Synthesis of 12-membered macrocyclic templates and library analogs for PPI

Article abstract of DOI:10.1016/j.tetlet.2013.04.058

We report novel syntheses of 12-membered macrocyclic templates and a library of 4000 macrocyclic analogs. The key macrocyclization step was performed at up to 100 g scale without resorting to syringe pumps, flow reactors or large volumes of solvent. An in

Full text of DOI:10.1016/j.tetlet.2013.04.058

Tetrahedron Letters 54 (2013) 3298–3301
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Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
Synthesis of 12-membered macrocyclic templates and library analogs for PPI
Jinshan Chen a, , Frank Rong b,c, Bo Shan b, Yuanwei Chen c, Yingfu Li c, Hua Yu c, Li Chen c,
⇑
Tongtao Kuang c, Shai Li c, Youpei Chen c, Jing Du c, Chaowu Ai c, Jian Li c, Xiaoyong Li c, Changqing Shi c,
Zhilong Jiang c, Yuanqiang Long c, Qiu Gao c, Zhibo Wang c, Kexin Xu c, Xin Ran b, Hua Yi b, Dapeng Zhao b,
Hongyun Qiao b, Junwei Shen b, Bo Liu b, Chonggang Liu b, Kai Wu b, Xi Geng b, Jieyang Tan b,
Dale McLeod a, Heather Frost a, Guoyun Bai a, Gilles Goetz a, James Federico III a, Carrie Whitney-Pickett a,
Matthew Troutman a, Mark C. Noe a, Cristiano Guimaraes a, David W. Piotrowski a, Thomas V. Magee a
a Worldwide Research and Development, Pfizer Inc., Eastern Point Road, Groton, CT 06340, United States
b ChemPartner, No. 5 Building, 998 Halei Road, Zhangjiang Hi-Tech Park, Pudong, Shanghai 201203, China
c ChemPartner, Building B3, Tianfu Life Science Park, No. 88 Keyuan South Road, Chengdu Hi-Tech Park, Chengdu 610041, China
a r t i c l e i n f o
a b s t r a c t
Article history:
We report novel syntheses of 12-membered macrocyclic templates and a library of 4000 macrocyclic
analogs. The key macrocyclization step was performed at up to 100 g scale without resorting to syringe
pumps, flow reactors or large volumes of solvent. An interesting observation of considerably different
permeability properties was made on diastereomeric analogs due to differences in intramolecular hydro-
gen bond interactions.
Received 14 March 2013
Revised 15 April 2013
Accepted 16 April 2013
Available online 24 April 2013
Ó 2013 Elsevier Ltd. All rights reserved.
Keywords:
Macrocyclization
12-Membered ring
Lactam cyclization
Intra-molecular hydrogen bond
Permeability
Introduction
which is required to be successful in generating leads for PPI inhi-
bition, we embarked on an effort to generate libraries of macrocy-
Protein–protein and peptide–protein interactions (PPI collec-
tively) are the basis for the majority of regulatory pathways in bio-
logical systems. As such they represent important targets of
intervention for the discovery of novel therapeutics. However, PPIs
are inherently complex, multivariate, and poorly understood.1 The
surface interactions tend to be large and uncontoured in relation to
small ligand–protein interactions; the binding partner can be any-
thing from large globular and/or intrinsically unstructured proteins
to a well-defined extruding peptide loop, tail, or small peptide; and
the binding affinity for the endogenous interaction can range from
femto- to millimolar. High-throughput screening has generally
been unsuccessful in identifying lead matter for PPIs due to the
rule-of-five centric existing screening collection.2 Mutational stud-
ies however suggest that ‘hotspots’ exist at the binding interface,
and small molecules with a more extended reach than those typi-
cally found in a screening file are more likely to bind productively.3
As part of our initial attempt to bridge the gap between what is
currently available in the screening compound collection and that
clic analogs. Herein we report the syntheses of 12-membered
macrocylic templates (1) and a library of macrocyclic analogs (2)
as shown in Scheme 1.4 Overall 16 templates (1) were synthesized
incorporating different amino acids into the ring and 3987 final
analogs (2) were elaborated using amide, sulfonamide, heteroaryl
or simple amine capping groups (R).
