New ion-exchange cum separation technique: A study for the synthesis of ω-guanidine containing peptides using ω-amino acid as surrogate

Article abstract of DOI:10.1080/00908320701230117

This article describes a systematic study for the introduction of ω-guanidine function at a late stage of synthesis using a protected amino group as a surrogate to improve overall yield. This concept was used to design and synthesize pseudo-peptides as GP IIb-IIIa receptor antagonist wherein glycine in endogenous ligand Arg-Gly-Asp (RGD) is replaced by 2-amino-thiazole-4-ylacetic acid (Tha) as a spacer. Further, we describe here a unique salt exchange cum purification technology based on reverse phase (RP-18) medium-pressure liquid chromatography. Copyright Taylor & Francis Group, LLC.

Full text of DOI:10.1080/00908320701230117

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New Ion‐Exchange cum Separation
Technique: A Study for the Synthesis of
ω‐Guanidine containing Peptides using
ω‐Amino Acid as Surrogate
Dr. Ashok K. Gangopadhyay a & Bansi Lal a
a Medicinal Chemistry , Nicholas Piramal Research Centre , Goregaon (East),
Mumbai, India
Published online: 12 Apr 2007.
To cite this article: Dr. Ashok K. Gangopadhyay & Bansi Lal (2007) New Ion‐Exchange cum Separation
Technique: A Study for the Synthesis of ω‐Guanidine containing Peptides using ω‐Amino Acid as Surrogate,
Synthetic Communications: An International Journal for Rapid Communication of Synthetic Organic Chemistry,
37:8, 1389-1419, DOI: 10.1080/00908320701230117
To link to this article: http://dx.doi.org/10.1080/00908320701230117
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Synthetic Communicationsw, 37: 1389–1419, 2007
Copyright # Taylor & Francis Group, LLC
ISSN 0039-7911 print/1532-2432 online
DOI: 10.1080/00908320701230117
New Ion-Exchange cum Separation
Technique: A Study for the Synthesis of
v-Guanidine containing Peptides using
v-Amino Acid as Surrogate
Ashok K. Gangopadhyay and Bansi Lal
Medicinal Chemistry, Nicholas Piramal Research Centre,
Goregaon (East), Mumbai, India
Abstract: This article describes a systematic study for the introduction of v-guanidine
function at a late stage of synthesis using a protected amino group as a surrogate to
improve overall yield. This concept was used to design and synthesize pseudo-
peptides as GP IIb–IIIa receptor antagonist wherein glycine in endogenous ligand
Arg-Gly-Asp (RGD) is replaced by 2-amino-thiazole-4-ylacetic acid (Tha) as a
spacer. Further, we describe here a unique salt exchange cum purification technology
based on reverse phase (RP-18) medium-pressure liquid chromatography.
Keywords: amino group as surrogate, GP IIb–IIa, RGD, salt exchange cum RP-18
purification, v-guanidine function
1
INTRODUCTION
Fibrinogen is a major blood glycoprotein. It plays an essential role in haemo-
stasis. Fibrinogen maintains blood viscosity and has a role in developing the
haemostatic plug.[1] Higher levels of fibrinogen have found to be associated
with increased age, high blood pressure, obesity, smoking, high stress, and
diabetes.
Received September 15, 2004
Address correspondence to Dr. Ashok K. Gangopadhyay, Nicholas Piramal
Research Centre, Nicholas Piramal India Ltd., 1A, 1B & 1C Nirlon Complex, off
Western Express Highway, Goregaon (East), Mumbai 400 063, India. Tel.: 91-22-
30818312; Fax: 91-22-30818036; E-mail: agangopadhyay@nicholaspiramal.co.in
1389

1390
A. K. Gangopadhyay and B. Lal
With the discovery of inhibitory effects of Arg-Gly-Asp (RGD) residue
against the binding of fibrinogen to GP IIb–IIIa, a plethora of information
accumulated in the literature describing synthetic mimics of RGD as GP
IIb–IIIa antagonists in both peptide and nonpeptide areas.[2–8] The essential
features identified during previous structure–activity studies indicate the
presence of a basic functional moiety such as guanidino, amino, or benzami-
dino and a carboxylic acid moiety appropriately oriented with respect to each
other. It is equally important to place them at a certain distance apart on a
suitable template.
We have designed our molecule for the synthesis of GP IIb–IIIa antagonist
based on the idea of replacing glycine with a small spacer. We had used the
2-amino-thiazole-4-yl-acetyl group as a glycine substitute. The assumption
was that the amino group of thiazole would make a pseudo-peptide linkage
while the CH2 group between the thiazole ring and carboxylic group, because
of its free rotation, would achieve the right orientation. We also believed that
replacement of arginine by other v-guanidino acids would help us to optimize
the critical distance between the basic and the acidic moiety as well as the role
of a-amino group of arginine. It is well established now that introduction of a
hydrophobic residue around C-terminus and amidation of a-carboxylic acid of
Asp residue increases GP IIb–IIIa antagonistic activity.[2,9] We had incorporated
these thoughts in our synthetic strategy. Thus we had decided to synthesize non-
peptides of the general structure shown in Fig. 1. Another objective of the present
study was to develop a synthetic strategy for the introduction of v-guadino func-
tionality at a late stage of synthesis.
2
RESULTS AND DISCUSSION
Scheme 1 describes our initial strategy to maintain Arg residue at N-terminus,
AspNHR as C-terminus, and 2-amino-thiazol-4-yl acetyl (Tha) as spacer. The
C-terminal intermediate Asp (OBzl) NHR was prepared in two steps. First,
Boc-Asp (OBzl)[10] was converted into the corresponding amide by mixed
anhydride procedure using isobutyl chloroformate and NMM.[11] In the next
step, the Boc group was removed by formic acid–anisole.[12] The amino
Figure 1. (R1 ¼ NH2 or H; X ¼ (CH2)n; n ¼ 0–3).

Ion-Exchange cum Separation Technique
1391
Scheme 1. i) DCC, HOBt, CH2Cl2, DMF; ii) 1-methyl morpholine, isobutyl chloro-
formate, CHCl3, 2158C; iii) 85% aqueous AcOH, D, 10 min; iv) trifluoromethanesul-
fonic acid, TFA; v) purification, HCl–ether.
group of Tha was protected by tritylation using trimethylsilyl as transient
acid protection as reported earlier[13] to obtain 1. The reaction of 1
with different Asp (OBzl)–NHR in the presence of DCC–HOBt[14] or
the mixed anhydride method resulted in compounds 2–10. The Trit group
from 2–10 was removed by treatment with 85% AcOH.[15] The resulting
amine component was coupled with tri-Z-Arg[16] by the mixed anhydride
procedure[11] to yield compounds 11–19. The yield of the coupling of
Arg residue was found to be poor in DMF (20–35%), however,
when THF–CH2Cl2 (or better CHCl3) replaced DMF, the yield improved
to 45–55%. The simultaneous removal of both Z and Bzl from 11–19
was carried out by treatment with trifluoromethanesulfonic acid in
TFA[17] followed by reverse-phase MPLC purification to give 20 to 28 in
good yield.
To examine the effect of chain length at N-terminus on activity, the Arg
residue was replaced by v-guanidino acid as shown in Scheme 2 (n ¼ 0 or 1;
R1 ¼ H, R2 ¼ Phe in Fig. 1) and Scheme 3 (n ¼ 5; R1 ¼ H, R2 ¼ nBu in
Fig. 1).
2.1 New Systematic Approach
As described previously, we have observed poor yield while coupling Arg
residue with the C-terminal fragment. Thereafter, we developed a novel

1392
A. K. Gangopadhyay and B. Lal
Scheme 2. i) DCC, HOBt, EtOAc; ii) 1N NaOH, MeOH; iii) H-Asp (OBzl-Phe-
OBzl . HCl, Et3N, DCC, HOBt; iv) Formic acid, anisole, room temperature; v) 85%
aqueous AcOH, 1008C, 10 min; vi) Et3N, dicartbobenzoxy-S-methyl isothiourea,
CH2C12, reflux; vii) trifluoromethanesulfonic acid, TFA, rt.
synthetic strategy in which the guanidino functionality was introduced at the
later stage once the coupling of all the fragments was over. The highly efficient
method for introduction of protected guanidino group to amino acid was
published by us[16b] and others.[16c] The protected amino group was used as
a precursor for guanidine. Thus Boc-Gly[18] or Trit-b-Ala[19] was coupled
with Tha-OEt[20] in the presence of DCC-HOBt to yield compounds 29 and
30 in high yield as shown in Scheme 2. The ethyl esters 29 and 30 were hydro-
lyzed with 1 N NaOH and MeOH to the corresponding acid 31 and 32 respect-
ively. The free acids 31 and 32 were coupled with Asp (OBzl) Phe-OBzl
[obtained by removing amino protection from Boc-Asp (OBzl)-Phe-OBzl
by treatment with formic acid–anisole] by DCC–HOBt to get 33 and 34 in
high yield. At this stage, the amino protecting groups from 33 and 34 were
removed by treatment with formic acid–anisole and 85% AcOH respectively.
The resulting amine salt (after neutralizing with Et3N) was refluxed with
dicarbobenzoxy-S-methyl isothiourea[16b,21] to get the Z-protected
guanidino compounds 35 and 36 in good yield. Finally, all the protecting
groups were removed by treating 35 and 36 with trifluoromethansulfonic
acid in TFA, which after purification as described next yielded final
compounds 37 and 38. The synthesis of compound 44, in which 1-gunidino
haxanoyl moiety replaced the Arg residue, was carried out according to
Scheme 3 using 1-amino acid as precursor. Thus Boc-1-Ahx[22] was

Ion-Exchange cum Separation Technique
1393
Scheme 3. i) DCC, HOBt, EtOAc; ii) 1N NaOH, MeOH; iii) H-Asp (OBzl)-OH, Et3N,
Tms-Cl; iv) N-methyl morpholine, isobutyl chloroformate, DMF; v) Formic acid, ani-
sole, rt; vi) Et3N, di-cartbobenzoxy-S-methyl isothiourea, CH2C12, CH3CN, reflux; vii)
Trifluoromethanesulfonic acid, TFA, rt, Purification and salt exchange; viii) HCl-ether.
coupled with Tha-OEt in the presence of DCC–HOBt to obtain 39. It was
hydrolyzed with 1N NaOH in MeOH, giving free acid 40. The a-carboxylic
group of Asp (OBzl) was protected in situ as trimethyl silyl ester using
Tms-Cl. The resulting ester hydrochloride was neutralized with triethyl
amine and then coupled with 40 in the presence of DCC–HOBt.
The temporary protection was removed by treatment with MeOH and
usual workup yielded Boc-1-Ahx-Tha-Asp (OBzl)-OH (41) in very good
yield (84%). The activation of 41 through the mixed anhydride method
followed by treatment with nBuNH2 afforded 42, which was converted to
the final compound 44 through the intermediate 43 by the same method as
described for the synthesis of compound 37.
The two protons attached to the b-carbon of Asp moiety in most cases
showed well separated jeminal coupling followed by vicinal coupling with
C-a-proton, resulting in two sets of doublets. Considering the general
structure in Fig. 1, the proton trans to C-a-H appeared upfield (d 2.6–2.8)
1
with higher vicinal coupling constant value (6–8 Hz) in H NMR spectrum
and is assigned Hb. On the other hand, the proton cis to C-a-H appears
downfield (d 2.8–3.05) with lower vicinal coupling constant value (4–5 Hz)
and is assigned Ha.

