The present invention provides processes for making a glucagon (Gcg) and GLP-1
dual agonist peptide, or a pharmaceutically acceptable salt thereof
5 Over the past several decades, the prevalence of diabetes has continued to rise.
Type 2 diabetes mellitus (T2D) is the most common form of diabetes accounting for
approximately 90% of all diabetes. T2D is characterized by high blood glucose levels
caused by insulin resistance. Uncontrolled diabetes leads to several conditions that
impact morbidity and mortality of patients. The leading cause of death for diabetic
10 patients is cardiovascular complications. One of the main risk factors for type 2 diabetes
is obesity. The majority of T2D patients ( ~ 90%) are overweight or obese. It is
documented that a decrease in body adiposity will lead to improvement in obesityassociated
co-morbidities including hyperglycaemia and cardiovascular events.
Therefore, therapies effective in glucose control and weight reduction are needed for
15 better disease management.
Gcg helps maintain the level of glucose in the blood by binding to Gcg receptors
on hepatocytes, causing the liver to release glucose- stored in the form of glycogenthrough
glycogenolysis. As these stores become depleted, Gcg stimulates the liver to
synthesize additional glucose by gluconeogenesis. This glucose is released into the
20 bloodstream, preventing the development of hypoglycaemia.
GLP-1 has different biological activities compared to Gcg. The actions ofGLP-
1 include stimulation of insulin synthesis and secretion, inhibition of Gcg secretion and
inhibition of food intake. GLP-1 has been shown to reduce hyperglycaemia in diabetics.
Several GLP-1 agonists have been approved for use in the treatment of T2D in humans,
25 including exenatide, liraglutide, lixisenatide, albiglutide and dulaglutide. Such GLP-1
agonists are effective in glycaemic control with favourable effects on weight without the
risk of hypoglycaemia. However, the weight loss is modest due to dose-dependent
gastrointestinal side-effects.
Gcg and GLP-1 dual agonist peptides that may be useful in the treatment of T2D
30 and obesity are described and claimed in US Patent No. 9,938,335 B2. A process for the
production of such Gcg and GLP-1 dual agonist peptides is described therein.
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There remains a need, however, for improved processes for production of Gcg and
GLP-1 dual agonist peptides, such processes having a combination of advantages
including commercially desired purity. Similarly, there is a need for efficient and
environmentally "green" processes, including stable compounds to provide Gcg and
5 GLP-1 dual agonist peptides with fewer or simpler purification steps. The preparation of
large-scale, pharmaceutically-elegant Gcg and GLP-1 dual agonist peptides presents a
number of technical challenges that may affect the overall yield and purity. There is also
a need for processes to avoid the use of harsh reaction conditions that are incompatible
with peptide synthesis.
10 The present invention seeks to meet these needs by providing novel processes
useful in the manufacture of a Gcg and GLP-1 dual agonist peptide (SEQ ID NO: 1), or a
pharmaceutically acceptable salt thereof The improved manufacturing processes of the
present invention provide compounds and process reactions embodying a combination of
advances, including an efficient route having fewer steps, while at the same time
15 maintaining high quality and purity. Importantly, the improved processes and compounds
decrease resource intensity.
The improved processes described herein provide various compounds useful for
production of a Gcg and GLP-1 dual agonist peptide.
In particular, there is provided a process for the preparation of a compound of the
20 following formula:
H2N-H-Aib-Q-G-T -F-T -S-D-Y -S-K-Y -L-D-E-K-K-A-K-E-F-V -E-W-L-L-E-GG-
P-S-S-G-NH2
25 wherein lysine (Lys/K) at position 20 is chemically modified by conjugation of
30
the epsilon-amino group of the lysine side chain with ([2-(2-aminoethoxy )-ethoxy ]acetyl)
2-(y-Glu)-CO-(CH2)1sC02H (SEQ ID NO: 1),
and wherein said process comprises the steps of:
(i) solid-phase synthesis of a compound of the following formula:
5
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... ·•··
wherein PG 1 is a base stable side-chain protecting group,
wherein the Thr at position 5 is optionally protected by PG1,
PCT/US2021/036914
and wherein PG2 is an ivDde, Dde or Alloc side-chain protecting group (SEQ ID
NO: 2);
(ii) selective acylation at Lys at position 20 (SEQ ID NO: 7) by selectively deprotecting
said lysine and coupling the resulting Lys-NH2 (SEQ ID NO: 5) with
tBuO-C2o-yGlu(~u)-AEEA-AEEA-OH;
10 (iii) cleavage of the compound from the solid support and removal of base stable sidechain
protecting groups; and
(iv) purification of the compound (SEQ ID NO: 1).
Conventional preparation of a peptide compound wherein a side chain (e.g. fatty
acid side chain) is built by individual couplings in a stepwise manner produce significant
15 amounts of addition and deletion by-products. This results in an unfavourable purity
profile that makes it challenging to purify the peptide compound of interest. Furthermore,
low yields are typical when AEEA spacers are part of a side-chain built by conventional
methods.
The selective deprotection ofLys at position 20 and subsequent acylation reaction
20 proceeds with the de-protected 1-34 Lys-20-NH2 peptide on resin backbone (SEQ ID NO:
4) coupled to the ~uO-C2o-yGlu(~u)-AEEA-AEEA-OH sidechain as an intact fragment.
This represents a novel on resin large fragment coupling. This approach provides an
efficient and robust process for acylation of a peptide or protein wherein the compound is
produced in high yield. Acylation occurs at lysine at position with> 99% selectivity and
25 minimal impurities. Selective deprotection and subsequent coupling results in a favorable
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impurity profile for the acylation reaction. Moreover, the improved acylation process
facilitates an easier purification and isolation of the desired acylated peptide product that
results in higher yields and purity.
Selective de-protection of the Lys at position 20 is facilitated by use of an ivDde,
5 Dde or Alloc side-chain protecting group at position 20 and base stable side-chain
protecting groups at other positions. De-protection conditions are selected wherein the
ivDde, Dde or Alloc side-chain protecting group at position 20 is removed but the basestable
side-chain protecting groups (PG 1) remain in place.
A variety of base-stable protecting groups are known in the art and may be used in
10 the process of the present invention. In an embodiment of the present invention, the basestable
side-chain protecting groups PG 1 used in the synthesis of the compound are (a)
tert-butyloxycarbonyl (Boc) for Trp and Lys, (b) tert-butyl ester (OtBu) for Asp and Glu,
(c) tert-butyl CBu) for Ser, Thr and Tyr, (d) triphenylmethyl (trityl)(Trt) for Gln, and (e)
Boc(Boc) or Boc(Dnp) for His.
15 In a preferred embodiment of the process of the present invention, the side-chain
protecting group at Lys at position 20 is ivDde.
In an alternative embodiment of the process of the present invention, the sidechain
protecting group at the Lys at position 20 is Dde.
Dde is a protecting group stable to most conventional bases and is, therefore,
20 stable to Fmoc removal conditions. ivDde is a derivative ofDde and is also stable to
Fmoc removal conditions. An additional advantage of ivDde is that its steric hindrance
makes it less prone to migrate to other free Lys residues. Both Dde and ivDde are
commonly removed by hydrazinolysis.
Preferably, when PG2 is ivDde or Dde, the Lys at position 20 is selectively de-
25 protected by contacting the compound with a solution comprising hydrazine hydrate.
Further preferably, the solution comprises 1%- 15% w/w hydrazine hydrate in
DMF, NMP, NBP orDMSO.
Still further preferably, the solution comprises 8% w/w hydrazine hydrate in
DMF.
30 In an alternative embodiment of the process of the present invention, the sidechain
protecting group at the Lys at position 20 is Alloc.
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Alloc is a base-labile protecting group. It is commonly removed by a
palladium catalyst in the presence of a scavenger to capture the generated carbocation.
The use of Alloc side-chain protecting group is compatible with the Boc/Bn and
Fmoc!Bu strategies and allows tandem removal-acylation reactions when the palladium-
5 catalyzed amino deblocking is performed in the presence of acylating agents. This
approach prevents diketopiperazine (DKP) formation.
Preferably, when the side-chain protecting group at Lys at position 20 is Alloc,
Lys at position 20 is selectively de-protected by contacting the compound with a
palladium catalyst in the presence of scavengers,
10 Further preferably, the Alloc side-chain protecting group at Lys at position
removed by contacting the compound with Pd(PPh3)4 in the presence ofH3N•BH3,
Me2NH•BH3, or PhSiH3.
