Simple and mild one-pot synthesis of dipeptidyl ureas via carbamoyl azides of α-N-protected amino acids

Article abstract of DOI:10.1080/00397911003710570

(Chemical Equation Presented) A simple and mild one-pot synthesis of potentially bioactive α-N-protected dipeptidyl ureas is reported. The procedure involves the reaction between the carbamoyl azide of an α-N-protected amino acid and an α-amino acid methyl ester. The reaction is fast (3 h at 45 °C), regardless of the nature of both the reagents, and racemization free. The reported protocol represents a valid alternative to existing methods. Copyright Taylor & Francis Group, LLC.

Full text of DOI:10.1080/00397911003710570

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Simple and Mild One-Pot Synthesis of
Dipeptidyl Ureas via Carbamoyl Azides of
α-N-Protected Amino Acids
Cesare Daniele Venneri a , Giancarlo Verardo a & Paolo Strazzolini a
a Department of Chemical Sciences and Technology, University of
Udine, Udine, Italy
Version of record first published: 03 Mar 2011.
To cite this article: Cesare Daniele Venneri, Giancarlo Verardo & Paolo Strazzolini (2011): Simple
and Mild One-Pot Synthesis of Dipeptidyl Ureas via Carbamoyl Azides of α-N-Protected Amino Acids,
Synthetic Communications: An International Journal for Rapid Communication of Synthetic Organic
Chemistry, 41:7, 1027-1041
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Synthetic Communications1, 41: 1027–1041, 2011
Copyright # Taylor & Francis Group, LLC
ISSN: 0039-7911 print=1532-2432 online
DOI: 10.1080/00397911003710570
SIMPLE AND MILD ONE-POT SYNTHESIS OF
DIPEPTIDYL UREAS VIA CARBAMOYL AZIDES OF
a-N-PROTECTED AMINO ACIDS
Cesare Daniele Venneri, Giancarlo Verardo, and
Paolo Strazzolini
Department of Chemical Sciences and Technology, University of Udine,
Udine, Italy
GRAPHICAL ABSTRACT
Abstract A simple and mild one-pot synthesis of potentially bioactive a-N-protected
dipeptidyl ureas is reported. The procedure involves the reaction between the carbamoyl
azide of an a-N-protected amino acid and an a-amino acid methyl ester. The reaction is fast
(3 h at 45 ꢀC), regardless of the nature of both the reagents, and racemization free. The
reported protocol represents a valid alternative to existing methods.
Keywords Amino acid methyl esters; amino acids; carbamoyl azide; Curtius rearrange-
ment; dipeptidyl ureas
INTRODUCTION
Over the years, intense research has been focused on chemical modifications of
peptide backbone[1] in order to obtain bioactive mimetics with high metabolic stab-
ility, good bioavailability, high receptor affinity and selectivity, and minimal side
effects. In the field of peptidomimetics, the urea linkage as a peptide bond surrogate
(denoted w[. . . .])[1a] has attracted much attention in recent years[2] because of its
metabolic stability and bonding properties. The first ureidopeptides with biological
activity were synthesized and studied by Chipens and coworkers 30 years ago.[3]
Since then, the urea moiety has been widely used as a structural element in the
synthesis of a number of bioactive molecules such as [Leu]enkephalin analogs,[4]
CCK-B receptors,[2f] endothelin[2e] and gastrin[5] antagonists, HIV-1 protease,[2a,6]
Received January 26, 2010.
Address correspondence to Giancarlo Verardo, Department of Chemical Sciences and Technology,
University of Udine, Via del Cotonificio 108, 33100 Udine, Italy. E-mail: Giancarlo.verardo@uniud.it
1027

1028
C. D. VENNERI, G. VERARDO, AND P. STRAZZOLINI
c-secretase,[7] aspartic acid protease,[8] aspartic peptidase[9] and microbial alkaline
proteinase[10] inhibitors, and growth hormone secretagog.[11] In this frame, the devel-
opment of efficient, simplified synthetic procedures suitable to introduce a ureido
connection between two neighboring a-amino acids is of particular interest.
Current strategies to synthesize ureidopeptides involve the corresponding
amino acid isocyanate derivatives (Scheme 1). The most employed protocol involves
the classical Curtius rearrangement of the pertinent a-N-protected amino acid azides,
and the crude isocyanates prepared in this way can be subsequently trapped with
pentafluorophenol,[12] 2,4,5-trichlorophenol,[13] 4-nitrophenol,[14] or N-hydroxysuc-
cinimide[2o,2p,15] to give the corresponding stable carbamates, which react rapidly
with suitably protected amino acid derivatives to afford the desired ureidopeptides.
Sureshbabu et al. have reported that the isocyanates of a-N-[(9-fluorenylmethyl
)oxy]carbonyl (Fmoc)[16] and 1,1-dioxobenzo[b]thiophene-2-ylmethyloxycarbonyl
(Bsmoc)[17] protected amino acids and peptides are stable as such and directly usable
in the synthesis of ureidopeptides and peptidyl ureas. More recently, the same
authors have described the interesting and straightforward synthesis of a-N-
Fmoc=Bsmoc=Boc=Z-protected ureido peptidomimetics starting from the a-N-
protected amino acid or peptide acid in the presence of diphenylphosphoryl azide
(DPPA) followed by the one-pot addition of the amino acid ester.[18] On the other
hand, the suitable isocyanate intermediates can also be prepared through the
oxidative Hofmann or Lossen rearrangements of the C-terminal of a-N-protected
Scheme 1. Overview of current approaches for the insertion of a ureido linkage w[NHCONH] between
two neighboring a-amino acids.

