Synthesis of sterically-hindered peptidomimetics using 4-(4,6-dimethoxy-1,3,5-triazine-2-yl)-4-methyl-morpholinium chloride

Article abstract of DOI:10.1016/j.tetlet.2008.06.119

Our study demonstrated that 4-(4,6-dimethoxy-1,3,5-triazine-2-yl)-4-methyl-morpholinium chloride (DMTMM) is a versatile coupling reagent for the synthesis of sterically-hindered peptidomimetics. It is superior to HBTU/HOBt and CDMT in controlling racemization and N-arylation, respectively.

Full text of DOI:10.1016/j.tetlet.2008.06.119

Tetrahedron Letters 49 (2008) 5359–5362
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Tetrahedron Letters
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Synthesis of sterically-hindered peptidomimetics using 4-(4,6-dimethoxy-
1,3,5-triazine-2-yl)-4-methyl-morpholinium chloride
b,
a
a
b
Wen-Chung Shieh a, , Zhuoliang Chen , Song Xue , Joe McKenna , Run-Ming Wang ,
*
*
a
ˇ a
Kapa Prasad , Oljan Repic
a Chemical and Analytical Development, Novartis Pharmaceuticals Corporation, East Hanover, NJ 07936, USA
b Novartis Institutes for Biomedical Research, Cambridge, MA 02139, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Our study demonstrated that 4-(4,6-dimethoxy-1,3,5-triazine-2-yl)-4-methyl-morpholinium chloride
(DMTMM) is a versatile coupling reagent for the synthesis of sterically-hindered peptidomimetics. It is
superior to HBTU/HOBt and CDMT in controlling racemization and N-arylation, respectively.
Ó 2008 Elsevier Ltd. All rights reserved.
Received 5 April 2008
Revised 23 June 2008
Accepted 25 June 2008
Available online 1 July 2008
This Letter is dedicated to Professor E. J.
Corey on the occasion of his 80th birthday
Keywords:
4-(4,6-Dimethoxy-1,3,5-triazine-2-yl)-4-
methyl-morpholinium chloride
DMTMM
Sterically-hindered peptide synthesis
Racemization control
To support our drug discovery program, we had to synthesize
several peptidomimetics that exhibit good in vitro activity in mul-
tiple cell lines. As shown in generic structure 1, these peptidomi-
metics contain two sterically-hindered motifs, namely the
pyrrolidine and cyclohexyl rings. Syntheses of sterically-hindered
peptides or chiral amides have been challenging since racemiza-
tion and side reactions are common competing pathways.
Although coupling reagents such as thiazolium1 or benzimidazo-
lium2 salts have been reported for the synthesis of sterically-
hindered peptides, they were not considered by us because of their
unavailability on large scale. To assemble the peptidomimetics
of our interest, we decided to start with a method employing O-
(benzotriazol-1-yl)-N,N,N0,N0-tetramethyluronium hexafluorophos-
phate (HBTU)3 and hydroxy-benzotriazole (HOBt), a combination
that is commonly used for peptide syntheses involving sterically-
hindered amino acids as reported by Knorr4 and Giralt.5 Our initial
efforts to couple sterically-hindered amine 9 and acid 5 using
HBTU/HOBt were unsatisfactory. We observed 25% of the unde-
sired diastereomer 7, which was caused by a racemization path-
way that occurred during the amide bond formation (Table 1,
H
N
R1
O
N
R
MeO
CH3
H
MeO
R2
R3
N
N
N
N
N+
N
N
O
-
Cl
OMe
MeO
2a, R = OH
2b, R = Cl
3
1
* Corresponding authors. Tel.: +1 8627786878; fax: +1 9737818434 (W.-C. Shieh).
E-mail addresses: wen.shieh@novartis.com (W.-C. Shieh), julian.chen@novartis.com (Z. Chen).