1394
A. K. Gangopadhyay and B. Lal
2.2 Simple New Method Developed for Salt Exchange and
Purification
Initially we had encountered difficulty in exchanging trifluoromethanesulfo-
nate salt of the final compounds into any other biologically acceptable
salt(s). Direct exchange with HCl or use of a different type of ion
exchanger failed to give desired results. Finally, we developed a unique
reverse-phase purification protocol. Thus the crude material was dissolved
in cold water basified the solution by adding cold dilute NaOH to make the
guanidino moiety free basified the solution. Because guanidine is a strong
base, use of organic bases or a weak inorganic base could not be used. The
solution was immediately loaded on RP-18 MPLC column. After initial
washing with water (to remove sodium trifluoromethanesulfonate), further
purification was carried out using the required eluting solvent that always
contained acetic acid (see Experimental). The pure product was obtained as
acetate salt. Thus we call this technique as “salt exchange cum purification.”
The acetate salt thus obtained was converted into hydrochloride salt by the
usual procedure. The same technique was utilized in all the cases where tri-
fluoromethanesulfonic acid salt had to be exchanged. Thus compounds 20–
28, 37, 38, and 44 could be obtained in reasonably good yields.
The compounds synthesized in the present series were tested for GP IIb–
IIIa receptor antagonism; however, they did not show any significant activity.
The introduction of Tha in place of Gly possibly has either altered the orien-
tation of N-terminus guanidine and C-terminus carboxylic groups or the
required length was not achieved for the RGD type of binding.
3
CONCLUSION
We have successfully synthesized the newly designed analogues of RGD,
wherein Tha, a heterocyclic to give metabolic stability, replaced the central
glycine part. However, this did not yield any positive result in terms of
FRA activity.
While executing the present study, we have developed a systematic approach
to synthesizing v-guanidino acid containing peptides/pseudo peptides by
carrying the surrogate amino acid untill the late stage of peptide assembly. This
approach enabled us to overcome the low yield associated with the coupling of
Arg or other guanidino acid. As far as we know, this is the first systematic
study wherein a protected v-guanidino group was introduced at a late stage of
the synthesis. There is another example in the literature where a Boc-protected
guanidino group was introduced at the final stage of synthesis for Daptomycin
analogues. However, they used f[tert-butoxycarbonylimino]-pyrazol-1-yl-
methylg-carbamic acid tert-butyl ester as guanidinylating agent.[23]
During the present investigation, we developed a unique reverse-phase
(RP-18) purification protocol whereby in one step, exchange of unwanted

Ion-Exchange cum Separation Technique
1395
trifluorosulfonate salt followed by purification of the desired compound could
be achieved. In principle, this can be applied to any salt exchange. We believe
this method may find wide application in purification technology.
4
EXPERIMENTAL
4.1 General Procedures
Melting points were determined with a Kofler hot-stage apparatus and are
uncorrected. IR spectra were determined with a Perkin-Elmer 157 spectropho-
tometer as KBr film unless otherwise mentioned. 1H NMR spectra were
recorded in CDCl3 unless otherwise mentioned on a Jeol FT-90 spectrometer
or Bruker ACP 300 spectrometer with TMS as internal standard, and
coupling constant values are expressed in Hertz. Light petroleum refers to
the fraction of bp 60–808C. For flash Column chromatography, silica gel
(finer than 0.08-mm particle size) was used. For reverse-phase MPLC purifi-
cation, RP-18 (30-m particle size) was used. Precoated (silica-gel 60 F254
)
TLC plates were used for checking purity of compounds. UV light at
366 nm and 254 nm as well as spray reagents such as 0.2% ninhydrin in
acetone, Draggendroff, and I2 vapor was used to detect the spots on TLC.
All compounds were homogeneous on TLC and gave proper spectral character-
istics. The yield of the reactions and physicochemical properties were
described in Table 1 for most of the compounds.
4.2 Boc-Asp (OBzl)-Val-OBzl
A solution of Boc-Asp (OBzl)-OH (4.8 g; 15 mmol) in DMF (30 ml) was
chilled to 2308C. 1-Methyl morpholine (1.65 ml; 15 mmol) and isobutyl-
chloroformate (1.95 ml; 15 mmol) were added to the solution in quick succes-
sion. In a separate round-bottomed flask, Val-OBzl–PTSA[24] (1.25 g;
16.5 mmol) was dissolved in DMF (20 ml) and chilled to 2308C and neutralized
with Et3N (2.31 ml; 16.5 mmol). The resulting free amine was added to the
mixed anhydride after 2 min. The reaction mixture was stirred at 2208C for
1 h. It was stored in a freezer overnight. Solvent was removed under reduced
pressure, and the residue was stirred with EtOAc (30 ml) and 1N NaHCO3
(10 ml) and transferred into a separating funnel. The organic layer was
separated and washed with water, 1N citric acid, and brine. It was dried over
anhydrous Na2SO4. Solvent was removed, and the residue was purified by crys-
tallization from EtOAc–light petroleum to yield 7.49 g (96.4%) of the title
compound. IR (KBr): 3360–3300 (br), 2950, 1730 (br), 1685, 1670 cm21; 1H
NMR (60 MHz): 0.84, 0.91 [2 ꢀ d, 6H, J ¼ 7.6, CH (CH3)2], 1.43 [s, 9H, C
(CH3)3], 2.85 [m, 2H, CH (CH2COO-)-], 4.4–4.7 (2 ꢀ m, 2 ꢀ CaH of Asp
and Val), 5.12 (s, 4H, 2 ꢀ PhCH2O), 7.32 (br, 10H, PhH).

Table 1. Yield and physicochemical properties for identification of compounds
Sub.
no.
Yield
(%)
Analysis MF; calc. (found)
(%)
Mp (8C)
162–64
IR (KBr) (cm21
)
1H NMR
3
62.6
3270 (br), 1730 (br),
1635 (br)
2.14, 2.21 (2 ꢀ d, 6H, J ¼ 7.1), 2.63 (dd, 1H,
Jgem ¼ 17.2, J ¼ 6.0), 3.03 (dd, 1H, Jgem ¼ 17.2,
J ¼ 5.06), 3.49 (s, 2H), 4.0 (heptate, 1H), 4.64–
4.86 (m, 1H), 5.0 (s, 2H), 6.10 (s, 1H), 6.29
(m, 1H), 6.86 (br, 1H), 7.29–7.40 (m, 20H), 8.37
(d, 1H)
C38H38N4O4S: C, 70.56; H, 5.92;
N, 8.66; S, 4.96 (C, 70.43; H,
5.94; N, 8.56; S, 4.83)
4
5
7
57.08
58.9
117–19
127–29
117–18
3260 (br), 2930 (br),
1740 (br), 1645 (br)
0.84 (t, 3H, J ¼ 6.1), 1.11–1.50 (m, 4H), 2.64 (dd, C39H40N4O4S: C, 70.88; H, 6.10;
1H, Jgem ¼ 17.2, J ¼ 6.08), 3.05 (dd, 1H,
Jgem ¼ 17.2, J ¼ 4.56), 3.17 (m, 2H), 3.46 (s, 2H),
4.80 (m, 1H), 4.97 (s, 2H), 6.07 (s, 1H), 6.81 (br,
1H), 7.24–7.35 (m, 20H) 8.43 (d, 1H)
N, 8.48; S, 4.85(C, 70.65; H,
6.18; N, 8.42; S, 4.71)
3280 (br), 2940 (br),
1735 (br), 1640 (br)
0.84, 0.91 (2 ꢀ s, 6H), 1.56–1.97 (m, 1H), 2.64 (dd, C39H40N4O4S: C, 70.88; H, 6.10;
1H, Jgem ¼ 17.2, J ¼ 7.1), 2.94–3.17 (m, 3H),
3.50 (s, 2H), 4.81 (m, 1H), 5.01 (s, 2H), 6.09
(s, 1H), 6.66 (br, 1H), 6.86 (br, 1H), 7.23–7.40
(m, 20H) 8.51 (d, 1H)
N, 8.48; S, 4.85 (C, 70.83; H,
6.14; N, 8.41; S, 4.96)
97.83
3280 (br), 2970, 2940, 0.76, 0.84 (2 ꢀ t, 6H), 1.10–1.57 (m, 4H), 2.60 (dd, C40H42N4O4S: C, 71.19; H, 6.27;
1740 (br), 1640 (br)
1H, Jgem ¼ 17.2, J ¼ 6.08), 3.05 (dd, 1H, Jgem
¼
N, 8.30; S, 4.75 (C, 70.91; H,
6.31; N, 8.25; S, 4.66)
17.2, J ¼ 4.05), 3.49 (s, 2H), 3.57–3.80 (m, 1H),
4.71–4.91 (m, 1H), 5.01 (s, 2H), 6.09 (s, 1H), 6.86
(br, 1H), 7.23–7.40 (m, 20H), 8.53 (d, 1H)