ONE-POT SYNTHESIS OF DIPEPTIDYL UREAS
1029
amino and peptide amides[6e,19] or hydroxamic acids,[20] respectively (Scheme 1).
Under these conditions, the amide C ! N migration is usually promoted by bis(tri-
fluoroacetoxy)iodobenzene (PIFA), whereas N,N-dicyclohexylcarbodiimide (DCC)
or, better, the water-soluble 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide
(EDC) have been used with hydroxamic acids. The proposed isocyanate intermedi-
ates obtained from these rearrangements are directly intercepted by an amine
nucleophile.
Among the reported methods, some require multistep processes, high tempera-
tures with potentially explosive substrates (e.g., acyl azides in the Curtius rearrange-
ment), and the preliminary preparation of suitable reagents (e.g., amides or
hydroxamic acids). Moreover, the reaction conditions employed can catalyze the
hydrolysis of the formed isocyanates or the a-N-protecting group (PIFA)[19] as well
as promote significant racemization (DPPA).
In this context, we recently proposed a fast and simple preparation of a-N-
protected carbamoyl azides[21] and their use as suitable building blocks in the syn-
thesis of unsymmetrical ureas.[22] In the present paper, we report an efficient use
of carbamoyl azides in the one-pot, racemisation-free synthesis of a-N-protected
ureidopeptides.
RESULTS AND DISCUSSION
The simple and convenient preparation of a-N-protected ureidopeptide esters 1
reported here has been successfully accomplished in a two-step, one-pot reaction
sequence (Fig. 1, Table 1); the first step of the process consisted of the preparation
of the a-N-protected carbamoyl azide 2, promptly obtained as previously
described.[22] In the second step, the free a-amino acid methyl ester 4 (1.4 equiv. with
respect to 3) was slowly added into the reaction mixture containing the crude carba-
moyl azide 2, and stirring was continued for 3 h at 45 ꢀC. After separation of the
organic phase and removal of tetrahydrofuran (THF), the obtained residue was
thoroughly washed with water and crystallized from water=MeOH (80:20) to afford
the pure a-N-protected dipeptidyl urea methyl ester 1 in 75–95% overall yield.
With the aid of electrospray ionization–mass spectrometry (ESI-MS) analysis,
we observed that the reaction rate was not influenced by the nature of either the car-
bamoyl azide 2 or the a-amino acid methyl ester 4 involved. This circumstance
might be explained by the high reactivity of the carbamoyl function as a result of
Figure 1. Synthesis of a-N-protected dipeptidyl urea methyl esters 1.

1030
C. D. VENNERI, G. VERARDO, AND P. STRAZZOLINI
Table 1. Analytical data of the synthesized a-N-protected dipeptidyl urea methyl esters 1
R1
R2
Mp (ꢀC)
[20]
[a]D (c, solvent)
1
R
aae
abe
ace
bad
baf
bbf
bcf
(CH3)2CH
(CH3)2CH
(CH3)2CH
(CH3)2CHCH2
(CH3)2CHCH2
(CH3)2CHCH2
(CH3)2CHCH2
Ph
Et
t-Bu
Bn
HO-C6H4-CH2
HO-C6H4-CH2
HO-C6H4-CH2
PhCH2
177–179
137–139
162–164
132–135
125–127
152–155
158–160
127–129
100–101
143–144
180–183
138–140
165–167
154–156
186–188
þ13.6 (0.2, DMSO)
ꢁ34.3 (0.3, DMSO)
þ27.8 (0.4, DMSO)
ꢁ58.8 (0.3, MeOH)
ꢁ26.7 (0.3, CHCl3)
þ37.5 (0.6, CHCl3)
þ16.4 (0.5, MeOH)
þ16.7 (0.3, MeOH)
þ12.8 (0.4, DMSO)
þ40.7 (0.3, CHCl3)
ꢁ28.0 (0.5, MeOH)
ꢁ66.6 (0.3, DMSO)
ꢁ46.7 (0.2, CHCl3)
ꢁ67.7 (0.3, CHCl3)
ꢁ28.0 (0.5, DMSO)
Et
Et
1H-indole-3-CH2
1H-indole-3-CH2
1H-indole-3-CH2
PhCH2
t-Bu
Bn
cad
cba
cbd
daa
dab
dac
dba
dca
Et
Ph
Ph
t-Bu
t-Bu
Et
(CH3)2CHCH2
PhCH2
PhCH2
PhCH2
(CH3)2CHCH2
MeOOC(CH2)2
Ph
Et
Et
PhCH2
PhCH2
PhCH2
t-Bu
Bn
(CH3)2CHCH2
(CH3)2CHCH2
the presence of internally hydrogen-bonded conformations, which may favor the
availability of the CON3 group (Fig. 2).
1
This hypothesis was confirmed by comparison of the H NMR spectra of N-
ethoxycarbonyl-L-phenylalanine carbamoyl azide (2da) carried out at different con-
centrations in dimethylsulfoxide (DMSO-d6; from 50 to 0.5 mg=mL): the chemical
shift of all of the signals, and in particular the doublet (J ¼ 7.8 Hz) at d ¼ 8.48 ppm
assigned to the NHCON3 proton, as well as the broad singlet at d ¼ 7.56 ppm corre-
sponding to the NHCOOEt proton, remained unchanged. Moreover, a similar
experiment carried out at different temperatures (from 40 ꢀC to 90 ꢀC) evidenced
an upfield shift (ca. 0.5 ppm) effect involving just the two N-H signals, attributable
to the weakening of intramolecular hydrogen bonds.[23]
Figure 2. Possible conformations of 2 promoting the availability of the CON3 group.

