Design and synthesis of peptide-based macrocyclic cyclophilin inhibitors

Article abstract of DOI:10.1016/j.bmcl.2016.09.039

The efficient assembly of an 18-membered macrocyclic peptide core was realized by a straightforward and convergent approach utilizing ring-closing metathesis of the corresponding linear tetrapeptides as the key transformation. This approach allowed for the facile preparation of a focused library of novel macrocycles that culminated in the discovery of a cyclophilin A inhibitor with a K_d= 5.4 μM.

Full text of DOI:10.1016/j.bmcl.2016.09.039

Bioorganic & Medicinal Chemistry Letters xxx (2016) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Design and synthesis of peptide-based macrocyclic cyclophilin
inhibitors
⇑
⇑
Brett A. Granger , Dean G. Brown
Infection Innovative Medicines Unit, AstraZeneca R&D Boston, Waltham, MA 02451, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
The efficient assembly of an 18-membered macrocyclic peptide core was realized by a straightforward
and convergent approach utilizing ring-closing metathesis of the corresponding linear tetrapeptides as
the key transformation. This approach allowed for the facile preparation of a focused library of novel
Received 15 August 2016
Revised 12 September 2016
Accepted 14 September 2016
Available online xxxx
macrocycles that culminated in the discovery of a cyclophilin A inhibitor with a Kd = 5.4 lM.
Ó 2016 Elsevier Ltd. All rights reserved.
Keywords:
Peptidyl-prolyl isomerases
Cyclophilin
Macrocycle
Ring-closing metathesis
cis,trans-Peptidyl-prolyl isomerases (PPIases) are ubiquitous
enzymes that catalyze the isomerization of prolyl peptide bonds
from the thermodynamically unfavorable cis-conformation to the
preferred trans-orientation.1 The PPIases are subdivided into three
families including the cyclophilins (Cyps), FK506-binding proteins
(FKBPs), and parvulins.2 The Cyps and FKBPs are collectively ter-
med immunophilins owing to their affinity towards the immuno-
favorable binding event due to ligand pre-organization.11 To this
end we designed compound 2 with functionalized serine residues
containing reactive groups X and Y. We hypothesized that ring-
closing metathesis would enable the synthesis of 18-membered
macrocyclic peptides related to 1 and thus decided to target an
analog of linear tetrapeptide 2 containing pendant alkene function-
ality (X and Y = CH2) and orthogonal protecting groups at R1 and R2.
Notably, among macrocyclic compounds produced in nature,
18-membered rings are particularly frequent.12 Synthetically, we
planned to first prepare two functionalized serine residues and
stitch the peptide together via standard coupling techniques.
suppressant agents cyclosporine
A
(CsA)3 and FK506,4
respectively. Although macrocyclic ligands of the Cyps which pos-
sess potent immunosuppressive activities are known, these
enzymes have also been proven as essential host factors for the
replication of HIV5 and HCV.6 This has triggered the development
of various non-immunosuppressive Cyp inhibitors for the treat-
ment of HCV infection, however there are currently no drugs
approved for this indication.6b
HO
O
O
X
O
Me
Me
NH
HN
R1HN
NH
O
N
O
N
The linear tetrapeptide suc-Ala-Gly-Pro-Phe-pNA (1) is a well-
O
O
O
known cyclophilin A (CypA) inhibitor with a Kd = 135 20
l
M7
cyclize
O
that is commonly used as a substrate in the chymotrypsin-coupled
PPIase activity assay (Fig. 1).8 Additionally, the structure of 1
bound to human CypA has been reported (PDB code: 1ZKF).9 We
thus sought to utilize 1 as a starting point for the design of
macrocyclic tetrapeptide Cyp inhibitors. Notably, macrocyclic
compounds often display more favorable physical properties, such
as permeability, when compared to their linear counterparts.10
Furthermore, macrocyclization can result in a more entropically
H
N
H
N
O2N
N
H
R2O
O
O
Y
O
2
1
Figure 1. The design of macrocyclic peptides for Cyp inhibition.
The synthesis of the tetrapeptide began with the construction of
the substituted serine derivatives. Accordingly, the known
⇑
Corresponding authors.
E-mail addresses: grangerba35@gmail.com (B.A. Granger), dean.brown@
protected
L
-serine 313 was alkylated under Tsuji–Trost conditions
to deliver allyl serine 4 in 79% yield (Scheme 1).14 The tert-butyl
astrazeneca.com (D.G. Brown).
http://dx.doi.org/10.1016/j.bmcl.2016.09.039
0960-894X/Ó 2016 Elsevier Ltd. All rights reserved.
