) νmax/cm 3322, 1601, 1506, 1436, 1393,
+
1
8
237, 1163, 1033; HRMS (ESI) m/z calculated for C H N NaO S [M+Na] 824.3300, found
42
51
5
9
24.3310.
α
ε
N
ꢀ(Alloc)ꢀLꢀPhenylalaninylꢀazasulfurylꢀ2ꢀnaphthylalaninylꢀ
N
ꢀ(Boc)ꢀLꢀlysine benzyl ester
(
20d) Employing the protocol described for azasulfurylꢀphenylalanine tripeptide 20a, 2ꢀ
(bromomethyl)naphthalene (90 mg, 0.41 mmol) was reacted with azasulfurylꢀglycine tripeptide 19
245 mg, 0.37 mmol). The residue was purified by flash chromatography by eluting hexanes/EtOAc
0:10 to afford azasulfurylꢀ2ꢀnaphthylglycine tripeptide 20d as a white solid (224 mg; 76% yield):
(
9
20
1
R 0.27 (80:20 hexanes/EtOAc); mp 99ꢀ101 °C; [
f
α]
7.6 (c 1.58, THF); H NMR (acetoneꢀd , 400
D 6
MHz)
δ 1.37ꢀ1.52 (4H, m), 1.40 (9H, s), 1.76ꢀ1.82 (2H, m), 2.78ꢀ2.82 (1H, m), 2.92ꢀ3.01 (1H, br),
3
.05 (2H, dd, J = 5.9, 11.6 Hz), 4.33ꢀ4.43 (4H, m), 4.66 (1H, d, J = 14.4 Hz), 4.75 (1H, d, J = 14.4
Hz), 5.09 (1H, d, J = 10.6 Hz), 5.16ꢀ5.27 (1H, m), 5.21 (2H, s), 5.76ꢀ5.85 (1H, m), 5.87ꢀ5.96 (1H,
br), 6.47ꢀ6.56 (1H, br), 6.79ꢀ6.89 (1H, br), 7.12ꢀ7.54 (13H, m), 7.84ꢀ7.90 (4H, m), 9.15ꢀ9.26 (1H,
13
br); C NMR (acetoneꢀd
6
, 100 MHz)
δ 172.9, 171.9, 156.8, 156.7, 138.2, 137.0, 134.2, 134.2,
1
1
34.0, 134.1, 130.1, 129.3, 129.1, 129.0 (2C), 129.0, 128.8, 128.8, 128.4, 127.9, 127.3, 126.9,
–
1
26.8, 117.3, 78.4, 67.5, 65.8, 57.2, 55.9, 55.4, 40.7, 38.2, 33.1, 30.3, 28.7, 23.0; IR (neat) νmax/cm
51 5 9
3259, 1744, 1682, 1520, 1454, 1346, 1249, 1162; HRMS (ESI) m/z calculated for C42H N NaO S
+
[
M+Na] 824.3300, found 824.3315.
α
ε
N
ꢀ(Alloc)ꢀLꢀPhenylalaninylꢀazasulfurylꢀphenylalaninylꢀN ꢀ(Boc)ꢀLꢀlysine (21a) A solution
α
ε
of N ꢀ(Alloc)ꢀLꢀphenylalaninylꢀazasulfurylꢀphenylalaninylꢀN ꢀ(Boc)ꢀLꢀlysine benzyl ester 20a (200
mg, 0.27 mmol) in 1,4ꢀdioxane (14 mL) was treated with a solution of LiOH (34 mg, 0.80 mmol) in
water (7 mL), stirred overnight at rt, diluted with water, and acidified to pH~1 using a 5% citric
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acid solution. The aqueous layer was extracted with EtOAc (40 mL x 3). The combined organic
phase was washed with brine (20 mL x 3), dried with MgSO , filtered and evaporated under
4
reduced pressure to provide acid 21a as white solid (138 mg; 77% yield): R
f
0.21 (98:2
20
1
DCM/MeOH); [
α]
2.9 (c 1.29, THF); H NMR (acetoneꢀd , 400 MHz)