ONE-POT SYNTHESIS OF DIPEPTIDYL UREAS
1031
To rule out any incursion of racemization under our experimental conditions,
Eoc-L-Phg-w[NHCONH]-L-Ph-OMe (1cad) was prepared by reaction of N-
ethoxycarbonyl-L-phenylglycine (3ca) with L-phenylalanine methyl ester (4d), and
its high-performance liquid chromatography (HPLC)–MS profile, as well as 1H
and 13C NMR spectra, were compared with those obtained from the reaction of
racemic N-ethoxycarbonyl-DL-phenylglycine (rac-3ca) with 4d. The ESI-HPLC-
MS analysis of the diastereoisomeric mixture of Eoc-D,L-Phg-w[NHCONH]-L-
Ph-OMe (diast-1cad) obtained from rac-3ca evidenced two distinct peaks at
Rt ¼ 12.1 and 14.3 min of the same area and presented an identical MSn fragmen-
tation pattern. Both ESI HPLC-MS peaks evidenced the following features: (i) the
sodium cationized molecule [M þ Na]þ at m=z ¼ 422, (ii) the MS2 spectrum of
[M þ Na]þ was characterized by the presence of three ions at m=z 333, 245, and
=
228 due to the loss of NH2COOEt (89 Da), Ph-CH NCOOEt (177 Da), and
PhCH(NH2)NHCOOEt (194 Da), respectively, and (iii) the fragmentation (MS3)
þ
=
of the ion at m=z ¼ 333 [PhCH NCONHCH(COOMe)CH2Ph þ Na] produced
the peak at m=z ¼ 228 [PhCH2CH(NCO)COOMe þ Na]þ. In addition, the H and
1
13C NMR spectra showed two sets of peaks for each proton and carbon signal,
respectively. On the other hand, when we carried out the reaction with 3ca, the cor-
responding H and 13C NMR spectra of 1cad did not exhibit a similar complexity
1
and the ESI-HPLC-MS analysis pointed out the presence of only one peak at
Rt ¼ 12.1 min. The analogous chromatographic and NMR behavior found in all
the synthesized dipeptidyl ureas 1 confirmed that the present protocol proceeds with
retention of configuration.
It is worth pointing out that when we attempted to synthesize Eoc-L-Phg-
w[NHCONH]-L-Ph-OMe (1cad) following the DPPA method proposed by
Sureshbabu et al.[18] the ESI-HPLC-MS analysis of the reaction mixture evidenced
the presence of an equimolar mixture of the two epimers 1cad and Eoc-D-Phg-
w[NHCONH]-L-Ph-OMe (epi-1cad), demonstrating that a complete racemization
had occurred. In addition, the same method employed in the reaction between
N-Boc-L-phenylglycine (3cb) and 4d afforded
w[NHCONH]-L-Ph-OMe (1cbd) and Boc-D-Phg-w[NHCONH]-L-Ph-OMe
(epi-1cbd) in the approximate ratio of 70:30.
a
mixture of Boc-L-Phg-
Some properties of dipeptidyl ureas 1 are reported in Table 1. All the products
1
obtained were fully characterized by H NMR, 13C NMR, MS, and infrared (IR)
spectra.
Taking into account the importance of mass spectrometry to elucidate the struc-
ture of peptides and peptidomimetics, we report some common features of the
ESI-MS spectra of a-N-protected ureidopeptide esters 1. In detail, the MS2 spectra
of sodium-cationized molecule [M þ Na]þ in the positive ion mode were characterized
(Scheme 2), in addition to the intense ion (100% relative intensity) due to the loss of
the carbamate moiety (NH2COOR1), by the presence of three additional peaks (10–
40% relative intensity) corresponding to the urea, the isocyanate, and the a-amino
methyl ester derived from the a-amino methyl ester moiety of 1. Moreover, the
MS2 spectra of [M þ Na]þ for the N-Boc-derivatives 1abc, 1bbf, 1cba, 1cbd and
1dba evidenced two fragment ions corresponding to the loss of isobutene and CO2.[22]
In summary, we have developed an efficient, mild, and simple one-pot synthesis
of ureidopeptide esters 1 starting from the carbamoyl azides of a-N-protected amino

1032
C. D. VENNERI, G. VERARDO, AND P. STRAZZOLINI
Scheme 2. Fragmentation pattern of a-N-protected ureidopeptide esters 1.
acids. The protocol proceeds with retention of configuration and represents a valid
alternative to the DPPA method. Moreover, the study of ESI-MSn spectra points out
some common fragmentation pathways involving both the amino acid residues
present in the ureidopeptide ester.
EXPERIMENTAL
All N-protected amino acids 3 were prepared by reported procedures.[24] All
solvents and reagents were purchased from Aldrich Chemical Company and were
used without further purification. Direct inlet mass spectra (DI-MS) and MSn experi-
ments were conducted with a Thermo Scientific Polaris Q ion trap mass spec-
trometer, working in the positive-ion 70-eV electron-impact mode. Spectra were
recorded in the range 35–450 u, and temperatures between 100 and 180 ꢀC were suit-
able to vaporize all the compounds into the ion source. The reactions were moni-
tored by ESI-MS in the positive-ion mode with a Finnigan LXQ (linear trap) by
simply diluting the intact reaction mixture with MeCN and directly infusing the
obtained solution into the ion source with the aid of a syringe pump. HPLC-ESI-
MS analyses were performed with the same instrument coupled with a Finnigan Sur-
veyor LC Pump Plus and equipped with a Finnigan Surveyor Autosampler Plus. The
LC separations were performed on a Zorbax SB C18 column (150 mm ꢂ 2.1 mm,
3.5 mm) operating at 30 ꢀC and isocratically at 0.2 mL=min with the mobile phase
was composed of MeOH=H2O (57:43, v=v). IR spectra were obtained with a Bruker
Vector 22 spectrophotometer using the KBr technique for solids and recorded in the
1
range 4000–400 cmꢁ1. H and 13C NMR spectra were recorded on a Bruker AC-F
200 spectrometer at 200 and 50 MHz, respectively, using DMSO-d6 at 40 ꢀC as sol-
1
vent. NMR peak locations are reported as d values from TMS. Some H multiplets
are characterized by the term app (apparent): this refers only to their appearance
and may be an oversimplification. Optical rotations were determined on suitable

