Bergman cyclization of acyclic amino acid derived enediynes leads to the formation of 2,3-dihydrobenzo[ f ]isoindoles

Article abstract of DOI:10.1021/jo101302n

Figure presented. Enediyne-peptide conjugates are recently recognized as useful tools in targeting various proteins, while the mechanism underlying the observed activity remains somewhat unclear. Addressing these issues, we have prepared acyclic amino acid derived enediynes and disclosed a novel thermally induced cyclization-elimination pathway. Initial formation of 1,4-benzene diradical and H-atom abstraction from an external donor is followed by S _N2 substitution leading to 2,3-dihydrobenzo[f]isoindoles. The proposed mechanism is supported by experimental and computational data. Additionally, we showed that amino acid side chains, although placed three bonds away from acetylene terminuses, have an appreciable influence on the reactivity of studied enediynes. These results demonstrate that amino acid or peptide parts of enediyne-peptide conjugates cannot be considered as recognition elements exclusively but may also participate in various reactions through amine functionality.

Full text of DOI:10.1021/jo101302n

pubs.acs.org/joc
Bergman Cyclization of Acyclic Amino Acid Derived Enediynes Leads
to the Formation of 2,3-Dihydrobenzo[f]isoindoles
†
Matija Gredicak, Ivana Matanovic, Boris Zimmermann, and Ivanka Jeric*
†
,†
ꢀ
ꢁ ‡
ꢁ
†Division of Organic Chemistry and Biochemistry and ‡Division of Physical Chemistry,
Ru{er Bosꢀkovicꢁ Institute, Bijenicꢀka 54, Zagreb, 10000-HR Croatia
ijeric@irb.hr
Received July 2, 2010
Enediyne-peptide conjugates are recently recognized as useful tools in targeting various proteins,
while the mechanism underlying the observed activity remains somewhat unclear. Addressing these
issues, we have prepared acyclic amino acid derived enediynes and disclosed a novel thermally
induced cyclization-elimination pathway. Initial formation of 1,4-benzene diradical and H-atom
abstraction from an external donor is followed by SN2 substitution leading to 2,3-dihydrobenzo-
[f]isoindoles. The proposed mechanism is supported by experimental and computational data.
Additionally, we showed that amino acid side chains, although placed three bonds away from
acetylene terminuses, have an appreciable influence on the reactivity of studied enediynes. These
results demonstrate that amino acid or peptide parts of enediyne-peptide conjugates cannot be
considered as recognition elements exclusively but may also participate in various reactions through
amine functionality.
Introduction
abstraction of two protons from the DNA backbone, result-
ing in the cleavage of the DNA and consequently death of
a cell. Total syntheses of natural enediynes brought solutions
for many challenging molecular architectures and made a
distinct contribution to the progress of synthetic methodo-
logies.3-6 Apart from their biological activity, enediynes are
key building blocks in the synthesis of helical polycyclic
aromatic hydrocarbons, interesting due to their optical and
electronic properties.7 Also, it was suggested that enediynes
are potentially useful mechanophores, stress-sensitive units
applied to the synthesis of polymers whose properties can be
altered by mechanical energy.8
Knowledge gathered in the field of enediyne chemistry
has greatly influenced research in different areas of organic
chemistry, spectroscopy, and material-oriented chemistry.
Isolated from soil bacteria in the 1980s, enediyne compounds
soon became recognized as important synthetic targets from
numerous points of view. Antitumor properties of naturally
occurring enediynes are attributed to the embedded enediyne
((Z)-3-hexene-1,5-diyne) moiety.1 After initial triggering
through intramolecular nucleophilic addition, the enediyne
group undergoes Bergman cyclization (BC).2 Stabilization
of thus formed 1,4-benzene diradical is achieved by the
(1) (a) Smith, A. L.; Nicolaou, K. C. J. Med. Chem. 1996, 39, 2103–2117.
(b) Edo, K.; Mizugaki, M.; Koide, Y.; Seto, H.; Furihata, K.; Otake, N.;
Ishida, N. Tetrahedron Lett. 1985, 26, 331–334. (c) Lee, M. D.; Dunne, T. S.;
Chang, C. C.; Ellestad, G. A.; Siegel, M. M.; Morton, G. O.; McGahren,
W. J.; Borders, D. B. J. Am. Chem. Soc. 1987, 109, 3466–3468. (d) Golik, J.;
Clardy, J.; Dubay, G.; Groenwold, G.; Kawaguchi, H.; Konishi, M.;
Krishnan, B.; Ohkuma, H.; Saitoh, K.; Doyle, T. W. J. Am. Chem. Soc.
1987, 109, 3461–3462. (e) Konishi, M.; Ohkuma, H.; Tsuno, T.; Oki, T.;
VanDuyne, G. D.; Clardy, J. J. Am. Chem. Soc. 1990, 112, 3715–3716.
(2) Bergman, R. G. Acc. Chem. Res. 1973, 6, 25–31.
(3) Guanti, G.; Riva, r. Org. Biomol. Chem. 2003, 1, 3967–3976.
(4) Komano, K.; Shimamura, S.; Inoue, M.; Hirama, M. J. Am. Chem.
Soc. 2007, 129, 14184–14186.
(5) Ren, F.; Hogan, P. C.; Anderson, A. J.; Myers, A. J. Am. Chem. Soc.
2007, 129, 5381–5383.
(6) Desrat, S.; van de Weghe, P. J. Org. Chem. 2009, 74, 6728–6734.
(7) Zhang, Y.; Petersen, J.; Wang, K. K. Tetrahedron 2008, 64, 1285–
1293.
(8) Hickenboth, C. R.; Rule, J. D.; Moore, J. S. Tetrahedron 2008, 64,
8435–8448.
DOI: 10.1021/jo101302n
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Published on Web 08/18/2010
J. Org. Chem. 2010, 75, 6219–6228 6219
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Structural complexity, nonselectivity, and poor stability of
natural enediynes urge scientists to find new molecules with
advantages of natural occurring ones but having less draw-
backs. With this aim, a number of cyclic and acyclic enediyne-
related molecules have been prepared and their activity has
been tested upon thermal activation,9 photochemical activa-
tion,10 or complexation with metals.11
SCHEME 1. Synthetic Path to 4a-ea
Our scope of research is the study of acyclic amino acid
derived enediynes. Amino acids, as carriers of the enediyne
moiety, have multiple advantages: they are good complexa-
tion ligands; an array of side chains provides easy change of
molecule properties (e.g., lipophilicity, acidity) and versati-
lity of functional groups enables binding of other mole-
cules. Therefore, enediyne-amino acid conjugates are lately
recognized as important tools in studies of Bergman cycliza-
tion. A series of photoactivated lysine-enediyne conjugates
have been used to demonstrate nonrandom double-stranded
DNA cleavage and the role of amino acid residue on the
selectivity of DNA cleavage.12 Also, some of these conjugates
were capable of recognizing the site of single-stranded DNA
cleavage and induce conversion of this damage into double-
stranded DNA cleavage. Moreover, the activity of lysine-
enediyne conjugates toward pH-controlled light-induced
Bergman cyclization was emphasized recently.12 It was shown
that enediyne-amino acid conjugates effectively target R-chy-
motrypsin and inhibit its activity,13 while enediyne-(Asp)3
conjugate showed high affinity binding to the H1 histone,
causing its degradation upon photoactivation.14 Cyclic analo-
gues with an enediyne moiety bonded to the amino acid
R-carbon have also been reported and their activity evaluated.15
Contrary to that, our approach was to retain properties of
natural amino acids by placing the enediyne moiety at the
N-terminus and benefit from the neighboring side chains
(compounds 4a-e, Scheme 1). Thus, C-terminuses can be easily
modified in defined steps of the synthesis (coupling with amino
acids or polyamines to enhance solubility or binding to biomole-
cules), while the enediyne skeleton will direct conformation of
peptide grown on both C-terminuses. We were also driven by
the question: can side chains distinctively influence the rate of
Bergman cyclization? Here, we answer this question and provide
evidence about the process following initial Bergman cyclization.
aReagents and conditions: (a) o-C6H4I2 (3.6mmol), piperidine (3 mmol),
CuI (0.3 mmol), Pd(PPh3)4 (0.03 mmol) in THF, 30 min, then 1 (3 mmol)
and piperidine (3 mmol) in THF, 2 h; (b) o-C6H4I2 (0.5 mmol), piperidine
(1 mmol), CuI (0.1 mmol), Pd(PPh3)4 (0.01 mmol) in THF, 30 min, then
1 (1 mmol) and piperidine, 10 h; (c) 2 (1 mmol), piperidine (1 mmol), CuI
(0.1 mmol), and Pd(PPh3)4 (0.01 mmol) in THF, 30 min, then 1 (1 mmol)
and piperidine (1 mmol), 10 h; (d) 3 (0.5 mmol), K2CO3 (1 mmol) and
PhSH (1 mmol), DMF, 1 h. bOverall yields; two reaction steps for
4a-4c and three reaction steps for 4d and 4e.