8
9
78.2
53.3
Oily
3350–3330, 2970,
1745 (br), 1735 (br),
1660 (br)
0.83–0.84 (m, 9H), 2.61 (dd, 1H, Jgem ¼ 17.2,
J ¼ 6.08), 2.81–3.13 (m, 3H), 3.49 (s, 2H), 4.73–
4.96 (m, 1H), 5.0 (s, 2H), 6.09 (s, 1H), 6.89 (br,
1H), 7.20–7.43 (m, 20H), 8.63 (m, 1H)
C40H42N4O4S: C, 71.19; H, 6.27;
N, 8.30; S, 4.75 (C, 71.01; H,
6.30; N, 8.13; S, 4.73)
113–15
3300–3260 (br), 2930, 0.85–1.77 (3 ꢀ m, 10H), 2.64 (dd, 1H, Jgem ¼ 17.2, C40H40N4O4S: C, 71.69; H, 6.16;
2850, 1740, 1645
(br)
J ¼ 6.08), 3.04 (dd, 1H, Jgem ¼ 17.2, J ¼ 4.05,
3.49 (s, 2H), 4.77 (m, 1H), 5.01 (s, 2H), 6.09
(s, 1H), 6.37, 6.83 (2 ꢀ br, 2H), 7.23–7.40
(m, 20H), 8.34 (m, 1H)
N, 8.16; S, 4.67 (C, 71.82; H,
6.34; N, 7.97; S, 4.58)
10
11
67.07
34.6
Oily
3300 (br), 2980 (br),
1740 (br), 1685 (br)
1655 (br)
0.80–0.97 (m, 6H), 1.86–2.31 (br, 1H), 2.68 (dd,
1H, Jgem ¼ 17.2, J ¼ 6.08), 3.05 (2 ꢀ quartet, 1H,
Jgem ¼ 17.2, J ¼ 4.05, 2.0), 3.49 (s, 2H), 4.49 (dd,
1H), 4.74–5.17 (m, 5H), 6.06 (s, 1H), 6.97
C47H46N6O4S: C, 71.01; H, 5.83;
N, 7.05; S, 4.03 (C, 71.14; H,
5.81; N, 6.98; S, 4.10)
(2 ꢀ br, 2H), 7.23–7.37 (m, 20H), 8.57 (m, 1H)
107–09,
3405, 3320 (br), 2980 (300 MHz): 0.73 (t, 3H), 1.30 (m, 2H), 1.62–1.82
C49H54N8O11S: C, 61.11; H,
5.65; N, 11.64; S, 3.33
(C, 60.93; H, 5.61; N, 11.49; S,
3.25)
EtOAc–
light
petroleum
(br), 1750 (br), 1660
(br)
(m, 4H), 2.64 (dd, 1H, Jgem ¼ 17.85, J ¼ 7.1),
2.87 (dd, 1H, Jgem ¼ 17.85, J ¼ 4.5), 3.0 (m, 2H),
3.49 (s, 2H), 3.88–4.0 (m, 2H), 4.50, 4.72, 4.90
(3 ꢀ m, 3H), 4.95–5.15 (m, 8H), 6.15, 6.46
(2 ꢀ m, 2H), 6.61 (s, 1H), 7.12–7.35 (m, 20H),
7.70, 9.28, 9.35 (3 ꢀ m, 3H)
(continued)

Table 1. Continued
Sub.
no.
Yield
(%)
Analysis MF; calc. (found)
(%)
Mp (8C)
IR (KBr) (cm21
)
1H NMR
12
13
14
52.07
170–72,
EtOAc–
light
3405, 3320 (br), 2980 (300 MHz): 0.90 (d, 6H), 1.0–1.9 (2 ꢀ m, 4H),
C49H54N8O11S: C, 61.11; H,
5.65; N, 11.64; S, 3.33
(C, 61.18; H, 5.61; N, 11.55; S,
3.40)
(br), 1750 (br), 1660
(br)
2.59 (dd, 1H, Jgem ¼ 17.6, J ¼ 6.0), 2.85 (dd,
1H, Jgem ¼ 17.6, J ¼ 4.0), 3.38 (m, 1H), 3.50
(s, 2H), 3.80 (m, 2H), 4.45, 4.65, 4.90 (3 ꢀ m,
3H), 4.90–5.10 (m, 8H), 6.05, 6.15 (2 ꢀ br, 2H),
6.65 (s, 1H), 7.08–7.30 (m, 20H), 7.75, 9.12,
9.30 (3 ꢀ m, 3H)
petroleum
29.5
84–86,
EtOAc–
light
3405, 3300 (br), 2975 (300 MHz): 0.76 (t, 3H), 0.95–1.30, 1.60–1.90
C50H56N8O11S: C, 61.46; H,
5.78; N, 11.47; S, 3.28
(C, 61.27; H, 5.88; N, 11.29; S,
3.22)
(br), 1730 (br), 1660
(br)
(2 ꢀ m, 8H), 2.60 (dd, 1H, Jgem ¼ 17.5, J ¼ 7.8),
2.85 (dd, 1H, Jgem ¼ 17.5, J ¼ 4.5), 3.05 (m, 2H),
3.52 (s, 2H), 3.90 (m, 2H), 4.52, 4.72, 4.98
(3 ꢀ m, 3H), 5.0–5.15 (m, 8H), 6.15, 6.48
(2 ꢀ br, 2H), 6.63 (s, 1H), 7.13–7.30 (m, 20H),
7.83, 9.25, 9.40 (3 ꢀ m, 3H)
petroleum
55.95
84–86,
EtOAc–
light
3400, 3310 (br), 2970 (300 MHz): 0.78, 0.88 (2 ꢀ d, 6H), 1.58–1.78
C50H56N8O11S: C, 61.46; H,
5.78; N, 11.47; S, 3.28
(C, 61.11; H, 6.06; N, 11.14; S,
3.11)
(br), 1750 (br),
1670–1650 (br)
(m, 5H), 2.70 (dd, 1H, Jgem ¼ 17.7, J ¼ 7.8),
2.89–3.08 (m, 3H), 3.58 (s, 2H), 3.95 (m, 2H),
4.55, 4.80, 5.0 (3 ꢀ m, 3H), 5.05–5.25 (m, 8H),
6.15, 6.63 (2 ꢀ br, 2H), 6.72 (s, 1H), 7.0 (br, 1H),
7.25–7.38 (m, 20H), 7.70, 8.21, 9.40 (3 ꢀ m, 3H)
petroleum

15
16
17
42.7
47.9
48
86–88,
EtOAc–
light
3400–3300 (br), 2960 (300 MHz): 0.85 (d, 6H), 1.2, 1.68 (2 ꢀ m, 6H), 1.45 C51H58N8O11S: C, 61.80; H,
(br), 1730 (br), 1690,
1645 (br)
(m, 1H), 2.65 (dd, 1H, Jgem ¼ 17.5, J ¼ 7.8), 3.00
(dd, 1H, Jgem ¼ 17.5, J ¼ 5.0), 3.08 (m, 2H), 3.58
(s, 2H), 3.92 (m, 2H), 4.52, 4.72, 4.98 (3 ꢀ m, 3H),
5.01–5.20 (m, 8H), 6.13, 6.40 (2 ꢀ br, 2H), 6.69
(s, 1H), 7.12–7.32 (m, 20H), 7.75, 9.29, 9.40
(3 ꢀ m, 3H)
5.89; N, 11.31; S, 3.23
(C, 61.65; H, 5.51; N, 10.93; S,
3.05)
petroleum
85–87,
3420, 3320 (br), 2950 (300 MHz): 0.85 (d, 6H), 1.36, 1.50, 1.79 (3 ꢀ m,
C51H58N8O11S: C, 61.80; H,
5.89; N, 11.31; S, 3.23
(C, 61.54; H, 5.58; N, 11.02; S,
3.27)
EtOAc–
light
petroleum
(br), 1740 (br), 1695,
1650 (br)
8H), 2.79 (dd, 1H, Jgem ¼ 17.75, J ¼ 7.8), 3.02
(dd, 1H, Jgem ¼ 17.75, J ¼ 5.07), 3.68 (s, 2H),
3.70 (m, 1H), 4.05 (m, 2H), 4.62, 4.92, 5.12
(3 ꢀ m, 3H), 5.12–5.30 (m, 8H), 6.15, 6.30
(2 ꢀ br, 2H), 6.79 (s, 1H), 7.32–7.50 (m, 20H),
8.05 (br, 1H), 9.39, 9.50 (2 ꢀ br, 2H)
89–90,
3430, 3335 (br), 2990 (300 MHz): 0.87 (s, 9H), 1.76–2.08 (2 ꢀ m, 4H),
C51H58N8O114S: C, 61.80; H,
5.89; N, 11.31; S, 3.23
(C, 61.72; H, 5.68; N, 11.27; S,
3.09)
EtOAc–
light
petroleum
(br), 1740 (br), 1695
(br), 1660 (br).
2.78 (dd, 1H, Jgem ¼ 17.2, J ¼ 7.8), 3.03 (m, 3H),
3.66 (s, 2H), 4.02 (m, 2H), 4.63, 4.88, 5.04
(3 ꢀ m, 3H), 5.13–5.28 (m, 8H), 6.22, 6.70
(2 ꢀ br, 2H), 6.77 (s, 1H), 7.27–7.45 (m, 20H),
7.88 (br, 1H), 9.32, 9.48 (2 ꢀ br, 2H)
(continued)

Table 1. Continued
Sub.
no.
Yield
(%)
Analysis MF; calc. (found)
(%)
Mp (8C)
88–90,
IR (KBr) (cm21
)
1H NMR
18
55
3420 (br), 3320 (br),
2960 (br), 1740 (br),
1700, 1660 (br)
(300 MHz): 0.88–1.20 (m, 10H), 2.10–2.48 (2 ꢀ m, C52H58N8O11S: C, 62.26; H,
EtOAc–
light
petroleum
4H), 2.70 (dd, 1H, Jgem ¼ 17.5, J ¼ 7.8), 2.95
(m, 1H), 3.45 (s, 2H), 3.57 (m, 1H), 3.97 (m, 2H),
4.60, 4.77, 5.03 [3 ꢀ m, 3H), 5.05–5.23 (m, 8H),
6.17, 6.37 (2 ꢀ br, 2H), 6.70 (s, 1H), 7.20–7.38
(m, 20H), 7.80 (br, 1H), 9.25, 9.40 (2 ꢀ br, 2H)
5.83; N, 11.17; S, 3.19
(C, 62.36; H, 5.87; N, 11.09; S,
3.22)
19
21
22
43.56
50.7
Glassy solid 3400–3300 (br), 2970 (300 MHz): 0.75–0.95 (m, 6H), 1.65–1.85 (m, 4H), C58H62N8O5S: C, 62.69; H, 5.62;
(br), 1740 (br), 1700
(br), 1675 (br)
2.15 (m, 1H), 2.70 (m, 1H), 3.05 (m, 1H), 3.60
(s, 2H), 4.0 (m, 2H), 4.50, 4.88, 4.96 (3 ꢀ m, 4H),
5.02–5.15 (m, 10H), 6.04 (m, 1H), 6.73 (s, 1H),
7.20–7.45 (m, 25H), 9.28, 9.42 (2 ꢀ br, 2H)
N, 10.08; S, 2.89 (C, 62.48; H,
5.39; N, 10.11; S, 2.72)
188–92
3500–2950 (br), 1730 (D2O; 300 MHz): 1.15 (m, 6H), 1.69, 2.07 (2 ꢀ m, C18H30N8O5S: 2HCl, 2H2O; C,
(br), 1665 (br
4H), 2.81 (dd, 1H, Jgem ¼ 17.5, J ¼ 7.5), 2.90 (dd,
1H, Jgem ¼ 17.5, J ¼ 4.5), 3.23 (t, 2H), 3.74
(s, 2H), 3.91 (m, 1H), 4.32, 4.62 (2 ꢀ t, 2H,
J ¼ 5.63, J ¼ 7.5), 7.05 (s, 1H)
36.17; H, 6.41; N, 18.75; Cl,
11.87; S, 5.37 (C, 35.97; H,
6.31; N, 18.59; Cl, 11.86; S,
5.26)
70.32
170–75 (d) 3400–3300 (br), 2940 (D2O; 300 MHz): 0.85 (t, 3H), 1.18, 1.42 (2 ꢀ m,
C19H31N8O5S; 2HCl, 3H2O: C,
37.31; H, 6.59; N, 18.32; Cl,
11.60; S, 5.24 (C, 35.44; H,
6.41; N, 18.18; Cl, 11.47; S,
5.32)
(br), 1720 (br), 1655
(br)
4H), 1.70, 2.06 (2 ꢀ m, 4H), 2.88 (m, 2H), 3.0–
3.12 (m, 4H), 3.72 (s, 2H), 4.35, 4.68 (2 ꢀ m, 2H),
7.08 (s, 1H)