The de-protected (at position 20) compound may be washed, de-swelled, isolated,
dried and packaged. The de-protected (at position 20) compound is re-swelled prior to
15 coupling with sidechain
In a preferred embodiment of the process of the present invention, PG 1 is Boc for
Trp and Lys, otBu for Asp and Glu, tBu for Ser, Thr and Tyr, Trt for Gln and Boc(Boc)
for His, PG2 is ivDde, and the solid-phase synthesis of the compound (SEQ ID NO: 3) of
step (i) is performed on a Fmoc amide resin solid support and comprises Fmoc
20 deprotection of the amide resin and sequential coupling of the following:
25
30
Fmoc-L-Gly-OH, Fmoc-L-SerCBu)-OH, Fmoc-L-Ser(~u)-OH, Fmoc-L-Pro-OH,
Fmoc-L-Gly-OH, Fmoc-L-Gly-OH, Fmoc-L-Glu(O~u)-OH, Fmoc-L-Leu-OH,
Fmoc-L-Leu-OH, Fmoc-L-Trp(Boc)-OH, Fmoc-L-Glu(OtBu)-OH, Fmoc-L-ValOH,
Fmoc-L-Phe-OH, Fmoc-L-Glu(O~u)-OH, Fmoc-Lys(ivDde)-OH, Fmoc-LAla-
OH, Fmoc-L-Lys(Boc)-OH, Fmoc-L-Lys(Boc)-OH, Fmoc-L-Glu(OtBu)-OH,
Fmoc-L-Asp(O~u)-OH, Fmoc-L-Leu-OH, Fmoc-L-Tyr(~u)-OH, Fmoc-LLys(
Boc)-OH, Fmoc-L-Ser(~u)-OH, Fmoc-L-Tyr(~u)-OH, Fmoc-L-Asp(O~u)OH,
Fmoc-L-SerCBu)-OH, Fmoc-L-Thr(~u)-OH, Fmoc-L-Phe-OH, Fmoc-GlyThr(I'Me,Mepro)-OH, Fmoc-L-Gln(Trt)-OH, Fmoc-Aib-OH; and Boc-L-His(Boc)OH.
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In an alternative embodiment of the process of the present invention, PG 1 is
Boc(Dnp) for His and the solid-phase synthesis of the compound of step (i) is performed
as described above.
5 Solid phase synthesis of the compound is performed on a Fmoc amide resin solid
support wherein the first step is Fmoc deprotection of the amide resin followed by
sequential coupling of the Fmoc amino acids of the peptide. A glycine-threonine
pseudoproline dipeptide is used in place of individual Fmoc-L-Gly and Fmoc-L-Thr
amino acids for coupling at positions 4 and 5. In these embodiments, the Thr residue at
10 position 5 is reversibly protected as a proline-like acid-labile oxazolidine. As such, there
is no requirement to protect that particular Thr residue with a PG 1. A substantial benefit
is realized in that the reaction proceeds to completion for the glycine-threonine
pseudoproline dipeptide. In contrast, coupling individual Fmoc-L-Gly and Fmoc-L-Thr
amino acids result in high levels of peptide impurities having a Thr5 deletion.
15 In an alternative preferred embodiment of the process of the present invention,
20
25
30
PG 1 is Boc for Trp and Lys, OIJ3u for Asp and Glu, 13u for Ser, Thr and Tyr, Trt for Gln,
and Boc(Dnp) for His, PG2 is ivDde, and the solid-phase synthesis of the compound
(SEQ ID NO: 4) of step (i) is performed on a Fmoc amide resin solid support and
comprises Fmoc deprotection of the amide resin and sequential coupling of the following:
Fmoc-L-Gly-OH, Fmoc-L-SerCBu)-OH, Fmoc-L-Ser(IJ3u)-OH, Fmoc-L-Pro-OH,
Fmoc-L-Gly-OH, Fmoc-L-Gly-OH, Fmoc-L-Glu(OIJ3u)-OH, Fmoc-L-Leu-OH,
Fmoc-L-Leu-OH, Fmoc-L-Trp(Boc)-OH, Fmoc-L-Glu(OtBu)-OH, Fmoc-L-ValOH,
Fmoc-L-Phe-OH, Fmoc-L-Glu(OIJ3u)-OH, Fmoc-Lys(ivDde)-OH, Fmoc-LAla-
OH, Fmoc-L-Lys(Boc)-OH, Fmoc-L-Lys(Boc)-OH, Fmoc-L-Glu(OtBu)-OH,
Fmoc-L-Asp(OIJ3u)-OH, Fmoc-L-Leu-OH, Fmoc-L-Tyr(IJ3u)-OH, Fmoc-LLys(
Boc )-OH, Fmoc-L-Ser(IJ3u )-OH, Fmoc-L-Tyr(IJ3u )-OH, Fmoc-L-Asp(OIJ3u )OH,
Fmoc-L-SerCBu)-OH, Fmoc-L-Thr(IJ3u)-OH, Fmoc-L-Phe-OH, and BocHis(
Dnp )-Aib-Gln(Trt)-Gly-ThrCBu)-OH.
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Solid phase synthesis of the compound is performed on a Fmoc amide resin solid
support wherein the first step is Fmoc deprotection of the amide resin followed by
sequential coupling of the Fmoc amino acids of the peptide. A Boc-His(Dnp)-AibGln(
Trt)-Gly-ThrCBu)-OH pentamer (SEQ ID NO: 14) is coupled as a single fragment to
5 Phe6 of the H2N-6-34 intermediate (SEQ ID NO: 10). A substantial benefit realized by
this preferred embodiment is improved purity due to minimization of histidine
racemization.
The compound of SEQ ID NO: 4 may be selectively de-protected at the lysine at
position 20 as described herein. The resulting compound has the following formula (SEQ
10 ID NO: 18):
~~1
;.
:(/
" ~cc-'l"i ~~H,-.Aib -rQ~- G.-T -.r-: r"- S -Dt-Y -S~ -~ -Y~ -l -tv-E -K)-'<~- A~~~,- /(·r~ -F-.V -E~-.Wc.L ,_L-®E-,~"- G-•P -Sili-u~ -G-:'i K £n:)
D~p lli~ ~ iE~ a,~~ Btl &u H & tau 8{t k~: : '9
The compound of SEQ ID NO: 18 may be coupled with the 13uO-C2o-yGlu(IJ3u)-
15 AEEA-AEEA-OH sidechain as an intact fragment as described herein. The resulting
compound has the following formula (SEQ ID NO: 19):
--·. ...-. __: .;,
( '·.f ,. \'}}~;,
·'
( ;;.;: "-" i"
'·· _...,_,. v···· ...- '·-···x:x:~-~~;-~-- 'C: ___ .-·'n:.-· t N ,. . ··-o·· ·.. }\/''-.:~li
>:cc H>: . , ;n . !; ;s'~ :tb ;fJ>J" }~ !B'i ~t -. ><··( : - ·. . ~~~ . t :E''' ·.. . -~~~ -.- :~ ('~
~~,, .. , N-~--Aib-0-G-T-> -l-B-0. Y-S--~-¥ ·'l...-0-E.JO<-A-~ _rE-F V-... -":1 ~-t-E.GG.P.S 7-0--N, .•
;):~p 1B:$ ·~li ~s~ ~:.~:::. tZ;t! ~~ :...: 1:9~1 s~~: t&:t;
In a further alternative preferred embodiment of the process of the present
20 invention, PG1 is: (a) Boc for Trp and Lys, (b) otBu for Asp and Glu, (c) 13u for Ser, Thr
5
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and Tyr, (d) Trt for Gln, and (e) Boc(Dnp) for His, PG2 is ivDde, and the solid-phase
synthesis of the compound (SEQ ID NO: 4) of step (i) is performed on a Fmoc amide
resin solid support and comprises Fmoc deprotection of the amide resin and sequential
coupling of the following:
Fmoc-L-Gly-OH, Fmoc-L-SerCBu)-OH, Fmoc-L-Ser(IJ3u)-OH, Fmoc-L-Pro-OH,
Fmoc-L-Gly-OH, Fmoc-L-Gly-OH, Fmoc-L-Glu(OIJ3u)-OH, Fmoc-L-Leu-OH,
Fmoc-L-Leu-OH, Fmoc-L-Trp(Boc)-OH, Fmoc-L-Glu(OtBu)-OH, Fmoc-L-ValOH,
Fmoc-L-Phe-OH, Fmoc-L-Glu(OIJ3u)-OH, Fmoc-Lys(ivDde)-OH, Fmoc-L-
10 Ala-OH, Fmoc-L-Lys(Boc)-OH, Fmoc-L-Lys(Boc)-OH, Fmoc-L-Glu(OtBu)-OH,
Fmoc-L-Asp(OIJ3u)-OH, Fmoc-L-Leu-OH, Fmoc-L-Tyr(IJ3u)-OH, Fmoc-LLys(
Boc )-OH, Fmoc-L-Ser(IJ3u )-OH, Fmoc-L-Tyr(IJ3u )-OH, Fmoc-L-Asp(OIJ3u )OH,
Fmoc-L-SerCBu)-OH, Fmoc-L-Thr(IJ3u)-OH, Fmoc-L-Phe-OH, Fmoc-LThr(
IJ3u)-OH; and Boc-His(Dnp)-Aib-Gln(Trt)-Gly-OH.