0040-4039/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2008.06.119

5360
W.-C. Shieh et al. / Tetrahedron Letters 49 (2008) 5359–5362
Table 1
Coupling of sterically-hindered amine 4 and acid 5
CH3
CH3
CH3
H3C
H
H3C
H3C
OMe
N
N
N
N
Boc
H
Boc
Boc
H
H
H
N
O
N
N
O
N
O
O
N
R
O
H
O
+
R
+
+
H
N
OMe
N
H
R
HO
N
R
N
4
7
5
8
6
Entry
Amine
Coupling method
HBTU/HOBt
% 6a
% 7b (racemization)
% 8a (N-arylation)
H
H
1
75
25
NA
N
N
H
9
2
3
4
9
9
9
CDMT
DMTMM
BOP-Cl
25
97.5
94.5
0
0.5
1.5
75
2
NAb
N
N
5
HBTU/HOBt
53
47
NA
N
H
10
6
10
DMTMM
90
0
10
a
Product distribution was determined by HPLC (UV absorption, area normalization) analysis of the reaction mixture or of the subsequent ‘Boc-free’ mixture after TFA
treatment.
b
A different, un-identified by-product (4%) was observed.
Table 2
entry 1). Recently, we reported a peptide synthesis protocol that
can significantly reduce racemization using 2-chloro-4,6-dime-
thoxy-1,3,5-triazine (CDMT, 2b) in a one-pot, one-step procedure.6
This methodology was applied to the amide formation between 9
and 5 and yielded racemization-free 6. However, a serious side
reaction involving N-arylation of amine 4 and CDMT contributed
to the formation of 8 as the major product in 75% HPLC yield (Table
1, entry 2).
Coupling of acid 5 with various sterically-hindered amine amines
Entry Amines
% 6a
% 7a
% 8a (N-
(isolated) (racemization) arylation)
CH3
N
N
1
96 (95)
3
1
F
To suppress formation of 8, we decided to probe the same cou-
pling reaction using 4-(4,6-dimethoxy-1,3,5-triazine-2-yl)-4-
methyl-morpholinium chloride (DMTMM, 3), an analogue of
CDMT. Since the discovery of DMTMM by Kaminski a decade
ago,7 several laboratories demonstrated its utility for the synthesis
of esters,8 amides,9 and simple peptides.10 DMTMM is a white so-
lid, commercially available, and stable under certain conditions.11
From a ‘green’ chemistry point of view, DMTMM is attractive. First,
the coupling reactions can be performed in the presence of water.12
Secondly, the byproduct 2-hydroxy-4,6-dimethoxy-1,3,5-triazine
(2a), generated during the reaction, can be easily separated and
recycled for DMTMM manufacturing.11 Since DMTMM can tolerate
water or alcohol,12 peptide synthesis involving serine does not
require the protection of its hydroxyl functionality.10 In a case
study, Kunishima reported the suppression of racemization for a
N
N
H
11
O
O
F
2
>97 (82) <1.0
2
4
N
N
H
12
3
91 (77)
5
0
F
N
H
13
tripeptide synthesis involving Z(OMe)-Gly-L-Ala-OH and H-L-Phe-
N
Obzl.13 Despite all the advantages mentioned above, DMTMM has
not been used extensively for peptide syntheses. Here, we disclose
another attractive utility of DMTMM, especially for the synthesis of
sterically-hindered peptidomimetics.
N
4
92 (79)
8
N
H
14
Gratifyingly, employing DMTMM (instead of CDMT) did afford
the desired peptide 6 in 97.5% yield, along with 0.5% of diastereo-

W.-C. Shieh et al. / Tetrahedron Letters 49 (2008) 5359–5362
5361
Table 2 (continued)
tion (entries 4 and 5). Encouraged by these good results, we
applied DMTMM to the syntheses of several other sterically-hin-
dered analogues to explore the generality. The results are summa-
rized in Table 2. Among five other amines examined, racemization
was determined to be in a range of 0–5%. For the competing N-ary-
lation pathway, adduct 8 was minimized to single-digit percent-
ages (entries 1–4). In one instance, crude product contained 12%
of 8, which was removed from 6 by chromatography without diffi-
culty due to their intrinsic difference in Rf values, and afforded the
desired amide 6 in 73% yield after purification (entry 5).