23
57
172–74
3450–3300 (br), 2970 (D2O; 300 MHz): 0.77, 0.80 (2 ꢀ d, 6H), 1.58–1.72 C19H31N8O5S; HCl, H2O: C,
(br), 1720 (br),
1680–1660 (br)
(m, 1H), 1.98–2.06 (2 ꢀ m, 4H), 2.49–3.25
(3 ꢀ m, 6H), 3.70 (s, 2H), 4.29 (t, 1H), 4.51
(m, 1H), 7.03 (s, 1H)
42.33; H, 6.50; N, 20.79; Cl,
6.57; S, 5.95 (C, 42.46; H,
6.54; N, 20.54; Cl, 6.42; S,
6.01)
24
25
77.2
74.1
160–63
3400–3200 (br), 2970 (D2O; 300 MHz): 0.83 (br, 6H), 1.28 (m, 2H), 1.43 C20H33N8O5S; 2HCl: C, 38.95;
(br), 1740 (br), 1670
(br)
(m, 1H), 1.79, 2.10 (2 ꢀ m, 4H), 2.78–2.95
(m, 2H), 3.16–3.29 (m, 4H), 3.70 (s, 2H), 4.32,
4.66 (2 ꢀ m, 2H), 7.06 (s, 1H)
H, 6.70; N, 18.17; Cl, 11.50; S,
6.20 (C, 38.79; H, 6.35; N,
17.85; Cl, 11.36; S, 5.29)
195–200
3350–3200 (br), 2990 (D2O; 300 MHz): 0.79 (m, 6H), 1.30, 1.51 (2 ꢀ m, C20H34N8O5S, 2HCl, H2O: C,
(br), 1745 (br),
1690–1670 (br)
4H), 1.73, 2.06 (2 ꢀ m, 4H), 2.77–3.02 (m, 2H),
3.21 (m, 2H), 3.55 (m, 1H), 3.73 (s, 2H), 4.33, 4.68
(2 ꢀ m, 2H), 7.09 (s, 1H)
40.74; H, 6.49; N, 19.09; Cl,
12.03; S, 5.44 (C, 40.87; H,
6.12; N, 18.88; Cl, 12.23; S,
5.41)
26
27
66
188–191
195–199
3500–3300 (br), 2980 (D2O; 300 MHz): 0.83 (s, 9H), 1.75, 2.05 (2 ꢀ m,
C20H34N8O5S, 2HCl, 2H2O: C,
39.53; H, 6.64; N, 18.44; Cl,
11.67; S, 5.28 (C, 39.57; H,
6.44; N, 18.32; Cl, 11.76; S,
5.36)
(br), 1740 (br),
1685–1660 (br)
4H), 2.80–3.03 (m, 2H), 3.0 (s, 2H), 3.28 (t, 2H),
3.72 (br, 2H), 4.35, 4.79 (2 ꢀ m, 2H), 7.08 (s, 1H)
66.67
3400–3250 (br), 2910 (D2O; 300 MHz): 1.0–1.30, 1.50–1.80, 2.01–2.12 C21H34N8O5S, 2HCl, 1.5 H2O:
(br), 1725 (br),
1670–1640 (br)
(3 ꢀ m, 15H), 2.80–2.98 (m, 2H), 3.25 (t, 2H),
3.58 (br, 2H), 4.31, 4.62 (2 ꢀ m, 2H), 7.05 (s, 1H).
C, 41.31; H, 6.44; N, 18.35;
Cl, 11.61; S, 5.25 (C, 41.22; H,
6.59; N, 18.08; Cl, 11.76; S,
4.98)
(continued)

Table 1. Continued
Sub.
no.
Yield
(%)
Analysis MF; calc. (found)
(%)
Mp (8C)
IR (KBr) (cm21
)
1H NMR
28
29
63.97
196–202
3400–3300 (br), 2960 (D2O; 300 MHz): 0.88 (m, 6H), 1.63–1.76 (m, 2H), C20H32N8O7S, 2HCl, 2H2O: C,
(br), 1725 (br),
1670–1640 (br)
2.01–2.19 (m, 3H), 2.83–2.95 (2 ꢀ dd, 2H,
Jgem ¼ 15.4, J ¼ 7.7, 6.6), 3.23 (t, 2H), 3.72 (br,
2H), 4.22, 4.31, 4.72 (3 ꢀ m, 3H), 7.05 (s, 1H)
37.68; H, 6.01; N, 17.58; Cl,
11.12; S, 5.03 (C, 37.92; H,
6.15; N, 17.35; Cl, 11.07; S,
4.87)
94.6
168–69
3300 (br), 3000–2930 1.21 (t, 3H), 1.46 (s, 9H), 3.63 (s, 2H), 4.08 (s, 2H), C14H21N3O5S: C, 48.96; H, 6.16;
(br), 1730, 1705,
1680 (br)
4.09 (quartet, 2H), 6.74 (s, 1H).
N, 12.24; S, 9.34 (C, 49.13; H,
6.24; N, 12.11; S, 9.57)
30
31
74.7
89.9
148–50
176–78
3000–2950 (br), 1745 1.31 (t, 3H), 2.63 (br, 4H), 3.74 (s, 2H), 4.09 (quartet, C29H29N3O3S: C, 69.71; H, 5.85;
(br), 1650 (br)
2H), 6.83 (s, 1H), 7.23–7.54 (m, 15H)
N, 8.41; S, 6.42 (C, 69.77; H,
5.92; N, 8.24; S, 6.67)
3365, 1685 (br), 1675 (CDCl3 þ DMSO-D6): 1.46 (s, 9H), 3.63 (s, 2H),
C12H17N3O5S: C, 45.70; H, 5.44;
N, 13.33; S, 10.17; (C, 45.76;
H, 5.52; N, 13.19; S, 10.40)
4.26 (d, 2H), 6.20 (br, 1H), 6.74 (s, 1H)
32
33
92.7
205–06
93–95
3210 (br), 3075 (br),
1745 (br), 1710,
1565 (br)
(CDCl3 þ DMSO-D6): 2.51, 2.57 (2 ꢀ t, 4H), 3.53 C27H25N3O3S: C, 68.77; H, 5.34;
(s, 2H), 6.73 (s, 1H), 7.11–7.50 (m, 15H)
N, 8.91; S, 6.80 (C, 68.66; H,
5.29; N, 8.92; S, 6.84)
77.24
3350 (br), 2980 (br),
1730 (br), 1675,
1665, 1655
(300 MHz): 1.45 (s, 9H), 2.69 (dd, 1H), 3.03 (d, 2H), C39H43N2O7S: C, 61.81; H, 5.72;
3.18 (dd, 1H), 3.56 (s, 2H), 3.95 (d, 2H), 4.88, 5.02
(2 ꢀ m, 2H), 5.12, 5.23 (2 ꢀ s, 4H), 6.61 (s, 1H),
7.03–7.40 (m, 10H)
N, 9.24; S, 4.23; (C, 61.75; H,
5.61; N, 9.07; S, 4.05)

34
36
87.2
79–81
74–75
3310 (br), 1750 (br),
1700 (br), 1670 (br)
(300 MHz): 2.43 (s, 4H), 2.62 (dd, 1H), 2.94 (d, 2H), C59H51N5O7S: C, 71.03; H, 5.52;
3.08 (dd, 1H), 3.51 (s, 2H), 4.83, 4.96 (2 ꢀ m, 2H),
N, 7.67; S, 3.51 (C, 70.85; H,
5.66; N, 7.54; S, 3.68)
5.0 (br, 4H), 6.60 (s, 1H), 6.93–7.38 (m, 25H)
67.88
3345 (br), 1755, 1745 (300 MHz): 2.60 (m, 2H), 2.65 (dd, 1H), 2.94
C52H51N7O11S: C, 63.59; H,
5.24; N, 9.98; S, 3.26;
(C, 63.42; H, 5.15; N, 9.94; S,
3.40)
(br), 1660 (br)
(d, 2H), 3.13 (dd, 1H), 3.50 (s, 2H), 3.72 (s, 2H),
4.83, 4.94 (2 ꢀ m, 2H), 5.04 (s, 6H), 5.12 (s, 2H),
6.63 (s, 1H), 6.93–7.44 (m, 20H)
38
39
65
135–38
3400–3100 (br),
1700–1650 (br)
(D2O, 300 MHz): 2.63–2.87 (m, 4H), 2.92, 3.08
(m, 2H), 3.45–3.68 (m, 4H), 4.39, 4.58 (2 ꢀ m,
2H), 6.90 (s, 1H), 7.06–7.30 (m, 5H)
C22H27N7O7S, HCl, 2H2O: C,
44.78; H, 5.47; N, 16.17; Cl,
6.01; S, 5.43 (C, 44.63; H,
5.49; N, 16.05; Cl, 6.17; S,
5.28)
87.5
Oily
(neat): 3400–3000 (br), 1.27 (t, 3H), 1.47 (s, 9H), 1.58–1.98 (m, 6H), 2.43 C18H29N3O5S: C, 54.12; H, 7.32;
3000–2940 (br),
1740, 1700 (br), 1680
(br)
(t, 2H), 3.11 (m, 2H), 3.67 (m, 2H), 4.17 (quartet,
2H), 6.77 (s, 1H)
N, 10.52; S, 8.02; (C, 54.36; H,
7.41; N, 1038; S, 5.16)
40
41
84.7
84
122–24
112–14
3390, 3370, 2970 (br), (CDCl3 þ DMSO-D6): 1.46 (s, 9H), 1.49–1.86
C16H25N3O5S: C, 51.74; H, 6.78;
N, 11.31; S, 8.63; (C, 51.69; H,
6.95; N, 11.47; S, 8.51)
1710–1680 (br)
(m, 6H), 2.46 (t, 2H), 3.37 (m, 2H), 3.66 (s, 2H),
6.74 (s, 1H)
3380–3300, 3000–
2950 (br), 1750 (br),
1705, 1690 (br)
(CD3OD): 1.18–1.65 (2 ꢀ m, 6H), 1.31 (s, 9H), 2.33 C27H36N4O8S: C, 56.24; H, 6.29;
(t, 2H), 2.79, 2.90 (2 ꢀ m, 4H), 3.50 (s, 2H), 4.68
(m, 1H), 4.96 (s, 2H), 6.72 (s, 1H), 7.20, 7.40,
7.60. 7.75 (4 ꢀ m, 5H)
N, 9.72; S, 5.56; (C, 56.15; H,
6.17; N, 9.65; S, 5.41)
(continued)