δ
1.29ꢀ1.58 (4H, m), 1.40
D
6
0
1
2
3
4
5
6
7
8
9
0
1
2
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4
5
6
7
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0
1
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7
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9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(9H, s), 1.76ꢀ1.93 (2H, m), 2.77 (1H, dd, J = 9.8, 13.6 Hz), 2.91ꢀ3.00 (1H, br), 3.02ꢀ3.18 (2H, br),
.22ꢀ4.30 (1H, br), 4.31ꢀ4.37 (1H, m), 4.42 (1H, d, J = 5.1 Hz), 4.51 (1H, d, J = 14.1 Hz), 4.60 (2H,
d, J = 16.0 Hz), 5.11 (1H, d, J = 10.4 Hz), 5.21 (1H, d, J = 17.6 Hz), 5.79ꢀ5.89 (1H, m), 5.91ꢀ6.01
4
1
3
(
1H, br), 6.42ꢀ6.55 (1H, br), 6.59ꢀ6.70 (1H, br), 7.16ꢀ7.38 (11H, m), 9.04ꢀ9.21 (1H, br); C NMR
(acetoneꢀd , 100 MHz) 173.8, 156.7, 156.7, 144.1, 138.3, 136.5, 134.2, 130.2, 130.0, 129.1,
28.9, 128.6, 127.3, 117.3, 78.4, 65.9, 57.0, 55.9, 55.2, 40.1, 38.2, 33.0, 32.3, 28.7, 23.0; IR (neat)
6
δ
1
–1
νmax/cm 3359, 2938, 1677, 1525, 1352, 1252, 1162, 1033; HRMS (ESI) m/z calculated for
+
C
31
H
43
N
5
NaO
9
S [M+Na] 684.2674, found 684.2672.
α
ε
N
ꢀ(Alloc)ꢀLꢀPhenylalanylꢀazasulfurylꢀ2ꢀnaphthylalaninylꢀN ꢀ(Boc)ꢀLꢀlysine (21d) Using the
α
representative procedure for the synthesis of acid 21a, N ꢀ(Alloc)ꢀLꢀphenylalanylꢀazasulfurylꢀ2ꢀ
ε
naphthylalaninylꢀN ꢀ(Boc)ꢀLꢀlysine benzyl ester 20d (209 mg, 0.26 mmol) was reacted with LiOH
(
33 mg, 0.78 mmol) to afford the acid 21d as white solid (150 mg; 81% yield): R 0.27 (98:2
f
20
1
D 6
DCM/MeOH); [α] 14.0 (c 1.13, THF); H NMR (acetoneꢀd , 400 MHz) δ 1.35ꢀ1.64 (4H, m),
1
5
5
.40 (9H, s), 1.76ꢀ1.96 (2H, m), 2.76 (1H, dd, J = 9.6, 13.7 Hz), 2.89ꢀ2.99 (1H, br), 3.10 (2H, d, J =
.6 Hz), 4.32ꢀ4.47 (4H, m), 4.63ꢀ4.80 (2H, m), 5.08 (1H, d, J = 10.5 Hz), 5.18 (1H, d, J = 17.2 Hz),
.76ꢀ5.85 (1H, m), 5.91ꢀ6.04 (1H, br), 6.41ꢀ6.56 (1H, br), 6.64ꢀ6.77 (1H, br), 7.15ꢀ7.56 (9H, m),
1
3
7.78ꢀ7.97 (4H, m), 9.17ꢀ9.29 (1H, br); C NMR (acetoneꢀd
6
, 100 MHz)
56.7, 138.2, 134.2, 134.2, 134.1, 134.0, 130.1, 129.1, 129.0, 128.9, 128.8, 128.8, 128.4, 127.9,
27.3, 126.8, 117.3, 78.4, 65.9, 57.0, 55.9, 55.4, 40.1, 38.2, 33.1, 32.3, 28.7, 23.0; IR (neat)
δ 173.9, 171.9, 156.8,
1
1
–
1
νmax/cm 3297, 2939, 1681, 1512, 1455, 1347, 1251, 1160, 1033; HRMS (ESI) m/z calculated for
+
C H N NaO S [M+Na] 734.2830, found 734.2823.
