(S)-3-(Carboxyformamido)-2-(3-(carboxymethyl)ureido)propanoic Acid as a Novel PSMA Targeting Scaffold for Prostate Cancer Imaging

Article abstract of DOI:10.1021/acs.jmedchem.9b02031

In an effort to seek novel agents targeting prostate-specific membrane antigen (PSMA), 16 ligands (L1-L16) with structural modifications in S1′ binding pocket were synthesized and evaluated for PSMA inhibition. (S)-3-(Carboxyformamido)-2-(3-(carboxymethyl)ureido)propanoic acids proved to be potent PSMA ligands with K_i values ranging from 0.08 nM to 8.98 nM, which are in the range of or are higher in potency compared to previously published urea-based ligands. Computational docking was performed to study the binding mode of the two most potent ligands discovered. FITC-conjugated L14 could selectively stain PSMA⁺ LNCaP cells over PSMA^- PC3 cells. IRDye800CW conjugated L16 can effectively image tumors in a murine xenograft model of prostate cancer.

Full text of DOI:10.1021/acs.jmedchem.9b02031

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Article
(S)-3-(Carboxyformamido)-2-(3-(carboxymethyl)ureido)propanoic Acid
as A Novel PSMA Targeting Scaffold for Prostate Cancer Imaging
Xiaojiang Duan, Futao Liu, Hongmok Kwon, Youngjoo Byun, Il Minn, Xuekang
Cai, Jingming Zhang, Martin G Pomper, Zhi Yang, Zhen Xi, and Xing Yang
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.9b02031 • Publication Date (Web): 24 Mar 2020
Downloaded from pubs.acs.org on March 28, 2020
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(
S)-3-(Carboxyformamido)-2-(3-(carboxymethyl)ureido)propanoic Acid as A
Novel PSMA Targeting Scaffold for Prostate Cancer Imaging
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Xiaojiang Duan , Futao Liu , Hongmok Kwon , Youngjoo Byun , Il Minn , Xuekang Cai ,
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Jingming Zhang , Martin G. Pomper , Zhi Yang , Zhen Xi , Xing Yang
1Department of Nuclear Medicine, Peking University First Hospital, Beijing, 100034, China.
2College of Pharmacy, Korea University, 2511 Sejong-ro, Sejong, 30019, Republic of Korea.
3Russell H. Morgan Department of Radiology and Radiological Science, Johns Hopkins Medical
Institutions, Baltimore, MD 21287, USA.
4Key Laboratory of Carcinogenesis and Translational Research (Ministry of Education/Beijing),
Department of Nuclear Medicine, Peking University Cancer Hospital & Institute, Beijing, 100142,
China.
5State Key Laboratory of Elemento-Organic Chemistry and Department of Chemical Biology,
National Pesticide Engineering Research Center (Tianjin), Nankai University, Tianjin, 300071,
China.
6Institute of Medical Technology, Peking University, Beijing, 100191. China
Corresponding Author:
Xing Yang, Tel: 86 10 83572928; Email: yangxing2017@bjmu.edu.cn
Zhen Xi, Tel: 86 22 23504782; E-mail: zhenxi@nankai.edu.cn
Zhi Yang, Tel: 86 10 88196196; Email: pekyz@163.com.
Xiaojiang Duan and Futao Liu contribute equally to this work.
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Key words: prostate-specific membrane antigen (PSMA), novel inhibitors, prostate cancer,
molecular imaging
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ABSTRACT
In an effort to seek novel agents targeting prostate-specific membrane antigen (PSMA), sixteen
ligands (L1-L16) with structural modifications in S1’ binding pocket were synthesized and
evaluated for PSMA inhibition. (S)-3-(Carboxyformamido)-2-(3-(carboxymethyl)ureido)
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propanoic acids proved potent PSMA ligands with K values ranging from 0.08 nM - 8.98 nM,
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which are in the range of or are higher in potency compared to previously published urea-based
ligands. Computational docking was performed to study the binding mode of the two most potent
+
ligands discovered. FITC-conjugated L14 could selectively stain PSMA LNCaP cells over
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PSMA PC3 cells. IRDye800CW conjugated L16 can effectively image tumors in a murine
xenograft model of prostate cancer.
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INTRODUCTION:
Prostate cancer (PCa) is the most common cancer and one of the leading causes of cancer-related
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death among men. A variety of options exist but metastatic, castration-resistant disease (mCRPC)
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continues to portend a poor prognosis. Recently, radiopharmaceuticals targeting prostate-specific
membrane antigen (PSMA) have showed significantly improved sensitivity and specificity over
the conventional techniques of computed tomography and bone scan for imaging both primary and
metastatic lesions.2, 3 Such PSMA-targeted agents have been further adapted for radioligand
therapy using therapeutic isotopes. Promising results have been reported and this new option may
4
provide an effective way to control mCRPC. The development of novel PSMA-targeted agents
with superior pharmacokinetic properties, namely, those with fewer off-target tissue binding, is
important to improve further the imaging and treatment of PCa, and has been an area of intensive
investigation.
PSMA, also known as glutamate carboxypeptidase II (GCPII) and folate hydrolase 1 (FOLH1), is
a type-II transmembrane protein and zinc-dependent metallopeptidase catalyzing the hydrolysis of
N-acetylaspartylglutamate (NAAG) or other glutamate derivatives.2, 5 Its expression is restricted
to a few normal tissues such as kidneys, proximal small intestine and, to a lesser extent, to salivary
glands, and has been reported to reach 1,000-fold enhancement in the epithelium of most PCa and
the neovasculature of other solid tumors. Those properties make PSMA one of the most attractive
6
targets for prostate cancer. Low-molecular-weight targeting agents have been developed that bind
tightly to the catalytic pocket of PSMA as inhibitors of its natural function. Many PSMA inhibitors
have been reported over the past 20 years, and the most successful ones for imaging and therapy
so far identified are those that provide modifications of the zinc-binding group in the catalytic
pocket mimicking NAAG. Moderate to high potency can be achieved with the scaffolds of
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phosphinates7, 8/phosphoramidates9 (1, 2), ureas10, 11 (3) and carbamates (4, 5) as well as
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thiols13, 14 and hydroxamates. Phosphoramidates and ureas have been well studied for imaging
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applications. A variety of low-molecular-weight ligands have been labeled with radionuclides ( C,
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18 64
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Ga, Y, 99mTc, 111In,125/124I, 177Lu, 161Tb and 225Ac) and studied for positron emission
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F, Cu,
tomography (PET), single photon emission computed tomography (SPECT) or targeted
radioligand therapy.16-31 With high binding affinity and synthetic simplicity, urea-based inhibitors
currently dominate in clinical trials for radiopharmaceutical imaging and radioligand therapy.
Nevertheless, new targeting scaffolds with improved pharmacokinetic properties are still in
demand to enhance non-targeting tissue clearance thereby decreasing potential toxicity in
radioligand therapy.
Compared with the extensive studies of the PSMA ligand at the zinc-binding site and S1 site, only
a few studies have reported modifications on the glutamate-like moiety, and those reported have
met with little success.11, 32 The S1’ binding pocket of PSMA, which binds to the glutamate-like
moiety, has strict structural requirements to enable productive interaction. To find a novel ligand
with improved pharmacokinetic properties and to understand better the binding mode of PSMA
ligands in S1’ pocket, we designed a series of compounds under the general structure of 6. There
were several considerations in our initial design: 1) The modified structures were more hydrophilic
than glutamate, which was expected to improve in vivo non-targeting clearance; 2) The ligands
could be easily accessed and diversified from 7. After we synthesized and evaluated 16 ligands,
we identified (S)-3-(carboxyformamido)-2-(3-(carboxymethyl)ureido) propanoic acid as a novel
PSMA targeting moiety with K values as low as 80 pM. Optical imaging was carried out as a
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proof-of-principle demonstration for its imaging potential.
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RESULTS:
Following our initial design, five compounds (L1 - L5) were synthesized with variations in the
PSMA S1’ pocket, and their syntheses are shown in Schemes 1 and 2. Commercially available 8
was first reacted with Fmoc-Cl to protect the free amino group and the  amino group was
deprotected through hydrogenation to give compound 10. After coupling of 10 with tert-butyl L-
leucinate, compound 11 was obtained in suitable yield after three steps. After Fmoc deprotection,
the intermediate 12 was applied to generate L1 - L4. The yield ranged from 38 to 48% after HPLC
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purification. L5 was synthesized in N -((benzyloxy)carbonyl)-L-lysine form to improve HPLC
purification process with UV absorbance at 254 nm, as shown in Scheme 2. Intermediate 14 could
be easily generated and used to access L5 with a yield of 41%. The PSMA inhibitory activities of
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L1 - L5 were measured by florescence-based NAALDase assay (Table 1). ZJ-43, a known
PSMA inhibitor, was used as control inhibiting PSMA activity with a K of 3.53 nM. The (S)-2-
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amino-3-(carboxyformamido)propanoic acid variation L1 showed comparable activity of 5.69 nM
with ZJ-43, but all other compounds failed to show reasonable activity.33
We chose (S)-3-(carboxyformamido)-2-(3-(carboxymethyl)ureido) propanoic acid as the scaffold
for further structural optimization. L6 - L15 were synthesized as shown in Scheme 3.