Results and Discussion
Synthesis. Derivatives 4a-e were prepared according to
the synthetic path shown in Scheme 1. The Sonogashira
coupling of fully protected N-propargylated derivatives of
alanine, valine, and leucine 116 with 1,2-diiodobenzene was
performed in THF in the presence of piperidine, CuI, and
Pd(PPh3)4. When coupled with an equimolar amount of
1,2-diiodobenzene (Scheme 1, path a), corresponding mono-
substituted derivatives 2 were obtained in around 50% yield
(slight excess of 1,2-diiodobenzene was used to ensure com-
plete consumption of acetylene component and facilitate
purification by column chromatography). Second, Sono-
gashira coupling with either the same or different derivative
1 gave enediyne-bridged symmetric Ala-Ala (3a), Val-Val
(3b), Leu-Leu (3c), and asymmetric Ala-Val (3d) and Val-
Gly (3e) sequences in good yields. Symmetric enediynes 3a-c
were also prepared according to path b, by coupling of 1 with
1.2 equiv of 1,2-diiodobenzene. Deprotection of o-nitro-
benzenesulfonyl group (oNbs) with PhSH and K2CO3 in
DMF gave aromatic enediynes 4 in good yields. All compounds
were characterized by NMR spectroscopy, mass spectrometry,
HRMS, and FT-IR spectroscopy (Experimental Section).
(9) (a) Shain, J. C.; Khamrai, U. K.; Basak, A. Tetrahedron Lett. 1997, 34,
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1446. (b) Breiner, B.; Schlatterer, J. C.; Kovalenko, S. V.; Greenbaum, N. L.;
Alabugin, I. V. Angew. Chem., Int. Ed. 2006, 45, 3666–3670. (c) Breiner, B.;
Schlatterer, J. C.; Kovalenko, S. V.; Greenbaum, N. L.; Alabugin, I. V. Proc.
Natl. Acad. Sci. U.S.A. 2007, 104, 13016–13021. (d) Yang, W.-Y.; Breiner, B.;
Kovalenko, S. V.; Ben, C.; Singh, M.; LeGrand, S. N.; Sang, Q-X. A.;
Strouse, G. F.; Copland, J. A.; Alabugin, I. J. Am. Chem. Soc. 2009, 131,
11458–11470.
(13) Dutta, S.; Basak, A.; Dasgupta, S. Bioorg. Med. Chem. 2009, 17,
3900–3908.
(14) Fouad, F. S.; Wright, J. M.; Plourde, G., II; Purohit, A. D.; Wyatt,
J. K.; El-Shafey, A.; Hynd, G.; Crasto, C. F.; Lin, Y.; Jones, G. B. J. Org.
Chem. 2005, 70, 9789–9797.
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Zhao, B.; Belkowski, S. M.; Reitz, A. B. Bioorg. Med. Chem. 2005, 13, 5936–
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Smits, J. M. M.; Butterworth, S.; Rutjes, F. P. J. T. Org. Biomol. Chem. 2009,
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Reactivity of Enediynes 4a-e. Thermal reactivity of ene-
diyne compounds toward the Bergman cyclization is tradition-
ally studied by the differential scanning calorimetry (DSC).
It can provide information about onset and maximum temp-
eratures and activation energies of a certain process. However,
DCS results have to be interpreted with caution when
numerous side reactions occur, capable of lowering activation
barriers.17 Also, the exact structural changes accompanying
the process cannot be determined by DSC. Contrary to that,
temperature-dependent FT-IR spectroscopy is an established
method for simple and rapid monitoring of thermally induced
physical and chemical rearrangements that simultaneously
provide temperature values and insight into structural changes.18
IR spectra of enediynes 4a-e have characteristic vibrational
bands of ester and amine groups. The most prominent bands
are associated with N-H stretching (3340 cm-1), CdO stretch-
ing (1730 cm-1), andC-O-C stretching (1190 cm-1), andthey
can be used for tracking structural changes within the molecule.
The weak band at 2225 cm-1 is associated with CꢀC stretching
vibrations and is essential for monitoring the Bergman cycliza-
tion by FT-IR technique. The FT-IR measurements performed
within the temperature range of 20-250 °C are consistent for
all five enedyines and indicate that a chemical process occurs at
approximately 170 °C (Figure 1). Disappearance of an acet-
ylene band at 2225 cm-1 is in accordance with anticipated
Bergman cycloaromatization. However, substantial intensity
changes of all spectral bands and disappearance of the amine
band at 3340 cm-1 point toward additional or parallel chemical
processes occurring.
Onset temperatures of cycloaromatization (the tempera-
tures at which process begins), obtained by baseline analysis
(see Supporting Information for more details), vary signifi-
cantly with respect to the bulkiness of side chains (Table 1).
Comparison of symmetric compounds 4a, 4b, and 4c (alanine,
valine, and leucine side chains, respectively) revealed the temp-
erature difference of 30 °C between compounds 4a and 4c. It
was demonstrated previously that properties of substituents at
the terminal acetylenes may contribute significantly to the
reactivity of enediynes.19 Here, it is evident that substituents
placed even three bonds away from reactive centers have a
considerable influence on the cyclization process. In this res-
pect, we were interested at looking at asymmetric derivatives 4d
and 4e. Although not notable, somewhat lower onset tempera-
tures for both compounds can be explained most likely by
better spatial accommodation of unequal side chains.
TABLE 1. Results of the FT-IR Measurements of Enediynes 4a-4ea
compound
Tonset/°C
Tmax/°C
4a
4b
4c
4d
4e
130
135
160
125
125
175
185
210
175
175
aMeasurements were performed within the temperature range of
20-250 °C under atmospheric pressure and at the heating rate of 1 K min-1
.
SCHEME 2. Bergman Cyclization of 4 (10 equiv of 1,4-CHD,
DMF, Δ)20
Isolation and Characterization of Products. The next step
was performing Bergman cyclization in solution and isola-
tion of products. Cycloaromatization of 4a was performed
with 10 equiv of 1,4-cyclohexadiene (1,4-CHD) in DMF at
175 °C (Scheme 2). Reaction mixture was checked by TLC
and after 8 h shows consumption of starting compound.
13C NMR spectrum of isolated and purified product showed
FIGURE 1. Temperature-dependent FT-IR measurement of 4a
(Ala-Ala): FT-IR spectra at 100 and 250 °C (top); MW 2D IR
correlation analysis as contour plot of the baseline-corrected data
(bottom) (analyzed spectral regions, 4000-2400, 2300-2100,
2000-400 cm-1; window, 10 spectra; window step, 1 spectrum;
heating rate, 1 K min-1; resolution, 4 cm-1; number of scans per
spectrum, 10).
(19) Nicolaou, K. C.; Smith, A. L.; Yue, E. W. Proc. Natl. Acad. Sci. U.S.A.
1993, 90, 5881–5888.
(20) Bergman cyclization of 4e, due to the high onset temperature, was
performed in N-methyl-pyrrolidone (NMP); Δ marks the Tmax obtained by
the FT-IR for selected derivative.
(17) Zeidan, T.; Kovalenko, S. V.; Manoharan, M.; Alabugin, I. V.
J. Org. Chem. 2006, 71, 962–975.
ꢁ
(18) Zimmermann, B.; Baranovic, G. Appl. Spectrosc. 2009, 63, 1152–
1161.