35 45
5
9
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9
α
in
N
ꢀ(Alloc)ꢀ
N
ꢀ(Boc)ꢀN’ꢀ(Phenylisopropyloxycarbonyl)ꢀLꢀtryptophan hydrazide (24)
A
solution of acid 22 (970 mg, 2.5 mmol) in 10 mL of DCM was treated with HOBt (371 mg, 2.75
mmol) followed by DCC (566 mg, 2.75 mmol), stirred for 30 min at rt, treated with a solution of
carbazate in 3 mL of DCM, and stirred overnight. The reaction mixture was filtered through a
fritted filter, and the filtrate was evaporated to furnish a residue, that was dissolved in cold EtOAc
from which additional N,N′ꢀdicyclohexylurea (DCU) precipitated. The precipitate was filtered. The
filtrate was washed with saturated aqueous solution of NaHCO and brine, dried over Na SO , and
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
3
2
4
evaporated to a residue that was purified by flash chromatography on silica gel eluting with 70:30
hexanes/EtOAc. Evaporation of the collected fractions gave hydrazide 24 (1.310 g; 82% yield) as a
20
1
f D
white solid: R 0.32 (70:30 hexanes/EtOAc); mp 75ꢀ77 °C; [α] 40.1 (c 1.04, THF); H NMR
(
CDCl , 400 MHz) δ 1.66 (9H, s), 1.80 (6H, s), 3.08 (1H, s), 3.31ꢀ3.15 (1H, m), 4.45 (2H, s), 4.61
3
(
1H, s), 5.02ꢀ5.37 (2H, m), 5.65ꢀ5.86 (2H, m), 6.93 (1H, s), 7.15ꢀ7.42 (7H, br), 7.44ꢀ7.63 (2H, m),
13
8
.13 (1H, s), 8.41 (1H, s); C NMR (CDCl
3
, 100 MHz) δ 170.9, 156.1, 154.6, 149.6, 145.5, 135.4,
1
32.3, 130.3, 128.3, 127.1, 124.5, 124.0, 122.6, 119.0, 117.9, 115.2, 83.6, 82.9, 66.0, 60.5, 53.6,
–
1
28.8, 28.2, 27.8, 21.0, 14.2; IR (neat) νmax/cm 3272, 2979, 2933, 1727, 1682, 1225, 745; HRMS
+
(
ESI) m/z calculated for C H N NaO [M+Na] 587.2476, found 587.2480.
30
36
4
7
α
in
N
ꢀ(Alloc)ꢀN ꢀ(Boc)ꢀLꢀTryptophan hydrazide (25) Diacyl hydrazine 24 (1.2 g, 2.3 mmol) was
treated with a solution of 1.5% TFA in DCM (40 mL) for 1 h. The volatiles were evaporated and
the residue was purified by flash chromatography in silica gel by eluting with 60:40
EtOAc/hexanes. Evaporation of the collected fractions gave hydrazide 25 (650 mg; 74% yield) as a
20
1
pale yellow solid: R
f D
0.09 (60:40 EtOAc/hexanes); mp 84ꢀ86 °C; [α] 7.2 (c 1.04, THF); H NMR
(
CDCl , 400 MHz) δ 1.66 (9H, s), 3.14 (1H, dd, J = 8.1, 15.0 Hz), 3.20 (1H, dd, J = 6.6, 14.8 Hz),
3
3.57ꢀ3.94 (2H, br), 4.48 (1H, dd, J = 7.4, 14.8 Hz), 4.55 (2H, d, J = 5.7 Hz), 5.21 (1H, d, J = 10.4
Hz), 5.27 (1H, d, J = 17.2 Hz), 5.42ꢀ5.64 (1H, br), 5.83ꢀ5.92 (1H, m), 7.20ꢀ7.58 (5H, br), 8.05ꢀ8.19
13
(
1H, br); C NMR (CDCl
3
, 100 MHz) δ 171.8, 155.0, 149.7, 135.5, 132.5, 130.2, 124.6, 124.3,
–1
1
22.6, 118.8, 118.0, 115.5, 115.2, 83.0, 66.0, 53.8, 28.3, 28.1; IR (neat) νmax/cm 3290, 1726, 1452,
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1
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1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
+
1367, 1252, 1154, 1085; HRMS (ESI) m/z calculated for C H N O [M+H] 403.1981, found
2
0
27
4
5
4
03.1981.4ꢀNitrophenylꢀ
chlorosulfate (2.58 g, 10.84 mmol) was dissolved in dry DCM (10 mL) and treated dropꢀwise with a
solution of amine 23 (2.08 g, 5.42 mmol), 4ꢀnitrophenol (2.261 g, 16.61 mmol) and NEt (4.5 mL,