Commercially available compound 8 first reacted with tert-butyl-2-chloro-2-oxoacetate and then
the protecting group was removed under hydrogenation to afford 16 in good yield. 16 could be
6
efficiently coupled with N -((benzyloxy)carbonyl)-L-lysine and after hydrogenation, compound
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8 could be obtained in gram scale for further functionalization. L6 - L15 were synthesized and
evaluated for PSMA binding affinity. The synthetic yield ranged from 21% to 66% after HPLC
purification. As shown in Table 1, L6 showed the most potent activity at a K of 0.08 nM and all
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other modifications L7 - L15 also showed good binding affinity to PSMA between 0.34 nM and
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.74
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which
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(S)-3-(carboxyformamido)-2-(3-
(carboxymethyl)ureido)propanoic acid ligand could be generally tagged with diversified
functional groups for imaging and drug delivery purpose, without diminishing its PSMA binding
potency.
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As a proof-of-principle application, we modified L12 with IRDye800CW dye and synthesized
optical imaging ligand L16. The synthesis is shown in Scheme 4. The Cbz-group of 20 was
removed under hydrogenation and then conjugated with a protected glutamic acid linker to afford
2
2. After the deprotection steps, reactive intermediate 24 was synthesized and purified by HPLC
in moderate yield. IRDye800CW NHS ester and 24 were allowed to react at room temperature for
2 h. L16 was prepared in 40% yield after purification. It inhibited PSMA at a K of 0.65 nM, which
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is suitable for further biological evaluation.
We performed molecular docking studies of compounds L1 and L6 with the X-ray crystal structure
1
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of PSMA (PDB ID: 3D7H) by using Surflex-Dock GeomX module of SYBYL-X 2.1.1 to
elucidate their potential binding modes at the active site of PSMA (Figure 2). Both ligands made
strong hydrogen-bonding interactions with Arg210 and Lys699, which are key amino acid residues
of S1’ pharmacophore subpocket. However, L6 made more hydrogen bonds with the amino acid
residue in the S1 subpocket such as Arg534, Arg 536, Asp 387 and Asp 453 as shown in Figure
2
B. Furthermore, the hydrophobic interaction of 4-iodobenzoyl group on L6 projected to S1
subpocket and enhanced its affinity towards PSMA, while L1 did not benefit from this because the
short residue of Leu on L1 could not fill the S1 subpocket effectively.
L14 with a green fluorescent dye (FITC) conjugated to the PSMA targeting moiety showed a Ki
+
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of 0.43 nM. It could selectively stain PSMA LNCaP cells over PSMA PC3 cells as shown in
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Figure 3. The fluorescent signal mostly located on the cell surface, consistent with prior reports.2
In addition, this fluorescent uptake could be successfully blocked with an established PSMA
inhibitor DCIBzL at a concentration of 100 μM.34
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The in vivo study was carried out with L16 with a K of 0.65 nM (Figure 4). The tumor could be
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clearly visualized at 4 h post-injection and the image quality was improved at 10 h and 24 h with
decreasing background signal. The ex vivo results further supported the observation, with tumor
and kidney accumulating the most intensive signal. In addition, the tumor and kidney uptake
could be significantly blocked with PSMA inhibitor DCIBzL.
DISCUSSION AND CONCLUSIONS:
PSMA inhibitors derived from glutamate-like scaffolds (1 – 5) have proved successful as targeting
agents for PCa. With their high tumor-to-background contrast, PSMA targeting agents have been
applied for radiopharmaceutical imaging and radioligand therapy in clinical studies. The potential
of those agents demonstrated to date has attacted higher demand for novel targeting agents with
improved pharmacokinetic properties, to improve further the imaging quality and reduce side
effects previously demonstrated during the radioligand therapy, such as xerostomia, resulting from
off-target effects. Compared with the active investigation of PSMA inhibitors deriving from the
key modification at the PSMA Zn binding site, little progress has been made on modifying the
glutamate moiety, due to strict structural requirements in the S1’ binding pocket. Kozikowski et
al. reported that incorporating even a methyl substitution into the glutamate weakened enzyme
inhibitory activity dramatically.35 Wang et al. systematically studied over 50 structural
modifications in the S1’ binding pocket and demonstrated that the allyl, alkynyl, furanyl, and
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thiophenyl moieties were among the most effective. But those inhibitors still displayed over 500-
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fold lower potency than glutamate analogues ZJ-43 and DCIBzL, respectively. To discover a
novel replacement for the glutamate moiety, we designed a series of novel inhibitors with the
general structure of 6 by introducing a nitrogen substitution on the backbone.
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L1, with a (S)-3-(carboxyform amido)-2-(3-(carboxymethyl)ureido)propanoic acid structure,
exhibited a similar potency with its close analogue ZJ-43 (K =5.69 nM vs 3.63 nM). That result
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suggested that the N-amide replacement could satisfy the stereoelectronic requirement in the
binding pocket and a slightly extended anionic carboxylate at the far end could also be tolerated.
In sharp contrast, L2 to L5, with further increasing of the size or extension of the anion showed
minimal inhibition of PSMA. This result again reflected the strict structural requirements of the
S1’ binding pocket. It is interesting to note that oxalyldiaminopropionic acid (ODAP) has been
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discovered as an effective natural product binding tightly to glutamate receptors, and the novel
ligand we discovered may reflect the biological similarity between Glu and ODAP. Computational
docking supported our hypothesis as shown in Figure 2. Both L1 and L6 featured with the key
interactions in the S1’ binding pocket, including Tyr552, Tyr700, Arg210 to the carboxylic acid
motif and Lys699 to oxalate motif, which were very similar. In the case of L6, the 4-iodobenzoic
substitution not only helped with an extra “-cation” interaction, but also improved the binding
efficiency of oxalate to Lys699 (1.98 Å for L1 vs 1.74 Å for L6). As a result, improved potency
was observed.
Given that L1 displayed high affinity to PSMA, we further synthesized and evaluated 10
derivatives to test whether (S)-3-(carboxyformamido)-2-(3-(carboxymethyl)ureido) propanoic
acid could be applied as a general PSMA targeting moiety. Key intermediate 18 could be
conveniently accessed from commercially available 8 in gram scale. From 18, L6-L15 were
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synthesized and tested for their PSMA binding potency. As a close analog of DCIBzL, one of the
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most potent PSMA inhibitors reported to date, L6 inhibited PSMA with a K of 0.08 nM,
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compared with DCIBzL 0.20 nM with our assay. L7-L9 substituted with the substitution of 4-
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fluorobenzamide, 6-fluoronicotinamide, 6-methoxy-2-naphthamide showed similar level or
slightly improved potency compared with L1. One order of magnitude improvement could be
achieved in L10 and L15, with 4'-methoxy-[1,1'-biphenyl]-4-carboxamide and N-(4-
3
8
bromobenzyl)acetamide substitution, which is also consistent with literature reports. Overall, our
results indicated that this novel scaffold could effectively target PSMA and be modified for
imaging and therapeutic purposes. To balance the strong PSMA binding and synthetic cost, L12
and L13 with functional groups (NH or COOH in protected form) for further bioconjugation
2
applications were synthesized and tested with 1 nM potency.
+
To evaluate the cell targeting property, L14 with a K of 0.43 nM was tested using PSMA LNCaP
i
-
and PSMA PC3 cells. L14 could selectively stain LNCaP cells with most of the signal located on
2
the cell surface, consistent with the known PSMA expression pattern. With this specificity proved,
we performed in vivo imaging for a preliminary evaluation, using IRDye800CW conjugated ligand
L16 with a K of 0.65 nM. The result indicated the novel scaffold could effectively and specifically
i
target tumor. At 4 h post injection, the tumor could be clearly visualized, with tumor and kidneys
accumulating the most intensive signal, which is in agreement with reports on glutamate-based
3
9
targeting agents (Figure 4). After the ligand cleared from normal tissue and organs, target-
specific imaging could be achieved at 10 h and 24 h. At 24 h post-injection, the mice were
sacrificed and ex vivo imaging was performed on the organs for quantification. The results further
confirmed the in vivo imaging with tumor and kidneys as the main organs for the signal uptake,
which significantly higher than other organs (tumor-to-muscle 19.0 and tumor-to-kidney 0.19, see
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details in supporting information Figure S4). Due to tumor model difference, direct comparison
with literature could not be accurately achieved, but the biodistribution of L16 at 24 h post-
injection suggesting it’s among the successful ones with high tumor-to-muscle and tumor-to-organ
ratio.39,40 Effort should be further spent on improving the tumor-to-kidney ratio and developing
radiopharmaceuticals for more accurate quantification.