Entry Amines
% 6a
% 7a
% 8a (N-
arylation)
(isolated) (racemization)
N
N
5
88 (73)
0
12
N
H
15
A typical experimental procedure follows. In a 100-mL, 4-
necked flask equipped with a mechanical stirrer, a thermometer,
and an addition funnel were charged amine 11 (1.4 g, 5 mmol),
acid 5 (1.7 g, 5 mmol), and ethyl acetate (20 mL). The resulting
mixture was cooled to ꢀ20 °C. DMTMM (1.5 g, 5.3 mmol) was
added, and the mixture was allowed to warm to ambient temper-
ature and stirred for an additional 45 min. Any precipitate was
removed by filtration and rinsed with ethyl acetate (10 mL). The
combined filtrate was washed with 0.5 M aqueous citric acid
(20 mL). The organic layer was separated, concentrated under vac-
uum at 25 °C, and purified by chromatography (silica gel) to obtain
the corresponding amide 6 as a foamy solid (2.8 g, 95% yield).15
A plausible pathway proposed in Scheme 1 could be used to ex-
plain the versatility of DMTMM in controlling both racemization
a
Product distribution was determined by HPLC (UV absorption, area normali-
zation) analysis of the reaction mixture or of the subsequent ‘Boc-free’ mixture after
TFA treatment.
mer 7 and 2% of 8 (N-arylation) (Table 1, entry 3). A comparison
study was performed using bis(2-oxo-3-oxazolidinyl)phosphinic
chloride (BOP-Cl) as the coupling reagent, which is known for sup-
pressing racemization on peptide synthesis involving sterically-
hindered amines.14 Coupling amine 9 with acid 5 using BOP-Cl,
we observed 1.5% racemization (Table 1, entry 4), which is compa-
rable to that of DMTMM. In another racemization comparison
study for the coupling of hindered amine 10 with acid 5, DMTMM
was significantly advantageous over HBTU/HOBt: the former con-
tributed an undetectable amount and the latter 47% of racemiza-
H
H
N
CH3
N+
OMe
R
MeO
N
N
N
N
O
N
H
Cl-
R
4
N
OMe
N
MeO
pathway B
slow
3
8
CH3
H3C
N
CH3
pathway A
fast
H
H3C
H
Boc
O
N
N
O
Boc
O
O
N
O
O
N
O
Boc
HO
H
Cl-
H
N
N+
H3C
H3C
CH3
5
D
O
5
4
5
OMe
CH3
pathway A2
H3C
H
CH3
N
N
N
H3C
Boc
N
N
O
O
A
MeO
N
H
OMe
N
Boc
O
4
N
O
+
O
N
pathway A1
fast
H
R
N
OH
MeO
N
2a
6
pathway A3
slow
CH3
CH3
CH3
H3C
N
H3C
N
H3C
N
N
N
N
H
Boc
Boc
Boc
racemization
4
O
O
O
O
H
R
N
O
O
B+H
7
B
C
(B = organic base)
Scheme 1. Plausible pathways for the amide bond formation.

5362
W.-C. Shieh et al. / Tetrahedron Letters 49 (2008) 5359–5362
7. Kamin´ ski, Z. J.; Paneth, P.; Rudzin´ ski, J. J. Org. Chem. 1998, 63, 4248–4255.
8. Kunishima, M.; Morita, J.; Kawachi, C.; Iwasaki, F.; Terao, K.; Tani, S. Synlett
1999, 1255–1256.
9. Kunishima, M.; Kawachi, C.; Iwasaki, F.; Terao, K.; Tani, S. Tetrahedron Lett.
1999, 40, 5327–5330.