Table 1. Continued
Sub.
no.
Yield
(%)
Analysis MF; calc. (found)
(%)
Mp (8C)
Oily
IR (KBr) (cm21
)
1H NMR
42
43
88
3370–3300, 3000–
2950 (br), 1755 (br),
1710, 1695 (br)
(CD3OD): 0.78 (t, 3H), 1.19, 1.32, 1.43, 1.69 (4 ꢀ m, C31H45N5O7S: C, 58.93; H, 7.18;
10H), 1.37 (s, 9H), 2.39 (t, 2H), 2.65, 2.95
(2 ꢀ dd, 2H), 3.02–3.13 (m, 4H), 3.54 (s, 2H),
4.55 (br, 1H), 4.77 (m, 1H), 5.06 (s, 2H), 6.61
(s, 1H), 7.26 (m, 5H), 8.24 (br, 1H)
N, 11.09; S, 5.07(C, 58.77; H,
7.02; N, 11.17; S, 5.25)
60
Oily
3500–3300, 1730 (br), (300 MHz): 0.79 [t, 3H, J ¼ 7.8, N(CH2)3CH3],
C45H51N7O9S: C, 61.34; H, 6.11;
N, 11.65; S, 3.81; (C, 61.07; H,
6.16; N, 11.44; S, 4.03)
1660 (br), 1650–
1630 (br)
1.15–1.78 [4 ꢀ m, 10H, NHCH2(CH2)2CH3,
-NHCH2(CH2)3CH2CO-], 2.37 (t, 2H, J ¼ 7.8,
(CH2)4CH2CO], 2.63, 2.95 [2 ꢀ dd, 2H,
Jgem ¼ 17.5, J ¼ 7.5, 5.5, CH(CH2COO) of Asp],
3.12–3.33 (2 ꢀ m, 4H, 2 ꢀ NCH2), 3.52 (s, 2H,
CH2CO of Tha), 4.73 (m, 1H, -CaH of Asp), 5.05
(m, 6H, 3 ꢀ OCH2Ph), 6.62 (s, 1H, H-5 of Tha),
7.18–7.37 (m, 5H, PhH), 8.14, 8.26 (br, 1H, NH)
(300 MHz, D2O): 0.79 [t, 3H, J ¼ 7.63,
44
60
172–75
3310, 2980, 1690–
1670 (br), 1655
C21H34N7O8S2F3: C, 40.12; H,
5.41; N, 15.47; F, 8.99; S,
10.12 (C, 39.96; H, 5.33; N,
15.22; F, 8.82; S, 9.99)
N(CH2)3CH3], 1.15, 1.48, 1.62, 1.73 [4 ꢀ m, 10H,
NHCH2(CH2)2CH3, -NHCH2(CH2)3CH2 CO-],
2.55 [t, 2H, J ¼ 7.62, (CH2)4CH2CO], 2.82, 2.88
[2 ꢀ dd, 2H, Jgem ¼ 17.5, J ¼ 7.6, 5.02, -
CH(CH2COOH)- of Asp], 3.12–3.19 (m, 4H,
2 ꢀ NCH2), 3.66 (s, 2H, CH2CO of Tha), 4.65
(m, 1H, -CaH of Asp), 6.96 (s, 1H, H-5 of Tha)

Ion-Exchange cum Separation Technique
1405
The same procedure was utilized to prepare amides of the general structure
Boc-Asp (OBzl)-NHR, starting from Boc-Asp (OBzl)-OH and the required
amine. The final product was characterized by 1H NMR (data not included).
4.3 General Method for Deblocking Boc-Asp (OBzl)-NHR
A clear solution of Boc-Asp (OBzl)–NHR (2.5 mmol) in formic acid (15 ml)
and anisole (0.5 ml) was kept at room temperature. TLC monitored the
progress of the reaction. When the reaction was over (ꢀ4 h), slight excess
of HCl in dry ether was added and stirred for 5 min to convert the formate
salt into hydrochloride salt. Solvent was removed under reduced pressure.
The residue was triturated with dry ether to obtain a white solid of hydrochlo-
ride salt. It was filtered and washed with dry ether. The solid was dried in
vacuum desiccators over KOH pellets overnight. The solid material was suffi-
ciently pure to carry out the coupling reaction.
4.4 Benzyl 3-S-Propylcarbamoyl-4-oxo-5-(2-tritylamino-1,-
3-thiazol-4-yl) Pentanoate (2)
A solution of Trit-Tha-OH (1.2 g; 3 mmol) and HOBt (0.41 g; 3.5 mmol) in
CH2Cl2 (5 ml) and DMF (15 ml) was chilled to 08C. To this solution, DCC
(0.72 g; 3.5 mmol) was added under vigorous stirring. After 5 min, Asp-NHPr
(n) was obtained by neutralizing AspNHPr (n). HCl (0.77 g; 2.55 mmol) by
adding Et3N (0.36 ml; 2.58 mmol) in DMF (2 ml) at 08C. The reaction
mixture was stirred at 08C for 3 h. It was kept in the freezer overnight. DCU
was filtered off. The filtrate was successively with cold 1N HCl, water, 1N
NaHCO3, and brine. The organic layer was dried over anhydrous Na2SO4.
Solvent was removed, and the residue was purified by flash chromatography
with 10% CH3CN in CHCl3 followed by 10% CH3CN þ 0.2% MeOH in
CHCl3. Yield 1.01 g (61.74%), semisolid; IR (KBr): 3300 (br), 3050 (br),
2960, 1745 (br), 1735, 1670 (br) cm21
;
1H NMR: 0.83 (t, 3H, J ¼ 7.1,
NHCH2CH2CH3), 1.23–1.60 (m, 2H, NHCH2CH2CH3), 2.66 [dd, 1H,
Jgem ¼ 17.2, J ¼ 6.1, -CH (CHaHbCOO-) of Asp], 2.95–3.26 [m, 3H, -CH
(CHaHbCOO-) of Asp & NHCH2CH2CH3], 3.46 (s, 2H, CH2CO of Tha), 4.79
(m, 1H, -CaH of Asp), 5.0 (s, 2H, PhCH2O), 6.09 (s, 1H, H-5 of Tha), 7.26–
7.39 (m, 20H, PhH); anal. calc. for C38H38O4N4S: C, 70.56; H, 5.92; N, 8.66;
S, 4.96; found: C, 70.51; H, 5.90; N, 8.58; S, 4.84%.
4.5 Benzyl 3-S-Isopropylcarbamoyl-4-oxo-5-(2-tritylamino-1,-
3-thiazol-4-yl) Pentanoate (3)
This compound was prepared by coupling 1 with Asp (OBzl)-NHPr (i)
following the method described for the synthesis of 2. The crude product

1406
A. K. Gangopadhyay and B. Lal
was purified by flash chromatography with 10% CH3CN in CHCl3 followed by
10% CH3CN þ 0.2% MeOH in CHCl3.
4.6 Benzyl 3-S-Butylcarbamoyl-4-oxo-5-(2-tritylamino-1,-
3-thiazol-4-yl) Pentanoate (4)
This was obtained by coupling 1 with Asp (OBzl)-NHBu (i) following the
method described for the synthesis of 2. The crude product was purified by
flash chromatography with 10% CH3CN in CHCl3 followed by 10%
CH3CN þ 0.2% MeOH in CHCl3.
4.7 Benzyl 3-S-Isobutylcarbamoyl-4-oxo-5-(2-tritylamino-1,-
3-thiazol-4-yl) Pentanoate (5)
This was obtained by coupling 1 with Asp (OBzl)-NHBu (i) following the
method described for the synthesis of 2. The crude product was purified by
flash chromatography with 10% CH3CN in CHCl3 followed by 10%
CH3CN þ 0.2% MeOH in CHCl3.
4.8 Benzyl 3-S-Isopentylcarbamoyl-4-oxo-5-(2-tritylamino-1,-
3-thiazol-4-yl) Pentanoate (6)
A solution of Trit-Tha-OH (2.2 g; 5.5 mmol) in DMF (20 ml) was chilled to
2308C and 1-methyl morpholine (0.61 ml; 5.5 mmol) and isobutyl chlorofor-
mate (0.72 ml; 5.5 mmol) were added under vigorous stirring. A solution of
HCl-Asp (OBzl) NHCH2CH2CHMe2 (1.64 g; 5 mmol) in DMF (10 ml) was
neutralized with Et3N (0.7 ml; 5 mmol) at 2308C and then added to the
mixed anhydride generated previously. The reaction mixture was stirred at
2208C to 2308C for 1 h and then kept in the freeze at 2158C overnight.
The solvent was removed, and the residue was partioned between EtOAc
(25 ml) and aqueous 1 N NaHCO3 (10 ml) and stirred for 5 min. The
organic layer was separated. It was washed successively with water, 1N
citric acid, and brine. The organic layer was dried over anhydrous Na2SO4.
Solvent was removed and the crude product was purified by flash chromato-
graphy with 10% CH3CN in CHCl3 followed by 10% CH3CN þ 0.2%
MeOH in CHCl3. Yield 74.4%; semisolid; IR (KBr): 3300 (br), 2970 (br),
1
1755 (br), 1745 (br) cm21; H NMR: 0.86, [d, 6H, J ¼ 6.1, -CH (CH3)2],
1.19–1.60 (m, 3H, NCH2CH2CHMe2), 2.62 [dd, 1H, Jgem ¼ 17.2, J ¼ 6.08,
-CH (CHaHbCOO-) of Asp], 3.04 [dd, 1H, Jgem ¼ 17.2, J ¼ 4.05, -CH
(CHaHbCOO-) of Asp], 3.09–3.30 (m, 2H, NHCH2CH2-), 3.49 (s, 2H,
CH2CO of Tha), 4.77 (m, 1H, -CaH of Asp), 4.99 (s, 2H, PhCH2O), 6.09
(s, 1H, H-5 of Tha), 6.53 (br, 1H, NH), 6.86 (br, 1H, NH), 7.23–7.39