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In summary, we discovered (S)-3-(carboxyformamido)-2-(3-(carboxymethyl)ureido) propanoic
acids as a novel class of compounds targeting PSMA with similar or higher potency, compared
with urea-based ligand 3. These ODAP-based ligands could selectively bind to PSMA over-
expressing LNCaP cells. As a proof-of-principle study, IRDye800CW conjugated ODAP-based
ligand succeeded in detecting tumor in vivo with high specificity. More detailed studies of this
novel ligand are currently under investigation.
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EXPERIMENTAL SECTION
General: Synthesis was carried out following Schemes 1-4. The final products and key
1
13
intermediates were characterized with H-NMR, C-NMR and HRMS. All the final products were
purified by HPLC to reach >95% purity before performing the binding assay. Generally, solvents
and chemicals purchased from commercial sources were of analytical grade or better and were
used without further purification. Analytical thin-layer chromatography (TLC) was performed
using Merck Aluminum-backed silica gel 60 F254 (Billerica, MA). NMR spectra were recorded
on a Bruker 400 spectrometer. Chemical shifts () were reported in ppm downfield by reference
to proton resonances resulting from incomplete deuteration of the NMR solvent. High resolution
ESI mass spectra were obtained on an Agilent 6545 Triple Quadrupole LC MS (Santa Clara, CA).
0
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2
HPLC analysis was performed using a Phenomenex C18 Luna 4.60  250 mm column on a
Shimadzu LC-20A chromatography system with solvent gradient A ( water with 0.1%
trifluoroacetic acid ) and gradient B ( acetonitrile with 0.1% trifluoroacetic acid ) flowing at 1
mL/min and all analysis HPLC retention times are given for an eluent gradient of 5% B for the
first 1 min, 5 – 95% B over 5 min, and 95% B for the final 10 min. HPLC purification was
2
performed using a Phenomenex C18 Luna 10.0  250 mm column on a Bonna-Agela
Technologies Co., Ltd FL-H050G preparative chromatography system (Tianjin, China). The
products were eluted using eluent A (water with 0.1% trifluoroacetic acid) and eluent B
(acetonitrile with 0.1% trifluoroacetic acid). The general procedures, purification and
characterization are provided in detail in the Supplemental Data.
Synthesis and Characterization:
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Synthesis of (S)-tert-butyl-3-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-2-(((benzyloxy)
carbonyl)amino)propanoate (9). A mixture of (S)-tert-butyl-3-amino-2-(((benzyloxy)carbonyl)
amino)propanoate hydrochloride 8 (1 g, 3.03 mmol) and Et N (1.23 g, 13.12 mmol) was mixed in
3
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anhydrous CH Cl (20 mL) at 0°C. Fmoc-Cl (862 mg, 3.34 mmol) was added and the reaction
2
2
mixture was stirred at room temperature for 6 h. Then, the solvent was evaporated under vacuum.
1
.09 g compound 9 was isolated by silica gel flash column chromatography (ethyl
1
acetate/petroleum ether, 0% to 25%, vol/vol) as a colorless thick oil. The yield is 70%. H NMR
(
400 MHz, CDCl ) δ 7.76 (d, J = 7.5 Hz, 2H), 7.58 (d, J = 7.4 Hz, 2H), 7.40 (t, J = 7.4 Hz, 2H),
3
7
.37 - 7.28 (m, 7H), 5.71 (s, 1H), 5.19 (s, 1H), 5.11 (s, 2H), 4.40 - 4.35 (m, 3H), 4.20 (t, J = 6.8
1
3
Hz, 1H), 3.63 (s, 2H), 1.46 (s, 9H). C NMR (100 MHz, CDCl ) δ 169.31, 156.71, 156.25, 143.99,
3
1
43.85, 141.41, 136.24, 128.65, 128.34, 128.29, 127.83, 127.20, 125.19, 120.10, 83.21, 67.25,
+
6
7.17, 55.17, 47.26, 43.21, 28.03. HRMS calcd. for C H N O [M+H] 517.2333, found
3
0
32
2
6
517.2331.
Synthesis
of
(S)-tert-butyl
3-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-2-amino
propanoate (10). Compound 9 (900 mg, 1.74 mmol) and 10% dry Pd/C (35 mg) were mixed in
MeOH (20 mL) under H and stirred at room temperature for 12 h. After filtration, the solvent was
2
removed under vacuum and 550 mg of product 10 was obtained after silica gel flash column
chromatography (methanol/dichloromethane, 0% to 10%, vol/vol) as a colorless oil. The yield was
1
8
3%. H NMR (400 MHz, CDCl ) δ 7.76 (d, J = 7.5 Hz, 2H), 7.59 (d, J = 7.4 Hz, 2H), 7.40 (t, J =
3
7.4 Hz, 2H), 7.31 (t, J = 7.4 Hz, 2H), 5.33 (s, 1H), 4.40 (d, J = 6.9 Hz, 2H), 4.22 (t, J = 6.8 Hz,
13
1
1
H), 3.57 - 3.47 (m, 2H), 3.31 - 3.24 (m, 1H), 1.47 (s, 9H). C NMR (100 MHz, CDCl ) δ
3
73.14, 156.61, 144.07, 141.45, 127.82, 127.18, 125.19, 120.11, 82.09, 66.93, 54.86, 47.37, 44.65,
+
28.15. HRMS calcd. for C H N O [M+H] 383.1965, found 383.1959.
2
2
26
2
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Synthesis of (6S,10S)-tert-butyl 6-(tert-butoxycarbonyl)-1-(9H-fluor-en-9-yl)-10-isobutyl-
3
,8-dioxo-2-oxa-4,7,9-triazaundecan-11-oate (11). Triphosgene (139 mg, 0.47 mmol) was
o
dissolved in anhydrous CH Cl (10 mL) at -10 C. (S)-tert-butyl 2-amino-4-methylpentanoate
2
2
0
1
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0
hydrochloride (300 mg, 1.35 mmol) and Et N (545 mg, 5.38 mmol) in anhydrous CH Cl (20 mL)
3
2
2
was added within 5 min. The reaction was stirred at -10°C for 2 h. Then, a mixture of compound
0 (514 mg, 1.35 mmol) and Et N (408 mg, 4.03 mmol) in anhydrous CH Cl (20 mL) was added
1
3
2
2
in 30 min, and the reaction was further stirred for 5 h. After the solvent was removed, 620 mg of
the product 11 was obtained as a colorless oil, after silica gel flash column chromatography
1
(methanol/dichloromethane, 0% to 10%, vol/vol). The yield is 77%.
H NMR (400 MHz, CDCl3)
δ 7.75 (d, J = 7.5 Hz, 2H), 7.63 (t, J = 7.9 Hz, 2H), 7.39 (t, J = 7.4 Hz, 2H), 7.30 (t, J = 7.4 Hz,
2
1
H), 5.68 (s, 1H), 5.43 (s, 1H), 5.26 (s, 1H), 4.49 (s, 1H), 4.42 - 4.17 (m, 4H), 3.64 - 3.49 (m, 2H),
1
3
.75 - 1.70 (m, 2H), 1.61 - 1.51 (m, 1H), 1.45 (s, 9H), 1.42 (s, 9H), 0.94 (d, J = 5.1 Hz, 6H). C
NMR (100 MHz, CDCl ) δ 173.79, 170.35, 157.47, 156.84, 144.11, 141.53, 127.76, 127.22,
3
1
2
25.44, 120.03, 82.90, 82.04, 67.24, 54.51, 52.48, 47.29, 43.93, 42.45, 28.14, 28.01, 24.99, 22.97,
+
2.22. HRMS calcd. for C H N O [M+H] 596.3330, found 596.3321.
3
3
45
3
7
Synthesis of (S)-tert-butyl 2-(3-((S)-3-amino-1-(tert-butoxy)-1-oxopropan-2-yl)ureido)-4-
methyl pentanoate (12). Compound 11 (400 mg, 0.67 mmol) and piperidine (2 mL) were
dissolved in DMF (10 mL) and the reaction was stirred for 4 h at room temperature. The solvent
was evaporated under vacuum. The crude product 12 was used for next step without purification.
+
HRMS calcd. for C H N O [M+H] 374.2649. Found 374.2632
1
8
35
3
5
Synthesis of tert-butyl 2-chloro-2-oxoacetate. Oxoacetate (2 g, 15.76 mmol) was dissolved in
anhydrous CH Cl (30 mL) and a tert-Butanol (1.17 g, 15.76 mmol) solution in CH Cl (20 mL)
2
2
2
2
was added dropwise in 2 h. The reaction mixture was stirred at 0°C for another 24 h. After the
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solvent was evaporated under vacuum, 2.08 g of 2-chloro-2-oxoacetate was obtained as a colorless
oil and used for next step without further purification. The yield is 80%.