10. Falchi, A.; Giacomelli, G.; Porcheddu, A.; Taddei, M. Synlett 2000, 275–277.
11. Kunishima, M.; Hioki, K.; Wada, A.; Kobayashi, H.; Tani, S. Tetrahedron Lett.
2002, 43, 3323–3326.
12. Kunishima, M.; Kawachi, C.; Hioki, K.; Terao, K.; Tani, S. Tetrahedron 2001, 57,
1551–1558.
and N-arylation for the synthesis of sterically-hindered peptidom-
imetics. As described in the experimental procedure above,
DMTMM (3), amine 4, and acid 5 were mixed at low temperature
(ꢀ20 °C) and allowed to warm to ambient temperature for the
completion of reaction. Amide 6 formation presumably went
through an activated-ester A (pathway A), which could react either
with amine 4 (pathway A1) affording 6 or with acid 5 (pathway A2)
furnishing symmetrical anhydride D.16 Intermediate D could react
with 4 to afford 1 equiv each of 6 and 5, which can be recycled to
generate A and subsequently more 6. This ‘one-stage’ procedure,
forming activated-ester A and immediately reacting with amine
4 or acid 5, can shorten the lifetime of A in the solution and min-
imize the probability of transforming it into oxazolones B and C
(pathway A3), which are responsible for the racemization of the
stereogenic center.17 Steric effect presumably is accountable for
the ability of DMTMM to suppress N-arylation. Interaction of bulky
DMTMM with hindered amine 4 leading to 8 (pathway B) was
unfavored (relative to pathway A) due to the pair’s severe steric
hindrance.
13. Kunishima, M.; Kitao, A.; Kawachi, C.; Watanabe, Y.; Iguchi, S.; Hioki, K.; Tani, S.
Chem. Pharm. Bull. 2002, 50, 549–550.
14. (a) Tung, R. D.; Rich, D. H. J. Am. Chem. Soc. 1985, 107, 4342–4343; (b) Tung, R.
D.; Dhaon, M. K.; Rich, D. H. J. Org. Chem. 1986, 51, 3350–3354.
15. Representative analytical data of amides: (a) Amide from 9 and 5: 1H NMR
(500 MHz, CDCl3) d 7.15–7.31 (m, 5H), 6.60 (br s, 1H), 4.64 (t, J = 8.6 Hz, 0.5H),
4.50 (t, J = 8.5 Hz, 0.5H), 4.30 (m, 0.5H), 4.06 (m, 0.5H), 3.72 (q, 9.5 Hz, 0.5H),
3.55–3.65 (m, 1H), 3.45 (m, 0.5H), 3.30 (m, 0.5H), 3.13 (m, 0.5H), 2.75–2.90 (m,
6H), 2.55–2.70 (m, 3H), 2.42 (m, 0.5H), 2.00–2.35 (m, 3.5H), 1.60–1.90 (m, 9H),
1.50 (s, 9H), 1.32 (m, 3H), 0.95–1.25 (m, 6H). Racemization assessment was
performed as follows. The Boc group of crude amide (9 + 5) was removed by
treating with TFA and analyzed with HPLC: Zorbax SB-C18, 3 ꢁ 150 mm, 3.5-
l
m, 40 °C, flow rate = 0.5 mL/min, mobile phase A: CH3CN, mobile phase B:
buffer (0.05 M NaH2PO4, pH 2.5 with H3PO4), equilibrated with A:B = 10:90 for
5 min then gradient: t0 min A:B = 10:90, t12 min A:B = 40:60, t15 min A:B = 90:10.