Ion-Exchange cum Separation Technique
1407
(m, 20H, PhH) 8.49 (d, 1H, NH, exchangeable); anal. calc. for C40H42O4N4S: C,
71.19; H, 6.27; N, 8.30; S, 4.75; found: C, 70.76; H, 6.21; N, 8.19; S, 4.63%.
4.9 Benzyl 3-S- (1-Ethylpropylcarbamoyl-4-oxo-5-(2-tritylamino-
1,3-thiazol-4-yl) Pentahyphennoate (7)
This compound was prepared by coupling 1 with Asp (OBzl)-NHCHEt2
following the method described for the synthesis of 6. The crude product
was purified by flash chromatography with 10% CH3CN in CHCl3 followed
by 10% CH3CN þ 0.2% MeOH in CHCl3.
4.10 Benzyl 3-S-Neopentylcarbamoyl-4-oxo-5-(2-tritylamino-1,-
3-thiazol-4-yl) Pentanoate (8)
This compound was prepared by coupling 1 with Asp (OBzl)-NHCH2CMe3
following the method described for the synthesis of 6. The crude product
was purified by flash chromatography with 10% CH3CN in CHCl3 followed
by 10% CH3CN þ 0.2% MeOH in CHCl3
4.11 Benzyl 3-S-Cyclohexylcarbamoyl-4-oxo-5-(2-tritylamino-1,-
3-thazol-4-yl) Pentanoate (9)
Compound 10 was prepared by coupling 1 with Asp (OBzl)-NHC6H11
following the method described for the synthesis of 2. The crude product
was purified by flash chromatography with 10% CH3CN in CHCl3 followed
by 10% CH3CN þ 0.2% MeOH in CHCl3.
4.12 Benzyl 3-S-(1-S-Benzyloxycarbonyl-2-methylpropylcarbamoyl-
4-oxo-5-(2-tritylami-no1,3-thazol-4-yl) Pentanoate (10)
This compound was prepared by coupling 1 with Asp (OBzl)-Val-OBzl
following the method described for the synthesis of 2. The crude product
was purified by flash chromatography with 10% CH3CN in CHCl3 followed
by 10% CH3CN þ 0.2% MeOH in CHCl3.
4.13 Benzyl 5-f2-[1-S-Benzyloxycarbonylamino-4-
benzyloxycarbonylamino(benzyloxycarbonylimino)-
methylaminobutylcarboxamido]-1,3-thazol-
4-yl)-4-oxo-3-S-propylcar-bamoylpentanoate (11)
Compound 2 (3.23 g; 5 mmol) was dissolved in 85% AcOH (40 ml) and
heated on a steam bath for 10 min. It was brought back to room

1408
A. K. Gangopadhyay and B. Lal
temperature, and 1 N HCl (5 ml) was added. It was stirred for 2 min.
Solvent was removed, and the residue was triturated with dry ether to
obtain a white solid. The solid was filtered and washed with dry ether.
The resulting hydrochloride salt was crystallized from dry MeOH–ether.
Before the next step, it was dried in vacuum desiccators over NaOH
pellets to give a yield of 2.0 g (90.5%).
Tri-Z-Arg (1.9 g; 2.3 mmol) was dissolved in CHCl3 (20 ml) and
chilled to 2208C. To this solution was added 1-methyl morpholine
(0.36 ml; 2.3 mmol) and isobutyl chloroformate (0.42 ml; 2.3 mmol)
under vigorous stirring conditions. Deprotected 2 (1.32 g; 3 mmol) was
dissolved in CHCl3 (10 ml) and chilled to 2208C. Et3N (0.42 ml;
3 mmol) was added under stirring. After 1 min, the free amine was added
to the reaction mixture containing the mixed anhydride. The reaction
mixture was stirred for 1 h at 2208C and then kept in the freezer
overnight. The reaction mixture was then stirred with 1N NaHCO3 for
5 min. The organic layer was separated and successively washed with
water, 1N HCl, and brine. After drying over anhydrous Na2SO4, the
solvent was removed. The residue was purified by flash chromatography
with 2% MeOH in CHCl3.
Compounds 12–19 were prepared from 3–10 after deblocking the trityl
group followed by coupling with tri-Z-Arg using a mixed anhydride
method. The experimental details for both the steps were same as described
for the preparation of 11.
4.14 Benzyl 5-(2-[1-S-Benzyloxycarbonylamino-4-
benzyloxycarbonylamino(benzyloxycarbonylimino)-
methylaminobutylcarboxamido]-1, 3-thazol-4-yl)-4-oxo-3-S-
isopropyl-carbamoylpentanoate (12)
This compound was prepared from 3 and Tri-Z-Arg using the mixed
anhydride procedure as described for the synthesis of 11. Two different
solvents were tried in this case. The residue was purified by flash chromato-
graphy with 5% MeOH in CHCl3. Yield was 21.8% when DMF was used
as solvent for mixed anhydride reaction and 52.07% when THF–CH2Cl2
2:1 was as solvent for coupling reaction.
4.15 Benzyl 5-(2-[1-S-Benzyloxycarbonylamino-4-
benzyloxycarbonylamino(benzyloxycarbonylimino)-
methylaminobutylcarboxamido]-1,3-thazol-4-yl)-4-oxo-3-S-
butylcarbamoylpentanoate (13)
This compound was prepared from 4 and Tri-Z-Arg using the mixed
anhydride procedure as described for the synthesis of 11. The reaction was

Ion-Exchange cum Separation Technique
1409
carried out in CH2Cl2. The residue was purified by flash chromatography with
2% MeOH in CHCl3.
4.16 Benzyl 5-(2-[1-S-Benzyloxycarbonylamino-4-
benzyloxycarbonylamino(benzyloxycarbonylimino)-
methylaminobutylcarboxamido]-1, 3-thazol-4-yl)-4-oxo-3-S-
isobutylcarbamoylpentanoate (14)
This compound was prepared from 5 and Tri-Z-Arg using the mixed
anhydride procedure as described for the synthesis of 11. The reaction was
carried out in CHCl3. The residue was purified by flash chromatography
with 2% MeOH in CHCl3.
4.17 Benzyl 5-(2-[1-S-Benzyloxycarbonylamino-4-
benzyloxycarbonylamino(benzyloxyca rbonylimino)-
methylaminobutylcarboxamido]-1, 3-thazol-4-yl)-4-oxo-3-S-
isopentyl-carbamoylpentanoate (15)
This compound was prepared from 6 and Tri-Z-Arg using the mixed
anhydride procedure as described for the synthesis of 11. The reaction was
carried out in CHCl3. The residue was purified by flash chromatography
with 2% MeOH in CHCl3.
4.18 Benzyl 5-(2-[1-S-Benzyloxycarbonylamino-4-
benzyloxycarbonylamino(benzyloxycarbonylimino)-
methylaminobutylcarboxamido]-1,3-thazol-4-yl)-4-oxo-3-(1-
ethylproplcarbamoyl) Pentanoate (16)
This was prepared from 7 and Tri-Z-Arg using the mixed anhydride procedure
as described for the synthesis of 11. The reaction was carried out in CHCl3.
The residue was purified by flash chromatography with 2% MeOH in CHCl3
4.19 Benzyl 5-(2-[1-S-Benzyloxycarbonylamino-4-
benzyloxycarbonylamino(benzyloxycarbonylimino)-
methylaminobutylcarboxamido]-1,3-thazol-4-yl)-4-oxo-3-S-
neopentyl-carbamoylpentanoate (17)
This was prepared from 8 and Tri-Z-Arg using the mixed anhydride procedure
as described for the synthesis of 11. The reaction was carried out in CHCl3.
The residue was purified by flash chromatography with 2% MeOH in CHCl3.

1410
A. K. Gangopadhyay and B. Lal
4.20 Benzyl 5-(2-[1-S-Benzyloxycarbonylamino-4-
benzyloxycarbonylamino(benzyloxycarbonylimino)-
methylaminobutylcarboxamido]-1,3-thazol-4-yl)-4-oxo-3-S-
cyclohexyl-carbamoylpentanoate (18)
This compound was prepared from 9 and Tri-Z-Arg using the mixed
anhydride procedure as described for the synthesis of 11. The reaction was
carried out in CHCl3. The residue was purified by flash chromatography
with 2% MeOH in CHCl3.
4.21 Benzyl 5-(2-[1-S-Benzyloxycarbonylamino-4-
benzyloxycarbonylamino(benzyloxycarbonylimino)-
methylaminobutylcarboxamido]-1,3-thazol-4-yl)-3-S-(1-S-
benzyloxycarbonyl-2-methylpropylcarbamoyl)-4-oxo-pentanoate (19)
The reaction was carried out in CHCl3. The residue was purified by flash
chromatography with 2% MeOH in CHCl3.
4.22 5-(2-[1-S-Amino-4-amino(imino)-
methylaminobutylcarboxamido]-1,3-thazol-4-yl)-4-oxo-3-S-
propylcarbamoylpentanoic Acid (20)
Compound 11 (0.6 g; 0.62 mmol) was dissolved in TFA (15 ml) and anisole
(0.3 ml). To this solution, trifluorosulfonic acid (0.8 ml) was added under
stirring at room temperature. After stirring at room temperature for 3 h,
excess dry ether was added. The precipitated solid (highly hygroscopic) was
filtered and washed with dry ether. It was dried under vacuum overnight.
The crude material was dissolved in ice-cold water (2 ml) and made alkaline
to pHꢀ10 by adding cold dilute NaOH. The mixture was immediately
loaded on an RP-18 MPLC column. The column was first eluted with water
followed by water–nBuOH–AcOH 90:2.5:2.5 (v/v). The fractions containing
the required compound were pulled together and evaporated to dryness. The
pure compound was dissolved in dry MeOH (2 ml), and HCl in ether was
added. The white solid was filtered and crystallized from dry MeOH–dry
ether. Yield 0.14 g (41.7%); mp 185–908C (d); IR (KBr): 3500–3300 (br),
2950 (br), 1745 (br), 1680–1660 (br) cm21; 1H NMR (D2O; 300 MHz): 0.78
(t, 3H, J ¼ 7.3, -NCH2CH2CH3), 1.43 (m, 2H, NCH2CH2CH3), 1.68, 2.01
(2 x m, 4H, b-CH2 and g-CH2 of Arg), 2.58 [dd, 1H, Jgem ¼ 14.5, J ¼ 7.3,
-CH(CHaHbCOO-) of Asp], 2.66 [dd, 1H, Jgem ¼ 14.5, J ¼ 4.8, -CH
(CHaHbCOO-) of Asp], 3.10, 3.20 (2 ꢀ m, 4H, d-CH2 of Arg þ NCH2CH2-
CH3), 3.59 (s, 2H, CH2CO of Tha), 4.28, 4.49 [2 ꢀ t, 2H, J ¼ 7.0, 2 ꢀ -
CaH- of Arg, Asp), 7.05 (s, 1H, H-5 of Tha); anal. calc. for C18H30O5N8S,