15
Solid phase synthesis of the compound is performed on a Fmoc amide resin solid
support wherein the first step is Fmoc deprotection of the amide resin followed by
sequential coupling of the Fmoc amino acids of the peptide. A Boc-His(Dnp)-AibGln(
Trt)-Gly-OH tetramer (SEQ ID NO: 16) is coupled as a single fragment to Thr5 of
20 the 2HN-5-34 intermediate (SEQ ID NO: 12). A substantial benefit realized by this
preferred embodiment is improved purity due to minimization of histidine racemization.
The compound of SEQ ID NO: 4 may be selectively de-protected at the lysine at
position 20 as described herein. The resulting compound has the formula of SEQ ID NO:
18.
25 The compound of SEQ ID NO: 18 may be coupled with the 13uO-C2o-yGlu(IJ3u)-
AEEA-AEEA-OH sidechain as an intact fragment as described herein. The resulting
compound has the formula of SEQ ID NO: 19.
In a preferred embodiment of the process of the present invention, the resin solid
support is a Fmoc amide resin solid support and the solid phase synthesis comprises Fmoc
30 deprotection of the resin.
Further preferably, the Fmoc amide resin solid support is a Sieber resin.
5
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In an embodiment of the present invention, step (iii) further comprises adjusting
the pH of a solution comprising the cleaved and deprotected compound to 7.0- 8.0,
stirring for 1-24 hours, subsequently adjusting the pH of the solution to 1.0- 3.0, and
stirring for 1-24 hours.
Adjusting the pH to 7.0- 8.0 neutralizes the solution and converts any depsipeptide
ester serine and threonine impurities to the desired compound.
Subsequent adjustment of the pH to 1. 0 - 3. 0 decarboxylates the Trp residue and
converts the Trp C02 salt to the desired product.
In an embodiment of the process of the invention, the purification of the
10 compound comprises subjecting the crude solution ofthe compound of step (iii) to
chromatographic purification.
Preferably, the chromatographic purification is HPLC or reverse phase HPLC.
Still further preferably, the purification further comprises the steps of (i) adding
the chromatographic eluent to a solution comprising aqueous sodium hydroxide or
15 aqueous sodium bicarbonate to form a sodium salt of the compound in solution, (ii)
precipitating the sodium salt of the compound from solution and (iii) filtering, washing
and drying the precipitated sodium salt of the compound.
The sodium salt imparts improved solubility of the compound relative to the
zwitterion or actetate forms. Furthermore, precipitation of the sodium salt of the
20 compound replaces expensive lyophilization procedures.
25
In a further aspect of the present invention, there is provided a process for the
preparation of a compound of the following formula:
FT . P.S\ :::'Gt %1 1":81 PGi ::\';i (' ?G1. K;•j PG~
PG1 -H~i-H-Aih-6-a-r-F-t -s-o-v -$-K-+-L -0-t-A-K-A-:r-·· ''::e--r~v-e-\\H. . -L -5-G-G-p-s-s-G···NH~\)
?L P~1 PS1 ?:L ~~ F~~j ;>:,-; :-: :S~:;·; P~! ?G1
wherein PG 1 is a base stable side-chain protecting group,
5
10
15
20
25
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wherein PG2 is an ivDde, Dde or Alloc side-chain protecting group (SEQ ID NO:
17),
and wherein said process comprises the steps of:
(i) solid-phase synthesis of a compound of the following formula:
... ,.·
•'·n< t PQt. F'fj . P~'i~ . ~';~ • ~~l P(\ .· . ..-( :\_sl /?j II ;>$1 .• . PG1 . ·. ,.,. ~/_....,.·~\:
t'-,,,, ... ,~---F--T--~-0--~ -S--K--Y--t--t:l--,5---K--~-A--M r,..--F.\ --E--¥1V-l--l--i-G-G--P-S----~-Gc-,,,,, .... ,::
: : : : : /~; ~ ::
(ii)
P~·{ ~\~·~ ~:n Pt:;~ ~~*1 .. . v\3.~ jPG~
wherein PG 1 is a base stable side-chain protecting group,
and wherein PG2 is an ivDde, Dde or Alloc side-chain protecting group
(SEQ ID NO: 9); and
coupling the compound of step (i) with a pentamer of the following
formula:
PG 1-His(PG 1 )-Aib-Gln(PG 1 )-Gly-Thr(PG 1 )-OH
wherein PG 1 is a base stable side-chain protecting group (SEQ ID NO:
13).
In a preferred embodiment of the process of the present invention, PG 1 is Boc for
Trp and Lys, otBu for Asp and Glu, tBu for Ser, Thr and Tyr, Trt for Gln, and Boc(Dnp)
for His.
In a further preferred embodiment of the process of the present invention, PG2 is
ivDde.
In an alternative preferred embodiment of the process of the process invention,
PG2 is Dde.
5
10
15
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In a further aspect of the present invention, there is provided a process for the
preparation of a compound of the following formula:
.... J
wherein PG 1 is a base stable side-chain protecting group,
and wherein PG2 is an ivDde, Dde or Alloc side-chain protecting group (SEQ ID
NO: 17)
said process comprising the steps of:
(i) solid-phase synthesis of a compound of the following formula:
HN
.J
PG.~ PG~ F?~ FCf~ F~1 F{3.; f ~·~ F~:~· PG1 P:~1
·P'G ~ .... ~-T~F j ~~-D-: -5-~-~--L-0--E-K-~-A --;·······\ jE-F-V-E-\~ -L-L-E-Gc_G-P -S-~-G-fJ~~
?G~ PGi ~~b.:~ ~~31 p~~· P~Gi .. 6 ~.s:; P~1
wherein PG 1 is a base stable side-chain protecting group,
and wherein PG2 is an ivDde, Dde or Alloc side-chain protecting group
(SEQ ID NO: 11); and
5
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(ii) coupling the compound of step (i) with a tetramer of the following
formula:
PG 1-His(PG 1 )-Aib-Gln(PG 1 )-Gly-OH
wherein PG 1 is a base stable side-chain protecting group (SEQ ID NO:
15).
In a preferred embodiment of the process of the present invention, PG 1 is Boc for
Trp and Lys, otBu for Asp and Glu, tBu for Ser, Thr and Tyr, Trt for Gln, and Boc(Dnp)
10 for His.
In a further preferred embodiment of the process of the present invention, PG2 is
ivDde.
In an alternative preferred embodiment of the process of the present invention,
PG2 is Dde.
15 In a further aspect of the present invention, there is provided a process for the
20
25
30
preparation of a sodium salt of the compound of the following formula:
H2N-H-Aib-Q-G-T -F-T -S-D-Y -S-K-Y -L-D-E-K-K-A-K-E-F-V -E-W-L-L-E-GG-
P-S-S-G-NH2
wherein lysine (Lys/K) at position 20 is chemically modified by conjugation of
the epsilon-amino group of the lysine side chain with ([2-(2-aminoethoxy )-ethoxy ]acetyl)
2-(y-Glu)-CO-(CH2)1sC02H (SEQ ID NO: 1)
said process comprising the steps of:
(i) adding aqueous sodium hydroxide or aqueous sodium bicarbonate to a
solution comprising the compound to form a sodium salt of the compound
in solution;
(ii) precipitating the sodium salt of the compound from solution; and
5
10
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-13-
(iii) filtering, washing and drying the precipitated sodium salt of the
compound.
In a further aspect of the present invention, there is provided a compound having
the following formula (SEQ ID NO: 3):
.. ·
T:t i&; ~B~ \8~. i3w Bt: !ht {} 'i<:·' "'-'•· ,,~ ..
"" HNtAib-!l-G-1-F-ts-D-Y-s-K ·hil-E-K~K-A ~f 17-fv £~1-tr~~p;~-"'-Q
D!\-o tS:J ~B;._~ &x tSu ~~ 'w~ ~B~ ~c tEt:
In a further aspect of the present invention, there is provided a compound having
the following formula (SEQ ID NO: 4):
Q • ,.,, , ••. Jrt . ;B\; . tBw. f'~ ;s~ ~B~ ~<: , .. /.(: . , .l~h •• ·~\i.. :s~~ . . K·" { :. wch,~-H-Aib~O-G-T-F-T-S-0-;-"'~-Y-L-D-7-K-K-A---q y-E-f-V-E.-~-l-L-E-G-G-P-5-S-G---:•~-~
t~1p ~&..~ ·!Bi.:: ta~~ ·S~"iC ·t8~ ~Boc C! ta:;.~ 8~· l8w
In a further aspect of the present invention, there is provided a compound having
the following formula (SEQ ID NO: 10):
5
10
15
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In a further aspect of the present invention, there is provided a compound having
the following formula (SEQ ID NO: 12):
16:~: ts~~ tE~ ·Bt~ B8 &~ {...: ~au ;st tBu taw
Fm~xJLr- F-T-S -0-Y-S -K-Y -L-0-E-.K-K-A-<'l.,,.·\\-E-F-V-E- W-L-l---'E~G-G-P -S-S-G-NH--Qt
~'' ~u k~, ~'x ~\i LK H '~' ~>l' ~3~ .