Retention time: (R,S)-diastereomer = 8.7 min, (S,S)-diastereomer = 9.2 min. (b)
Amide from 11 and 5: 1H NMR (500 MHz, CDCl3) d 8.1 (d, J = 5.0, 1H), 7.23 (dd,
J = 8.9, 5.0 Hz, 2H), 7.1 (t, J = 8.9 Hz, 2H), 6.40 (dd, J = 5.4, 1.3 Hz, 1H), 6.30 (s,
1H), 4.91 (dd, J = 8.2, 4.1 Hz, 1H), 4.60 (dd, J = 8.4, 7.2 Hz, 1H), 3.91 (m, 1H), 3.66
(m, 1H), 3.42 (s, 3H), 2.79 (s, 3H), 2.21 (m, 1H), 1.97 (m, 2H), 1.55–1.80 (m, 7H),
1.48 (s, 9H), 1.32 (d, J = 7.3 Hz, 3H), 1.06–1.26 (m, 4H), 0.90–1.00 (m, 2H); 13C
NMR (125 MHz, CDCl3): d 171.3, 170.4, 161.3, 159.3, 159.0, 147.9, 142.7, 128.4,
128.3, 128.2, 116.6, 116.5, 110.3, 105.6, 60.7, 60.6, 55.0, 48.1, 41.0, 38.9, 35.5,
30.1, 30.0, 28.0, 26.1, 26.0, 25.8, 24.2. MS (ES+ & AP+): m/z = 596 (M+H). (c)
Amide from 13 and 5: 1H NMR (500 MHz, CDCl3) d 8.78 (d, J = 1.9 Hz, 1H), 8.67
(d, J = 2.2 Hz, 1H), 7.92 (t, J = 2.2 Hz, 1H), 7.85 (dd, J = 5.4, 3.5 Hz, 2H), 7.20 (t,
J = 8.5 Hz, 2H), 5.22 (dd, J = 4.7, 3.5 Hz, 1H), 4.64 (t, J = 7.3 Hz, 1H), 4.03 (dd,
J = 10.1, 7.3 Hz, 1H), 3.78–3.87 (m, 1H), 2.78 (s, 3H), 2.36–2.45 (m, 1H), 2.03–
2.13 (m, 2H), 1.87–1.96 (m, 1H), 1.50–1.74 (m, 6H), 1.46 (s, 9H), 1.32 (d,
J = 6.9 Hz, 3H), 0.87–1.21 (m, 6H).
In conclusion, our study demonstrated that 4-(4,6-dimethoxy-
1,3,5-triazine-2-yl)-4-methyl-morpholinium chloride (DMTMM)
is a versatile coupling reagent for the synthesis of sterically-hin-
dered peptidomimetics. It is superior to HBTU/HOBt and CDMT in
controlling racemization and N-arylation, respectively.
References and notes
1. Xu, J. C.; Li, P. Tetrahedron Lett. 1999, 40, 8301–8304.
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Ziegler, J.-C.; Gross, B. Tetrahedron Lett. 1978, 19, 1269–1272; (b) Dourtoglou,
V.; Cross, B. Synthesis 1984, 572–574; (c) Carpino, L. A.; Imazumi, A.; El-Faham,
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C.; Wenschuh, H.; Klose, J.; Beyermann, M.; Bienert, M. Angew. Chem., Int. Ed.
2002, 41, 442–445.
4. Knorr, R.; Trzeciak, A.; Bannwarth, W.; Gillessen, D. Tetrahedron Lett. 1989, 30,
1927–1930.
5. Jou, G.; González, I.; Albericio, F.; Lloyd-Williams, P.; Giralt, E. J. Org. Chem.
1997, 62, 354–366.
16. For symmetric anhydride formation during peptide synthesis, see: Bailey, P. D.
In An Introduction to Peptide Chemistry; John Wiley & Sons: Chichester, 1992;
pp 131–132.
17. (a) For the discussion of racemization of peptide synthesis via oxazolone
intermediate, see: The Peptides; Gross, E., Meienhofer, J., Eds.; Academic Press:
New York, 1979; Vol. 1, Chapter 7; (b) Bailey, P. D. An Introduction to Peptide
Chemistry; John Wiley & Sons: Chichester, 1992; pp 125–129.
6. Garrett, C. E.; Jiang, X.; Prasad, K.; Repic, O. Tetrahedron Lett. 2002, 43, 4161–
4165.