This type of 12-membered macrocylic skeleton has been re-
ported in the literature. The synthesis of four apicidin analogs (3)
in mg scale was reported by Ladlow and coworkers as shown in
Figure 1.5 These analogs all have a cyclic amino acid in the ring
skeleton, proline or piperidine carboxylic acid, which led to an
entropic factor favoring the ring formation. The cyclization at
milligram scale was reported to be boosted by incorporating a
(2-nitrophenyl)sulfonyl group (nosyl) as a ‘back bone hinge’ on
the nitrogen atom opposite to the ring closing amidation point.
This key step was carried out at a concentration as high as
20 mg/mL (ꢀ20 mM), as opposed to the less than 1 mg/mL typical
for solution phase macrocyclization.6 Detailed experimental proce-
dures of this effort are not available in the two peer reviewed
publications.5a,b The macrocyclization was performed at no more
than 100 mg scale according to the published patent application. 5c
⇑
Corresponding author. Tel.: +1 860 715 4671; fax: +1 860 686 6191.
E-mail address: michael.j.chen@pfizer.com (J. Chen).
0040-4039/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.tetlet.2013.04.058

J. Chen et al. / Tetrahedron Letters 54 (2013) 3298–3301
3299
O
P2
N
O
R2
N
N
N
O
P1
R1
OH
N
H
NH
N
H
NH
A
A
O
NH
P3
HN
N
H
O
NH
R3
N
H
NH2
O
P = orthogonal protecting groups
R = amides, sulfonamides, etc.
1
2
Scheme 1. Templates and library analogs of 12-membered macrocycles.
Nosyl
Nosyl
O
O
R' =
N
O
N
O
N
N
O
R''
R''
R'
3
R'
NH
NH
HO2C
R'' =
O
N
H
H2N
N
H
3a
Figure 1. Macrocyclization for apicidin analogs.
O
Nosyl
N
Nosyl
O
Nosyl
N
HO2C
NH2
N
O
N
P1
Fmoc
N
H
NH
N
H
O
A
A
N
HN
O
O
NH
P3
NH
N
H
N
H
NH
O
Cbz
Cbz
5
4
Scheme 2. Retrosynthesis of macrocyclic templates.
We decided to adopt the same nosyl protecting group for P2 in
our templates (1, P2 = nosyl). Our cyclization would not have the
entropy advantage of a cyclic amino acid residue in the ring sys-
tem. In addition, we would have to form the ring via activation
of the carboxylic acid of glycine, instead of the diamino propionic
acid (DAP) which corresponds to the cyclization point used by Lad-
low and coworkers. This represented an unprecedented cyclization
approach but necessary due to concerns of potential racemization
associated with activation of chiral amino acids.7 At our scale, race-
mization could represent particularly troublesome purification
consequences. Furthermore, even at the high 20 mg/mL literature
concentration, we would require ꢀ80 L of solvent for the cycliza-
tion step of each of the 16 templates at 100 g scale. For practical
considerations, we would have to develop conditions to further re-
duce the amount of solvents required for the cyclization.
Once settled on activation of the glycine residue for the key
cyclization step and nosyl protecting group for P2, retrosynthetic
analysis then led us to the cyclization substrate 4 and a triamine
carboxylate intermediate 5 as indicated in Scheme 2. Intermediate
5 would serve as a common precursor from which standard amino
acid chemistry would be applied to generate the macrocyclization
substrates of all 16 templates, thus maximizing synthesis effi-
ciency via synergy.
presence of a t-butyl ester using a 1:5 mixture of TFA/DCM10 led
to intermediate 5. This seven-step sequence required only one col-
umn chromatography purification (step f) and furnished about 8 kg
of intermediate 5 in ꢀ20% overall yield, using roughly 8 kg of
hydroxyethyl ethylene diamine. Compounds 8, 10 and 5 in this
scheme are novel entities and have never been reported in the lit-
erature to the best of our knowledge.
This was followed with sequential incorporation of Boc-amino
acids and again selective Boc removal in the presence of the t-butyl
ester using TFA in DCM. Eventual deprotection of both t-butyl car-
boxylate and Boc-amino groups at the penultimate step was
accomplished with HCl/THF. This sequence enabled us to reliably
generate ꢀ100 g of each of the 16 cyclization substrates.