ONE-POT SYNTHESIS OF DIPEPTIDYL UREAS
1033
solutions (g=100 mL) at 20 ꢀC using an AP-300 automatic polarimeter purchased
from Atago (Japan). Elemental analyses were performed with a Carlo Erba Mod.
1106 elemental analyzer. Melting points were determined with an automatic Mettler
(Mod. FP61) melting-point apparatus and are not corrected.
General Procedure for the Synthesis of Dipeptidyl Ureas 1
N-Methylmorpholine (NMM, 0.30 mL, 2.73 mmol) was slowly added into a
stirred solution of a-N-protected amino acid 3 (2.50 mmol) in THF (15 mL). After
5 min, isobutyl chloroformate (IBCF, 0.36 mL, 2.73 mmol) was slowly added into
the reaction mixture, previously cooled to ꢁ20 ꢀC, and stirring was continued for
20 min at the same temperature. The reaction mixture was subsequently warmed
to 0 ꢀC, and a solution of KH2PO4 (0.17 g, 1.25 mmol) in H2O (1 mL) was added
in one lot, followed after 5 min by a solution of KH2PO4 (1.70 g, 12.50 mmol) and
NaN3 (0.41 g, 6.25 mmol) in H2O (9 mL). After stirring for 30 min at 0 ꢀC, the mix-
ture was warmed to 45 ꢀC for 1.5 h, the free amino acid ester 4 (3.5 mmol) was slowly
added, and the resulting mixture was stirred for an additional 3 h at the same tem-
perature. THF was finally removed under reduced pressure, and the crude residue
was washed with water and crystallized from water=MeOH (80:20), affording the
product in a 75–95% overall yield.
Spectral Data of a-N-Protected Dipeptidyl Urea Methyl Esters 1
Eoc-L-Val-w[NHCONH]-L-Tyr-OMe (1aae). Yield: 80%; IR nmax (cmꢁ1):
3368 (br), 3293 (br), 2954, 1756, 1687, 1634, 1539, 1513, 1459, 1254, 1154, 1038,
1
817, 614; H NMR: d 0.80 and 0.81 [2 d, J ¼ 6.7, 6.7, 6 H, (CH3)2CH], 1.15 (t,
J ¼ 7.3, 3 H, CH3CH2), 1.85 [app sext, J ¼ 6.4, 1 H, (CH3)2CH], 2.74 and 2.84
(AB of ABX, J ¼ 14.1, 5.9, 5.6, 2 H, Ar-CH2), 3.58 (s, 3 H, OCH3), 3.96 (q,
ꢃ
J ¼ 7.3, 2 H, OCH2), 4.35 (app q, J ¼ 6.7, 1 H, CHCOO), 4.81 (app q, J ¼ 9.1, 1
H, NH ꢃ CHNH), 6.33 (br t, J ¼ 9.1, 2 H, NHCONH), 6.65 (app d, J ¼ 8.2, 2 H,
Ar-H), 6.92 (app d, J ¼ 8.2, 2 H, Ar-H), 7.24 (br s, 1 H, NHCOO), 9.23 (br s, 1
H, OH); 13C NMR: d 15.5, 18.9, 19.2, 33.3, 38.0, 52.4, 54.9, 60.3, 63.8, 115.9,
127.7, 130.9, 156.1, 156.9, 157.1, 173.8; EI-MS m=z: 293 (13), 292 (10), 249 (70),
233 (9), 196 (29), 178 (52), 147 (22), 144 (100), 116 (23), 107 (47), 98 (6), 72 (48);
ESI-MS m=z: 404 [M þ Na]þ ! (MS2) 315, 261, 244, 218. Anal. calcd. for
C18H27N3O6 (381.43): C, 56.68; H, 7.13; N, 11.02. Found: C, 56.43; H, 7.00; N,
11.12.
Boc-L-Val-w[NHCONH]-L-Tyr-OMe (1abe). Yield: 75%; IR nmax (cmꢁ1):
3364 (br), 3291 (br), 2978, 1749, 1688, 1654, 1563, 1515, 1450, 1368, 1247, 1208,
1
1015, 780; H NMR: d 0.78 and 0.80 [2 d, J ¼ 6.5, 6.5, 6 H, (CH3)2CH], 1.37 [s, 9
H, (CH3)3C], 1.79 [app sext, J ¼ 6.5, 1 H, (CH3)2CH], 2.73 and 2.83 (AB of ABX,
J ¼ 13.2, 6.7, 6.5, 2 H, Ar-CH2), 3.57 (s, 3 H, OCH3), 4.34 (app q, J ¼ 6.8, 1 H,
ꢃCHCOO), 4.75 (app q, J ¼ 8.2, 1 H, NH ꢃ CHNH), 6.28 (br d, J ¼ 9.1, 1 H,
NHCONH), 6.42 (br d, J ¼ 7.6, 1 H, NHCONH), 6.65 (app d, J ¼ 8.5, 2 H,
Ar-H), 6.92 (app d, J ¼ 8.5, 2 H, Ar-H), 7.02 (br s, 1 H, NHCOO), 9.19 (br s, 1
H, OH); 13C NMR: d 18.9, 19.2, 29.0, 33.2, 38.0, 52.3, 54.9, 63.9, 79.9, 115.8,