Ion-Exchange cum Separation Technique
1411
2HCl, 3H2O: C, 36.17; H, 6.41; N, 18.75; Cl, 11.87; S, 5.37; found: C, 36.03; H,
6.33; N, 18.58; Cl, 12.07; S, 5.15%.
The final compounds 21–28 were obtained from 12–19 by following the
same procedure including purification, formation of hydrochloride salt, and
final crystallization as described for the synthesis of compound 20.
4.23 5-(2-[1-S-Amino-4-amino(imino)-
methylaminobutylcarboxamido]-1,3-thazol-4-yl)-4-oxo-3-S-
isopropylcarbamoylpentanoic Acid (21)
This compound was prepared from 12 as described for the synthesis of 20. The
crude product was purified in a similar manner.
4.24 5-(2-[1-S-Amino-4-amino(imino)-
methylaminobutylcarboxamido]-1,3-thazol-4-yl)-4-oxo-3-S-
butylcarbamoylpentanoic Acid (22)
This compound was prepared from 13 as described for the synthesis of 20. The
crude product was purified in a similar manner.
4.25 5-(2-[1-S-Amino-4-amino(imino)-
methylaminobutylcarboxamido]-1,3-thazol-4-yl)-4-oxo-3-S-
isobutylcarbamoylpentanoic Acid (23)
This compound was prepared from 14 as described for the synthesis of 20. The
crude product was purified in a similar manner.
4.26 5-(2-[1-S-Amino-4-amino(imino)-
methylaminobutylcarboxamido]-1,3-thazol-4-yl)-4-oxo-3-S-
isopentylcarbamoylpentanoic Acid (24)
This compound was prepared from 15 as described for the synthesis of 20. The
crude product was purified in a similar manner.
4.27 5-(2-[1-S-Amino-4-amino(imino)-
methylaminobutylcarboxamido]-1,3-thazol-4-yl)-4-oxo-3-S-
(1-ethylpropylcarbamoyl)pentanoic Acid (25)
This compound was prepared from 16 as described for the synthesis of 20.
The crude product was purified in a similar manner.

1412
A. K. Gangopadhyay and B. Lal
4.28 5-(2-[1-S-Amino-4-amino(imino)-
methylaminobutylcarboxamido]-1,3-thazol-4-yl)-4-oxo-3-S-
neopentylcarbamoylpentanoicAcid (26)
This compound was prepared from 17 as described for the synthesis of 20. The
crude product was purified in a similar manner.
4.29 5-(2-[1-S-Amino-4-amino(imino)-
methylaminobutylcarboxamido]-1,3-thazol-4-yl)-4-oxo-3-S-
cyclohexylcarbamoylpentanoic Acid (27)
This compound was prepared from 18 as described for the synthesis of 20. The
crude product was purified in a similar manner.
4.30 5-(2-[1-S-Amino-4-amino(imino)-
methylaminobutylcarboxamido]-1,3-thazol-4-yl)-3-S-(1-S-
carboxy-2-methylcarbamoyl)-4-oxo-pentanoic Acid (28)
This compound was prepared from 19 as described for the synthesis of 20. The
crude product was purified in a similar manner. Yield 63.97%; mp 196–2028C
(d); IR (KBr): 3400–3300 (br), 2960 (br), 1725 (br), 1670–1640 (br) cm21
;
1H NMR (D2O; 300 MHz): 0.88 (m, 6H, 2 ꢀ CH3), 1.63–1.76 (2H), 2.01–
2.19 (3H) [2 ꢀ m, 5H, -CHMe2, and b-CH2 and g-CH2 of Arg), 2.83–2.95
[2 ꢀ dd, 2H, Jgem ¼ 15.4, J ¼ 7.7, 6.6, CH(CH2COOH)], 3.23 (t, 2H,
J ¼ 6.7, d-CH2 of Arg], 3.72 (br, 2H, CH2CO of Tha), 4.22, 4.31, 4.72
(3 ꢀ m, 3H, 3 ꢀ -CaH- of Val, Arg and Asp), 7.05 (s, 1H, H-5 of Tha);
anal. calc. for C20H32O7N8S, 2HCl, 2H2O: C, 37.68; H, 6.01; N, 17.58; Cl,
11.12; S, 5.03; found: C, 37.92; H, 6.15; N, 17.35; Cl, 11.07; S, 4.87%.
4.31 Ethyl 2-(2-Tertiarybutyloxycarbonylaminomethylcarboxamido-1,-
3-thiazol-4-yl)acetate (29)
A solution of Boc-Gly (7.0 g; 40 mmol) and HOBt (6.8 g; 50 mmol) in
CH2Cl2 (50 ml) was chilled at 08C. To this solution, DCC (10.3 g;
50 mmol) in CH2Cl2 (10 ml) was added under vigorous stirring. After
5 min, ethyl-2-amino-thiazol-4-yl-acetate (9.3 g; 50 mmol) was added, and
stirring continued for 2 h at 08C. The reaction mixture was kept in the
freezer overnight. DCU was filtered off. The filtrate was washed successively
with 1N citric acid, water, 1 N NaHCO3, and brine. The organic layer was
dried over anhydrous Na2SO4. Solvent was removed, and the residue was
crystallized from CH2Cl2–light petroleum.

Ion-Exchange cum Separation Technique
1413
4.32 Ethyl 2-[2-(2-Tritylaminoethylcarboxamido)-1,3-thiazol-4-
yl)acetate (30)
This compound was prepared from Trit-b-Ala and Tha-OEt using the same
procedure as described previously. The reaction was carried out in EtOAc–
DMF instead of CH2Cl2. The crude product was purified by flash chromatography
with 2% MeOH in CHCl3. It was crystallized from EtOAc–light petroleum.
4.33 2-(2-Tertiarybutyloxycarbonylaminomethylcarboxamido)-
1,-3-thiazol-4-yl)acetic Acid (31)
Compound 29 (10.29 g; 30 mmol) was suspended in MeOH (132 ml), and 1N
NaOH (33 ml; 33 mmol) was added at room temperature under vigorous
stirring conditions. Within 2 min, the entire solid was dissolved. The stirring
continued for 1 h. The solvent was removed. The solid was filtered, and the
filtrate was extracted with EtOAc (2 ꢀ 10 ml) and discarded. The aqueous
layer was acidified and extracted with EtOAc (3 ꢀ 30 ml). The EtOAc layer
was washed with brine and dried over anhydrous Na2SO4. Solvent was
removed. The residue was crystallized from hot EtOAc–light petroleum.
4.34 2-[2-(2-Tritylaminoethylcarboxamido)-1,3-thiazol-4-yl]-
acetic Acid (32)
Compound 30 was hydrolyzed according to the method described for the prep-
aration of 31. The residue was crystallized from hot EtOAc.
4.35 Boc-Asp(OBzl)-Phe-OBzl
To a solution of Boc-Asp (OBzl)-OH (3.23 g; 10 mmol) and HOBt (1.62 g;
12 mmol) in CH2Cl2 (60 ml) chilled at 08C, DCC (2.47 g; 12 mmol) was
added under vigorous stirring conditions. In a separate round-bottomed flask
HCl-PheOBzl[24] (3.5 g; 12 mmol) was dissolved in CH2Cl2 (20 ml) and
DMF (5 ml) and neutralized with Et3N (1.7 ml; 12 mmol) at 08C. After
5 min, the amine component was added to the reaction mixture and stirring
continued for 1 h at 08C and stored in the freezer overnight. DCU was
filtered off. The filtrate was successively washed with 1N NaHCO3, water,
1N citric acid, and brine. It was dried over anhydrous Na2SO4. Solvent was
removed, and the residue was purified by flash chromatography using 5%
CH3CN in CHCl3. The pure compound was crystallized from EtOAc–light
petroleum. Yield 4.0 (71.4%), mp 102–48C; IR (KBr): 3380, 3220, 1770,
1755, 1705, 1695 cm21; 1H NMR: 1.43 [s, 9H, C (CH3)3], 2.64, 3.14 (2 ꢀ dd,
2H, Jgem ¼ 17.5, J ¼ 6.08, Js ¼ 5.06, CH (CH2COO-)], 3.06 (d, 2H, J ¼ 6.08,
CH (CH2Ph)], 4.51, 4.80 (2 ꢀ m, 2H, 2 ꢀ CaH), 5.09 (s, 4H, 2 ꢀ OCH2Ph),

1414
A. K. Gangopadhyay and B. Lal
6.83–7.34 (m, 15H, PhH); anal. calc. for C32H36N2O7: C, 68.55; H, 6.47; N,
4.99; found: C, 68.34; H, 6.41; N, 4.69%.
4.36 Benzyl 3-(2-Tertiarybutyloxycarbonylaminomethylcarboxamido-
1,3-thiazol-4-yl-methylcarboxamido)-4-(1-benzyloxycarbonyl-2-
phenylethylamino)-4-pentanoate (33)
This was prepared by treating 31 with Asp (OBzl)-Phe (OBzl)-HCl by DCC–
HOBt as described for the preparation of 2. The crude product was purified by
flash chromatography with 2.5% MeOH in CHCl3. It was crystallized from
EtOAc–light petroleum
4.37 Benzyl 4-(1-benzyloxycarbonyl-2-phenylethylamino)-3-[2-(2-
tritylaminoethylcarbohyphen;xamido)-1,3-thiazol-4-yl-
methylcarboxamido)-4-pentanoate (34)
This was prepared by coupling 32 with Asp (OBzl)-Phe (OBzl)-HCl by DCC–
HOBt as described for the preparation of 2. The crude product was purified by
flash chromatography with 2.5% MeOH in CHCl3. It was crystallized from
EtOAc–light petroleum.
4.38 Benzyl 4-(1-Benzyloxycarbonyl-2-phenylethylamino)-3-[2-
benzyloxycarbonylamino(benzyloxycarbonylimino)-
methylaminomethylcarboxamido-1,3-thiazol-4-yl-methyl-
carboxamido]-4-pentanoate (35)
A solution of compound 33 (1.52 g; 2 mmol) in formic acid (23 ml) and anisole
(0.75 ml) was kept at room temperature for 3 h. Dry HCl in ether (1 ml) was
added, and stirring continued for 5 min. The solvent was removed under
reduced pressure. The oily residue was triturated with dry ether, and the solid
obtained was filtered. It was dried in vacuum desiccators over KOH pellets
for 12 h. Yield 1.28 g (92.2%). The amine hydrochloride (1.0 g; 1.44 mmol)
was dissolved in dichloromethane (15 ml) and acetonitrile (5 ml). After neutra-
lizing with Et3N (0.21 ml; 1.5 mmol), dicarbobenzoxy-S-methylisothiourea
(0.53 g; 1.5 mmol) was added, and the mixture was refluxed for 12 h. The
solvent was distilled off. The residue was taken up in EtOAc and washed
with water. The organic layer was dried over anhydrous Na2SO4. Solvent was
removed. The residue was purified by flash chromatography with 2% MeOH
in CHCl3. The crude product was crystallized from MeOH. Yield 0.86 g,
1
(63.9%); mp 75–788C; IR (KBr): 3320 (br), 1735 (br), 1640 (br) cm21; H
NMR (300 MHz): 2.65 [dd, 1H, Jgem ¼ 17.5, Jtrans ¼ 6.5, -CH (CHaHbCOO)
of Asp], 2.95 [d, 2H, J ¼ 6.7, -CH (CH2Ph)- of Phe], 3.02 [dd, 1H,