Initial cyclization attempts were made using 50 mg of substrate
11 in 5 mL solvent volumes (10 mg/mL, ꢀ10 mM, Scheme 4,
A = benzyl). The desired product 12 in our case was indeed formed
but only in low yield, with significant dimer formation observed
even at this concentration under various coupling agent/solvent
conditions. For example, with PyBop/DCM the ratio of desired
product versus dimer was 0.8 to 1.0. This improved somewhat
when HATU was used, with HATU/DMF giving a better ratio of
1.8 to 1.0. This result is significantly different from the reported
cyclization of apicidin analogs where cyclization of 3a was
achieved at 2 and 20 mg/mL concentrations with 80–90% isolated
yields.5a Our result may be attributed to an unfavorable entropy
factor derived from the lack of a cyclic amino acid residue in the
ring skeleton as noted earlier.
Results and discussion
Common intermediate 5 was synthesized using solution phase
chemistry following Scheme 3.8 Cbz protection of hydroxyethyl
ethylene diamine9 at the terminal amine followed by Boc protec-
tion and Mitsunobu reaction led to compound 8. Hydrazinolysis
followed by the installation of the nosyl group, selective alkylation
of the nosyl nitrogen atom and selective removal of Boc in the
Efforts were then made to alter the sequence of reagent addi-
tion, resulting in substantial dimer suppression when the coupling
substrate was slowly introduced into a solution of the coupling
reagent and base. For example, when 50 mg substrate 11 was
introduced into
a 2.5 mL DMF solution of HATU and DIEA

3300
J. Chen et al. / Tetrahedron Letters 54 (2013) 3298–3301
OH
OH
OH
O
O
N
a
b
c
HN
N
HN
NH
Boc
NH
NH2
N
8
Cbz
Cbz
NH
Boc
6
7
Cbz
Nosyl
O
Nosyl
HN
NH2
N
O
e
f, g
d
N
N
HN
NH
Cbz
NH
Cbz
Boc
Boc
NH
Cbz
9
10
5
Scheme 3. Synthesis of common intermediate 5. Reagents and conditions: (a) CbzOSu, DCM, 0 °C; (b) (Boc)2O, DCM; (c) Ph3P, DIAD, phthalimide, THF; (d) NH2NH2, EtOH; (e)
nosyl-Cl; (f) BrCH2CO2t-Bu, ACN, K2CO3; (g) TFA/DCM (1:5).
HO
O
N
O
Nosyl
N
DIEA, 0 oC, 2 h
50 mg / 5 mL
N
O
Nosyl
N
NH
Fmoc
N
H
NH2
+ cyclic dimer
A
Fmoc
N
H
A
N
H
NH
Cbz
O
NH
Cbz
O
N
H
O
11 (A = benzyl)
12 (A = benzyl)
scale
(mg)
concentration
condition
product : dimer isolated yield
(mg/mL)
50
50
10
10
PyBop/DCM
HATU/DMF
0.8 : 1
1.8 :1
3:1
NA
NA
50
20
HATU/DMF, slow addn
HATU/DMF, slow addn
HATU/DMF/ACN, slow addn
NA
1,000
10,000
50
>10:1
>10:1
70%
78%
125
Scheme 4. Macrocyclization for template synthesis.
O
Nosyl
O
Nosyl
N
O
N
O
NH
R1
NH
Fmoc
N
H
A
N
H
A
N
N
N
H
NH
O
NH
O
N
H
Cbz
Cbz
O
R2
N
O
R2
N
N
N
NH
R1
NH
R1
N
A
N
H
H
A
N
H
NH
R3
O
NH
O
N
H
O
O
Cbz
Scheme 5. Final analog synthesis from macrocyclic templates.
(20 mg/mL), the ratio of desired product to dimer increased to 3:1.