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FIGURE 2. MS/MS spectra of the molecular [M þ H]þ ions of 4a and 6a.
disappearance of acetylenic carbons, a new signal in the aro-
matic region, and a large downfield shift of CH2 carbon adjacent
to the acetylene (from 37.6 to 55.4 ppm). The 1H NMR spectrum
pointed toward a symmetric molecule with six aromatic protons
and only one alanine moiety attached. Also, mass spectrum
showed a signal at m/z 270 but not m/z 387 corresponding to the
Bergman product 5a. Looking at the fragmentation patterns of
the molecular [M þ H]þ ions of both 4a and the product, we
noticed that the [M þ H]þ ion at m/z 270 and its product ions at
m/z 155 and 196 represent 2 Da mass gain to the product ions of
4a (m/z268, 153, and 194) (Figure 2). In the MS/MS spectrum of
the molecular [M þ H]þ ion m/z 385 of 4a, the presence of
product ion at m/z 268 corresponds to the elimination of alanine
residue (117 Da).
that account for low yields of isolated products (around 20%).
It was demonstrated previously by using IR, solid-state NMR,
UV-vis spectroscopy, and MS that formation of different poly-
mer products in the Bergman cycloaromatization reaction of
simple aromatic enediyne can be explained by a set of inter-
molecular radical reactions.21
Isoindole-related compounds are not unknown in enediyne
chemistry; they have been obtained from the cobalt complex of a
strained nine-membered enediyne ring.22 Upon deprotection, this
compound underwent a spontaneous Bergman cyclization at 0 °C.
Benzo[f]isoindoles display some interesting properties; they are
farnesyl transferase inhibitors23 and EP4 receptor agonists.24
(21) Johnson, J. P.; Bringley, D. A.; Wilson, E. E.; Lewis, K. D.; Beck,
L. W.; Matzger, A. J. J. Am. Chem. Soc. 2003, 125, 14708–14709.
(22) Kumar Roy, S.; Basak, A. Chem. Commun. 2006, 1646–1648.
(23) Pichon, N.; Harrison-Marchand, A.; Mailliet, P.; Maddaluno, J.
J. Org. Chem. 2004, 69, 7220–7227.
All together, FT-IR, NMR, and MS analysis undoubtedly
confirmed the structure of 2,3-dihydrobenzo[f]isoindole 6a.
The very same result was obtained with other two symmetric
derivatives, 4b and 4c. Formation of isoindoles was accom-
panied by polymerization reactions (poorly soluble material)
(24) Claessens, S.; Jacobs, J.; Aeken, S. V.; Tehrani, K. A.; De Kimpe, N.
J. Org. Chem. 2008, 73, 7555–7559.
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To the best of our knowledge, this is the first example of isoindoles
obtained by the Bergman cyclization of acyclic enediynes.
Two paths of the mechanism, both of them based on SN2, are
possible (Scheme 3). In path A, the amino group of one amino
acid performs nucleophilic attack on the methylene carbon
atom placed between acetylene and the amino group of the
second amino acid. Newly formed nine-membered ring is highly
unstable and spontaneously undergoes Bergman cyclization
resulting in 6. In path B, Bergman cyclization is the first step;
it brings amines closer, facilitating SN2 attack. Methylene
carbon atom placed between acetylene and the amino group
(path A) or attached to the aromatic ring (path B) is deficient in
electrons and, consequently, susceptible to nucleophilic attack.
To prove which path is genuine, 4a was heated under the
same conditions as before, but without 1,4-CHD. If path A is
probable, then the result should be decomposition of substrate
since nine-membered rings are extremely unstable19 and there is
no H-atom donor to quench the Bergman diradical. If the
mechanism goes by path B, the substrate should remain intact.
After 48 h of heating, substrate 4a remains intact based on TLC
and MS and was quantitatively retrieved. Furthermore, it
survived heating up to 240 °C for the same time period. This
led us to conclude that the formation of the isoindole derivative
takes place through path B, the Bergman cyclization followed
by intramolecular nucleophilic substitution with consequent
amino acid elimination.25
SCHEME 3. Two Possible Mechanism Paths to 6
FIGURE 3. Path B. (a) Reaction energy pathway for the Bergman
cycloaromatization of enediyne 4a with 1,4-cyclohexadiene as a
H-atom donor ending with the Bergman product 5a. (b) Reaction
energy pathway for the elimination reaction of Bergman product 5a,
giving the isoindole product 6a. All energies are calculated at the
UB3LYP/6-31G(d,p) level of theory and given in kcal/mol relative
to the energy of the 4a (a) and 5a (b).
theory (DFT) with the UB3LYP26 and UBLYP27 exchange-
correlation functional and the 6-31G(d,p) basis set as implemen-
ted in the Gaussian03 quantum chemical software package.28
Computed energy profiles for two paths are shown in Figures 3
and 4. As proposed above, the first step in path B is the formation
of a diradical followed by two hydrogen abstraction steps from
the 1,4-CHD as a H-atom donor. The activation barrier for the
cyclization step is calculated to be 37.2 kcal/mol at the UB3LYP/
6-31G(d,p) level of theory and 29.1 kcal/mol at the UBLYP/6-
31G(d,p) level (Figure 3a). The energy of the transition state is
just slightly above the energy of the diradical intermediate, which
is less stable for 36.2 kcal/mol (27.1 0 kcal/mol at UBLYP/
6-31G(d,p) level) than the enediyne reactant 4a. Calculated
energy of diradicals should be regarded with care as it is known
Additional support was searched from quantum chemistry
approaches as a complement to experimental studies. Gas
phase geometries of intermediates, transition states, reactants,
and products in paths A and B for the formation of isoindole-
related compounds were obtained using the density functional
(25) It is worth noting that we tried to “catch” the Bergman product
before elimination by closer monitoring of the reaction. 4a was dissolved in
DMF with 100 equiv of 1,4-CHD; aliquots were placed into melting point
tubes, frozen by liquid nitrogen, degassed under high vacuum, sealed, and
placed into an oil bath at 160 °C. Capillaries were withdrawn in different time
periods and analyzed by ESI MS. However, only disappearance of 4a
(m/z 385) and appearance of 6a (m/z 270) was observed.
(26) (a) Becke, A. D. Phys. Rev. A 1988, 38, 3098–3100. (b) Lee, C.; Yang,
W.; Parr, R. G. Phys. Rev. B 1988, 37, 785–789. (c) Vosko, S. H.; Wilk, L.;
Nusair, M Can. J. Phys. 1980, 58, 1200–1211. (d) Stephens, P. J.; Devlin,
F. J.; Chabalowski, C. F.; Frisch, M. J. J. Phys. Chem. 1994, 98, 11623–
11627.
(27) (a) Becke, A. D. J. Chem. Phys. 1996, 104, 1040–1046. (b) Lee, C.;
Yang, W.; Parr, R. G. Phys. Rev. B 1988, 37, 785–789.
(28) Frisch, M. J.; et al. Gaussian 03, revision B.05; Gaussian, Inc.:
Pittsburgh, PA, 2003.
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The substitution step is followed by the formation of a
diradical with the barrier of 17.3 kcal/mol. The diradical
formation step is also highly endothermic and thus reversible.
We believe that two energetically unfavorable starting steps
in path A can explain the experimental finding that
2,3-dihydrobenzo[f]isoindoles form via mechanism B where only
one small barrier exists.
With the cycloaromatization-elimination mechanism of
enediynes 4a-c proven, we turned our attention to asym-
metric derivatives. Therefore, compounds 4d and 4e were
heated in the presence of 1,4-CHD, and they both yielded
(based on TLC and MS) only valine-related isoindole 6b,
although in the latter, the major event was decomposition of
substrate, accompanied by traces of 6b. Reaction mixture of
Ala-Val enediyne 4d was also checked by HPLC-MS. TIC
chromatogram (Figure 5) clearly points to the formation of
valine-related isoindole 6b (RT = 4.82 min), while alanine-
related isoindole 6a is detected only in traces among numerous
components eluted at RT = 2.40 min (mass spectra shown in
Supporting Information).