Synthesis of (S)-2-(3-((S)-1-carboxy-2-(carboxyformamido)ethyl)ureido)-4-methylpentanoic
1
1
1
1
1
1
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0
acid (L1). Compound 12 (50 mg, 0.13 mmol) and Et N (136 mg, 1.34 mmol) were mixed in
3
anhydrous DCM (15 mL) at 0°C and tert-butyl 2-chloro-2-oxoacetate (66 mg, 0.40 mmol) was
added dropwise in anhydrous DCM (5 mL). The reaction was stirred at room temperature for 5 h.
After the solvent was removed under vacuum, CF COOH (5 mL) was added and the solution was
3
stirred at room temperature for 3 h. After CF COOH was removed under vacuum, the product was
3
purified by preparative HPLC. 20 mg of L1 was obtained as a white solid with a yield of 44%.
1
The analysis HPLC retention time t = 10.7 min. H NMR (400 MHz, MeOD) δ 4.35 (t, J = 5.9
R
Hz, 1H), 4.16 (q, J = 5.0 Hz, 1H), 3.53 (d, J = 5.9 Hz, 2H), 1.67 - 1.59 (m, 1H), 1.53 - 1.37 (m,
1
3
2
H), 0.83 (t, J = 7.3 Hz, 6H). C NMR (100 MHz, MeOD) δ 177.22, 173.84, 162.36, 160.52,
159.95, 53.90, 52.69, 42.58, 42.48, 25.98, 23.39, 22.01. HRMS calcd. for C H N O
8
1
2
19
3
+
[
M+H] 334.1245, found 334.1239.
Synthesis of 4-(((S)-2-carboxy-2-(3-((S)-1-carboxy-3-methylbutyl)ureido)ethyl)carbamoyl)
benzoic acid (L2). 4-(tert-butoxycarbonyl)benzoic acid (29 mg, 0.13 mmol), DIPEA (55 mg, 0.43
mmol) and HATU (82 mg, 0.21 mmol) were mixed in DMF (20 mL) and stirred for 20 min at
room temperature. Compound 12 (40 mg, 0.11 mmol) was then added and the reaction mixture
was stirred for 5 h at room temperature. After the solvent was removed under vacuum, CF COOH
3
(6 mL) was added and stirred at room temperature for another 5 h. The CF COOH was removed
3
under vacuum and 21 mg L2 was obtained by preparative HPLC as a white solid. The yield is
1
4
8%. The analysis HPLC retention time t = 10.8 min. H NMR (400 MHz, DMSO) δ 8.74 (t, J =
R
5.6 Hz, 1H), 8.01 (d, J = 8.3 Hz, 2H), 7.89 (d, J = 8.3 Hz, 2H), 6.53 (d, J = 8.2 Hz, 1H), 6.32 (d, J
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=
8.2 Hz, 1H), 4.35 (q, J = 7.0 Hz, 1H), 4.08 (q, J = 8.5 Hz, 1H), 3.55 - 3.49 (m, 2H), 1.67 - 1.59
1
3
(m, 1H), 1.52 - 1.33 (m, 2H), 0.86 (dd, J = 10.9, 6.6 Hz, 6H). C NMR (100 MHz, DMSO) δ
175.06, 172.90, 166.85, 166.06, 157.38, 138.18, 133.03, 129.28, 127.53, 52.59, 50.92, 41.43, 41.16,
0
1
2
3
4
5
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8
9
0
1
2
3
4
5
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7
8
9
0
1
2
3
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8
9
0
1
2
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6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
+
2
4.28, 22.85, 21.69. HRMS calcd. for C H N O [M+H] 410.1558, found 410.1552.
1
8
23
3
8
Synthesis of 2-(((S)-2-carboxy-2-(3-((S)-1-carboxy-3-methylbutyl)ureido)ethyl)carbamoyl)
benzoic acid (L3). Compound 12 (40 mg, 0.11 mmol), Et N (109 mg, 1.07 mmol) and
3
isobenzofuran-1,3-dione (48 mg, 0.32 mmol) were mixed in CH Cl (20 mL) and reacted at room
2
2
temperature for 24 h. After the solvent was evaporated under vacuum, CF COOH (6 mL) was
3
added, and stirred at room temperature for 5 h. 20 mg of L3 was obtained as a white solid after
preparative HPLC purification. The yield is 47%. The analysis HPLC retention time t = 10.8 min.
R
1H NMR (400 MHz, DMSO) δ 8.39 (t, J = 5.8 Hz, 1H), 7.77 - 7.71 (m, 1H), 7.58 - 7.47 (m, 2H),
7
.42 (d, J = 7.4 Hz, 1H), 6.49 (d, J = 8.1 Hz, 1H), 6.29 (d, J = 8.2 Hz, 1H), 4.32 (q, J = 7.0 Hz,
1
H), 4.11 (q, J = 8.0 Hz, 1H), 3.44 - 3.38 (m, 2H), 1.71 - 1.59 (m, 1H), 1.50 - 1.37 (m, 2H), 0.87
13
(dd, J = 11.5, 6.6 Hz, 6H). C NMR (100 MHz, DMSO) δ 175.08, 172.92, 168.90, 167.93, 157.28,
1
38.26, 131.15, 130.68, 129.24, 129.11, 127.94, 52.61, 50.93, 41.18, 41.04, 24.28, 22.83, 21.77.
+
HRMS calcd. for C H N O [M+H] 410.1558, found 410.1552.
1
8
23
3
8
Synthesis of (S)-2-(3-((S)-1-carboxy-2-(1H-1,2,3-triazole-4-carboxamido)ethyl)ureido)-4-
methylpentanoic acid (L4). 1H-imidazole-2-carboxylic acid (20 mg, 0.17 mmol)，DIPEA (173
mg, 1.34 mmol) and HATU (102 mg, 0.27 mmol) were mixed and stirred in DMF (20 mL) for 10
min at room temperature. Compound 12 (50 mg, 0.13 mmol) was then added and the reaction
mixture was stirred for 5 h. After the solvent was evaporated under vacuum, CF COOH (6 mL)
3
was added and stirred at room temperature for another 5 h. The solvent was then removed under
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vacuum, and 18 mg L4 was obtained as a white solid after preparative HPLC purification. The
1
yield is 38%. The analysis HPLC retention time t = 10.6 min. H NMR (400 MHz, MeOD) δ 8.22
R
(s, 1H), 4.53 (t, J = 5.8 Hz, 1H), 4.29 (q, J = 5.1 Hz, 1H), 3.80 (d, J = 3.7 Hz, 2H), 1.81 - 1.72 (m,
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
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2
3
4
5
6
7
8
9
0
13
1
1
H), 1.65 - 1.52 (m, 2H), 0.96 (t, J = 6.9 Hz, 6H). C NMR (100 MHz, MeOD) δ 175.22, 174.14,
74.06, 163.14, 160.01, 142.91, 54.36, 52.69, 42.47, 42.04, 29.11, 25.95, 23.38, 22.01. HRMS
+
calcd. for C H N O [M+H] 357.1517, found 357.1505.
13 20
6
6
Synthesis of (9S,13S)-tert-butyl 13-(((((9H-fluoren-9-yl)methoxy)carbonyl)amino)methyl)-9-
(tert-butoxycarbonyl)-3,11-dioxo-1-phenyl-2-oxa-4,10,12-triazatetradecan-14-oate(13).
o
Triphosgene (84 mg, 0.28 mmol) was dissolved in anhydrous CH Cl (20 mL) at -10 C. To the
2
2
solution, a mixture of (S)-tert-butyl 2-amino-6-(((benzyloxy)carbonyl)amino)hexanoate
hydrochloride (300 mg, 0.81 mmol) and Et N (326 mg, 3.23 mmol) in anhydrous CH Cl (10 mL)
3
2
2
was added in 5 min. The reaction mixture was stirred at -10°C for 2 h, and then a solution of
compound 10 (308 mg, 0.81 mmol) and Et N (163 mg,1.61 mmol) in anhydrous CH Cl (10 mL)
3
2
2
was added over 30 min. The reaction was stirred for another 5 h. After the solvent was removed
under vacuum, 430 mg of product 13 was obtained after silica gel flash column chromatography
1
(
methanol/dichloromethane, 0% to 10%, vol/vol) as a colorless oil. The yield is 72%. H NMR
(
400 MHz, CDCl ) δ 7.74 (d, J = 7.5 Hz, 2H), 7.60 (dd, J = 7.3, 3.0 Hz, 2H), 7.38 - 7.27 (m, 9H),
3
5
.66 – 5.57 (m, 2H),5.09 (s, 2H), 4.48 (q, J = 5.5 Hz, 1H), 4.37 - 4.26 (m, 3H), 4.19 (t, J = 7.1 Hz,
1H), 3.56 - 3.53 (m, 2H), 3.20 - 3.10 (m, 2H), 1.76 - 1.73 (m, 1H), 1.63 - 1.50 (m, 5H), 1.42 (s,
13
1
1
5
8H). C NMR (100 MHz, CDCl ) δ 172.89, 170.56, 157.51, 156.77, 144.10, 144.07, 141.36,
3
36.78, 128.60, 128.19, 128.16, 128.12, 127.76, 127.19, 125.35, 120.03, 82.94, 82.08, 67.15, 66.71,
4.31, 53.45, 47.26, 43.90, 40.72, 32.61, 29.44, 28.11, 28.02, 22.36. HRMS calcd. for C H N O
9
41 52
4
+
[
M+H] 745.3807, found 745.3818
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5
6
Synthesis of (9S,13S)-tert-butyl 13-(aminomethyl)-9-(tert-butoxyc-arbonyl)-3,11-dioxo-1-
phenyl-2-oxa-4,10,12-triazatetradecan-14-oate (14). Compound 13 (400 mg, 0.54 mmol) was
mixed with piperidine (2 mL) in DMF (10 mL) and the reaction was stirred for 4 h at room
temperature. The solvent was removed under vacuum to get 241 mg crude product 14 as a yellow
solid and used for next step without purification. The yield is 86%. HRMS calcd. for C H N O
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
6
42
4
7
+
[
M+H] 523.3126, found 523.3135.