On larger scale, the control of substrate addition rate became more
practical and formation of dimer was further suppressed. Thus at
1 g scale in 20 mL DMF (50 mg/mL), slow substrate addition led
to further improved product dimer ratio of >10:1 and an isolated
yield of ꢀ70%. Further optimization resulted in a 10 g scale reac-
tion using only 80 mL solvent (125 mg/mL) with isolated product
in ꢀ80% yield after simple purification procedures. Ultimately,
these procedures were performed on 16 substrates at scales up
to 100 g with isolated desired product in high yield.11
From the templates, the final analogs were elaborated as shown
in Scheme 5. Fmoc was first removed followed with elaboration of
the R1 diversification. The nosyl group was then removed (PhSNa,
K2CO3, DMF) with the introduction of the R2 diversification. The

J. Chen et al. / Tetrahedron Letters 54 (2013) 3298–3301
3301
O
analog
A
Papp AB
Average TC
O
O
O
N
Ph
Ph
S
N
H
NH
5.11x10-6 cm/s -1.5 ppb/K
0.92x10-6 cm/s -4.8 ppb/K
13
14
A
O
O
N
S
O
N
H
N
H
O
Figure 2. Permeability and intramolecular hydrogen bond interaction of diastereomers 13 and 14.
Curr. Opin. Struct. Biol. 2007, 17, 67–76; (c) London, N.; Movshovitz-Attias, D.;
Schueler-Furman, O. Structure 2010, 18, 188–199.
2. Lipinski, C. A.; Lombardo, F.; Dominy, B. W.; Feeney, P. J. Adv. Drug Deliv. Rev.
1997, 23, 3–25.
Cbz group was finally removed (TMSI, DCM) and the last diversifi-
cation R3 was incorporated. When R1 is a simple amine cap from
aldehyde reductive amination, Boc was introduced to protect the
newly formed secondary amine. Diversification was performed
using standard chemistry, i.e. amide coupling, sulfonamide forma-
tion, heteroaryl SNAr and reductive amination. Overall 3987 final
analogs were generated in this effort out of a target of 5488 analog
library (16 Â 7 Â 7 Â 7).
3. (a) Wells, J. A.; McClendon, C. L. Nature 2007, 450, 1001–1009; (b) Yin, H.;
Hamilton, A. D. Angew. Chem., Int. Ed. 2005, 44, 4130–4163; (c) Arkin, M. R.;
Wells, J. A. Nat. Rev. Drug Disc. 2004, 3, 301–317; (d) Sillerud, L. O.; Larson, R. S.
Curr. Protein Pept. Sci. 2005, 6, 151–169; (e) Wilson, A. J. Chem. Soc. Rev. 2009,
38, 3289–3300.
4. Our analogs incorporate an ethylene unit into the ring thus differentiating
themselves from any cyclic tetrapeptide analogs. Synthetic efforts toward a
cyclic tetrapeptide library has been disclosed recently. Lim, A.; Ong, A.; Chia, B.
In Peptide Science 2010: Proceedings of the Fifth International Peptide
Symposium; Fijii, N., Kiso, Y., Eds.; The Japanese Peptide Society: Osaka,
2011; p 216.
5. (a) Berst, F.; Ladlow, M.; Holmes, A. Chem. Commun. 2002, 508–509; (b) Murray,
P.; Kranz, M.; Ladlow, M.; Taylor, S.; Berst, F.; Holmes, A.; Keavey, K.; Jaxa-
Chamiec, A.; Seale, P.; Stead, P.; Upton, R.; Croft, S.; Clegg, W.; Elsegood, M.
Bioorg. Med. Chem. Lett. 2001, 11, 773–776; (c) Jaxa-Chamiec, A.; Kranz, M.;
Murray, P. International Patent Application WO 00/47608; (d) Also see Yau, E.
K.; Theodore, L. J.; Gustavson, L. M. U.S. Patent 5,541,287. The same ring
scaffold was reported in this patent where compound 15 was synthesized at
2 g scale using a syringe pump (61% yield). Note the same ring closing point
with TFA protecting group instead of nosyl at the position corresponding to Ref.
4. There was no discussion about the optical purity of the cyclization product in
the patent.
A subset of these analogs was evaluated in permeability assays.
Interestingly, a pair of diastereomers, analogs 13 and 14 exhibited
considerably different permeability (Fig. 2), with MDCKII-LE Papp
AB 5.11 Â 10À6 cm/s for analog 13 and 0.92 Â 10À6 cm/s for analog
14.12 This correlation of permeability and stereochemistry,
although not universal for all the library macrocyclic analogs we
made, was observed on a number of other pairs of diastereomers.