2.52 mmol) in dry DCM (50 mL) at –78 °C, stirred at –78 °C for 2 h, and allowed to warm up to rt
Oꢀ(tertꢀbutyl)ꢀLꢀtyrosine 2,4ꢀdimethoxybenzyl ester (26) 4ꢀNitrophenyl
3
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
3
with stirring for 1 h. The reaction mixture was evaporated to a residue, dissolved in DCM (20 mL),
washed with saturated aqueous solution of NaHCO (3 x 25 mL), dried over MgSO , filtered, and
3
4
evaporated to a residue that was purified by flash chromatography eluting with 80:20
hexanes/EtOAc to afford the sulfamidate 26 (2.13 g; 56% yield) as a pale yellow solid: R 0.30
, 400 MHz)
.41 (9H, s), 3.14 (2H, dd, J = 5.0, 14.8), 3.83 (6H, s), 5.10 (2H, s), 6.46ꢀ6.48 (2H, m), 6.94 (2H, d,
J = 7.8), 7.20ꢀ7.30 (2H, m), 7.34 (1H, dt, J = 1.1, 7.2), 7.48 (2H, s), 7.44 (2H, d, J = 7.8), 8.15
f
2
0
1
(80:20 hexanes/EtOAc); mp 110ꢀ113 °C; [
α]
D
6.2 (c 0.97, CHCl
3
); H NMR (CDCl
3
δ
1
13
(
2H, m); C NMR (CDCl , 100 MHz) δ 170.7, 161.9, 159.2, 154.8, 154.6, 145.9, 132.2, 130.4,
3
128.1, 125.4, 124.4, 124.0, 122.1, 115.9, 104.1, 98.5, 63.4, 57.9, 55.7, 55.4, 38.0, 28.5; IR (neat)
–1
νmax/cm
2975, 1736, 1487, 1346, 1155, 864, 728; HRMS (ESI) m/z calculated for
+
α
in
C H N NaO S [M+Na] 611.1669, found 611.1670.
N
ꢀ(Alloc)ꢀ
N ꢀ(Boc)ꢀLꢀTryptophanylꢀ
28
32
2
10
azasulfurylꢀglycinylꢀ
Oꢀ(tertꢀbutyl)ꢀLꢀtyrosine 2,4ꢀdimethoxybenzyl ester (27) Hydrazide 25
(265 mg, 0.66 mmol) and sulfamidate 26 (352 mg, 0.60 mmol) were dissolved in MeCN (6 mL) in
a 10ꢀ20 mL microwave vessel. Triethylamine (93 ꢁL, 0.66 mmol) was added to the mixture, at
which point the solution turned yellow. The microwave vessel was sealed and the solution was
heated at 60 °C under microwave irradiation for 3 h. The vessel was opened. The mixture was
evaporated to a residue, that was purified by flash chromatography eluting with 70:30
hexanes/EtOAc to afford sulfamide 27 (305 mg; 60% yield) as a pale yellow solid: R
f
0.23 (70:30
, 400 MHz) δ 1.31
9H, s), 1.66 (9H, s), 3.02ꢀ3.09 (4H, m), 3.82 (6H, s), 4.35ꢀ4.40 (1H, m), 4.42ꢀ4.48 (1H, m), 4.50ꢀ
20
1
D 3 3
hexanes:EtOAc); mp 101ꢀ104 °C; [α] –8.3; (c 1.10, CHCl ); H NMR (CDCl
(
4.53 (2H, m), 5.09 (2H, s), 5.15 (1H, d, J = 10.5), 5.19 (1H, d, J = 10.5), 5.23 (1H, d, J = 17.2), 5.62
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(1H, d, J = 7.7), 5.80ꢀ5.90 (1H, m), 6.41ꢀ6.45 (2H, m), 6.76ꢀ6.78 (2H, m), 6.96 (1H, s), 6.98 (1H, d,
13
J = 7.8), 7.19ꢀ7.21 (2H, m), 7.23ꢀ7.25 (2H, m), 7.32ꢀ34 (2H, m) 8.05ꢀ8.10 (2H, m); C NMR
CDCl , 100 MHz) 172.9, 171.9, 170.2, 161.7, 159.1, 155.8, 154.5, 154.3, 149.8, 132.3, 130.9,
30.0, 129.5, 125.0, 124.7, 123.9, 122.9, 118.9, 118.0, 115.4, 114.6, 104.8, 98.7, 84.1, 78.4, 66.1,
(
3
δ
1
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
64.2, 63.5, 57.1, 57.1, 55.5, 53.5, 37.8, 30.9, 29.7, 28.7; LCꢀMS monitoring [40ꢀ90% MeOH (0.1%
FA) in water (0.1% FA) over 14 min, t = 8.50 min] on Sunfire C18 column (100Å, 3.5 ꢁm, 4.6
R
–1
mm X 100 mm); IR (neat) νmax/cm 3296, 2941, 1717, 1615, 1511, 1452, 1368, 1255, 1152;
+
α
HRMS (ESI) m/z calculated for C42
H
53
N
5
NaO12S [M+Na] 874.3303, found 874.3295.