FIGURE 4. Path A. Reaction energy pathway calculated at the
UB3LYP/6-31G(d,p) level of theory. All energies are given in kcal/
mol and relative to the energy of the enediyne 4a.
that higher multiconfigurational levels of theory are needed to
accurately describe multiconfigurational electronic structure of
singlet diradicals.29 However, an unrestricted broken-symmetry
approach has proven to be a practical tool for the study of
enediyne and enyne-allene cyclizations,30,31 and thus the UDFT
method should provide a correct qualitative picture of a Berg-
man cyclization process. Two highly exothermic hydrogen
abstraction steps follow the endothermic cyclization with energy
gains of 56.1 and 38.1 kcal/mol, and the overall Bergman cyclo-
aromatization is an exothermic process. Unfortunately, all
attempts to locate the transition states in the two hydrogen
abstraction steps failed and led to either a reactant or product
structure. However, previous detailed theoretical studies of the
Bergman cyclization mechanism demonstrated that barriers
to hydrogen abstraction are only a few kcal/mol less than one-
tenth of the overall energy change.30 The second step in path B
(Figure 3b) includes a nucleophilic substitution reaction resulting
in the elimination of the amino acid and the formation of
isoindole-related product 6a. Here, we should point out that
several conformers of the Bergman product 5a, as well as of the
enediyne 4a, were located due to the significant floppiness of
amino acid chains. The schemes in Figure 4 include only the ones
with the largest stability. The most stable conformer of struc-
ture 5a is the one in which the C-C-C-N dihedral angle
(in Figure 4b denoted with an asterisk) is 2.7°. However, the
nucleophilic attack by the amino group on the methylene carbon
atom occurs in the conformer with the C-C-C-N dihedral
angle of 56.5°. So, the final step in path B proceeds only after the
rotation of the amino acid backbone around the C-C bond by
53.8°. The barrier for this rotation is only 5.3 kcal/mol, while the
substitution step has a barrier of 33.3 kcal/mol. In the course of
the Bergman cycloaromatization reaction, 58.6 kcal/mol of
energy was released and the overall reaction is still exothermic.
In path A, the elimination reaction precedes the Bergman
cyclization. The calculated energy barrier for the nucleo-
philic attack in enediyne 4a is 42.0 kcal/mol (Figure 4).
FIGURE 5. TIC chromatogram of the reaction mixture of ene-
diyne 4d.
Finally, further insight into cyclization-elimination pro-
cesses of asymmetric enediynes was sought by using NMR
spectroscopy. We were aware of its limitations in this particular
case: formation of multiple compounds present in low quan-
tities and polymerization reaction hamper conclusions about
kinetics. Therefore, we focused on parallel experiments: en-
ediynes 4a and 4d were dissolved in deuterated DMF (0.043 M)
with 2 equiv of 1,4-CHD and heated at 175 °C. NMR spectra
were recorded at different time points (every 5 h). If formation
of isoindole-related Bergman products 6a and 6b is accompa-
nied by the release of H-Ala-OEt, as we have proposed, NMR
spectra should reflect this resemblance in the mechanism.
Indeed, aliphatic region of spectra recorded after 5 and 10 h
showed the appearance of new signals that can be assigned as R
and β protons of alanine and CH3 protons of the ethyl ester
moiety (Figure 6). Unfortunately, protons of enediynes and
products have quite similar chemical shifts in DMF-d7; the
aromatic part of the spectrum is covered by benzene formed
upon proton transfer from 1,4-CHD to the diradical, and
formation of numerous products is visible in broadening of
the present signals and appearance of new ones, especially after
10 h of heating. However, the presence of the same pattern in
symmetric 4a and asymmetric enediyne 4d supported our initial
findings about products of the Bergman cyclization of asym-
metric enediynes.
(29) Zeidan, T. A; Manoharan, M.; Alabugin, I. V. J. Org. Chem. 2006,
71, 954–961.
(30) Pickard, F. C., IV; Shepherd, R. L.; Gillis, A. E.; Dunn, M. E;
Feldgus, S.; Kirschner, K. N.; Shields, G. C.; Manoharan, M.; Alabugin,
I. V. J. Phys. Chem. A 2006, 110, 2517–2526.
It is known that branching of the amino acid R-carbon
atom increases electron density in the system (branched
(31) Schreiner, P. R.; Navarro-Vazquez, A.; Prall, M. Acc. Chem. Res.
2005, 38, 29–37.
6224 J. Org. Chem. Vol. 75, No. 18, 2010

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FIGURE 6. Parts of NMR spectra of 4a and 4d obtained after heating at 175 °C at different time points (t1 = 5 h, t2 = 10 h).
alkanes are better electron donors than linear ones) and
makes amine more nucleophilic. Therefore, it is reasonable
to assume that increased nucleophilicity of amines carrying
branched side chains accounts for the formation valine-
related product 6b with the elimination of amino acid carry-
ing less branched side chains. Of course, two asymmetric
derivatives are not enough to allow more than hypothesis,
and additional studies are undertaken to bring more general
conclusions.
Conclusions
In summary, we have synthesized a series of acyclic amino
acid derived enediynes and tested them under conditions of
the Bergman cyclization. FT-IR analysis clearly showed that
the bulkiness of the amino acid side chain increases the
temperature of the Bergman reaction and also indicated
significant structural changes during the process. Experi-
mental and computational data support the mechanism
comprising cycloaromatization followed by the nucleophilic
substitution with subsequent amino acid elimination yielding
2,3-dihydrobenzo[f]isoindoles. Also, the structure of amino
acid determines the structure of isoindole formed. The influence
of amine properties on the formation of 2,3-dihydrobenzo-
[f]isoindoles and the importance of activation energies and
amino acid side chain will be investigated to better under-
stand the mechanism. Finally, the presented results opened two
important subjects: (a) amino acids with different side chains
can be considered for fine-tuning of reactivity, especially in
unstable enediyne systems, due to the established correlation
between bulkiness and onset temperature of Bergman cycliza-
tion; and (b) reactivity and participation of amino groups in
reactions following initial Bergman cyclization is to be taken
into account when dealing with enediyne-peptide conjugates.
Enediyne-derived 1,4-diyl radical can react in several
ways; abstraction of protons from DNA is only one of them.
It has been proposed that self-decomposition of C-1027
includes H-abstraction from apoprotein and subsequent
formation of glycyl radical.32 Glycyl radical can further
undergo different reactions, depending on environment,
leading to peptide scission, formation of cross-linking, or
adducts. Therefore, formation of diyl radical can be con-
sidered as a trigger for numerous fragmentation and rear-
rangement reactions. Recently, a detailed study on model
enediynes mimicking members of esperamicine and calchia-
micine families revealed the importance of intramolecular
H-atom abstraction and provided evidence about different
products formed, depending on reaction conditions and
substitution pattern.33 Our results made a contribution to
an array of Bergman cyclization products and had some
enlightening new moments. Absence of an external H-atom
donor hampers both cyclization and fragmentation in
enediynes 4a-e, contrary to enediynes bearing tetrahydro-
pyran and tetrahydrofuran rings, where fragmentation pro-
ducts were detected in 30-40% yields.32 However, the amino
acid moiety took part in a further process, nucleophilic sub-
stitution reaction. Formation of 2,3-dihydrobenzo[f]isoindoles
triggered by the Bergman cyclization is the first such example
and raises questions about reactivity and the role of amino
groups in enediyne-peptide conjugates. Addressing these
questions is important in light of recent increased usage of such
conjugates in targeting proteins instead of DNA.13,14
Experimental Section
The synthesis and characterization data of products are
provided below. Synthesis and characterization of amino acid
derivatives1 havebeen publishedpreviously.16 For further details
on general methods, computational details, FT-IR, NMR, and
MS spectra, consult the Supporting Information.
General Procedure for the Synthesis of 2. 1,2-Diiodobenzene
(3.6 mmol), piperidine (3 mmol), CuI (0.3 mmol), and Pd(PPh3)4
(0.03 mmol) are mixed in THF under argon at room tempera-
ture for 30 min. 1 (3 mmol) and piperidine (3 mmol) are mixed in
THF under argon and added dropwise to the initial solution.
The solution is stirred until all substrate is consumed, approxi-
mately 2 h. Solvent is evaporated and the product extracted with
EtOAc, washed with brine and water, and purified by flash
column chromatography.
(32) Inoue, M.; Usuki, T.; Lee, N.; Hirama, M.; Tanaka, T.; Hosoi, F.;
Ohie, S.; Otani, T. J. Am. Chem. Soc. 2006, 128, 7896–7903.
(33) Baroudi, A.; Mauldin, J.; Alabugin, I. V. J. Am. Chem. Soc. 2010,
132, 967–979.