In a further aspect of the present invention, there is provided a compound having
the following formula (SEQ ID NO: 13):
PG 1-His(PG 1 )-Aib-Gln(PG 1 )-Gly-Thr(PG 1 )-OH
wherein PG 1 is a base stable side-chain protecting group.
Preferably, PG 1 is 13u for Thr, Trt for Gln, and Boc(Dnp) for His.
In a further aspect of the present invention, there is provided a compound having
the following formula (SEQ ID NO: 15):
PG 1-His(PG 1 )-Aib-Gln(PG 1 )-Gly-OH
wherein PG 1 is a base stable side-chain protecting group.
Preferably, PG 1 is Trt for Gln and Boc(Dnp) for His.
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DETAILED DESCRIPTION
As used herein, the following abbreviations have the meanings as set forth herein:
"SPPS" means Solid Phase Peptide Synthesis, "Fmoc" means
5 fluorenylmethyloxycarbonyl chloride, "Boc" means tert-butyloxycarbonyl, "OtBu" means
tert-butyl ester, "~u" means tert-butyl, "Trt" means triphenylmethyl or trityl, "Dnp"
means 2,4-dinitrophenyl, "ivDde" means 1-(4,4-Dimethyl-2,6-dioxocyclohex-1-ylidene)-
3-methylbutyl, "Dde" means (1-(4,4-Dimethyl-2,6-dioxocyclohex-1-ylidene)-3-ethyl),
"Alloc" means allyloxycarbonyl, "Pip" means piperidine, "DIC'' means
10 diisopropylcarbodiimide, "Oxyma" means Ethyl cyanohydroxyiminoacetate, "DCM"
means dichloromethane, "IP A'' means isopropanol, "MTBE" means methyl-tert-butyl
ether, "TF A" means trifluoroacetic acid, "TIPS" means triisopropylsilane, "DTT" means
dithiothreitol, "UPLC" means Ultra High Performance Liquid Chromatography, "HATU"
means (1-[bis( dimethylamino )methylene ]-1H-1,2,3-triazolo[ 4,5-b ]pyridinium 3-oxide
15 hexafluorophosphate, "HFIP" means hexafluoroisopropanol, "CTC" means chlorotrityl,
"AEEA" means 17-amino-10-oxo-3,6, 12,15 tetraoxa-9-aza heptadecanoic acid "TMSA"
means trimethylsilyalmide, "HOBt" means hydroxybenzotriazole, and "API" means
active pharmaceutical ingredient, "PyBOP" means (benzotriazol-1-
yloxy)tripyrrolidinophosphonium hexafluorophosphate), "~uO-C2o-yGlu(~u)-AEEA-
20 AEEA-OH" means (3,6,12,15-Tetraoxa-9,18-diazatricosanedioic acid, 22-[[20-(1,1-
dimethylethoxy)-1,20-dioxoeicosyl]amino ]-1 0, 19-dioxo-, 2,3-(1, 1-dimethylethyl) ester,
(22S)), and "AEEA" means (8-amino-3,6-dioxaoctanoic acid).
The amino acid sequences of the present invention contain the standard single
letter or three letter codes for the twenty naturally occurring amino acids. Additionally,
25 "Aib" is alpha amino isobutyric acid.
The present invention is generally directed to a process for the preparation of a
Gcg and GLP-1 dual agonist compound wherein the compound is synthesized by SPPS.
SPPS incorporates several basic steps that are repeated as additional amino acids are
added to a growing peptide chain. The "solid phase" refers to resin particles to which
30 initial amino acids- and then the growing peptide chains- are at attached. Because the
chains are attached to particles, the chains can be handled as if they were a collection of
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solid particles (particularly for washing and separation-e.g., filtration-steps), and thus
making the overall process easier in many cases than pure solution synthesis.
There are several suitable resins for building the peptide compounds presented
herein. For example, Sieber and Rink amide resins are well known for preparing
5 peptides. Alternative resins, however, may be selected for the preparation of peptides
described herein. For example, but not limited to, 2-CTC and related resins may be used
to prepare a target peptide, followed by a C terminus amidation step.
10
15
20
The repeated steps of SPPS include deprotection, activation and coupling:
(i) Deprotection: before each cycle starts, the last acid on the peptide chain
(ii)
remains "protected". As used herein, the term "protected" means that a
protecting group is attached to at the indicated position, i.e., its "amino"
end is connected to a functional group that protects the acid from
unwanted reactions. A variety of protecting groups are well known, and
alternative protecting groups may be suitable for a particular process. The
"protecting group" is removed (the "deprotection" step) when the next
amino acid is about to be added;
Activation: a compound ("activator") is added to the reaction to produce an
intermediate amino acid species that is more likely to couple to the
deprotected acid on the peptide chain.
(iii) Coupling: the activated species connects to the existing peptide chain.
One of the most commonly used and studied activation methods for peptide
synthesis is based on the use of carbodiimides. A carbodiimide contains two slightly basic
nitrogen atoms which will react with the carboxylic acid of an amino acid derivative to
form a highly reactive 0-acylisourea compound. The formed 0-acylisourea can then
25 immediately react with an amine to form a peptide bond. Alternatively, the 0-acylisourea
can be converted into other reactive species. Some of these alternative reactions of 0-
acylisourea, however, promote undesirable pathways that may or may not lead to peptide
bond formation. Conversion to the unreactive N-acylurea prevents coupling, while
epimerization of an activated chiral amino acid can occur through oxazolone formation. A
30 more desirable highly reactive symmetrical anhydride can be formed by using excess
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amino acid compared to the carbodiimide. This approach, however, undesirably
consumes an additional amino acid equivalent.
A significant improvement for carbodiimide activation methods occurred with the
incorporation of 1-hydroxybenzotriazole (HOBt) as an additive during carbodiimide
5 activation. HOBt quickly converts the 0-acylisourea into an OBt ester that is highly
reactive, but avoids undesirable N-acylisourea and oxazolone formation. HOBt is a
hazardous reagent that is undesirable for use in large scale commercial manufacturing.
Other additives can be used in place ofHOBt such as ethyl 2-cyano-2-
(hydroxyimino)acetate (Oxyma, OxymaPure, ECHA) or 1-hydroxy-2,5-pyrrolidinedione
10 (NHS).
In respect of the processes of the present invention, the preferred activation
system is DIC/Oxyma in DMF. Preferably, the ratio of amino acid: Oxyma: DIC is
2.0:2.0:2.2. All charges are based on the limiting reagent which is the amide resin. The
Oxyma based system improves purity and eliminates downstream aggregation and
15 impurity issues observed in the purification step, in particular chromatographic
purification. Suitable solvents include DMF, NMP and NBP. DMF is the preferred
solvent system as it is significantly cheaper.
More generally in respect of the processes of the present invention, the SPPS
builds are preferably accomplished using standard Fmoc peptide chemistry techniques
20 employing sequential couplings with an automated peptide synthesizer. The preferred
resin is a Sieber amide resin. DMF is the preferred solvent system and the resin is swelled
with DMF. De-protected of the resin is preferably achieved using 20% piperidine
(Pip )/DMF (3 x 30 min). Subsequent Fmoc de-protections preferably use 20% Pip/DMF
(9 ml/g resin) 3 x 30 min treatments. 4 x 30 min treatments are preferably used for more
25 difficult couplings. After deprotection, the resin is washed with preferably 6 x 2 min, 10
volume DMF washes. Amino acid pre-activation preferably uses DIC/Oxyma/DMF
solutions at room temp for 30 min. Coupling of the activated amino acid to the resin
bound peptide occurs for a specified time for each individual amino acid. Solvent
washing with preferably 6 x 2 min 10 volumes DMF is performed after each coupling.
30 For isolation of the final product, the resin bound product is preferably washed 5 x
2 min with 10 volume DCM to remove DMF. The resin is preferably washed with 2 x 2
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min 10 volume IPA to remove DCM, washed 5 x 2 min 10 volume methyl-tert-butyl ether
(MTBE), then the product is dried at 40 oc under vacuum. The resin bound product is
stored cold ( -20°C).
For analysis, the peptide is cleaved from the resin with an acidic cocktail
5 preferably consisting of TF A/H20/TIPS/DTT in the following ratio:
(0.93v/0.04v/0.03v/0.03w). The resin is preferably swelled with DCM ( 4-5 mL, 3 x 30
min) and drained. The cleavage cocktail (4-5 mL) is added to the pre-swelled resin and
the suspension is stirred for 2 hr at room temp. The solution is filtered then the resin is
preferably washed with a small amount ofDCM and combined with the cleavage
10 solution. The resulting solution is preferably poured into 7-10 volumes of cold (0°C)
methyl-tert-butyl ether (MTBE). The suspension is preferably aged for 30 min at 0°C then
the resulting precipitate is centrifuged and the clear solution is decanted. The residue is
preferably suspended in the same volume ofMTBE, and the resulting suspension is again
centrifuged and decanted. After decanting the clear MTBE solution of the precipitated
15 peptide is dried in vacuo at 40 oc overnight.