1034
C. D. VENNERI, G. VERARDO, AND P. STRAZZOLINI
127.7, 130.8, 155.5, 156.9, 157.1, 173.9; EI-MS m=z: 366 (3), 293 (10), 292 (15), 233
(8), 196 (25), 178 (100), 172 (10), 147 (39), 116 (16), 107 (89), 98 (10), 72 (40), 56 (34);
ESI-MS m=z: 432 [M þ Na]þ ! (MS2) 376, 332, 315, 261, 244, 218. Anal. calcd. for
C20H31N3O6 (409.48): C, 58.66; H, 7.63; N, 10.26. Found: C, 58.77; H, 7.54; N,
10.20.
Z-L-Val-w[NHCONH]-L-Tyr-OMe (1ace). Yield: 77%; IR nmax (cmꢁ1): 3377
1
(br), 3286 (br), 2958, 1744, 1692, 1634, 1571, 1537, 1257, 1047; H NMR: d 0.81
and 0.82 [2 d, J ¼ 6.7, 6.5, 6 H, (CH3)2CH], 1.82 [app sext, J ¼ 6.5, 1 H, (CH3)2CH],
2.74 and 2.84 (AB of ABX, J ¼ 12.9, 7.3, 7.0, 2 H, Ar-CH2), 3.58 (s, 3 H, OCH3),
ꢃ
4.37 (app q, J ¼ 7.0, 1 H, CHCOO), 4.87 (app q, J ¼ 8.5, 1 H, NH ꢃ CHNH), 5.01
(app s, 2 H, PhCH2), 6.41 (br d, J ¼ 8.2, 2 H, NHCONH), 6.66 (app d, J ¼ 8.5, 2
H, Ar-H), 6.93 (app d, J ¼ 8.8, 2 H, Ar-H), 7.30–7.43 (m, 5 H, Ph-H), 7.53 (br s, 1
H, NHCOO), 9.21 (br s, 1 H, OH); 13C NMR: d 18.8, 19.2, 33.1, 38.0, 52.4, 54.9,
63.9, 65.9, 115.9, 127.6, 128.5, 129.0, 129.1, 130.8, 138.0, 155.9, 156.9, 157.1,
173.7; EI-MS m=z: 400 (5), 292 (7), 249 (23), 233 (5), 206 (15), 178 (52), 162 (27),
147 (21), 116 (10), 107 (100), 98 (3), 91 (90), 79 (31), 77 (30), 72 (19); ESI-MS m=z:
466 [M þ Na]þ ! (MS2) 315, 261, 244, 218. Anal. calcd. for C23H29N3O6 (443.50):
C, 62.29; H, 6.59; N, 9.47. Found: C, 62.20; H, 6.68; N 9.36.
Eoc-L-Leu-w[NHCONH]-L-Phe-OMe (1bad). Yield: 84%; IR nmax (cmꢁ1):
3354 (br), 3283 (br), 2985, 2956, 1749, 1696, 1658, 1562, 1530, 1467, 1359, 1241,
1
1210, 1046, 702; H NMR: d 0.90 [app d, J ¼ 5.9, 6 H, (CH3)2CH], 1.18 (t, J ¼ 7.3,
3 H, CH2CH3), 1.50–1.73 (m, 3 H, CHCH2), 2.99 and 3.11 (AB of ABX, J ¼ 13.8,
6.7, 6.5, 2 H, PhCH2), 3.64 (s, 3 H, OCH3), 4.00 (q, J ¼ 7.0, 2 H, OCH2), 4.71
ꢃ
(app q, J ¼ 6.5, 1 H, CHCOO), 5.13 (app quint, J ¼ 7.0, 1 H, NH ꢃ CHNH), 6.20
(br d, J ¼ 9.0, 1 H, NHCONH), 6.40 (br d, J ¼ 7.1, 1 H, NHCONH), 7.05–7.34
(m, 6 H, Ph-H þ NHCOO); 13C NMR: d 14.8, 22.5, 22.6, 25.1, 38.7, 44.0, 52.3,
54.5, 58.2, 61.2, 127.0, 128.6, 129.6, 136.9, 156.7, 157.2, 173.4; EI-MS m=z: 322
(44), 291 (37), 290 (86), 247 (51), 231 (41), 199 (27), 180 (100), 162 (91), 158 (93),
130 (15), 120 (73), 117 (75), 91 (81); ESI-MS m=z: 402 [M þ Na]þ ! (MS2) 313,
245, 228, 202. Anal. calcd. for C19H29N3O5 (379.46): C, 60.14; H, 7.70; N, 11.07.
Found: C, 60.18; H, 7.68; N, 11.15.
Eoc-L-Leu-w[NHCONH]-L-Trp-OMe (1baf). Yield: 85%; IR nmax (cmꢁ1):
3369 (br), 3259 (br), 2958, 2928, 1753, 1693, 1643, 1531, 1440, 1372, 1341, 1240,
1
1020, 740; H NMR: d 0.86 [app d, J ¼ 5.6, 6 H, (CH3)2CH], 1.15 (t, J ¼ 7.0, 3 H,
CH2CH3) 1.32–1.68 (m, 3 H, CHCH2), 3.02 and 3.12 (AB of ABX, J ¼ 13.5, 7.3,
5.9, 2 H, Ar-CH2), 3.97 (q, J ¼ 7.1, 2 H, OCH2), 4.49 (app q, J ¼ 6.5, 1 H, ꢃCHCOO),
5.10 (app quint, J ¼ 7.9, 1 H, NH ꢃ CHNH), 6.37 (br d, J ¼ 7.3, 1 H, NHCONH),
6.39 (br d, J ¼ 8.2, 1 H, NHCONH), 6.87–7.19 (m, 3 H, Ar-H), 7.21–7.57 (m, 3
H, 2 ꢂ Ar-H þ NHCOO), 10.87 (br s, 1 H, Ar-NH); 13C NMR: d 15.1, 23.1, 23.2,
25.2, 28.9, 45.1, 52.5, 54.3, 57.5, 60.3, 110.0, 112.2, 119.0, 119.2, 122.0, 124.5,
128.2, 137.0, 156.0, 156.9, 174.1; EI-MS m=z: 361 (2), 330 (4), 329 (8), 286 (5), 270
(8), 244 (3), 219 (7), 201 (49), 170 (8), 158 (7), 130 (100); ESI-MS m=z: 441
[M þ Na]þ ! (MS2) 352, 284, 267, 241. Anal. calcd. for C21H30N4O5 (418.49): C,
60.27; H, 7.23; N, 13.39. Found: C, 60.25; H, 7.28; N, 13.45.