(oNbs)[3-(2-Iodophenyl)propargyl]-L-alanine ethyl ester (2a):
Yield 911 mg (56%) (yellow oil); Rf 0.71 (petrol ether/EtOAc 2:1);
J. Org. Chem. Vol. 75, No. 18, 2010 6225

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Gredicak et al.
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[RD] -45.0 (c 1.0, EtOAc); 1H NMR (600 MHz, CDCl3) δ
8.25-8.23 (m, 1H, H3 oNbs), 7.79-7.77 (m, 1H, H6 oNbs),
7.65-7.60 (m, 3H, H3 2-iodophenyl; H4, H5 oNbs), 7.26-
7.24 (m, 2H, H5, H6 2-iodophenyl), 7.00-6.97 (m, 1H, H4,
and the product extracted with EtOAc, washed with brine and
water, and purified by flash column chromatography.
3,30-(1,2-Phenylene)bis(prop-2-yne-1-yl)bis[(2-nitrobenzensulfonyl)-
L-alanine ethyl ester] (3a): Yield 377 mg (50%) (yellowoil);Rf 0.37
3
1
2-iodophenyl), 4.87 (q, 1H, R Ala, JRβ = 7.4 Hz), 4.40, 4.74
(petrol ether/EtOAc 2:1); [RD] -57.0 (c 1.0, MeOH); H NMR
(d, 2H, CH2 propargyl, 2JHH = 18.7 Hz), 4.12 (q, 2H, CH2 OEt,
3JHH = 7.2 Hz), 1.74 (d, 3H, β Ala, 3JRβ = 7.4 Hz), 1.20 (t, 3H,
CH3 OEt, 3JHH = 7.2 Hz); 13C NMR (150 MHz, CDCl3) δ 170.8
(CO Ala), 147.5 (C2 oNbs), 138.2 (C3 2-iodophenyl), 133.5
(C1 oNbs), 132.4, 133.0 (C6 2-iodophenyl, C3 oNbs), 131.1,
131.3 (C4, C5 oNbs), 128.3 (C1 2-iodophenyl), 127.2, 129.9,
(C4, C5 2-iodophenyl), 123.8 (C6 oNbs), 99.8 (C2 2-iodophenyl),
87.5 (C2 propargyl), 86.1 (C3 propargyl), 61.1 (CH2 OEt), 55.5 (R
Ala), 35.1 (C1 propargyl), 16.4 (β Ala), 13.5 (CH3 OEt); HRMS
(MALDI) m/z [M þ K]þ calcd for C20H19IN2O6SK, 580.9640;
found 580.9639.
(300 MHz, CDCl3) δ8.23-8.20(m, 2H, H3oNbs),7.64-7.58 (m,
6H, H4,5,6 oNbs), 7.29-7.20 (m, 4H, H3,6, H4,5 phenylene),
4.86 (q, 2H, R Ala, 3JRβ = 7.2 Hz), 4.38, 4.46 (d, 4H, CH2 pro-
pyniyl, 2JHH = 19.0 Hz), 4.08 (q, 4H, CH2 OEt, 3JHH = 7.2 Hz),
1.70 (d, 6H, β Ala, 3JRβ = 7.2 Hz), 1.15 (t, 6H, CH3 OEt, 3JHH
=
7.2 Hz); 13C NMR (75 MHz, CDCl3) δ 171.1 (CO Ala), 147.9
(C2 oNbs), 133.7 (C1 oNbs), 133.5 (C5 oNbs), 131.4, 131.8, 132.0
(C3, C4 oNbs; C3,6 phenylene), 128.2 (C4,5 phenylene), 124.8
(C1,2 phenylene), 124.2 (C6 oNbs), 88.5 (C220 propynyl), 83.0
(C330 propynyl), 61.6 (CH2 OEt), 55.9 (R Ala), 35.7 (C110 pro-
pynyl), 16.7 (β Ala), 13.9 (CH3 OEt); HRMS (MALDI) m/z [M þ
Na]þ calcd for C34H34NaN4O12S2, 777.1506; found, 777.1471.
3,30-(1,2-Phenylene)bis(prop-2-yne-1-yl)bis[(2-nitrobenzensulfonyl)-
L-valine ethyl ester] (3b): Yield 390 mg (48%) (yellow oil); Rf 0.69
(oNbs)[3-(2-Iodophenyl)propargyl]-L-valine ethyl ester (2b):
Yield 775 mg (45%) (yellow oil); Rf 0.59 (petrol ether/EtOAc
2:1); [RD] -40.0 (c 0.5, EtOAc); 1H NMR (300 MHz, CDCl3) δ
8.24-8.22 (m, 1H, H3 oNbs), 7.84-7.80 (m, 1H, H6 oNbs),
7.66-7.60 (m, 3H, H3 2-iodophenyl; H4, H5 oNbs), 7.35-
7.26 (m, 2H, H5, H6 2-iodophenyl), 7.04-6.98 (m, 1H, H4,
2-iodophenyl), 4.69, 4.72 (d, 2H, CH2 propargyl, 2JHH = 18.6
Hz), 4.27 (d, 1H, R Val, 3JRβ = 10.3 Hz), 4.04 (q, 2H, CH2 OEt,
3JHH = 7.1 Hz), 2.53-2.41 (m, 1H, β Val), 1.12 (t, 3H, CH3 OEt,
1
(petrol ether/EtOAc 1:1); [RD] -67.0 (c 1.0, MeOH); H NMR
(300 MHz, CDCl3) δ 8.24-8.20 (m, 2H, H3 oNbs), 7.64-7.58
(m, 6H, H4,5,6 oNbs), 7.29-7.21 (m, 4H, H3,6, H4,5 phenylene),
=
4.56-4.74 (m, 2H, R Val), 4.25 (d, 4H, CH2 propynyl, 2JHH
19.0 Hz), 4.09-3.96 (m, 2H, β Val), 4.02 (q, 4H, CH2 OEt,
3JHH = 7.1 Hz), 1.09 (t, 6H, CH3 OEt, 3JHH = 7.1 Hz), 1.03, 1.14
(d, 12H, γγ0 Val, 3Jβγ = 6.7 Hz); 13C NMR (150 MHz, CDCl3)
δ 170.1 (CO Val), 148.2 (C2 oNbs), 133.6 (C5 oNbs), 133.2 (C1
oNbs), 13.9 (CH3 OEt), 131.5, 131.6, 132.0, (C3, C4 oNbs; C3,6
phenylene), 128.1 (C4,5 phenylene), 125.0 (C1,2 phenylene),
123.9 (C6 oNbs), 88.8 (C220 propynyl), 82.7 (C330 propynyl),
65.6 (R Val), 61.1 (CH2 OEt), 35.7 (C110 propynyl), 28.9 (β Val),
19.5, 19.9 (γγ0 Val); HRMS (MALDI) m/z [M þ Na]þ calcd for
C38H42NaN4O12S2, 833.2132; found, 833.2158.
3JHH = 7.1 Hz), 1.04, 1.14 (d, 6H, γγ0 Val, 3Jβγ = 6.6 Hz); 13
C
NMR (75 MHz, CDCl3) δ 170.2 (CO Val), 147.3 (C2 oNbs),
138.7 (C3 2-iodophenyl), 133.3 (C1 oNbs), 133.1, 133.6 (C6
2-iodophenyl, C3 oNbs), 131.5, 131.6 (C4, C5 oNbs), 129.0
(C1 2-iodophenyl), 127.7, 129.7, (C4, C5 2-iodophenyl), 124.0
(C6 oNbs), 100.3 (C2 2-iodophenyl), 88.4 (C2 propargyl), 86.1
(C3 propargyl), 65.5 (R Val), 61.1 (CH2 OEt), 35.5 (C1
propargyl), 19.5, 20.2 (γγ0 Val), 29.0 (β Val), 14.0 (CH3 OEt);
HRMS (MALDI) m/z [M þ Na]þ calcd for C22H23IN2O6SNa,
593.0214; found, 593.0228.