The present invention is directed to novel compounds and processes useful for the
synthesis of compounds disclosed herein, or a pharmaceutically acceptable salt thereof, in
particular a sodium salt. The novel processes and compounds are illustrated in the
Examples below. The reagents and starting materials are readily available to one of
20 ordinary skill in the art. It is understood that these Examples are not intended to be
limiting to the scope of the invention in any way.
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Example 1: Preparation ofthe compound of SEQ ID NO: 1
Synthesis of Preparation 1
~~'t 1B~ t8t~ s~~: ~E~: tBw a~ f...- St~ tau ifki
~~-:H\t.H-Aib-0-G-T -FJ--S-0-Y -S-K-Y-L-0--E--K-K-A--N_ .... -\rE-f-V -£_ W -L_,L-E-G-G-P-S-5-G,.--MH{J
~m: ia,, k~ &<: la:J ~cc H b ta::; ~x \au
5 SEQIDNO: 3
A Fmoc Sieber resin (0.6- 0.8 mmol/g) is charged to a reactor, is swelled with DMF,
stirred for 2 hours, then DMF filtered off from the resin. The resin is then washed with
DMF twice. The Fmoc-protected resin is then de-protected using 20% Pip/DMF
10 treatments at 9 ml/g resin. Sampling to verify Fmoc removal is performed after the last
Pip/DMF treatment to confirm >99% Fmoc removal via UV analysis (IPC target <1%
Fmoc remaining). After the final 20% w/w Pip/DMF treatment, the resin bed is washed
multiple times with DMF (e.g. 6 x 2 min, 10 volume DMF washes at 9 ml/g resin). The
peptide backbone is built out using the following conditions for each amino acid coupling
15 and deprotection:
Cycle Amino acid SPPS conditions
(i) 3/4 x 30 min De-Fmoc cycles,
(ii) 6 x 2 min post-dep DMF washes (9 ml/g resin),
1 Fmoc-L-Gly-OH
(iii) 2.0 AA/2.2 DIC/ 2.0 Oxyma, in DMF (7.25 ml/g resin), room
temperature (rt),
(iv) 6 x 2 min, 10 volumes DMF (9 ml/g resin) post-coupling
washes
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Cycle Amino acid SPPS conditions
(i) 3/4 x 30 min De-Fmoc cycles,
Fmoc-L-
(ii) 6 x 2 min post-dep DMF washes (9 ml/g resin),
2 (iii) 2.0 AA/2.2 DIC/ 2.0 Oxyma, in DMF (7.25 ml/g resin), rt.,
Ser(~Bu)-OH
(iv) 6 x 2 min, 10 volumes DMF (9 ml/g resin) post-coupling
washes
(i) 3/4 x 30 min De-Fmoc cycles,
Fmoc-L-
(ii) 6 x 2 min post-dep DMF washes (9 ml/g resin),
3 (iii) 2.0 AA/2.2 DIC/ 2.0 Oxyma, in DMF (7.25 ml/g resin), rt.,
Ser(~Bu)-OH;
(iv) 6 x 2 min, 10 volumes DMF (9 ml/g resin) post-coupling
washes
(i) 3/4 x 30 min De-Fmoc cycles,
(ii) 6 x 2 min post-dep DMF washes (9 ml/g resin),
4 Fmoc-L-Pro-OH (iii) 2.0 AA/2.2 DIC/ 2.0 Oxyma, in DMF (7.25 ml!g resin), rt.,
(iv) 6 x 2 min, 10 volumes DMF (9 ml/g resin) post-coupling
washes
(i) 3/4 x 30 min De-Fmoc cycles,
(ii) 6 x 2 min post-dep DMF washes (9 ml/g resin),
5 Fmoc-L-Gly-OH (iii) 2.0 AA/2.2 DIC/ 2.0 Oxyma, in DMF (7.25 ml/g resin), rt.,
(iv) 6 x 2 min, 10 volumes DMF (9 ml/g resin) post-coupling
washes
(i) 3/4 x 30 min De-Fmoc cycles,
(ii) 6 x 2 min post-dep DMF washes (9 ml/g resin),
6 Fmoc-L-Gly-OH (iii) 2.0 AA/2.2 DIC/ 2.0 Oxyma, in DMF (7.25 ml/g resin), rt.,
(iv) 6 x 2 min, 10 volumes DMF (9 ml/g resin) post-coupling
washes
(i) 3/4 x 30 min De-Fmoc cycles,
Fmoc-L-
(ii) 6 x 2 min post-dep DMF washes (9 ml/g resin),
7 (iii) 2.0 AA/2.2 DIC/ 2.0 Oxyma, in DMF (7.25 ml/g resin), rt.,
Glu(OtBu)-OH
(iv) 6 x 2 min, 10 volumes DMF (9 ml/g resin) post-coupling
washes
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Cycle Amino acid SPPS conditions
(i) 3/4 x 30 min De-Fmoc cycles,
(ii) 6 x 2 min post-dep DMF washes (9 ml/g resin),
8 Fmoc-L-Leu-OH (iii) 2.0 AA/2.2 DIC/ 2.0 Oxyma, in DMF (7.25 ml/g resin), rt.,
(iv) 6 x 2 min, 10 volumes DMF (9 ml/g resin) post-coupling
washes
(i) 3/4 x 30 min De-Fmoc cycles,
(ii) 6 x 2 min post-dep DMF washes (9 ml/g resin),
9 Fmoc-L-Leu-OH (iii) 2.0 AA/2.2 DIC/ 2.0 Oxyma, in DMF (7.25 ml/g resin), rt.,
(iv) 5 x 2 min, 10 volumes DMF (9 ml/g resin) post-coupling
washes
(i) 3/4 x 30 min De-Fmoc cycles,
Fmoc-L-
(ii) 6 x 2 min post-dep DMF washes (9 ml/g resin),
10 (iii) 2.0 AA/2.2 DIC/ 2.0 Oxyma, in DMF (7.25 ml!g resin), rt.,
Trp(Boc)-OH
(iv) 6 x 2 min, 10 volumes DMF (9 ml/g resin) post-coupling
washes
(i) 3/4 x 30 min De-Fmoc cycles,
Fmoc-L-
(ii) 6 x 2 min post-dep DMF washes (9 ml/g resin),
11 (iii) 2.0 AA/2.2 DIC/ 2.0 Oxyma, in DMF (7.25 ml/g resin), rt.,
Glu(OtBu)-OH
(iv) 6 x 2 min, 10 volumes DMF (9 ml/g resin) post-coupling
washes
(i) 3/4 x 30 min De-Fmoc cycles,
(ii) 6 x 2 min post-dep DMF washes (9 ml/g resin),
12 Fmoc-L-Val-OH (iii) 2.0 AA/2.2 DIC/ 2.0 Oxyma, in DMF (7.25 ml/g resin), rt.,
(iv) 6 x 2 min, 10 volumes DMF (9 ml/g resin) post-coupling
washes
(i) 3/4 x 30 min De-Fmoc cycles,
(ii) 6 x 2 min post-dep DMF washes (9 ml/g resin),
13 Fmoc-L-Phe-OH (iii) 2.0 AA/2.2 DIC/ 2.0 Oxyma, in DMF (7.25 ml/g resin), rt.,
(iv) 6 x 2 min, 10 volumes DMF (9 ml/g resin) post-coupling
washes
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Cycle Amino acid SPPS conditions
(i) 3/4 x 30 min De-Fmoc cycles,
Fmoc-L-
(ii) 6 x 2 min post-dep DMF washes (9 ml/g resin),
14 (iii) 2.0 AA/2.2 DIC/ 2.0 Oxyma, in DMF (7.25 ml/g resin), rt.,
Glu(OtBu)-OH
(iv) 6 x 2 min, 10 volumes DMF (9 ml/g resin) post-coupling
washes
(i) 3/4 x 30 min De-Fmoc cycles,
Fmoc-
(ii) 6 x 2 min post-dep DMF washes (9 ml/g resin),
15 (iii) 8% hydrazine/DMF (9 ml/g resin),
Lys(ivDde )-OH
(iv) 6 x 2 min, 10 volumes DMF (9 ml/g resin) post-coupling
washes
(i) 3/4 x 30 min De-Fmoc cycles,
(ii) 6 x 2 min post-dep DMF washes (9 ml/g resin),
16 Fmoc-L-Ala-OH (iii) 2.0 AA/2.2 DIC/ 2.0 Oxyma, in DMF (7.25 ml!g resin), rt.,
(iv) 6 x 2 min, 10 volumes DMF (9 ml/g resin) post-coupling
washes
(i) 3/4 x 30 min De-Fmoc cycles,
Fmoc-L-
(ii) 6 x 2 min post-dep DMF washes (9 ml/g resin),
17 (iii) 2.0 AA/2.2 DIC/ 2.0 Oxyma, in DMF (7.25 ml/g resin), rt.,
Lys(Boc)-OH
(iv) 6 x 2 min, 10 volumes DMF (9 ml/g resin) post-coupling
washes
(i) 3/4 x 30 min De-Fmoc cycles,
Fmoc-L-
(ii) 6 x 2 min post-dep DMF washes (9 ml/g resin),
18 (iii) 2.0 AA/2.2 DIC/ 2.0 Oxyma, in DMF (7.25 ml/g resin), rt.,