Synthesis of (9S,13S)-3,11,16-trioxo-1-phenyl-2-oxa-4,10,12,15-tetraazaoctadecane-9,13,18-
tricarboxylic acid (L5). Compound 14 (50 mg, 0.10 mmol), Et N (101 mg, 0.10 mmol) and
3
dihydrofuran-2,5-dione (29 mg, 0.29 mmol) were mixed in CH Cl (20 mL) and stirred at room
2
2
temperature for 24 h. After the solvent was removed under vacuum, CF COOH (6 mL) was added.
3
The solution was stirred at room temperature for 5 h and the solvent removed under vacuum. 20
mg of L5 was obtained as a white solid after preparative HPLC purification. The yield is 41%.
1
The analysis HPLC retention time t = 10.9 min. H NMR (400 MHz, DMSO) δ 7.94 (t, J = 5.8
R
Hz, 1H), 7.39 - 7.28 (m, 5H), 7.23 (t, J = 5.5 Hz, 1H), 6.50 (d, J = 7.8 Hz, 1H), 6.24 (d, J = 8.0
Hz, 1H), 5.00 (s, 2H), 4.15 (q, J = 6.8 Hz, 1H), 4.03 (q, J = 7.1 Hz, 1H), 3.39 - 3.26 (m, 2H), 2.96
(q, J = 6.5 Hz, 2H), 2.40 (t, J = 6.7 Hz, 2H), 2.31 (t, J = 6.7 Hz, 2H), 1.68 - 1.60 (m, 1H), 1.56 -
1
3
1
1
3
.47 (m, 1H), 1.43 - 1.36 (m, 2H), 1.31 - 1.25 (m, 2H). C NMR (100 MHz, DMSO) δ 174.53,
73.85, 172.91, 171.51, 157.25, 156.12, 137.32, 128.40, 127.76, 65.15, 52.79, 52.38, 40.56, 31.82,
+
0.01, 29.15, 22.51. HRMS calcd. for C H N O [M+H] 511.3025, found 511.3027.
22 30
4
10
Synthesis of (S)-tert-butyl 2-(((benzyloxy)carbonyl)amino)-3-(2-(tert-butoxy)-2-oxoacet
amido)propanoate (15). (S)-tert-butyl 2-amino-4-methylpentanoate hydrochloride (2.0 g, 6.08
mmol) and Et N (3.08 g, 30.40 mmol) were mixed in anhydrous CH Cl (30 mL) at 0°C. Tert-
3
2
2
butyl 2-chloro-2-oxoacetate (2.58 g, 15.76 mmol) in anhydrous CH Cl (10 mL) were added
2
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dropwise over 30 min and the reaction mixture was stirred at room temperature for 10 h. After the
solvent was removed under vacuum, 2.0g of 17 was obtained as a colorless oil after silica gel flash
column chromatography (ethyl acetate/petroleum ether, 0% to 30%, vol/vol). The yield is 78%.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
H NMR (400 MHz, CDCl ) δ 7.63 (s, 1H), 7.35 - 7.28 (m, 5H), 5.76 (d, J = 6.9 Hz, 1H), 5.10 (s,
3
1
3
2
H), 4.38 (q, J = 6.1 Hz, 1H), 3.72 - 3.64 (m, 2H), 1.52 (s, 9H), 1.44 (s, 9H). C NMR (100 MHz,
CDCl ) δ 168.89, 159.16, 158.00, 156.31, 136.10, 128.54, 128.22, 128.15, 84.59, 83.35, 67.16,
3
+
5
4.39, 42.15, 27.87,27.71. HRMS calcd. for C H N O [M+H] 423.2126, found 423.2121.
2
1
30
2
7
Synthesis of (S)-tert-butyl 2-amino-3-(2-(tert-butoxy)-2-oxoacetam-ido)propanoate (16). A
mixture of compound 15 (2 g, 4.74 mmol) and 10% dry Pd/C (15 mg) was stirred in MeOH (20
mL) under H for 12 h at room temperature. The reaction mixture was filtered and washed with
2
MeOH (10 mL) and CH Cl (10 mL). The solvent was removed under vacuum to give compound
2
2
1
1
6 (1.27 mg, yield 93%) as a colorless oil. H NMR (400 MHz, CDCl ) δ 7.58 (s, 1H), 3.65 -
3
1
3
3.58 (m, 1H), 3.51 - 3.47 (m, 1H), 3.37 - 3.29 (m, 1H), 1.72 (s, 2H), 1.52 (s, 9H), 1.45 (s, 9H). C
NMR (100 MHz, CDCl ) δ 172.77, 159.51, 157.76, 84.57, 82.33, 54.27, 43.16, 28.06, 27.80.
3
+
HRMS calcd. for C H N O [M+H] 289.1758, found 289.1741.
1
3
24
2
5
Synthesis of (9S,13S)-tri-tert-butyl 3,11,16-trioxo-1-phenyl-2-oxa-4,10,12,15-tetraazahexa
decane-9,13,16-tricarboxylate (17). Triphosgene (360 mg, 1.22 mmol) was dissolved in
anhydrous CH Cl (30 mL) at -10°C. To the solution, a mixture of (S)-tert-butyl 2-amino-6-
2
2
(
((benzyloxy)carbonyl)amino)hexanoate hydrochloride (1.29 mg, 3.47 mmol) and Et N (1.41 g,
3
13.88 mmol) in anhydrous CH Cl (15 mL) was added in 5 min. The reaction mixture was stirred
2
2
at -10°C for 2 h, and then a solution of compound 16 (1 g, 3.47 mmol) and Et N (0.70 g, 6.94
3
mmol) in anhydrous CH Cl (20 mL) was added over 30 min. The reaction was stirred for another
2
2
5 h. After the solvent was removed under vacuum, 1.62 mg of product 17 was obtained after silica
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2
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6
gel flash column chromatography (methanol/dichloromethane, 0% to 10%, vol/vol) as a colorless
1
oil. The yield is 72%. H NMR (400 MHz, CDCl ) δ 7.96 (t, J = 4.8 Hz, 1H), 7.35 - 7.25 (m, 5H),
3
5.87 (d, J = 6.7 Hz, 1H), 5.77 (d, J = 7.9 Hz, 1H), 5.10 - 5.50 (m, 2H), 4.49 (q, J = 6.9 Hz, 1H),
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
4
3
1
2
.38 - 4.31 (m, 1H), 3.68 - 3.51 (m, 2H), 3.20 - 3.09 (m, 2H), 1.77 - 1.68 (m, 1H), 1.64 - 1.33 (m,
1
3
2H). C NMR (100 MHz, CDCl ) δ 172.49, 170.09, 159.13, 158.09, 157.62, 156.70, 136.73,
3
28.51, 128.09, 128.06, 84.33, 83.12, 81.79, 66.58, 53.46, 53.35, 43.28, 40.70, 32.61, 29.32, 28.05,
+
7.92, 27.74, 22.31. HRMS calcd. for C H N O [M+H] 651.3600, found 651.3577.
3
2
50
4
10
Synthesis of (S)-tert-butyl 6-amino-2-(3-((S)-1-(tert-butoxy)-3-(2-(tert-butoxy)-2-oxoacet
amido)-1-oxopropan-2-yl)ureido)hexanoate (18). A mixture of compound 17 (1 g, 1.54 mmol)
and 10% dry Pd/C (50 mg) was stirred in MeOH (30 mL) under H for 12 h at room temperature.
2
The reaction mixture was filtered and washed with MeOH (10 mL) and CH Cl (10 mL). The
2
2
solvent was removed under vacuum to get 714 mg crude product 18 as a colorless oil and used for
+
next step without purification. The yield is 90%. HRMS calcd. for C H N O [M+H] 517.3232,
2
4
44
4
8
found 517.3241.