3,30-(1,2-Phenylene)bis(prop-2-yne-1-yl)bis[(2-nitrobenzensulfonyl)-
L-leucine ethyl ester] (3c): Yield 394 mg (47%) (yellow oil); Rf 0.68
1
(petrol ether/EtOAc 1:1); [RD] -16.0 (c 1.0, MeOH); H NMR
(oNbs)[3-(2-Iodophenyl)propargyl]-L-leucine ethyl ester (2c):
Yield 860 mg (49%) (yellow oil); Rf 0.69 (petrol ether/EtOAc
2:1); [RD] -35.0 (c 1.0, EtOAc); 1H NMR (600 MHz, CDCl3) δ
8.20-8.19 (m, 1H, H3 oNbs), 7.81-7.80 (m, 1H, H6 oNbs),
7.65-7.60 (m, 3H, H3 2-iodophenyl; H4, H5 oNbs), 7.35-7.26
(m, 2H, H5, H6 2-iodophenyl), 7.01-6.98 (m, 1H, H4,
2-iodophenyl), 4.76-4.74 (m, 1H, R Leu), 4.50, 4.65 (d, 2H,
(300 MHz, DMSO-d6) δ 8.23-8.11 (m, 2H, H3 oNbs), 7.75-7.63
(m, 6H, H4,5,6 oNbs), 7.47-7.35 (m, 4H, H3,6, H4,5 phenylene),
=
4.78-4.67 (m, 2H, RLeu), 4.36, 4.20 (d, 4H, CH2 propynyl, 2JHH
3
19.0 Hz), 4.03 (q, 4H, CH2 OEt, JHH = 7.2 Hz), 2.06-1.71
(m, 6H, γ Leu, ββ0 Leu), 1.13 (t, 6H, CH3 OEt, 3JHH = 7.2 Hz),
0.83, 0.90 (d, 12H, δδ0 Leu, 3JHH = 6.5 Hz); 13C NMR (75 MHz,
DMSO-d6) δ 171.3 (CO Leu), 147.4 (C2 oNbs), 134.8 (C5 oNbs),
131.2 (C1 oNbs), 130.7, 131.5, 132.2 (C3, C4 oNbs; C3,6
phenylene), 128.8 (C4,5 phenylene), 124.5 (C1,2 phenylene),
124.0 (C6 oNbs), 89.8 (C220 propynyl), 81.3 (C330 propynyl),
60.9 (CH2 OEt), 58.3 (R Leu), 38.0 (β Leu), 35.6 (C110 propynyl),
23.9 (γ Leu), 21.6, 22.6 (δδ0 Leu), 13.4 (CH3 OEt); HRMS
(MALDI) m/z [M þ Na]þ calcd for C40H46NaN4O12S2, 861.2451;
found, 861.2411.
CH2 propargyl, 2JHH = 19.0 Hz), 4.03 (q, 2H, CH2 OEt, 3JHH
=
7.0 Hz), 1.84, 2.01 (m, 2H, ββ0 Leu), 1.95 (m, 1H, γ Leu), 1.12
3
(t, 3H, CH3 OEt, JHH = 7.0 Hz), 0.95, 0.99 (d, 6H, δδ0 Leu,
3Jγδ = 6.7 Hz); 13C NMR (150 MHz, CDCl3) δ 170.7 (CO Leu),
147.6 (C2 oNbs), 138.2 (C3 2-iodophenyl), 132.8 (C1 oNbs),
132.5, 133.0 (C6 2-iodophenyl, C3 oNbs), 130.8, 131.1 (C4,
C5 oNbs), 128.5 (C1 2-iodophenyl), 127.2, 129.2 (C4, C5
2-iodophenyl), 123.7 (C6 oNbs), 99.9 (C2 2-iodophenyl), 87.9
(C2 propargyl), 85.6 (C3 propargyl), 60.9 (CH2 OEt), 58.1 (R
Leu), 38.6 (β Leu), 35.2 (C1 propargyl), 24.2 (γ Leu), 21.0, 22.þ3
(δδ0 Leu), 13.5 (CH3 OEt); HRMS (MALDI) m/z [M þ Na]
calcd for C23H25IN2O6SNa, 607.0370; found, 607.0366.
(2-Nitrobenzensulfonyl)[3-(2-(3-(N-((S)-1-ethoxy-1-oxopropan-
2-yl)-2-nitrophenylsulfonamido)prop-1-ynyl)phenyl)prop-2-ynyl]-
L-valine ethyl ester (3d): Yield 400 mg (51%) (yellow oil); Rf 0.36
(petrol ether/EtOAc 2:1); [RD] -47.0 (c 1.0, MeOH); 1H NMR (600
MHz, CDCl3) δ 8.26-8.18 (m, 2H, H3 oNbs Ala,Val), 7.65-7.59
(m, 6H, H4,5,6 oNbs Ala,Val), 7.27-7.25 (m, 2H, H3,6 phenyl),
7.22-7.20 (m, 2H, H4,5 phenyl), 4.86 (q, 1H, RAla, 3JRβ = 6.9 Hz),
4.71-4.57, 4.41-4.37 (m, 4H, CH2 prop-2-ynyl, CH2 prop-1-ynyl),
4.22 (d, 1H, R Val, 3J,β = 10.0 Hz), 4.11-3.96 (m, 4H, CH2 OEt,
Ala,Val), 2.44-2.37 (m, 1H, β Val), 1.71 (d, 3H, β Ala, 3JRβ = 6.9
Hz), 1.17-1.02 (m, 12H, CH3 OEt Ala,Val, γγ0 Val); 13C NMR
(150 MHz, CDCl3) δ 169.5, 170.7 (CO Ala,Val), 147.4, 147.7
(C2 oNbs Ala,Val), 132.9, 133.3 (C1 oNbs Ala,Val), 132.7, 133.1
(C5 oNbs Ala,Val), 131.0, 131.1, 131.3, 131.4, 131.5, 131.6 (C3,4
oNbs Ala,Val, C3,6 phenyl), 127.6, 127.7 (C4,5 phenyl), 124.3, 124.5
(C1,2 phenyl), 123.4, 123.6 (C6 oNbs Ala,Val), 88.0, 88.3 (C2 prop-
2-ynyl, C2 prop-1-ynyl), 82.1, 82.6 (C3 prop-2-ynyl, C1 prop-1-
ynyl), 65.1 (R Val), 60.6, 61.1 (CH2 OEt Ala,Val), 55.5 (R Ala), 35.1,
General Procedure for the Synthesis of 3. 2 (1 mmol), piper-
idine (1 mmol), CuI (0.1 mmol), and Pd(PPh3)4 (0.01 mmol) are
mixed in THF under argon at room temperature for 30 min. 1
(1 mmol) and piperidine (1 mmol) are mixed in THF under
argon and added dropwise to the initial solution. The solution is
stirred overnight. Solvent is evaporated and the product extracted
with EtOAc, washed with brine and water, and purified by flash
column chromatography. Symmetric compounds 3a-c can be
prepared alternatively: 1,2-diiodobenzene (0.5 mmol), piperidine
(1 mmol), CuI (0.1 mmol), and Pd(PPh3)4 (0.01 mmol) are mixed
in THF under argon at room temperature for 30 min. 1 (1 mmol)
and piperidine (1 mmol) are mixed in THF under argon and
added dropwise to the initial solution. The solution is stirred until
all substrate is consumed, approximately 2 h. Solvent is evaporated
6226 J. Org. Chem. Vol. 75, No. 18, 2010

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35.2 (C1 prop-2-ynyl, C3 prop-1-ynyl), 28.4 (β Val), 18.9, 19.4
(γγ0 Val), 16.0 (β Ala), 13.4, 13.5 (CH3 OEt Ala,Val); HRMS
(MALDI) m/z [M þ Na]þ calcd for C36H38N4O12S2Na, 805.1819;
found, 805.1840.
21.6 (δδ0 Leu), 13.2 (CH3 OEt); IR (KBr, 25 °C) 3336, 2233, 1732,
1481, 1466, 1445, 1194, 1148, 1026, 762; HRMS (MALDI) m/z
[M þ Na]þ calcd for C28H40N2O4Na, 491.2880; found, 491.2897.