ONE-POT SYNTHESIS OF DIPEPTIDYL UREAS
1035
Boc-L-Leu-w[NHCONH]-L-Trp-OMe (1bbf). Yield: 77%; IR nmax (cmꢁ1):
3386 (br), 3326 (br), 3265 (br), 2930, 1718, 1687, 1663, 1577, 1522, 1459, 1365,
1
1303, 1258, 1170, 1044, 866, 757; H NMR: d 0.84 [app d, J ¼ 5.6, 6 H, (CH3)2CH],
1.37 [s, 9 H, (CH3)3C], 1.38–1.59 (m, 3 H, CHCH2), 3.06 and 3.91 (AB of ABX,
J ¼ 13.8, 7.3, 6.2, 2 H, Ar-CH2), 3.56 (s, 3 H, OCH3), 4.47 (app q, J ¼ 6.2, 1 H,
ꢃCHCOO), 5.04 (app quint, J ¼ 7.6, 1 H, NH ꢃ CHNH), 6.28 (br d, J ¼ 9.1, 1 H,
NHCONH), 6.36 (br d, J ¼ 7.9, 1 H, NHCONH), 6.91–7.15 (m, 4 H,
3 ꢂ Ar-H þ NHCOO), 7.32 (app d, J ¼ 7.9, 1 H, Ar-H), 7.44 (app d, J ¼ 7.6, 1 H,
Ar-H), 10.85 (br s, 1 H, Ar-NH); 13C NMR: d 23.2, 23.3, 25.3, 28.9, 29.2, 45.2,
52.6, 54.3, 57.2, 78.6, 110.1, 112.3, 119.1, 119.3, 121.9, 124.6, 128.2, 137.0, 155.4,
157.0, 174.3; EI-MS m=z: 389 (2), 330 (4), 286 (3), 270 (8), 244 (7), 219 (4), 201
(67), 186 (4), 170 (8), 130 (100); ESI-MS m=z: 469 [M þ Na]þ ! (MS2) 413, 369,
352, 284, 267, 241. Anal. calcd. for C23H34N4O5 (446.55): C, 61.86; H, 7.67; N,
12.55. Found: C, 61.90; H, 7.65; N, 12.50.
Z-L-Leu-w[NHCONH]-L-Trp-OMe (1bcf). Yield: 87%; IR nmax (cmꢁ1): 3408
(br), 3319 (br), 3293 (br), 2953, 1731, 1695, 1647, 1571, 1536, 1456, 1433, 1338,
1
1232, 738, 697; H NMR: d 0.85 [app d, J ¼ 5.6, 6 H, (CH3)2CH], 1.31–1.67 (m, 3
H, CHCH2), 3.02 and 3.11 (AB of ABX, J ¼ 13.4, 7.1, 5.6, 2 H, Ar-CH2), 3.56 (s,
ꢃ
3 H, OCH3), 4.49 (app q, J ¼ 6.2, 1 H, CHCOO), 5.00 (app s, 2 H, PhCH2), 5.13
(app quint, J ¼ 7.4, 1 H, NH ꢃ CHNH), 6.33 (br d, J ¼ 7.7, 1 H, NHCONH), 6.40
(br d, J ¼ 8.5, 1 H, NHCONH), 6.91–7.12 (m, 3 H, Ar-H), 7.27–7.56 (m, 8 H,
2 ꢂ Ar-H þ 5 ꢂ Ph-H þ NHCOO), 10.85 (br s, 1 H, Ar-NH); 13C NMR: d 23.0,
23.1, 25.2, 28.9, 45.3, 52.5, 54.2, 57.6, 66.0, 110.0, 112.2, 119.0, 119.2, 121.8, 124.5,
128.2, 128.6, 129.0, 129.4, 137.0, 138.0, 155.8, 156.9, 174.0; EI-MS m=z: 423 (2),
329 (4), 286 (2), 270 (6), 244 (3), 220 (2), 219 (4), 201 (23), 170 (4), 130 (100), 91
(28); ESI-MS m=z: 503 [M þ Na]þ ! (MS2) 352, 284, 267, 241. Anal. calcd. for
C26H32N4O5 (480.56): C, 64.98; H, 6.71; N, 11.66. Found: C, 64.88; H, 6.80; N,
11.71.
Eoc-L-Phg-w[NHCONH]-L-Phe-OMe (1cad). Yield: 92%; IR nmax (cmꢁ1):
3392 (br), 3295 (br), 2982, 1731, 1697, 1641, 1566, 1528, 1259, 1068, 1037, 748, 696;
1H NMR: d 1.14 (t, J ¼ 6.8, 3 H, CH2CH3), 2.88 and 3.01 (AB of ABX, J ¼ 13.5,
8.2, 7.6, 2 H, Ph-CH2), 3.62 (s, 3 H, OCH3), 3.97 (q, J ¼ 7.0, 2 H, OCH2), 4.43
ꢃ
(app q, J ¼ 7.6, 1 H, CHCOO), 6.19 (t, J ¼ 8.5, 1 H, NH ꢃ CHNH), 6.54 (br d,
J ¼ 9.1, 1 H, NHCONH), 6.83 (br d, J ¼ 8.8, 1 H, NHCONH), 7.10–7.42 (m, 10 H,
Ar-H), 7.97 (br s, 1 H, NHCOO); 13C NMR: d 15.5, 38.4, 52.7, 55.1, 60.7, 61.1,
126.9, 127.6, 128.3, 129.2, 129.3, 130.1, 137.9, 142.4, 157.1, 157.2, 173.8; EI-MS m=z:
311 (20), 310 (15), 267 (5), 266 (40), 251 (28), 250 (10), 219 (17), 208 (8), 180 (10),
178 (22), 162 (77), 150 (15), 132 (100), 106 (55), 91 (90), 77 (45); ESI-MS m=z: 422
[M þ Na]þ ! (MS2) 333, 245, 228, 202. Anal. calcd. for C21H25N3O5 (399.45): C,
63.15; H, 6.31; N, 10.52. Found: C, 63.05; H, 6.33; N, 10.47.
Boc-L-Phg-w[NHCONH]-L-Leu-OMe (1cba). Yield: 95%; IR nmax (cmꢁ1):
3401 (br), 3315 (br), 2962, 1748, 1687, 1653, 1564, 1528, 1366, 1254, 1172, 698; H
1
NMR: d 0.88 and 0.90 [2 d, J ¼ 6.5, 6.7, 6 H, (CH3)2CH], 1.37 [s, 9 H, (CH3)3C],
1.40–1.74 (m, 3 H, CHCH2), 3.62 (s, 3 H, OCH3), 4.19 (app q, J ¼ 6.5, 1 H,
ꢃCHCOO), 6.16 (t, J ¼ 8.8, 1 H, NH ꢃ CHNH), 6.62 (br d, J ¼ 7.9, 2 H, NHCONH),