Lys(Boc)-OH
(iv) 6 x 2 min, 10 volumes DMF (9 ml/g resin) post-coupling
washes
(i) 3/4 x 30 min De-Fmoc cycles,
Fmoc-L-
(ii) 6 x 2 min post-dep DMF washes (9 ml/g resin),
19 (iii) 2.0 AA/2.2 DIC/ 2.0 Oxyma, in DMF (7.25 ml/g resin), rt.,
Glu(OtBu)-OH
(iv) 6 x 2 min, 10 volumes DMF (9 ml/g resin) post-coupling
washes
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Cycle Amino acid SPPS conditions
(i) 3/4 x 30 min De-Fmoc cycles,
Fmoc-L-
(ii) 6 x 2 min post-dep DMF washes (9 ml/g resin),
20 (iii) 2.0 AA/2.2 DIC/ 2.0 Oxyma, in DMF (7.25 ml/g resin), rt.,
Asp(OtBu)-OH
(iv) 6 x 2 min, 10 volumes DMF (9 ml/g resin) post-coupling
washes
(i) 3/4 x 30 min De-Fmoc cycles,
(ii) 6 x 2 min post-dep DMF washes (9 ml/g resin),
21 Fmoc-L-Leu-OH (iii) 2.0 AA/2.2 DIC/ 2.0 Oxyma, in DMF (7.25 ml/g resin), rt.,
(iv) 6 x 2 min, 10 volumes DMF (9 ml/g resin) post-coupling
washes
(i) 3/4 x 30 min De-Fmoc cycles,
Fmoc-L-
(ii) 6 x 2 min post-dep DMF washes (9 ml/g resin),
22 (iii) 2.0 AA/2.2 DIC/ 2.0 Oxyma, in DMF (7.25 ml!g resin), rt.,
Tyr(lBu)-OH
(iv) 6 x 2 min, 10 volumes DMF (9 ml/g resin) post-coupling
washes
(i) 3/4 x 30 min De-Fmoc cycles,
Fmoc-L-
(ii) 6 x 2 min post-dep DMF washes (9 ml/g resin),
23 (iii) 2.0 AA/2.2 DIC/ 2.0 Oxyma, in DMF (7.25 ml/g resin), rt.,
Lys(Boc)-OH
(iv) 6 x 2 min, 10 volumes DMF (9 ml/g resin) post-coupling
washes
(i) 3/4 x 30 min De-Fmoc cycles,
Fmoc-L-
(ii) 6 x 2 min post-dep DMF washes (9 ml/g resin),
24 (iii) 2.0 AA/2.2 DIC/ 2.0 Oxyma, in DMF (7.25 ml/g resin), rt.,
Ser(lBu)-OH
(iv) 6 x 2 min, 10 volumes DMF (9 ml/g resin) post-coupling
washes
(i) 3/4 x 30 min De-Fmoc cycles,
Fmoc-L-
(ii) 6 x 2 min post-dep DMF washes (9 ml/g resin),
25 (iii) 2.0 AA/2.2 DIC/ 2.0 Oxyma, in DMF (7.25 ml/g resin), rt.,
Tyr(lBu)-OH
(iv) 6 x 2 min, 10 volumes DMF (9 ml/g resin) post-coupling
washes
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Cycle Amino acid SPPS conditions
(i) 3/4 x 30 min De-Fmoc cycles,
Fmoc-L-
(ii) 6 x 2 min post-dep DMF washes (9 ml/g resin),
26 (iii) 2.0 AA/2.2 DIC/ 2.0 Oxyma, in DMF (7.25 ml/g resin), rt.,
Asp(OtBu)-OH
(iv) 6 x 2 min, 10 volumes DMF (9 ml/g resin) post-coupling
washes
(i) 3/4 x 30 min De-Fmoc cycles,
Fmoc-L-
(ii) 6 x 2 min post-dep DMF washes (9 ml/g resin),
27 (iii) 2.0 AA/2.2 DIC/ 2.0 Oxyma, in DMF (7.25 ml/g resin), rt.,
Ser(~Bu)-OH
(iv) 6 x 2 min, 10 volumes DMF (9 ml/g resin) post-coupling
washes
(i) 3/4 x 30 min De-Fmoc cycles,
Fmoc-L-
(ii) 6 x 2 min post-dep DMF washes (9 ml/g resin),
28 (iii) 2.0 AA/2.2 DIC/ 2.0 Oxyma, in DMF (7.25 ml!g resin), rt.,
Thr(~Bu)-OH
(iv) 6 x 2 min, 10 volumes DMF (9 ml/g resin) post-coupling
washes
(i) 3/4 x 30 min De-Fmoc cycles,
(ii) 6 x 2 min post-dep DMF washes (9 ml/g resin),
29 Fmoc-L-Phe-OH (iii) 2.0 AA/2.2 DIC/ 2.0 Oxyma, in DMF (7.25 ml/g resin), rt.,
(iv) 6 x 2 min, 10 volumes DMF (9 ml/g resin) post-coupling
washes
(i) 3/4 x 30 min De-Fmoc cycles,
Fmoc-Gly- (ii) 6 x 2 min post-dep DMF washes (9 ml/g resin),
30 Thr( 'I'Me,Mepro )- (iii) 2.0 AA/2.2 DIC/ 2.0 Oxyma, in DMF (7.25 ml/g resin), rt.,
OH (iv) 6 x 2 min, 10 volumes DMF (9 ml/g resin) post-coupling
washes
(i) 3/4 x 30 min De-Fmoc cycles,
Fmoc-L-
(ii) 6 x 2 min post-dep DMF washes (9 ml/g resin),
31 (iii) 2.0 AA/2.2 DIC/ 2.0 Oxyma, in DMF (7.25 ml/g resin), rt.,
Gln(Trt)-OH
(iv) 6 x 2 min, 10 volumes DMF (9 ml/g resin) post-coupling
washes
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Amino acid SPPS conditions
(i) 3/4 x 30 min De-Fmoc cycles,
(ii) 6 x 2 min post-dep DMF washes (9 ml/g resin),
32 Fmoc-Aib-OH (iii) 2.0 AA/2.2 DIC/ 2.0 Oxyma, in DMF (7.25 ml/g resin), rt.,
(iv) 6 x 2 min, 10 volumes DMF (9 ml/g resin) post-coupling
washes
(i) 3/4 x 30 min De-Fmoc cycles,
Boc-L-His(Boc)-
(ii) 6 x 2 min post-dep DMF washes (9 ml/g resin),
33 (iii) 2.0 AA/2.2 DIC/2.0 Oxyma, in DMF (7.25 ml/g resin), rt.,
OH
(iv) 6 x 2 min, 10 volumes DMF (9 ml/g resin) post-coupling
washes
Fmoc Deprotection:
Resin in the peptide reactor is treated with either three or four charges of the 20% v/v
Pip/DMF solution. Each treatment is stirred on the resin for 30 min followed by filtration
S to complete Fmoc protecting group removal. After the final20% v/v PIP/DMF treatment,
the resin bed is washed a minimum of six times with DMF at the pre-specified DMF
volume charge.
Amino Acid Activation:
10 A pre-prepared solution of 12% w/w Oxyma Pure/DMF is charged to a reactor. The
selected Fmoc amino acid is then added. The mixture is stirred at 20 ±soc until the
Fmoc amino acid has completely dissolved. The Fmoc-AA/Oxyma Pure/DMF solutions
are then cooled to 1S ± 3°C prior to activation to ensure the minor exothermic activation
reaction is controlled and the resulting solution temperature is maintained in the range
1S specified of20 ± S°C. The amino acid solution is activated by DIC addition. The
activated ester solution is stirred for 20-30 minutes prior to transfer of the solution to the
reactor containing the peptide on resin compound.
Coupling:
20 Upon completion of the activation step, the activated ester solution is transferred to the
reactor containing deprotected peptide on resin to initiate the coupling reaction. The
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peptide coupling reaction is stirred at 20 ± soc for at least 4 hours. After the required stir
time, the resin slurry is sampled for coupling completion (IPC). Sampling is repeated at
specific intervals as needed until a passing IPC result is obtained. Re-coupling operations
are performed, if necessary. When the coupling is complete, the peptide reactor solution
5 contents are filtered then the peptide on resin compounds are washed several times with
DMF to prepare for the next coupling.
10
A Gly-Thr pseudoproline dipeptide is used in place of individual Fmoc-L-Gly and
Fmoc-L-Thr amino acids for coupling at positions 4 and 5. Fmoc-Gly-Thr['P(Me,Me)Pro]OH
is coupled to Phe (6) using the above-described coupling conditions.