[3-(2-(3-((S)-1-Ethoxy-1-oxopropan-2-ylamino)prop-1-ynyl)-
phenyl)prop-2-ynyl]-L-valine ethyl ester (4d): Yield 140 mg (68%)
(yellow oil); Rf 0.34 (petrol ether/EtOAc 1:1); [RD] -22 (c 0.5
MeOH); 1H NMR (600 MHz, CDCl3) δ 7.41-7.39 (m, 2H, H4,5
phenyl), 7.23-7.21 (m, 2H, H3,6 phenyl), 4.22-4.13 (m, 4H, CH2
OEt Ala,Val), 3.76-3.63 (m, 5H, CH2 prop-2-ynyl, CH2 prop-1-
ynyl, R Ala), 3.27 (d, 1H, R Val, 3JRβ = 5.42 Hz), 2.03-1.97 (m,
1H, β Val), 1.36 (d, 3H, β Ala, 3JRβ = 7.12 Hz), 1.25, 1.27 (t, 6H,
(2-Nitrobenzensulfonyl)[3-(2-(3-(N-(2-ethoxy-2-oxoethyl)-
2-nitrophenylsulfonamido)prop-1-ynyl)phenyl)prop-2-ynyl]-L-valine
ethyl ester (3e): Yield 453 mg (59%) (yellow oil); Rf 0.60 (petrol
ether/EtOAc 1:1); [RD] -10 (c 0.5, MeOH); 1H NMR (300 MHz,
CDCl3) δ 8.23-8.14 (m, 2H, H3 oNbs Val,Gly), 7.67-7.58
(m, 6H, H4,5,6 oNbs Val,Gly), 7.25-7.21 (m, 4H, H3,4,5,6
phenyl), 4.61, 4.64 (s, 4H, CH2 prop-2-ynyl, CH2 prop-1-ynyl),
4.41 (s, 2H, R Gly), 4.26-4.10 (m, 4H, CH2 OEt Val,Gly),
4.08-3.94 (m, 1H, R Val), 2.45-2.34 (m, 1H, β Val), 1.26-1.01
(m, 12H, CH3 OEt Val,Gly, γγ0 Val); 13C NMR (75 MHz, CDCl3)
δ 170.0, 168.5 (CO Val,Gly), 148.0, 148.2 (C2 oNbs Val,Gly),
133.6, 133.7 (C5 oNbs Val,Gly), 133.1, 133.2 (C1 oNbs Val,Gly),
128.0, 128.2, 128.3, 131.1, 131.5, 131.6, 131.9, 132.0 (C4,5, C3,6
phenyl, C3,4 oNbs Val,Gly), 124.5, 125.0 (C1,2 phenyl), 123.9,
124.2 (C6 oNbs Val,Gly), 88.8, 88.9 (C2 prop-2-ynyl, C2 prop-1-
ynyl), 82.6, 84.4 (C3 prop-2-ynyl, C1 prop-1-ynyl), 65.5 (R Val),
61.1, 61.6 (CH2 OEt Val,Gly), 47.7 (R Gly), 35.6, 35.7 (C1 prop-
2-ynyl, C3 prop-1-ynyl), 28.9 (β Val), 19.4, 19.8 (γγ0 Val), 13.9,
14.0 (CH3 OEt Val,Gly); HRMS (MALDI) m/z [M þ Na]þ calcd
for C35H36N4O12S2Na, 791.1663; found, 791.1673.
CH3 OEt Ala,Val, 3JHH = 7.02 Hz), 0.96, 0.99 (d, 6H, γγ0 Val, 3Jβγ
=
6.75 Hz); 13C NMR (150 MHz, CDCl3) δ 174.8, 175.2 (CO Ala,
Val), 132.2, 132.3 (C3,6 phenyl), 127.9, 128.0 (C4,5 phenyl), 125.6,
125.8 (C1,2 phenyl), 90.9, 91.4 (C2 prop-2-ynyl, C2 prop-1-ynyl),
82.4, 82.7 (C130 prop-2-ynyl, C1 prop-1-ynyl), 66.2 (R Val), 60.8,
61.1 (CH2 OEt Ala,Val), 55.2 (R Ala), 37.6, 37.8 (C1 prop-2-ynyl,
C3 prop-1-ynyl), 31.9 (β Val), 18.7, 19.2 (γγ0 Val), 18.4 (β Ala),
14.4, 14.5 (CH3 OEt Ala,Val); IR (KBr, 25 °C) 3337, 2232, 1730,
1481, 1466, 1445, 1188, 1148, 1026, 760; HRMS (MALDI) m/z
[M þ Na]þ calcd for C24H32N2O4Na, 435.2254; found, 435.2251.
[3-(2-(3-(2-Ethoxy-2-oxoethylamino)prop-1-ynyl)pheny)prop-
2-ynyl]-L-valine ethyl ester (4e): Yield 133 mg (67%) (yellow oil);
1
Rf 0.52 (EtOAc/petrol ether 2:1); [RD] -5 (c 0.5 MeOH); H
General Procedure for the Synthesis of 4. Enediyne 3 (0.5 mmol),
K2CO3 (1 mmol), and PhSH (1 mmol) are mixed in DMF at
room temperature for 1 h. Solvent is evaporated and the product
extracted with EtOAc, washed with brine and water, and purified
by flash column chromatography.
NMR (300 MHz, CDCl3): δ 7.43-7.38 (m, 2H, H4,5 phenyl),
7.24-7.21 (m, 2H, H3,6 phenyl), 4.24-4.11 (m, 4H, CH2 OEt
Val,Gly), 3.77-3.63 (m, 6H, CH2 prop-2-ynyl, CH2 prop-1-
ynyl, R Gly), 3.27 (d, 1H, R Val, 3JRβ = 5.44 Hz), 2.09-1.97 (m,
1H, β Val), 1.25, 1.28 (t, 6H, CH3 OEt Val,Gly, 3JHH = 7.2 Hz),
0.96, 0.99 (d, 6H, γγ0 Val, 3Jβγ = 6.9 Hz); 13C NMR (75 MHz,
CDCl3) δ 172.0, 174.6 (CO Val,Gly), 131.9, 132.1 (C4,5 phenyl),
127.7, 127.8 (C3,6 phenyl), 125.4, 125.6 (C1,2 phenyl), 90.5, 91.2
(C2 prop-2-ynyl, C2 prop-1-ynyl), 82.2, 82.9 (C3 prop-2-ynyl,
C1 prop-1-ynyl), 66.0 (R Val), 60.6, 60.9 (CH2 OEt Val,Gly),
49.3 (R Gly), 38.5, 38.6 (C1 prop-2-ynyl, C3 prop-1-ynyl), 18.4,
19.2 (γγ0 Val), 14.2, 14.3 (CH3 OEt Val,Gly); IR (KBr, 25 °C)
3336, 2232, 1730, 1481, 1466, 1445, 1190, 1148, 1024, 760; HRMS
(MALDI) m/z [M þ H]þ calcd for C23H30N2O4, 399.2278; found,
399.2262.
General Procedure for the Synthesis of 6. 4 (0.25 mmol) was
dissolved in DMF in a test tube, and 1,4-cyclohexadiene (2.5
mmol) was added. The reaction mixture was degassed, closed
under high vacuum, and placed in a preheated oil bath at Tmax
determined by FT-IR spectroscopy for selected derivative. The
solution was heated until all substrate was consumed (moni-
toring by TLC and MS), approximately 24 h. Solvent was
evaporated and the product purified by HPLC.
3,30-(1,2-Phenylene)bis(prop-2-yne-1-yl)bis[L-alanine ethyl ester]
(4a): Yield 144 mg (75%) (yellow oil); Rf 0.42 (EtOAc); [RD] -29.0
1
(c 1.0, EtOAc); H NMR (300 MHz, CD3OD) δ 7.43-7.39
(m, 2H, H3,6 phenylene), 7.29-7.27 (m, 2H, H4,5 phenylene),
4.19 (q, 4H, CH2 OEt, 3JHH = 7.0 Hz), 3.71 (br s, 6H, R Ala, CH2
propynyl), 1.35 (d, 6H, β Ala, 3JRβ = 6.7 Hz), 1.26 (t, 6H, CH3
OEt, 3JHH = 7.0 Hz); 13C NMR (75 MHz, CD3OD) δ 176.0 (CO
Ala), 133.1 (C3,6 phenylene), 129.2 (C4,5 phenylene), 126.7 (C1,2
phenylene), 91.2 (C220 propynyl), 83.7 (C110 propynyl), 62.1 (CH2
OEt), 56.0 (R Ala), 37.6 (C330 propynyl), 18.5 (β Ala), 14.5 (CH3
OEt); IR (KBr, 25 °C) 3332, 2230, 1732, 1481, 1445, 1194, 1149,
1068, 1022, 760; HRMS (MALDI) m/z [M þ H]þ calcd for
C22H28N2O4, 385.2122; found, 385.2117.