1036
C. D. VENNERI, G. VERARDO, AND P. STRAZZOLINI
7.22–7.38 (m, 5 H, Ar-H), 7.58 (br s, 1 H, NHCOO); 13C NMR: d 22.5, 23.5, 25.1,
29.0, 41.7, 51.9, 52.2, 60.6, 79.0, 126.7, 128.1, 128.9, 142.7, 157.0, 157.1, 174.8;
EI-MS m=z: 277 (20), 276 (7), 233 (12), 217 (88), 206 (59), 174 (35), 165 (58), 150
(60), 146 (28), 132 (91), 128 (8), 106 (100), 57 (11); ESI-MS m=z: 416
[M þ Na]þ ! (MS2) 360, 316, 299, 211, 194, 168. Anal. calcd. for C20H31N3O5
(393.48): C, 61.05; H, 7.94; N, 10.68. Found: C, 61.19; H, 7.89; N, 10.65.
Boc-L-Phg-w[NHCONH]-L-Phe-OMe (1cbd). Yield: 86%; IR nmax (cmꢁ1):
3387 (br), 3320 (br), 2985, 1735, 1699, 1646, 1568, 1521, 1447, 1362, 1254, 1215,
1
1165, 1096, 1068, 1027, 747, 699; H NMR: d 1.37 [s, 9 H, (CH3)3C], 2.87 and
3.00 (AB of ABX, J ¼ 13.5, 7.9, 7.9, 2 H, Ph-CH2), 3.60 (s, 3 H, OCH3), 4.42 (app
ꢃ
q, J ¼ 7.7, 1 H, CHCOO), 6.14 (t, J ¼ 8.2, 1 H, NH ꢃ CHNH), 6.58 (br d, J ¼ 7.9,
1 H, NHCONH), 6.76 (br d, J ¼ 8.5, 1 H, NHCONH), 7.12–7.47 (m, 10 H,
Ar-H), 7.67 (br s, 1 H, NHCOO); 13C NMR: d 29.1, 38.5, 52.7, 55.1, 60.5, 79.0,
126.9, 127.6, 128.2, 129.1, 129.2, 130.1, 137.9, 142.7, 157.1, 157.2, 173.8; EI-MS
m=z: 311 (15), 310 (33), 267 (26), 266 (57), 251 (38), 250 (21), 219 (32), 206 (55),
180 (16), 162 (81), 150 (56), 132 (100), 106 (33), 91 (86); ESI-MS m=z: 450
[M þ Na]þ ! (MS2) 394, 350, 333, 245, 228, 202. Anal. calcd. for C23H29N3O5
(427.50): C, 64.62; H, 6.84; N, 9.83. Found: C, 64.64; H, 6.90; N, 9.80.
Eoc-L-Phe-w[NHCONH]-L-Leu-OMe (1daa). Yield: 80%; IR nmax (cmꢁ1):
3398 (br), 3311 (br), 2960, 1723, 1693, 1644, 1568, 1523, 1298, 1265, 999, 757, 700;
1H NMR: d 0.80 and 0.85 [2 d, J ¼ 6.5, 6.5, 6 H, (CH3)2CH], 1.11 (t, J ¼ 7.1, 3 H,
CH2CH3), 1.29–1.60 (m, 3 H, CHCH2), 2.86 (br app d, J ¼ 6.8, 2 H, Ph-CH2),
3.60 (s, 3 H, OCH3), 3.92 (q, J ¼ 7.0, 2 H, OCH2), 4.14 (app q, J ¼ 7.9, 1 H,
ꢃCHCOO), 5.15 (app quint, J ¼ 7.7, 1 H, NH ꢃ CHNH), 6.43 (br t, J ¼ 7.9, 2 H,
NHCONH), 7.08–7.32 (m, 5 H, Ar-H), 7.64 (br s, 1 H, NHCOO); 13C NMR: d
15.6, 22.5, 23.7, 25.1, 42.0, 42.2, 51.6, 52.6, 60.4, 127.1, 129.0, 130.1, 138.7, 156.7,
157.0, 175.0; EI-MS m=z: 291 (6), 290 (19), 288 (53), 258 (20), 247 (8), 231 (24),
202 (13), 192 (21), 191 (23), 146 (70), 145 (55), 128 (15), 120 (42), 119 (70), 118
(65), 117 (100), 91 (37), 89 (47); ESI-MS m=z: 402 [M þ Na]þ ! (MS2) 313, 211,
194, 168. Anal. calcd. for C19H29N3O5 (379.46): C, 60.14; H, 7.70; N, 11.07. Found:
C, 60.28; H, 7.65; N, 11.19.
Eoc-L-Phe-w[NHCONH]-L-Glu-(OMe)2 (1dab). Yield: 78%; IR nmax (cmꢁ1):
3396 (br), 3304 (br), 2979, 1732, 1700, 1641, 1575, 1553, 1513, 1297, 1266, 1237,
1
1053, 951, 705; H NMR: d 1.11 (t, J ¼ 6.8, 3 H, CH2CH3), 1.62–2.05 (m, 2 H,
ꢃCHCH2CH2), 2.28 (app t, J ¼ 7.3, 2 H, CH2COO), 2.89 (br app d, J ¼ 6.7, 2 H,
Ph-CH2), 3.58 (s, 3 H, CH2COOCH3),ꢃ3.62 (s, 3 H, ꢃCHCOOCH3), 3.93 (q,
J ¼ 7.0, 2 H, OCH2), 4.10–4.24 (m, 1 H, CHCOO), 5.16 (app quint, J ¼ 7.6, 1 H,
NH ꢃ CHNH), 6.40 (br d, J ¼ 8.5, 1 H, NHCONH), 6.47 (br d, J ¼ 8.2, 1 H,
NHCONH), 7.08–7.34 (m, 5 H, Ar-H), 7.46 (br s, 1 H, NHCOO); 13C NMR: d
15.4, 28.1, 30.3, 41.3, 52.2, 52.5, 60.4, 60.5, 61.8, 127.0, 128.9, 130.0, 138.7, 156.2,
156.9, 173.4, 173.9; EI-MS m=z: 321 (10), 320 (9), 318 (45), 286 (84), 277 (8), 261
(8), 246 (8), 192 (57), 191 (50), 176 (53), 158 (7), 145 (24), 144 (31), 120 (39), 119
(41), 118 (31), 117 (100), 91 (24), 89 (24); ESI-MS m=z: 432 [M þ Na]þ ! (MS2)
343, 241, 224, 198. Anal. calcd. for C19H27N3O7 (409.44): C, 55.74; H, 6.65; N,
10.26. Found: C, 55.70; H, 6.70; N, 10.36.