Synthesis of (4S,8S)-14-(4-iodophenyl)-1,6,14-trioxo-2,5,7,13-tetraazatetradecane-1,4,8-
tricarboxylic acid (L6). A mixture of compound 2,5-dioxopyrrolidin-1-yl 4-iodobenzoate (50 mg,
0
.15 mmol)，Et N (59 mg, 0.58 mmol) and compound 18 (50 mg, 0.10 mmol) was stirred in DMF
3
(20 mL) for 12 h at room temperature. The solvent was removed under vacuum, and then
CF COOH (6 mL) was added. The reaction mixture was stirred at room temperature for 5 h and
3
the solvent was evaporated under vacuum. 27 mg of L6 was obtained as a white solid after
preparative HPLC purification. The yield is 48%. The analysis HPLC retention time t = 10.9 min.
R
1H NMR (400 MHz, DMSO) δ 8.76 (t, J = 6.0 Hz, 1H), 8.50 (t, J = 5.5 Hz, 1H), 7.83 (d, J = 8.4
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Hz, 2H), 7.61 (d, J = 8.4 Hz, 2H), 6.50 (d, J = 8.1 Hz, 1H), 6.33 (d, J = 8.1 Hz, 1H), 4.26 (q, J =
6
.7 Hz, 1H), 4.06 (q, J = 7.8 Hz, 1H), 3.41 (t, J = 6.3 Hz, 2H), 3.21 (q, J = 6.7 Hz, 3H), 1.70 - 1.60
1
3
(m, 1H), 1.60 - 1.46 (m, 3H), 1.38 - 1.26 (m, 2H). C NMR (100 MHz, DMSO) δ 174.47, 172.68,
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
65.47, 161.72, 158.34, 157.26, 137.15, 134.11, 129.21, 98.57, 52.34, 52.20, 40.92, 31.89, 28.76,
+
2.70. HRMS calcd. for C H IN O [M+H] 579.0582, found 579.0580.
1
9
23
4
9
Synthesis of (4S,8S)-14-(4-fluorophenyl)-1,6,14-trioxo-2,5,7,13-tetraazatetradecane-1,4,8-
tricarboxylic acid (L7). A mixture of 4-fluorobenzoic acid (20 mg, 0.14 mmol), DIPEA (18 mg,
1
.43 mmol) and HATU (109 mg, 0.29 mmol) was stirred in DMF (20 mL) for 20 min at room
temperature. Then, compound 18 (50 mg, 0.10 mmol) was added and the reaction was stirred for
5 h. The solvent was evaporated under vacuum and CF COOH (6 mL) was added. The reaction
3
was stirred at room temperature for 5 h. After the solvent was evaporated under vacuum, 18 mg of
L7 was obtained by preparative HPLC as a white solid and the yield is 40%. The analysis HPLC
1
retention time t = 10.7 min. H NMR (400 MHz, DMSO) δ 8.76 (t, J = 5.9 Hz, 1H), 8.46 (t, J =
R
5
.5 Hz, 1H), 7.98 - 7.83 (m, 2H), 7.27 (t, J = 8.9 Hz, 2H), 6.51 (d, J = 7.3 Hz, 1H), 6.33 (d, J = 7.7
Hz, 1H), 4.27 (q, J = 6.1 Hz, 1H), 4.13 - 4.02 (m, 1H), 3.41 (t, J = 6.3 Hz, 2H), 3.22 (q, J = 6.8
1
3
Hz, 2H), 1.71 - 1.65 (m, 1H), 1.59 - 1.49 (m, 3H), 1.36 - 1.21 (m, 2H). C NMR (100 MHz,
DMSO) δ 174.48, 172.69, 165.11, 165.03, 162.56, 161.72, 158.36, 157.27, 131.18, 131.16, 129.84,
1
29.75, 115.27, 115.06, 52.35, 52.21, 40.93, 31.91, 28.83, 22.71. HRMS calcd. for C H FN O
19 23 4 9
+
[
M+H] 471.1522, found 471.1514.
Synthesis of (4S,8S)-14-(6-fluoropyridin-3-yl)-1,6,14-trioxo-2,5,7,13-tetraazatetradecane-
,4,8-tricarboxylic acid (L8). A mixture of 6-fluoronicotinic acid (20 mg, 0.14 mmol)，DIPEA
1
(18 mg, 1.43 mmol) and HATU (109 mg, 0.29 mmol) was stirred in DMF (20 mL) for 20 min at
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2
2
2
2
2
2
2
3
3
3
3
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3
3
3
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3
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4
4
4
4
4
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4
4
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5
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5
5
6
room temperature. Then, compound 18 (50 mg, 0.10 mmol) was added and the reaction was stirred
for 5 h. The solvent was evaporated under vacuum, and then CF COOH (6 mL) was added. The
3
reaction was stirred at room temperature for 5 h. After the solvent was evaporated in vacuum, 15
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
mg of compound L8 was obtained as a white solid after preparative HPLC purification and the
1
yield is 33%. The analysis HPLC retention time t = 10.5 min. H NMR (400 MHz, DMSO) δ 8.76
R
(
t, J = 5.9 Hz, 1H), 8.68 - 8.65 (m, 2H), 8.36 (td, J = 8.3, 2.5 Hz, 1H), 7.28 (dd, J = 8.6, 2.6 Hz,
H), 6.51 (d, J = 8.1 Hz, 1H), 6.33 (d, J = 8.1 Hz, 1H), 4.26 (q, J = 6.6 Hz, 1H), 4.07 (q, J = 7.8
Hz, 1H), 3.41 - 3.40 (m, 2H), 3.28 - 3.23 (m, 2H), 1.70 - 1.62 (m, 1H), 1.59 - 1.50 (m, 3H), 1.37 -
1
1
3
1
.25 (m, 2H). C NMR (100 MHz, DMSO) δ 174.45, 172.66, 165.32, 163.53, 162.94, 161.70,
158.32, 157.24, 147.32, 147.16, 141.38, 141.29, 128.92, 128.87, 109.56, 109.19, 52.29, 52.18,
+
4
0.90, 31.86, 28.66, 22.66. HRMS calcd. for C H FN O [M+H] 472.1474, found 472.1477.
1
8
22
5
9
Synthesis of 2,5-dioxopyrrolidin-1-yl 6-methoxy-2-naphthoate. A mixture of 6-methoxy-2-
naphthoic acid (100 mg, 0.50 mmol)，1-hydroxypyrrolidine-2,5-dione (85 mg, 0.74 mmol) and
EDCI (285 mg, 1.49 mmol) was stirred in DMF (20 mL) for 20 h at room temperature. After the
solvent was evaporated under vacuum, 119 mg of product was obtained as a white solid by silica
gel flash colomn chromatography (ethyl acetate/petroleum ether, 0% to 50%, vol/vol) and the yield
1
is 75%. H NMR (400 MHz, CDCl ) δ 8.66 (s, 1H), 8.04 (dd, J = 8.7, 1.7 Hz, 1H), 7.85 (d, J = 9.0
3
Hz, 1H), 7.80 (d, J = 8.7 Hz, 1H), 7.22 (dd, J = 9.0, 2.5 Hz, 1H), 7.16 (d, J = 2.3 Hz, 1H), 3.95 (s,
1
3
3H), 2.92 (s, 4H). C NMR (100 MHz, CDCl ) δ 169.57, 162.25, 160.57, 138.30, 132.81, 131.36,
3
1
27.80, 127.53, 126.01, 120.36, 119.89, 105.95, 55.60, 25.84. HRMS calcd. for C H NO
16 13 5
+
[
M+Na] 322.0686, found 322.0683.
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Synthesis of (4S,8S)-14-(6-methoxynaphthalen-2-yl)-1,6,14-trioxo-2,5,7,13-tetraazatetra
decane-1,4,8-tricarboxylic acid (L9). A mixture of compound 2,5-dioxopyrrolidin-1-yl 6-
methoxy-2-naphthoate (44 mg, 0.15 mmol)，Et N (98 mg, 0.97 mmol) and compound 18 (50
3
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
mg, 0.10 mmol) was stirred in DMF (10 mL) for 12 h at room temperature. The solvent was
evaporated under vacuum, and then CF COOH (6 mL) was added. The reaction was stirred at
3
room temperature for 5 h. After the solvent was evaporated in vacuum, 20 mg of L9 was obtained
as a white solid by preparative HPLC and the yield is 38%. The analysis HPLC retention time tR
1
=
10.9 min. H NMR (400 MHz, DMSO) δ 8.73 (s, 1H), 8.53 (t, J = 5.4 Hz, 1H), 8.35 (s, 1H), 7.95
- 7.83 (m, 3H), 7.36 (s, 1H), 7.22 (dd, J = 8.9, 2.4 Hz, 1H), 6.52 (d, J = 8.0 Hz, 1H), 6.35 (d, J =
7
1
.9 Hz, 1H), 4.27 (q, J = 6.9 Hz, 1H), 4.07 (q, J = 7.8 Hz, 1H), 3.89 (s, 3H), 3.38 - 3.21 (m, 4H),
1
3
.78 - 1.67 (m, 1H), 1.62 - 1.53 (m, 3H), 1.42 - 1.33 (m, 2H). C NMR (100 MHz, DMSO) δ
174.47, 172.67, 166.24, 161.70, 158.46, 157.25, 135.67, 130.41, 129.77, 127.50, 127.20, 126.59,
1
24.74, 119.31, 105.86, 55.33, 52.37, 52.21, 40.91, 31.89, 28.94, 22.75. HRMS calcd. for
+
C H N O [M+H] 533.1878, found 533.1852.