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2
B. A. Granger, D. G. Brown / Bioorg. Med. Chem. Lett. xxx (2016) xxx–xxx
carbamate in 4 could be selectively cleaved over the tert-butyl
ester utilizing the procedure of Han and co-workers to give amine
salt 5 in 89% yield.15
that upon hydrogenation afforded the 18-membered macrocycle
16 in 71% yield.
O
O
FmocHN
O
O
N
O
O
FmocHN
PyBOP
CH2Cl2, Et3N
95%
H
N
N
NHBoc
OH
NHBoc
O
O
O
+
5
t-BuO
t-BuO
Pd(PPh3)4, THF, 60 °C
79%
t-BuO
O
HO2C
O
3
4
12
13
O
O
4 M HCl/Dioxane
NH2•HCl
O
t-BuO
H2N
O
N
0 °C → rt
20% piperidine
DMF
PyBOP
H
N
11
+
89%
Et3N, CH2Cl2
59% (2 steps)
5
t-BuO
O
O
Scheme 1. Synthesis of allyl serine 5.
14
Me
FmocHN Me
The tetrasubstituted allyl serine 11 was prepared in 6 steps
from -serine methyl ester hydrochloride (6) (Scheme 2). Conden-
sation of 6 with pivaldehyde, followed by formylation with acetic
formic anhydride provided an intermediate ester that was subse-
quently methylated in a diastereoselective fashion to give the
known oxazolidine 7.16,17 Treatment of 7 with anhydrous hydrogen
H
N
O
NH
FmocHN
O
O
D
O
N
O
O
O
O
N
1) Grubbs' II, CH2Cl2
H
N
2) Pd/C, H2, EtOH
71%
HN
O
t-BuO
O
chloride in methanol delivered the tetrasubstituted
D-serine
O
O
Ot-Bu
hydrochloride 8, which was then protected as its 9-fluorenyl-
methyl carbamate 9 in 90% yield over the two steps. Allylation of
16
15
9 with the p–allyl cation complex formed from the reaction of allyl
Scheme 3. Macrocyclization of linear tetrapeptide 15.
methyl carbonate with Pd(PPh3)4 gave allyl serine 10 in 80% yield.
Notably, this transformation required an increased reaction tem-
perature as compared to that of serine 3, presumably due to the
relatively hindered nature of the primary hydroxyl group in 9.
Finally, cleavage of the methyl ester in 10 with lithium iodide in
refluxing ethyl acetate generated the desired tetrasubstituted allyl
serine 11 in 90% yield. Attempts to hydrolyze the methyl ester in
10 with lithium hydroxide resulted in concomitant cleavage of
the Fmoc carbamate, while reactions of 10 with boron trichloride
resulted in allyl group cleavage to give predominately alcohol 9.
Importantly, 1H and 19F NMR analysis of the Mosher’s amides
derived from amines 5 and 8 revealed that each amine was >95%
enantiomerically pure.18
With facile access to macrocycle 16 we began to unmask the
orthogonal protecting groups in order to further derivatize.
Accordingly, treatment of 16 with piperidine in DMF delivered
the amine 17 in 69% yield (Scheme 4). Alternatively, removing
the tert-butyl ester first by reaction of 16 with TFA gave an inter-
mediate acid, that could be further deprotected to give amino acid
18 in 54% overall yield. Notably, in a time resolved FRET CypA com-
petition assay compound 18 had a Kd = 127 l
M.20 At this juncture,
we designed and synthesized a focused library of macrocycles for
testing in this assay.
H2N Me
H
N
O
O
O
refs. 16 & 17
O
O
HCl, MeOH
NH2•HCl
OH
N
N
20% piperidine
MeO
CO2Me
Me
O
DMF
69%
HN
O
6
7
O
O
Ot-Bu
O
O
Fmoc-Cl, i-Pr2NEt
17
NH2•HCl
OH
NHFmoc
OH
16
MeO
MeO
CH2Cl2, 0 °C
90% (2 steps)
Me
Me
TFA•H2N Me
8
9
H
N
O
O
O
O
O
O
N
1) TFA, CH2Cl2
NHFmoc
NHFmoc
O
O
MeO
LiI
EtOAc, Δ
90%
HO
2) 20% piperidine, DMF
54%
Me
O
Me
O
Pd(PPh3)4
1,4-dioxane, 90 °C
80%
HN
O
O
10
11
O
OH
18
Scheme 2. Synthesis of tetrasubstituted allyl serine 11.