There are four amide or sulfonamide hydrogen atoms that may
serve as hydrogen bond donors in these molecules. NMR studies
revealed there is a higher degree of intramolecular hydrogen bond-
ing (IMHB) in analog 13 compared with compound 14, as mea-
sured by temperature coefficients (TC) of these individual
hydrogen atoms. In CDCl3 a TC higher than À3.0 ppb/K is typically
associated with strong IMHB.13,14 The average TCs for the four
hydrogen atoms in 13 is À1.5, with three of the four higher than
À3.0 ppb/K. On the other hand, the average TCs for the four hydro-
gen atoms of analog 14 is À4.8, with only one of the four higher
than À3.0. This appears to indicate a substantially higher level of
IMHB interaction for 13 compared with 14 which is consistent
with the observed permeability difference.14
O
O
N
CF3
NH
O2N
NH
O
N
H
O
15
6. White, C. J.; Yudin, A. K. Nat. Chem. 2011, 3, 509–524.
7. Joullie, M. M.; Lassen, K. M. Arkivoc 2010, 8, 189–250.
Summary and conclusions
8. Unlike Ladlow and coworkers,5 we focused solely on solution phase strategies
because solid phase would be too cumbersome at the 100 g scale for our
templates.
We described here a novel synthesis pathway for the 12-mem-
bered macrocyclic templates (1) and the elaboration of a library of
macrocyclic analogs (2) thereof. The key macrocyclization step was
performed at up to 100 g scale without resorting to syringe pumps,
flow reactors, large volume of solvent or solid phase chemistry. An
interesting observation of considerably different permeability
properties was made on diastereomeric analogs apparently due
to intramolecular hydrogen bond interactions.
9. Adamczyk, M.; Fishpaugh, J.; Heuser, K. Org. Prep. Proced. Int. 1998, 30(3), 339–
348.
10. Selective Boc-removal in the presence of t-butyl ester has been reported using
p-toluenesulfonic acid or dry HCl. See (a) Goodacre, J.; Ponsford, R.; Stirling, I.
Tetrahedron Lett. 1975, 42, 3609–3612; (b) Gibson, F.; Bergmeier, S.; Rapoport,
H. J. Org. Chem. 1994, 59, 3216–3218.
11. Detailed optimized macrocyclization of 11 at 10 g scale: to a stirred solution of
HATU (8.1 g, 21 mmol) and DIEA (2.7 g, 21 mmol) in 20 mL DMF was added a
solution of the cyclization substrate 11 (10 g, 11 mmol) in 60 mL MeCN
dropwise over a period of 6 h at 0 °C under argon. The resulting mixture was
stirred at 0 °C for 30 min. and then poured into 200 mL ice-water with stirring.
A precipitate formed and was collected via filtration. The solid was dried in a
lyophilizer to furnish the desired cyclized product 12 as a pale powder, 7.7 g
(78% yield).
12. Di, L.; Whitney-Pickett, C.; Zhang, H.; Zhang, X.; Gebhard, D. F.; Lai, Y.; Federico,
J. J., III; Davidson, R. E.; Smith, R.; Revner, E. L.; Lee, C.; Feng, B.; Rotter, C.;
Varma, M. V.; Kempshall, S.; Fenner, K.; El-Kattan, A. F.; Liston, T. E.; Troutman,
M. D. J. Pharm. Sci. 2011, 100(11), 4974–4985.
Acknowledgments
We acknowledge Dr. J. Starr and Dr. M. Shapiro for critical read-
ing of this manuscript. L. Chen, Y. Wang and Y. Pu of ChemPartner
provided analytical chemistry and purification support.
References and notes
13. Cierpicki, T.; Otlewski, J. J. Biomol. NMR 2001, 21, 249–261.
14. Alex, A.; Millan, D.; Perez, M.; Wakenhut, F.; Whitlock, G. Med. Chem. Commun.
2011, 2, 669–674.
1. (a) Fuller, J. C.; Burgoyne, N. J.; Jackson, R. M. Drug Discovery Today 2009, 14,
155–161; (b) Reichmann, D.; Rahat, O.; Cohen, M.; Neuvirth, H.; Schreiber, G.