CLAIMS:
1. A process for the preparation of a compound of the following formula:
H2N-H-Aib-Q-G-T -F-T -S-D-Y -S-K-Y -L-D-E-K-K-A-K-E-F-V -E-W-L-L-E-G-
5 G-P-S-S-G-NH2
wherein Lys at position 20 is chemically modified by conjugation of the
epsilon-amino group ofthe Lys side chain with ([2-(2-aminoethoxy)-ethoxy]acetyl)
2-(y-Glu)-CO-(CH2)1sC02H (SEQ ID NO: 1),
10 said process comprising the steps of:
15
20
(i) solid-phase synthesis of a compound of the following formula
P=G2
>iG\ PG1 P(;·: PGl PG1 ~<3~; ::;:;~ ( ;:lGl p;·i . PG1
PG1 -HN-H-Aib-Q-G-r -F-+-s-O-Y -S-K-Y-L-D-E-K-K-A-~/ >-E-F-v-e-w-L -L-E-G-G-P-s -s-G "N~
p;L P;~j pci~ ?~-~ 1=~1 F\;) PG1 " b PG1 Pb1 . Pt1
(ii)
wherein PG 1 is a base stable side-chain protecting group,
wherein Thr at position 5 is optionally protected by PG1,
and wherein PG2 is an ivDde, Dde or Alloc side-chain protecting group
(SEQ ID NO: 2)
selectively acylating the compound at the Lys at position 20 (SEQ ID NO:
7) by selectively de-protecting said Lys and coupling the resulting LysNH2
(SEQ ID NO: 5)with 13uO-C2o-yGluCBu)-AEEA-AEEA-OH; and
(iii) cleaving the acylated compound from the solid support and removal of the
25 remaining side chain protecting groups; and
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(iv) purifying the compound.
2. A process according to claim 1, wherein PG1 is:
(a) Boc for Trp and Lys;
5 (b) otBu for Asp and Glu;
(c) tBu for Ser, Thr and Tyr;
(d) Trt for Gln; and
(e) di-Boc for His.
10 3. A process according to claim 1 or claim 2, wherein PG2 is ivDde.
4.
5.
15
6.
20 7.
A process according to claim 1 or claim 2, wherein PG2 is Dde.
A process according to claim 3 or claim 4, wherein the Lys at position 20 is
selectively de-protected by reaction with a solution comprising hydrazine hydrate.
A process according to claim 5, wherein the solution comprises 1%- 15% w/w
hydrazine hydrate in DMF, NMP, NBP or DMSO.
A process according to claim 5 or 6, wherein the solution comprises 8% w/w
hydrazine hydrate in DMF.
8. A process according to claim 1 or claim 2, wherein PG2 is Alloc.
25 9. A process according to claim 5, wherein the Lys at position 20 is selectively deprotected
by reaction with Pd(PPh3)4 in the presence of scavengers, preferably
H3N•BH3, Me2NH•BH3, or PhSiH3.
10. A process according to any one of claims 1-7, wherein PG 1 is:
30 (a)
(b)
Boc for Trp and Lys;
otBu for Asp and Glu;
5
10
15
20
25
30
wo 2021/252829
(c) tBu for Ser, Thr and Tyr;
(d) Trt for Gln; and
(e) di-Boc for His,
wherein PG2 is ivDde,
PCT/US2021/036914
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wherein the solid-phase synthesis of the compound (SEQ ID NO: 3) of
step (i) is performed on a Fmoc amide resin solid support and comprises Fmoc
deprotection of the amide resin and sequential coupling of the following:
(01) Fmoc-L-Gly-OH;
(02) Fmoc-L-Ser(~u )-OH;
(03) Fmoc-L-Ser(~u )-OH;
(04) Fmoc-L-Pro-OH;
(05) Fmoc-L-Gly-OH;
(06) Fmoc-L-Gly-OH;
(07) Fmoc-L-Glu(OtBu )-OH;
(08) Fmoc-L-Leu-OH;
(09) Fmoc-L-Leu-OH;
(10) Fmoc-L-Trp(Boc )-OH;
(11) Fmoc-L-Glu(OtBu )-OH;
(12) Fmoc-L-Val-OH;
(13) Fmoc-L-Phe-OH;
(14) Fmoc-L-Glu(OtBu )-OH;
(15) Fmoc-Lys(ivDde )-OH;
(16) Fmoc-L-Ala-OH;
(17) Fmoc-L-Lys(Boc )-OH;
(18) Fmoc-L-Lys(Boc )-OH;
(19) Fmoc-L-Glu(OtBu )-OH
(20) Fmoc-L-Asp(O~u)-OH
(21) Fmoc-L-Leu-OH;
(22) Fmoc-L-TyrCBu )-OH;
(23) Fmoc-L-Lys(Boc )-OH;
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(24) Fmoc-L-Ser(IJ3u )-OH;
(25) Fmoc-L-TyrCBu )-OH;
(26) Fmoc-L-Asp(OIJ3u)-OH;
(27) Fmoc-L-Ser(IJ3u )-OH;
5 (28) Fmoc-L-ThrCBu )-OH;
(29) Fmoc-L-Phe-OH;
(30) Fmoc-Gly-Thr(\I'Me,Mepro )-OH;
(31) Fmoc-L-Gln(Trt)-OH;
(32) Fmoc-Aib-OH; and
10 (33) Boc-L-His(Boc )-OH.
11. A process according to any one of claims 1-7, wherein PG1 is:
(a) Boc for Trp and Lys;
(b) otBu for Asp and Glu;
15 (c) tBu for Ser, Thr and Tyr;
(d) Trt for Gln; and
(e) Boc(Dnp) for His,
wherein PG2 is ivDde,
wherein the solid-phase synthesis of the compound (SEQ ID NO: 4) of
20 step (i) is performed on a Fmoc amide resin solid support and comprises Fmoc
deprotection of the amide resin and sequential coupling of the following:
(01) Fmoc-L-Gly-OH;
(02) Fmoc-L-Ser(IJ3u )-OH;
25 (03) Fmoc-L-Ser(IJ3u )-OH;
(04) Fmoc-L-Pro-OH;
(05) Fmoc-L-Gly-OH;
(06) Fmoc-L-Gly-OH;
(07) Fmoc-L-Glu(OtBu )-OH;
30 (08) Fmoc-L-Leu-OH;
(09) Fmoc-L-Leu-OH;
5
10
15
20
25
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(10) Fmoc-L-Trp(Boc)-OH;
(11) Fmoc-L-Glu(OtBu)-OH;
(12) Fmoc-L-Val-OH;
(13) Fmoc-L-Phe-OH;
(14) Fmoc-L-Glu(OtBu)-OH;
(15) Fmoc-Lys(ivDde)-OH;
(16) Fmoc-L-Ala-OH;
(17) Fmoc-L-Lys(Boc)-OH;
(18) Fmoc-L-Lys(Boc)-OH;
(19) Fmoc-L-Glu(OtBu)-OH
(20) Fmoc-L-Asp(O~u)-OH
(21) Fmoc-L-Leu-OH;
(22) Fmoc-L-TyrCBu)-OH;
(23) Fmoc-L-Lys(Boc)-OH;
(24) Fmoc-L-Ser(~u)-OH;
(25) Fmoc-L-TyrCBu)-OH;
(26) Fmoc-L-Asp(O~u)-OH;
(27) Fmoc-L-Ser(~u)-OH;
(28) Fmoc-L-ThrCBu)-OH;
(29) Fmoc-L-Phe-OH;
PCT/US2021/036914
(30) Boc-His(Dnp )-Aib-Gln(Trt)-Gly-Thr(~u)-OH.
12. A process according to any one of claims 1-7, wherein PG1 is:
(a) Boc for Trp and Lys;
(b) otBu for Asp and Glu;
(c) tBu for Ser, Thr and Tyr;
(d) Trt for Gln; and
(e) Boc(Dnp) for His,
wherein PG2 is ivDde,
5
10
15
20
25
30
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wherein the solid-phase synthesis of the compound (SEQ ID NO: 4) of
step (i) is performed on a Fmoc amide resin solid support and comprises Fmoc
deprotection of the amide resin and sequential coupling of the following:
(01) Fmoc-L-Gly-OH;
(02) Fmoc-L-Ser(IJ3u )-OH;
(03) Fmoc-L-Ser(IJ3u )-OH;
(04) Fmoc-L-Pro-OH;
(05) Fmoc-L-Gly-OH;
(06) Fmoc-L-Gly-OH;
(07) Fmoc-L-Glu(OtBu )-OH;
(08) Fmoc-L-Leu-OH;
(09) Fmoc-L-Leu-OH;
(10) Fmoc-L-Trp(Boc )-OH;
(11) Fmoc-L-Glu(OtBu )-OH;
(12) Fmoc-L-Val-OH;
(13) Fmoc-L-Phe-OH;
(14) Fmoc-L-Glu(OtBu )-OH;
(15) Fmoc-Lys(ivDde )-OH;
(16) Fmoc-L-Ala-OH;
(17) Fmoc-L-Lys(Boc )-OH;
(18) Fmoc-L-Lys(Boc )-OH;
(19) Fmoc-L-Glu(OtBu )-OH
(20) Fmoc-L-Asp(OIJ3u)-OH
(21) Fmoc-L-Leu-OH;
(22) Fmoc-L-TyrCBu )-OH;
(23) Fmoc-L-Lys(Boc )-OH;
(24) Fmoc-L-Ser(IJ3u )-OH;
(25) Fmoc-L-TyrCBu )-OH;
(26) Fmoc-L-Asp(OIJ3u)-OH;
(27) Fmoc-L-Ser(IJ3u )-OH;
5
10
15
20
25
30
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(28) Fmoc-L-ThrCBu)-OH;
(29) Fmoc-L-Phe-OH;
(30) Fmoc-L-ThrCBu)-OH; and
(31) Boc-His(Dnp )-Aib-Gln(Trt)-Gly-OH.