CypA Kd = 127 μM
With fragments 5 and 11 in hand, we sought to complete the
synthesis of the requisite linear tetrapeptide. Accordingly, the
known Gly-Pro fragment 1219 was coupled with amine 5 under
standard conditions to deliver the tripeptide 13 in 95% yield
(Scheme 3). Cleavage of the Fmoc carbamate in 13, followed by
coupling of the resultant amine 14 with acid 11 afforded the key
linear tetrapeptide 15 in 59% overall yield from 13. We were
pleased to find that treatment of 15 with Grubbs’ 2nd generation
catalyst proceeded smoothly to give a mixture of E/Z olefin isomers
Scheme 4. Synthesis of amine 17 and amino acid 18.
Acylation or reductive alkylation of amine 17 with acid
chlorides or aldehydes, respectively, followed by cleavage of the
tert-butyl ester led to the formation of amides 19–22 and amines
23–29 (Scheme 5, Table 1). Amines 23–29 were most conveniently
isolated as their hydrochloride salts. Our library also included
amide derivatives of the acid moiety in 18, which were constructed
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B. A. Granger, D. G. Brown / Bioorg. Med. Chem. Lett. xxx (2016) xxx–xxx
3
using standard coupling conditions, however these amides did not
exhibit appreciable CypA inhibition.
The physiochemical properties of this focused library of com-
pounds were evaluated, and were generally favorable, however
these macrocycles invariably suffered from poor permeability as
exemplified by analog 28 (Fig. 2). Compound 28 has a logD value
of À1.8 and excellent aqueous solubility. Notably, compound 28
does not bind to human plasma proteins, and has a very low rate
of hepatic clearance. Finally, although these compounds are rela-
tively weak CypA binders, they show good selectivity across a wide
H
N
R1
X
H2N Me
H
N
Me
O
H
N
O
O
1) R1COCl, Et3N
CH2Cl2
O
O
N
O
N
or
array of targets (CEREP panel 100+ targets greater than 100 lM).
R1CHO
HN
O
HN
O
NaBH(OAc)3, 1,2-DCE
O
O
2) HCl(aq)
O
Ot-Bu
O
OH
19-29
X = O or H2
17
HN Me
H
N
O
Scheme 5. Synthesis of macrocycles 19–29.
O
O
N
Compounds were tested for CypA activity in a time-resolved
FRET assay, and the results are summarized in Table 1. The positive
control in this assay was cyclosporine A (CsA) which displayed a
Kd = 10 nM. With the exception of the compact hydrophobic
cyclobutyl analog 21, amides did not show any appreciable CypA
binding in this assay. As exemplified by compounds 19, 20, and
22 short or long chain alkyl amides and substituted aromatic
amides were not active.
Amines that were formed through reductive amination gener-
ally displayed greater affinity towards CypA. With a one carbon lin-
ker, the Kd value increased significantly from cyclopentyl
(compound 23) to cyclohexyl (compound 24) and finally cyclooctyl
(compound 25). Interestingly the pyran 26 and benzyl substituted
compound 27 were completely inactive in this assay. Furthermore,
HN
O
O
O
OH
28
μ
CypA Kd = 10.5
logD = -1.8
Sol(aq) > 1000
M
μ
M
Caco2 (A to B Papp 1x10-6 cm/s) = 0.10
Caco2 (B to A Papp 1x10-6 cm/s) = 0.17
hppb (% free) = 100%
μ
hClintmic. ( L/min/mg) < 3
μ
Secondary pharm > 100 M @ 100+ targets
Figure 2. Physiochemical properties of 28.
the phenethyl analog 28 showed a Kd = 10.5 lM, while having a
three carbon linker as in 29 was detrimental to activity. Taken
together, this data supports that CypA binding is driven by
hydrophobic interactions, and even small structural variations
can disrupt this relationship.21
We have discovered a novel series of peptide-based macrocyclic
CypA inhibitors which were synthesized by a straightforward and
convergent route utilizing RCM of the corresponding linear
tetrapeptide 15 as the key transformation. A focused library of
compounds was prepared which demonstrated that small to
medium hydrophobic groups increase affinity for CypA. These
compounds generally possess favorable physiochemical properties,
and display an excellent selectivity profile, but tend to suffer from
poor permeability.
Table 1
Inhibition of CypA by macrocyclic peptides
Compound
X
R1
CypA Kd (l
M)a
CsA
18
19
0.010
127
>600
N/A
O
H
Me
Supplementary data
20
21
O
O
>600
139
5
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2016.09.
039.
Cl
22
O
>600
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