ONE-POT SYNTHESIS OF DIPEPTIDYL UREAS
1037
Eoc-L-Phe-w[NHCONH]-L-Phg-OMe (1dac). Yield: 79%; IR nmax (cmꢁ1):
3381 (br), 3306 (br), 2984, 1737, 1697, 1645, 1553, 1514, 1297, 1234, 1053, 699; H
1
NMR: d 1.12 (t, J ¼ 7.0, 3 H, CHCH3), 2.90 (br app d, J ¼ 6.8, 2 H, Ph-CH2),
3.61 (s, 3 H, OCH3), 3.92 (q, J ¼ 6.8, 2 H, OCH2), 5.19 (app quint, J ¼ 7.6, 1 H,
NH ꢃ CHNH), 5.28 (d, J ¼ 7.6, 1 H, ꢃCHCOO), 6.52 (br d, J ¼ 7.9, 1 H, NHCONH),
6.96 (br d, J ¼ 7.6, 1 H, NHCONH), 7.12–7.48 (m, 10 H, Ar-H), 7.55 (br s, 1 H,
NHCOO); 13C NMR: d 15.3, 41.8, 52.9, 57.5, 60.4, 60.6, 127.0, 128.0, 128.9,
129.5, 129.6, 130.0, 138.2, 138.6, 156.0, 156.7, 172.7; EI-MS m=z: 340 (9), 311
(10), 310 (26), 308 (47), 278 (51), 276 (23), 267 (8), 251 (47), 192 (24), 191 (36),
166 (97), 145 (76), 120 (39), 119 (100), 118 (69), 117 (83), 91 (53), 89 (40); ESI-MS
m=z: 422 [M þ Na]þ ! (MS2) 333, 231, 214, 188. Anal. calcd. for C21H25N3O5
(399.45): C, 63.15; H, 6.31; N, 10.52. Found: C, 63.18; H, 6.25, N, 10.50.
Boc-L-Phe-w[NHCONH]-L-Leu-OMe (1dba). Yield: 85%; IR nmax (cmꢁ1):
3396 (br), 3323 (br), 2958, 1728, 1694, 1648, 1555, 1517, 1368, 1299, 1262, 1172,
1
1011, 701; H NMR: d 0.80 and 0.85 [2 d, J ¼ 6.2, 6.5, 6 H, (CH3)2CH], 1.33 [s, 9
H, (CH3)3C], 1.32–1.65 (m, 3 H, CHCH2), 2.86 (br app d, J ¼ 6.8, 2 H, Ph-CH2),
ꢃ
3.60 (s, 3 H, OCH3), 4.13 (app q, J ¼ 7.3, 1 H, CHCOO), 5.10 (app quint, J ¼ 7.6,
1 H, NH ꢃ CHNH), 6.40 (br d, J ¼ 7.6, 1 H, NHCONH), 6.50 (br d, J ¼ 8.2, 1 H,
NHCONH), 7.07–7.38 (m, 5 H, Ar-H), 7.60 (br s, 1 H, NHCOO); 13C NMR: d
22.5, 23.6, 25.1, 29.1, 41.7, 41.9, 51.6, 52.6, 60.2, 78.8, 127.0, 128.9, 130.1, 138.7,
156.8, 157.1, 175.0; EI-MS m=z: 316 (10), 291 (7), 290 (7), 260 (38), 247 (5), 231
(18), 220 (8), 216 (100), 164 (11), 146 (92), 128 (8), 120 (44), 119 (34), 118 (23), 91
(18), 89 (12); ESI-MS m=z: 430 [M þ Na]þ ! (MS2) 374, 330, 313, 211, 194, 168.
Anal. calcd. for C21H33N3O5 (407.51): C, 61.90; H, 8.16; N, 10.31. Found: C,
61.85; H, 8.10; N, 10.42.
Z-L-Phe-w[NHCONH]-L-Leu-OMe (1dca). Yield: 85%; IR nmax (cmꢁ1): 3400
(br), 3318 (br), 2951, 1727, 1694, 1636, 1570, 1535, 1453, 1295, 1266, 1087, 1044,
1020, 745, 699; 1H NMR: d 0.81 and 0.85 [2 d, J ¼ 6.2, 6.2, 6 H, (CH3)2CH],
ꢃ
1.30–1.68 (m, 3 H, CHCH2), 2.89 (br app d, J ¼ 7.1, 2 H, Ph-CH2 CH), 3.60 (s, 3
ꢃ
H, OCH3), 4.16 (app q, J ¼ 7.9, 1 H, CHCOO), 4.98 (app s, 2 H, PhCH2), 5.20
(app quint, J ¼ 7.5, 1 H, NH ꢃ CHNH), 6.43 (br d, J ¼ 6.8, 2 H, NHCONH),
7.07–7.45 (m, 10 H, Ar-H), 7.68 (br s, 1 H, NHCOO); 13C NMR: d 22.4, 23.5,
25.0, 41.3, 41.9, 51.5, 52.4, 60.5, 65.9, 127.0, 128.3, 128.5, 128.9, 129.1, 130.0,
137.9, 138.5, 156.7, 156.9, 174.8; EI-MS m=z: 350 (28), 291 (6), 290 (13), 258 (16),
254 (10), 247 (8), 231 (24), 179 (27), 146 (38), 145 (47), 128 (10), 120 (20), 119
(42), 118 (35), 91 (100); ESI-MS m=z: 464 [M þ Na]þ ! (MS2) 313, 211, 194, 168.
Anal. calcd. for C24H31N3O5 (441.53): C, 65.29; H, 7.08; N, 9.52. Found: C,
65.35; H, 7.12; N, 9.40.
ACKNOWLEDGMENTS
The authors are grateful to Dr. S. Turco for recording 1H and 13C NMR spec-
tra, Dr. I. Duse for MS measurements, and Mr. P. Padovani for expert instrumental
maintenance.

1038
C. D. VENNERI, G. VERARDO, AND P. STRAZZOLINI
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