2
4
28
4
10
Synthesis of 2,5-dioxopyrrolidin-1-yl 4'-methoxy-[1,1'-biphenyl]-4-carboxylate. A mixture of
'-methoxy-[1,1'-biphenyl]-4-carboxylic acid (66 mg, 0.29 mmol), 1-hydroxypyrrolidine-2,5-
4
dione (50 mg, 0.43 mmol) and EDCI (111 mg, 0.58 mmol) was stirred in DMF (20 mL) for 20 h
at room temperature. After the solvent was evaporated under vacuum, 66 mg of product was
obtained as a white solid by silica gel flash column chromatography (ethyl acetate/petroleum ether,
1
0
7
% to 50%, vol/vol) and the yield is 70%. H NMR (400 MHz, CDCl ) δ 8.16 (d, J = 8.2 Hz, 2H),
3
.68 (d, J = 8.2 Hz, 2H), 7.59 (d, J = 8.5 Hz, 2H), 7.01 (d, J = 8.5 Hz, 2H), 3.86 (s, 3H), 2.91 (s,
1
3
4H). C NMR (100 MHz, CDCl ) δ 169.47, 161.95, 160.36, 147.35, 131.87, 131.25, 128.62,
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Journal of Medicinal Chemistry
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26.93, 123.07, 114.62, 55.52, 25.82. HRMS calcd. for C H NO [M+H] 326.1023, found
18 15 5
26.1012.
Synthesis
of
(4S,8S)-14-(4'-methoxy-[1,1'-biphenyl]-4-yl)-1,6,14-trioxo-2,5,7,13-
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tetraazatetra decane-1,4,8-tricarboxylic acid (L10) A mixture of compound 2,5-
dioxopyrrolidin-1-yl 4'-methoxy-[1,1'-biphenyl]-4-carboxylate (49 mg, 0.15 mmol)，Et N (98
3
mg, 0.97 mmol) and compound 18 (50 mg, 0.10 mmol) was stirred in DMF (10 mL) for 12 h at
room temperature. The solvent was evaporated in vacuum, and then CF COOH (6 mL) was added.
3
The reaction was stirred at room temperature for 5 h. After the solvent was evaporated under
vacuum, 28 mg of L10 was obtained as a white solid after preparative HPLC and the yield is 52%.
1
The analysis HPLC retention time t = 11.1 min. H NMR (400 MHz, DMSO) δ 8.76 (t, J = 5.9
R
Hz, 1H), 8.46 (t, J = 5.4 Hz, 1H), 7.89 (d, J = 8.3 Hz, 2H), 7.69 (dd, J = 10.9, 8.6 Hz, 4H), 7.04
(d, J = 8.7 Hz, 2H), 6.52 (d, J = 8.0 Hz, 1H), 6.35 (d, J = 8.1 Hz, 1H), 4.27 (q, J = 6.6 Hz, 1H),
4
.07 (q, J = 7.8 Hz, 1H), 3.80 (s, 3H), 3.44 - 3.37 (m, 2H), 3.25 (q, J = 6.5 Hz, 2H), 1.75 - 1.63
1
3
(
m, 1H), 1.62 - 1.50 (m, 3H), 1.38 - 1.33 (m, 2H). C NMR (100 MHz, DMSO) δ 174.47, 172.68,
1
65.83, 161.74, 159.37, 158.44, 157.27, 142.23, 132.75, 131.52, 128.02, 127.83, 125.84, 114.49,
+
5
5.26, 52.37, 52.21, 40.92, 31.92, 28.91, 22.73. HRMS calcd. for C H N O [M+H] 559.2035,
2
6
30
4
10
found 559.2017.
Synthesis of (9S,13S)-3,11,16-trioxo-1-phenyl-2-oxa-4,10,12,15-tetraazahexadecane-9,13,16-
tricarboxylic acid (L11). Compound 18 (40 mg, 0.06 mmol) was dissolved in CF COOH (6 mL)
3
and stirred at room temperature for 5 h. After the solvent was removed under vacuum, 18 mg of
L11 was obtained as a white solid after preparative HPLC purification and the yield is 60%. The
1
analysis HPLC retention time t = 11.0 min. H NMR (400 MHz, DMSO) δ 8.76 (t, J = 6.0 Hz,
R
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H), 7.39 - 7.28 (m, 5H), 7.23 (t, J = 5.6 Hz, 1H), 6.49 (d, J = 8.1 Hz, 1H), 6.33 (d, J = 8.1 Hz,
H), 5.00 (s, 2H), 4.26 (q, J = 6.6 Hz, 1H), 4.07 - 4.02 (m, 1H), 3.43 - 3.93 (m, 2H), 2.96 (q, J =
1
3
6.6 Hz, 2H), 1.66 - 1.61 (m, 1H), 1.54 - 1.49 (m,1H), 1.41 - 1.36 (m, 2H), 1.31 - 1.25 (m, 2H). C
1
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NMR (100 MHz, DMSO) δ 174.43, 172.65, 161.70, 158.32, 157.23, 156.10, 137.30, 128.38,
+
1
4
27.75, 65.13, 52.34, 52.18, 41.90, 31.83, 29.13, 22.46. HRMS calcd. for C H N O [M+H]
20 26 4 10
83.1722, found 483.1705.
Synthesis of tert-butyl 4-(2-(((benzyloxy)carbonyl)amino)ethoxy)benzoate. A mixture of tert-
butyl 4-hydroxybenzoate (2 g, 10.31 mmol) and K CO (2.14 g, 15.46 mmol) was stirred in DMF
2
3
(20 mL) at 60°C for 1 h. Benzyl (2-bromoethyl)carbamate (2.66 g, 10.31 mmol) was then added
and the reaction was stirred for 7 h at 60°C. After the solvent was evaporated under vacuum, 3.17g
product was obtained as a white solid after silica gel flash column chromatography (ethyl
1
acetate/petroleum ether, 0% to 20%, vol/vol) and the yield is 83%. H NMR (400 MHz, CDCl ) δ
3
7.93 (d, J = 8.8 Hz, 2H), 7.39 - 7.29 (m, 5H), 6.86 (d, J = 8.7 Hz, 2H), 5.11 (s, 2H), 4.08 (t, J =
13
5
1
.0 Hz, 2H), 3.62 (q, J = 5.3 Hz, 2H), 1.58 (s, 9H). C NMR (100 MHz, CDCl ) δ 165.61, 161.92,
3
56.52, 136.43, 131.55, 128.68, 128.33, 128.28, 125.05, 113.93, 80.75, 67.13, 67.05, 40.58, 28.36.
+
HRMS calcd. for C H NO [M+Na] 394.1625, found 394.1609.
21 25
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Synthesis of 4-(2-(((benzyloxy)carbonyl)amino)ethoxy)benzoic acid. Tert-butyl 4-(2-
((benzyloxy)carbonyl)amino)ethoxy)benzoate (2 g, 5.39 mmol) was dissolved in CF COOH (15
(
3
mL) and stirred for 5 h at room temperature. After the solvent was removed under vacuum, 1.44 g
1
product was obtained as a white solid without further purification. The yield is 85%. H NMR (400
MHz, DMSO) δ 12.60 (s, 1H), 7.88 (d, J = 8.6 Hz, 2H), 7.53 (t, J = 5.4 Hz, 1H), 7.39 - 7.29 (m,
1
3
5
H), 7.01 (d, J = 8.7 Hz, 2H), 5.03 (s, 2H), 4.07 (t, J = 5.5 Hz, 2H), 3.40 (q, J = 5.4 Hz, 2H). C
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NMR (100 MHz, DMSO) δ 167.01, 162.04, 156.32, 137.13, 131.38, 128.38, 127.79, 123.10,
+
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14.31, 66.62, 65.38, 39.83. HRMS calcd. for C H NO [M+H] 316.1179, found 316.1108.