3,30-(1,2-Phenylene)bis(prop-2-yne-1-yl)bis[L-valine ethyl ester]
(4b): Yield 147 mg (67%) (yellow oil); Rf 0.49 (EtOAc/EtOH
10:1); [RD] -10.0 (c 0.5, MeOH); 1H NMR (300 MHz, CD3OD) δ
7.42-7.39 (m, 2H, H3,6), 7.29-7.26 (m, 2H, H4,5 phenylene),
3
4.17 (q, 4H, CH2 OEt, JHH = 7.1 Hz), 3.67 (d, 4H, CH2
propynyl, 2JHH = 16.9 Hz), 3.36 (d, 2H, R Val, 3JRβ = 5.6 Hz),
2.07-1.96 (m, 2H, β Val), 2.00 (m, 2H, β Val), 1.23 (t, 6H, CH3
OEt, 3JHH = 7.1 Hz), 0.97, 0.99 (d, 12H, γγ0 Val, 3Jβγ = 2.4 Hz);
13C NMR (75 MHz, CD3OD): δ 174.2 (CO Val), 131.7 (C3,6
phenylene), 127.6 (C4,5 phenylene), 125.4 (C1,2 phenylene), 90.4
(C220 propynyl), 82.1 (C110 propynyl), 65.6 (R Val), 60.4 (CH2
OEt), 37.4 (C330 propynyl), 31.4 (β Val), 17.6, 17.9 (γγ0 Val), 13.2
(CH3 OEt); IR (KBr, 25 °C) 3330, 2228, 1730, 1479, 1467, 1439,
1186, 1148, 1024, 756; HRMS (MALDI) m/z [M þ H]þ calcd for
C26H36N2O4, 441.2747; found, 441.2739.
3,30-(1,2-Phenylene)bis(prop-2-yne-1-yl)bis[L-leucine ethyl ester]
(4c): Yield 166 mg (71%) (yellow oil); Rf 0.36 (toluene/EtOAc 1:1);
[RD] -4.5 (c 0.5, MeOH); 1H NMR (300 MHz, CD3OD) δ
7.42-7.24 (m, 4H, H3,6, H4,5 phenylene), 4.16 (q, 4H, CH2
OEt, 3JHH = 7.2 Hz), 3.99 (s, CH2, propynyl), 3.62 (br d, 2H, R
Leu), 1.80-1.69 (m, 2H, γ Leu), 1.54 (m, 4H, β Leu), 1.25 (t, 6H,
CH3 OEt, 3JHH = 7.2 Hz), 0.95 (d, 12H, δδ0 Leu, 3Jγδ = 4.6 Hz);
13C NMR (75 MHz, CD3OD) δ 174.8 (CO Leu), 131.5, (C3,6
phenylene), 127.7 (C4,5 phenylene), 125.4 (C1,2 phenylene), 89.2
(C220 propynyl), 81.5 (C110 propynyl), 60.6 (CH2 OEt), 59.1
(R Leu), 42.0 (β Leu), 36.7 (C330 propynyl), 24.6 (γ Leu), 21.4,
(S)-Ethyl-2-(1H-benzo[f]isoindol-2(3H)-yl)propanoate (6a): Yield
12 mg (18%); Rf 0.76 (EtOAc/petrol ether 2:1); H NMR (600
1
MHz, CDCl3) δ 7.83 (dd, 2H, H3,6, 3JHH = 6.1 Hz, 4JHH = 3.4
Hz), 7.74 (br s, 2H, H2,7), 7.51 (dd, 2H, H4,5, JHH = 6.2 Hz,
3
4JHH = 3.3 Hz), 5.05, 4.88 (br d, 4H, CH2 isoindole), 4.49 (q, 1H,
R Ala, 3JRβ = 7.1 Hz), 4.24 (q, 2H, CH2 OEt, 3JHH = 7.1 Hz), 1.71
3
3
(d, 3H, β Ala, JRβ = 7.1 Hz), 1.28 (t, 3H, CH3 OEt, JHH
=
7.1 Hz); 13C NMR (150 MHz, CDCl3) δ 171.3 (CO Ala), 138.1
(C1,8), 133.3 (C9, 10), 128.0 (C3,6), 125.5 (C4,5), 120.8 (C2,7), 70.1
(R Ala), 63.6 (CH2 OEt), 55.4 (CH2 isoindole), 17.0 (β Ala), 14.3
(CH3 OEt); HRMS (MALDI) m/z[Mþ H]þ calcd for C17H19NO2,
270.1488; found, 270.1481.
(S)-Ethyl-2-(1H-benzo[f]isoindol-2(3H)-yl)-3-methylbutanoate (6b):
Yield 12.6 mg (17%); Rt 14.3 min (Eurospher 100 RP C-18 column;
150 ꢁ 4.5 mm i.d.; 5 μm; flow rate = 0.5 mL/min; mobile phase =
54% MeOH/0.1% TFA); 1H NMR (600 MHz, CD3OD) δ8.12(brs,
2H, H2,7), 7.90 (br s, 2H, H3,6), 7.54 (dd, 2H, H4,5, 3JHH = 6.2 Hz,
4JHH = 3.3 Hz), 4.87, 4.81 (m, 2H, CH2 isoindole), 4.41 (d, 1H, RVal,
3JRβ = 5.3 Hz), 4.20 (q, 2H, CH2 OEt, 3JHH = 7.1 Hz), 2.17 (m, 1H,
3
β Val), 1.27 (t, 3H, CH3 OEt, JHH = 7.1 Hz), 0.94, 0.96 (d, 6H,
3
γγ0 Val, Jβγ = 7.0 Hz); 13C NMR (150 MHz, CD3OD) δ 172.8
J. Org. Chem. Vol. 75, No. 18, 2010 6227

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Gredicak et al.
JOCArticle
(CO Val), 135.2 (C9, 10), 129.3 (C3,6), 128.2 (C4,5), 123.1 (C2,7), 73.7
(RVal), 62.4 (CH2 OEt), 49.0 (CH2 isoindole), 32.0 (βVal), 18.2, 19.6
(γγ0 Val), 14.6 (CH3 OEt); HRMS (MALDI) m/z[Mþ H]þ calcd for
C19H23NO2, 298.1801; found, 298.1797.
(βLeu), 26.1, (γLeu), 23.3, 22.0 (δδ0 Leu), 14.6 (CH3 OEt); HRMS
(MALDI) m/z [M þ H]þ calcd for C20H25NO2, 312.1958; found,
312.1960.
(S)-Ethyl 2-(1H-benzo[f]isoindol-2(3H)-yl)-4-methylpentanoate
(6c): Yield 11.7 mg (15%); Rt 17.5 min (Eurospher 100 RP C-18
column; 150 ꢁ 4.5 mm i.d.; 5 μm; flow rate = 0.5 mL/min; mobile
phase = 54% MeOH/0.1% TFA); 1H NMR (600 MHz, CD3OD)
δ 8.06, (br s, 2H, H2,7), 7.87 (br s, 2H, H2,7), 7.51 (dd, 2H, H4,5,
3JHH = 6.3 Hz, 4JHH = 3.4 Hz), 4.80, 4.75 (m, 2H, CH2 isoindole),
4.50 (m, 1H, R Leu), 4.15 (q, 2H, CH2 OEt, 3JHH = 7.2 Hz), 1.65
Acknowledgment. This research has been supported by
the Croatian Ministry of Science, Education and Sports,
Grant Nos. 098-0982933-2936, 098-0982904-2927, and
098-0352851-2921.
Supporting Information Available: Materials and methods,
computational details, FT-IR measurements, NMR spectra
of all compounds, mass spectra of 4a-4e, and products of
Bergman cyclization. This material is available free of charge via
the Internet at http://pubs.acs.org.
(m, 1H, γ Leu), 1.58 (m, 2H, β Leu), 1.24 (t, 3H, CH3 OEt, 3JHH
=
7.2 Hz), 0.93, 0.90 (d, 6H, 3Jγδ = 6.5 Hz); 13C NMR (150 MHz,
CDCl3) δ 171.7 (CO Leu), 129.3 (C3,6), 128.3 (C4,5), 123.2,
(C2,7), 67.0 (R Leu), 62.5 (CH2 OEt), 49.0 (CH2 isoindole), 41.8
6228 J. Org. Chem. Vol. 75, No. 18, 2010