Ion-Exchange cum Separation Technique
1415
Jgem ¼ 17.5, Jcis ¼ 5.3, -CH (CHaHbCOO-) of Asp], 3.48 (s, 2H, CH2CO of
Tha), 4.21 (s, 2H, NHCH2CO), 4.80, 4.93 (2 ꢀ m, 2H, 2 ꢀ CaH of Asp and
Phe), 4.96, 5.0, 5.12 (m), 5.18 (m, 8H, 4 ꢀ PhCH2O-), 6.62 (s, 1H, H-5 of
Tha), 6.98–7.43 (m, 20H, PhH); anal. calc. for C51H49N7O11S: C, 63.27; H,
5.10; N, 10.13; S, 3.31; found: C, 62.97; H, 4.96; N, 9.94; S, 3.63%.
4.39 Benzyl 4-(1-Benzyloxycarbonyl-2-phenylethylamino)-3-f2-[2-
benzyloxycarbonylamino(benzyloxycarbonylimino)-
methylaminoethylcarboxamido]-1,3-thiazol-4-ylmethyl-
carboxamidog-4-pentanoate (36)
Compound 34 (2 g; 2.19 mmol) was dissolved in 85% aqueous AcOH and
heated on a steam bath for 10 min. Acetic acid was removed, and the
residue was triturated with ether. The solid obtained was filtered and
washed with dry ether. The dried material was crystallized from MeOH–
dry ether to yield 1.47 g (91.87%). The amine acetate obtained previously
was reacted with dicarbobenzoxy-S-methylisothiourea as described for the
synthesis of compound 35. The crude product was purified by flash chromato-
graphy with 2% MeOH in CHCl3 and was crystallized from MeOH.
4.40 3-[2-Amino(imino)methylaminomethylcarboxamido]-1,-
3-thiazol-4-yl-methylcarboxamido]-4-(1-carboxy-2-
phenylethylamino)-4-pentanoic Acid (37)
Compound 35 (0.74 g; 0.76 mmol) was dissolved in TFA (20 ml). Anisole
(0.3 ml) and trifluoromethansulfonic acid (1 ml) were added at room tempera-
ture under vigorous stirring. After 3 h excess dry ether was added. The solid
separated was filtered and was washed with dry ether. The hygroscopic solid
was dissolved in water (2 ml), had its pH adjusted to ꢀ10, was filtered
through celite, and was loaded on a RP-18 MPLC column. The column was
first eluted with water (to remove trifluoromethansulfonate salt) followed by
nBuOH: AcOH–water: 5:5:90. The fractions containing the required
compound were pulled together and evaporated to dryness. The residue was
dissolved in dry MeOH, and dry HCl in ether was added followed by excess
ether. The white solid was filtered, washed with ether, and finally crystallized
from dry MeOH–dry ether. Yield 0.236 g (55.53 %); mp 186–898C; IR
1
(KBr): 3400–3300 (br), 1750–1700, 1685–1660 (br) cm21; H NMR (D2O,
300 MHz): 2.72, 2.83 [2 ꢀ dd, 2H, Jgem ¼ 17.5, J ¼ 6.4, 5.2, -CH (CH2Ph)-
of Phe], 2.8, 3.10 [2 ꢀ dd, 2H, Jgem ¼ 16.5, J ¼ 7.7, 5.2, -CH (CH2COOH)-
of Asp], 3.59 (br, 2H, CH2CO of Tha), 4.18 (br, 2H, NHCH2CO), 4.50, 4.62
(2 ꢀ m, 2H, 2 ꢀ CaH of Asp and Phe), 6.93 (s, 1H, H-5 of Tha), 7.09–7.28
(m, 5H, PhH); anal. calc. for C21H25N7O7S.HCl, 2H2O: C, 42.60; H, 5.10; N,
16.56; Cl, 5.99; S, 5.42; found: C, 42.69; H, 4.96; N, 16.48; Cl, 6.17; S, 5.64%.

1416
A. K. Gangopadhyay and B. Lal
4.41 3-f2-[2-Amino(imino)methylaminoethyl-1-carboxamido]-1,-
3-thiazol-4-yl-methylcarboxamidog-4-(1-carboxy-2-
phenylethylamino)-4-pentanoic Acid (38)
This compound was prepared from 36 by following the method described for
compound 37. The purification procedure was also same with only exception
being the use of solvent system nBuOH: AcOH:–water 2.5: 2.5: 95 v/v.
4.42 Ethyl 2-[2-(5-Tertiarybutyloxycarbonylaminopentylcarboxamido)-
1,3-thiazol-4-yl]acetate (39)
This compound was prepared from Boc-protected 6-amino hexanoic acid and
Tha-OEt in a similar fashion as described for the synthesis of compound 2.
The crude product was purified by flash chromatography with 2% MeOH in
CHCl3.
4.43 2-[2-(5-Tertiarybutyloxycarbonylaminopentylcarboxamido)-1,-
3-thiazol-4-yl]acetic Acid (40)
Compound 39 was hydrolyzed according to the method described for the
synthesis of compound 31. The crude product was crystallized from EtOAc.
4.44 Benzyl 3-Carboxy-3-[2-(5-
tertiarybutyloxycarbonylaminopentylcarboxamido-1,3-thiazol-4-
ylmethylcarboxamido)propanoate (41)
To a suspension of Asp (OBzl)-OH (6.2 g; 28 mmol) in CH2Cl2 (50 ml), tri-
methylsilyl chloride (3.6 ml; 28 mmol) was added under vigorous stirring
(5 min). After 2 h, the reaction mixture was chilled to 08C, and Et3N (4.2 ml;
30 mmol) was added to generate free amines. This was added after 10 min to
a well-stirred solution of 40 (5.19 g; 14 mmol), DCC (3.3 g; 16 mmol), and
HOBt (2.2 g; 14 mmol) in CH2Cl2 (50 ml) at 08C. The resulting reaction
mixture was stirred at 08C for 1 h and then stored in the freezer overnight.
Excess methanol (ꢀ10 ml) was added to the reaction mixture under stirring
at room temperature (5 min). Solvent was removed under reduced pressure,
and the residue was partitioned between EtOAc and aqueous 1N NaHCO3.
The aqueous layer was seperated and acidified with dil. NaHSO4 solution.
The separated oily compound was extracted with EtOAc (3 ꢀ 15 ml). The
combined organic layer was washed with brine and dried over anhydrous
Na2SO4, and the solvent was evaporated. The crude product was purified by
flash chromatography with 10% MeOH þ 0.1% AcOH in CHCl3. It was
finally crystallized from EtOAc–light petroleum.

Ion-Exchange cum Separation Technique
1417
4.45 Benzyl 3-Butylcarbamoyl-3-[2-(5-
tertiarybutyloxycarbonylaminopentylcarboxamido-1,3-thiazol-4-
yl-methylcarboxamido)propionate (42)
A solution of compound 41 (1.26 g; 2.18 mmol) in DMF (10 ml) was chilled
to 2308C, and 1-methyl morpholine (0.24 ml; 2.18 mmol) was added,
followed by isobutylchloroformate (0.28 ml; 2.18 mmol), under vigorous
stirring. After stirring for 5 min, nBuNH2. (0.4 ml; 4.96 mmol) was added,
and the bath temperature was raised to 2208C. The mixture was kept in the
freezer overnight after stirring at 2208C for 1 h. It was treated with 1N
NaHCO3 solution to decompose unreacted mixed anhydride. DMF was
removed, and the residue was extracted with EtOAc. The EtOAc layer was
washed successively with water, dilute 1N citric acid, and brine. It was
dried over anhydrous Na2SO4. Solvent was removed, and the residue was
purified by flash chromatography with 2% MeOH in CHCl3. Yield 1.15 g
(oily). IR (KBr): 3370–3300, 3000–2950 (br), 1755 (br), 1710, 1695 (br)
1
cm21; H NMR (CD3OD): 0.78 [t, 3H, J ¼ 8.0, N (CH2)3CH3], 1.19, 1.32,
1.43, 1.69 [4 ꢀ m, 10H, NHCH2 (CH2)2CH3, -NHCH2 (CH2)3CH2CO-], 1.37
[s, 9H, -C (CH3)3], 2.39 (t, 2H, J ¼ 8.0, (CH2)4CH2CO], 2.65, 2.95 [2 ꢀ dd,
2H, Jgem ¼ 17.5, J ¼ 7.5, 5.5, -CH (CH2COO)- of Asp], 3.02–3.13 (m, 4H,
2 ꢀ NCH2), 3.54 (s, 2H, CH2CO of Tha), 4.55 (br, 1H, NH), 4.77 (m, 1H,
-CaH of Asp), 5.06 (s, 2H, OCH2Ph), 6.61 (s, 1H, H-5 of Tha), 7.26
(m, 5H, PhH), 8.24 (br, 1H, NH); anal. calc. for C31H45N5O7S: C, 58.93;
H, 7.18; N, 11.09; S, 5.07; found: C, 58.77; H, 7.02; N, 11.17; S, 5.25%.
4.46 Benzyl 3-f2-[5-Benzyloxycarbonylamino-
(benzyloxycarbonylimino)methylaminopentylcarboxamido]-1,3-
thiazol-4-yl-methylcarboxamidog-3-butylcarbamoylpropanoate (43)
This was prepared from 42 after deblocking the Boc group and subsequent
refluxing with dicarbobenzoxy-S-methylisothiourea in CH2Cl2–CH3CN 2:1,
as described for the synthesis of 35. The crude product was purified by flash
chromatography with 2.5% MeOH in CHCl3.
4.47 3-f2-[5-Amino(imino)methylaminopentylcarboxamido]-1,-
3-thiazol-4-yl-methylcarbohyphen;xamidog-3-
butylcarbamoylpropanoic Acid (44)
This was obtained by deprotection of carbobenzoxy and benzyl groups from
43 as described for the synthesis of 37. The crude product was crystallized
from dry MeOH–ether. This compound was characterized as trifluorometha-
nesulfonic acid salt.