13. A process according to any one of claims 10-12, wherein the resin solid support is
a Fmoc amide resin solid support and the solid phase synthesis comprises Fmoc
deprotection of the resin.
14.
15.
16.
17.
18.
A process according to claim 13, wherein the Fmoc amide resin solid support is a
Sieber resin.
A process according to any one of claims 1-14, wherein step (iii) further
comprises adjusting the pH of a solution comprising the cleaved and deprotected
compound to 7.0- 8.0, stirring for 1-24 hours, subsequently adjusting the pH of
the solution to 1.0- 3.0, and stirring for 1-24 hours.
A process according to any one of claims 1-15, wherein the purification of the
compound comprises subjecting the compound produced by step (iii) to
chromatographic purification.
A process according to claim 16, wherein the chromatographic purification is
HPLC or reverse phase HPLC.
A process according to claim 16 or claim 17 wherein the purification further
comprises the steps of (i) adding the chromatographic eluent to a solution
comprising aqueous sodium hydroxide or aqueous sodium bicarbonate to form a
sodium salt of the compound in solution, (ii) precipitating the sodium salt of the
compound from solution and (iii) filtering, washing and drying the precipitated
sodium salt of the compound.
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19. A process for the preparation of a compound of the following formula:
wherein PG 1 is a base stable side-chain protecting group,
5 wherein PG2 is an ivDde, Dde or Alloc side-chain protecting group (SEQ ID NO:
17),
10
15
and wherein said process comprises the steps of:
(i) solid-phase synthesis of a compound of the following formula:
(ii)
wherein PG 1 is a base stable side-chain protecting group,
and wherein PG2 is an ivDde, Dde or Alloc side-chain protecting group
(SEQ ID NO: 9); and
coupling the compound of step (i) with a pentamer of the following
formula:
PG 1-His(PG 1 )-Aib-Gln(PG 1 )-Gly-Thr(PG 1 )-OH
5
10
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wherein PG 1 is a base stable side-chain protecting group (SEQ ID NO:
13).
20. A process according to claim 19, wherein PG1 is
(a)
(b)
Boc for Trp and Lys;
otBu for Asp and Glu;
(c) tBu for Ser, Thr and Tyr;
(d) Trt for Gln; and
(e) Boc(Dnp) for His.
21. A process according to claim 19 or claim 20, wherein PG2 is ivDde.
22. A process according to claim 19 or claim 20, wherein PG2 is Dde.
15 23. A process for the preparation of a compound of the following formula:
20
?G2
. . . ~! "" .. ?r.i:l F~~ ?8': PGi PGi p:j J) P~l p~~ F~~ /::,
P~1-HN-H-Aib-O-G-I-r-T--S-0-Y--S-K-Y-L-0-E-:K~-A-N'. ·rE-F-V--E-W-L-L-E-G-G-?--S-S-G---1-l~
PGl p~·l ~i~ PL PGi FG'l F~1 H 6 k·: ~:;1 p;jl -
wherein PG 1 is a base stable side-chain protecting group,
wherein PG2 is an ivDde, Dde or Alloc side-chain protecting group (SEQ ID NO:
17),
and wherein said process comprises the steps of:
(i) solid-phase synthesis of a compound of the following formula:
5
10
15
20
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PGt
-~· ~ . . PGl.~ 7~~ PG\ • ~'~1 .. ~Gi ~'Gl . " ....... (· \G1 /G;. . ~G~ . ·. F~j .. .· "' {'
N! -":<-l-F-l-7·-D-Y--5--~-Y--L'"D-E-K--K -A-q rE-F-v -E-W-L-L-t-G.cG-P -S-s~G-f'H--w
~~3~1 PG~: Ft·} Ps~ PG:·~: P~J1 t.J Ps1 PG1
wherein PG 1 is a base stable side-chain protecting group,
and wherein PG2 is an ivDde, Dde or Alloc side-chain protecting group
(SEQ ID NO: 11); and
(ii) coupling the compound of step (i) with a tetramer of the following
formula:
PG 1-His(PG 1 )-Aib-Gln(PG 1 )-Gly-OH
wherein PG 1 is a base stable side-chain protecting group (SEQ ID NO:
15).
24. A process according to claim 23, wherein Pis
(a)
(b)
Boc for Trp and Lys;
otBu for Asp and Glu;
(c) tBu for Ser, Thr and Tyr;
(d) Trt for Gln; and
(e) Boc(Dnp) for His.
25. A process according to claim 23 or claim 24, wherein PG2 is ivDde.
26. A process according to claim 23 or claim 24, wherein PG2 is Dde.
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27. A process for the preparation of a sodium salt of the compound of the following
formula:
HlN-H-Aib-Q-G-T-F-T-S-D-Y-S-K-Y-L-D-E-K-K-A-K-E-F-V-E-W-L-L-E-G-
5 G-P-S-S-G-NH2
10
15
20
wherein lysine (Lys/K) at position 20 is chemically modified by
conjugation of the epsilon-amino group of the lysine side chain with ([2-(2-
aminoethoxy)-ethoxy ]-acetyl)2-(y-Glu)-CO-(CH2)1sC02H (SEQ ID NO: 1)
said process comprising the steps of:
(i) adding aqueous sodium hydroxide or aqueous sodium bicarbonate to a
solution comprising the compound of SEQ ID NO: 1 to form a sodium salt
of the compound in solution;
(ii) precipitating the sodium salt of the compound from solution; and
(iii) filtering, washing and drying the precipitated sodium salt of the compound
of SEQ IDNO: 1.
28. A compound having the following formula (SEQ ID NO: 3):
~~:~ :-"
~1~/-·"'· .. f:~';_J·
: ~ :~ ~,.,. \.-'\,./'•v· ~~:l::O
0t:N ~~
~ ,,~. . , :rt . ~ fu ~~~t ~ ;e-~ -~u Tee . ~ .. /( 1 ~w .• ~u ~ 1Eu . , .... t'':,
"' "~Ulb-Q,G. T~ -1-S-0-~ -S-1<-Y-1 .-il-E--K-K-A ,1 t~H EW t-L E~uP S~G-!i~
~c~. iR ~Bi~ &~ ttl~ ~oc M b tBli ~t:e ~u .
29. A compound having the following formula (SEQ ID NO: 4):
5
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,.··
' ~~-," :"1 h~1~ -Ai~ b-:Q•- G~r.- F-T~- S -D~-d; -Su-~ -Y~-L -0~-"-- K~-~ -A,-'~ ,(ll-E -F -V' -E~-W '- -L•-: Lr-t-G- G-P -58<-S -G"-' :'~(r.,\.
tt:p ~t:~ ~8;:; tB~ ~~ ~B~~ Bac ~: ~E~~ S:;{. tBt~
30. A compound having the following formula (SEQ ID NO: 10):
t8w t3iJ tSu lBl: $l1 Sc.: (' tSi.i t8s..~ ib tBt:
,.,._fLF-t- S-0-T-S -K-t -i-0-7-K-~-A-('JE-F-v- UJ-l.-i.E-G-G~-5-s--G-NH-\)
~g~ tsw Soc. te~ &::~t ·..,' Bee ~E~:
31. A compound having the following formula (SEQ ID NO: 12):
:tE~· ~~··- tS::..~ tSu i8L~ &-t ~/ ,;~., ~:;:;~, i~i·, ~;:
'""'~tr -r t -s.-K'v S-K-Y L&.H-K-A-~'\lFvi~uiG-G-P L~-G-NH-\1
t8w tS~i !sw ~~t ts~ em; :: B:lt !Bti
10 32. A compound having the following formula (SEQ ID NO: 13):
wo 2021/252829 PCT/US2021/036914
5 33.
34.
10
35.
15
-73-
PG 1-His(PG 1 )-Aib-Gln(PG 1 )-Gly-Thr(PG 1 )-OH
wherein PG 1 is a base stable side-chain protecting group.
A compound according to claim 32, wherein PG1 is 13u for Thr, Trt for Gln, and
Boc(Dnp) for His ..
A compound having the following formula (SEQ ID NO: 15):
PG 1-His(PG 1 )-Aib-Gln(PG 1 )-Gly-OH
wherein PG 1 is a base stable side-chain protecting group.
A compound according to claim 34, wherein PG 1 is Trt for Gln and Boc(Dnp) for
His.