N ꢀ(Alloc)ꢀ
in
N
ꢀ(Boc)ꢀLꢀTryptophanylꢀ(4ꢀchloroꢀbutyl)ꢀazasulfurylꢀglycinylꢀOꢀ(tertꢀbutyl)ꢀLꢀtyrosine 2,4ꢀ
dimethoxybenzyl ester (28) A stirred solution of sulfamide 27 (288 mg, 0.74 mmol) in THF (14
mL) at 0 °C was treated with BTPP (226 µL, 0.74 mmol) for 30 min, treated with a bolus injection
of 1ꢀbromoꢀ4ꢀchlorobutane (170 µL, 1.48 mmol) and stirred for 4 h The reaction mixture was
evaporated under vacuum to residue and purified by flash chromatography eluting with 70:20
hexanes:EtOAc to afford the chloride 28 (210 mg; 66% yield) as white powder: R
f
0.34 (70:30
, 400 MHz) 1.33
9H, s), 1.40ꢀ1.50 (2H, d, J = 3.0 Hz), 1.68 (9H, s), 1.70ꢀ1.80 (2H, d, J = 5.6 Hz), 3.06 (2H, d, J =
.6 Hz), 3.13ꢀ3.26 (4H, m), 3.47 (2H, t, J = 6.5 Hz), 3.84 (6H, d, J = 6.1 Hz), 4.35ꢀ4.49 (2H, m),
4.55 (2H, d, J = 5.0 Hz), 5.05ꢀ5.33 (4H, m), 5.44 (1H, d, J = 7.2 Hz), 5.90 (1H, ddd, J = 16.1, 10.9,
20
1
hexane/EtOAc); mp 88ꢀ91 °C; [
α]
D
–7.1 (c 1.10, CHCl
3
); H NMR (CDCl
3
δ
(
5
5
.7 Hz), 6.41ꢀ6.54 (4H, m), 6.84 (2H, t, J = 5.5 Hz), 6.99 (2H, d, J = 8.4 Hz), 7.24ꢀ7.35 (4H, ddd, J
13
=
30.7, 19.0, 7.7 Hz), 7.54ꢀ7.70 (2H, m), 8.15 (1H, d, J = 8.2 Hz); C NMR (CDCl
3
, 100 MHz)
δ
1
70.3, 161.6, 159.1, 154.5, 151.1, 135.2, 132.8, 132.0, 130.1, 129.9, 124.7, 124.2, 123.9, 122.8,
118.9, 117.5, 115.6, 115.0, 112.2, 104.0, 98.4, 83.6, 67.3, 63.2, 60.2, 57.0, 55.4, 55.0, 53.5, 50.6,
4.4, 37.9, 30.9, 29.1, 28.8, 28.5, 28.1, 24.2, 21.8, 14.2; LCꢀMS monitoring [40ꢀ90% MeOH (0.1%
FA) in water (0.1% FA), 14 min, t = 7.8 min] on Sunfire C18 column (100Å, 3.5 ꢁm, 4.6 mm X
00 mm); IR (neat) νmax/cm 3280, 1727, 1453, 1366, 1255, 1155, 1087; HRMS (ESI) m/z
4
R
–
1
1
+
α
in
calculated for C46
H61ClN
5
O
12S [M+H] 942.3715, found 942.3705.
N
ꢀ(Alloc)ꢀ
N
ꢀ(Boc)ꢀLꢀ
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