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Synthesis of 2,5-dioxopyrrolidin-1-yl 4-(2-(((benzyloxy)carbonyl)amino)ethoxy)benzoate. A
mixture of 4-(2-(((benzyloxy)carbonyl)amino)ethoxy)benzoic acid (1 g, 3.17 mmol), 1-
hydroxypyrrolidine-2,5-dione (475 mg, 4.13 mmol) and EDCI (908 mg, 4.76 mmol) was stirred
in DMF (20 mL) for 20 h at room temperature. After the solvent was evaporated under vacuum,
0
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0
9
20 mg of product was obtained as a white solid after silica gel flash column chromatography
1
(
ethyl acetate/petroleum ether, 0% to 50%, vol/vol) and the yield is 71%. H NMR (400 MHz,
CDCl ) δ 8.06 (d, J = 8.8 Hz, 2H), 7.37 - 7.30 (m, 5H), 6.93 (d, J = 8.7 Hz, 2H), 5.28 (s, 1H), 5.11
3
1
3
(s, 2H), 4.11 (t, J = 4.9 Hz, 2H), 3.63 (q, J = 5.2 Hz, 2H), 2.87 (s, 4H). C NMR (100 MHz,
CDCl ) δ 169.58, 163.87, 161.49, 156.50, 136.38, 133.03, 128.69, 128.37, 128.29, 117.64, 114.71,
3
+
6
7.38, 67.08, 40.45, 25.77. HRMS calcd. for C H N O [M+Na] 435.1163, found 435.1155.
2
1
20
2
7
Synthesis of (4S,8S)-tri-tert-butyl 14-(4-(2-(((benzyloxy)carbonyl)-amino)ethoxy)phenyl)-
1
,6,14-trioxo-2,5,7,13-tetraazatetradecane-1,4,8-tricarboxylate (20). A mixture of compound
,5-dioxopyrrolidin-1-yl 4-(2-(((benzyloxy)carbonyl)amino)ethoxy)benzoate (1.20 g, 2.91 mmol)，
2
Et N (784 mg, 7.75 mmol) and compound 18 (1 g, 1.94 mmol) was stirred in DMF (20 mL) for 12
3
h at room temperature. After the solvent was evaporated under vacuum, 945 mg of product 20 was
obtained as
a
colorless oil after silica gel flash column chromatography
1
(methanol/dichloromethane, 0% to 6%, vol/vol) and the yield is 60%. H NMR (400 MHz, CDCl )
3
δ 7.93 (s, 1H), 7.77 (d, J = 8.7 Hz, 2H), 7.40 - 7.29 (m, 5H), 6.87 (d, J = 8.5 Hz, 2H), 6.65 (s, 1H),
5
.76 (s, 1H), 5.66 (s, 1H), 5.33 (s, 1H), 5.11 (s, 2H), 4.43 - 4.37 (m, 1H), 4.33 - 4.28 (m, 1H), 4.07
(t, J = 4.9 Hz, 2H), 3.71 - 3.55 (m, 4H), 3.45 - 3.41 (m, 2H), 3.16 - 3.10 (m, 1H), 1.87 - 1.70 (m,
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H), 1.68 - 1.58 (m, 4H), 1.51 (s, 9H), 1.43 (s, 9H), 1.42 (s, 9H). C NMR (100 MHz, CDCl ) δ
3
1
72.38, 169.97, 167.42, 161.14, 159.19, 158.21, 157.62, 156.56, 136.48, 129.09, 128.68, 128.33,
128.29, 127.52, 114.21, 84.60, 83.35, 82.02, 67.11, 67.03, 53.69, 53.45, 43.31, 40.59, 32.67, 29.82,
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8.94, 28.14, 27.90, 27.84, 22.62. HRMS calcd. for C H N O [M+H] 814.4233, found
4
1
59
5
12
14.4254.
Synthesis of (4S,8S)-14-(4-(2-(((benzyloxy)carbonyl)amino)eth-oxy)phenyl)-1,6,14-trioxo-
,5,7,13-tetraazatetradecane-1,4,8-tricarboxylic acid (L12). Compound 20 (40 mg, 0.05 mmol)
2
was dissolved in CF COOH (6 mL) and stirred at room temperature for 5 h. After the solvent was
3
evaporated under vacuum, 19 mg of L12 was obtained as white solid after preparative HPLC
1
purification and the yield is 60%. The analysis HPLC retention time t = 10.4 min. H NMR
R
(
400 MHz, DMSO) δ 8.74 (t, J = 5.8 Hz, 1H), 8.29 (t, J = 5.5 Hz, 1H), 7.79 (d, J = 8.7 Hz, 2H),
.49 (t, J = 5.5 Hz, 1H), 7.38 - 7.29 (m, 5H), 6.97 (d, J = 8.7 Hz, 2H), 6.51 (d, J = 8.0 Hz, 1H),
6.35 (d, J = 8.1 Hz, 1H), 5.03 (s, 2H), 4.26 (q, J = 6.6 Hz, 1H), 4.09 - 4.04 (m, 3H), 3.43 - 3.37
7
(
m, 4H), 3.21 (d, J = 6.1 Hz, 2H), 1.72 - 1.62 (m, 1H), 1.60 - 1.43 (m, 3H), 1.35 - 1.25 (m, 2H).
13C NMR (100 MHz, DMSO) δ 174.48, 172.68, 165.58, 161.77, 160.57, 158.70, 157.27, 156.33,
1
2
37.14, 128.99, 128.40, 127.83, 127.80, 127.05, 113.95, 66.51, 65.36, 52.37, 52.22, 40.90, 31.90,
+
8.97, 22.73. HRMS calcd. for C H N O [M+H] 646.2355, found 646.2338.
4
1
59
5
12
Synthesis of tert-butyl 4-(2-(benzyloxy)-2-oxoethoxy)benzoate. A mixture of tert-butyl 4-
hydroxybenzoate (2 g, 10.31 mmol) and K CO (2.14 g, 15.46 mmol) was stirred in DMF (20 mL)
2
3
at 60°C for 1 h. Benzyl 2-bromoacetate (2.36 g, 10.31 mmol) was then added and the reaction was
stirred for 7 h at 60°C. After the solvent was evaporated under vacuum, 3.00 g product was
obtained as a white solid after silica gel flash column chromatography (ethyl acetate/petroleum
1
ether, 0% to 20%, vol/vol). H NMR (400 MHz, CDCl ) δ 7.93 (d, J = 9.0 Hz, 2H), 7.38 - 7.29 (m,
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5
H), 6.88 (d, J = 9.0 Hz, 2H), 5.23 (s, 2H), 4.70 (s, 2H), 1.58 (s, 9H). C NMR (100 MHz, CDCl )
3
δ 168.36, 165.45, 161.08, 135.12, 131.56, 128.78, 128.75, 128.63, 125.71, 114.14, 80.82, 67.28,
+
65.30, 28.35. HRMS calcd. for C H O [M+H-56] 287.0914, found 287.0910.
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Synthesis of 4-(2-(benzyloxy)-2-oxoethoxy)benzoic acid.
Tert-butyl 4-(2-(benzyloxy)-2-
oxoethoxy)benzoate (2 g, 5.85 mmol) was dissolved in CF COOH (15 mL) and stirred at room
3
temperature for 5 h. After the solvent was evaporated under vacuum, 1.46 g product was obtained
as a white solid and used for next step without further purification. The yield is 87%.
Synthesis of 2,5-dioxopyrrolidin-1-yl 4-(2-(benzyloxy)-2-oxoethoxy) benzoate. 4-(2-
(
benzyloxy)-2-oxoethoxy)benzoic acid (1 g, 3.50 mmol), 1-hydroxypyrrolidine-2,5-dione (804 mg,
6.99 mmol) and EDCI (2.01 g, 10.49 mmol) were stirred in DMF (20 mL) for 20 h at room
temperature. After the solvent was evaporated under vacuum, 0.83 g product was obtained as a
white solid after silica gel flash chromatography (ethyl acetate/petroleum ether, 0% to 50%, vol/vol)
1
and the yield is 62%. H NMR (400 MHz, CDCl ) δ 8.07 (d, J = 8.9 Hz, 2H), 7.47 - 7.34 (m, 5H),
3
13
6
1
6
.95 (d, J = 8.9 Hz, 2H), 5.24 (s, 2H), 4.74 (s, 2H), 2.89 (s, 4H). C NMR (100 MHz, CDCl ) δ
3
69.49, 167.91, 162.99, 161.41, 134.98, 132.04, 128.87, 128.83, 128.71, 118.40, 114.93, 67.47,
+
5.32, 25.80. HRMS calcd. for C H NO [M+Na] 406.0897, found 406.0893.
20 17
7
Synthesis of (4S,8S)-14-(4-(2-(benzyloxy)-2-oxoethoxy)phenyl)-1,6,14-trioxo-2,5,7,13-
tetraazatetradecane-1,4,8-tricarboxylic acid (L13). A mixture of compound 2,5-
dioxopyrrolidin-1-yl 4-(2-(benzyloxy)-2-oxoethoxy)benzoate (57 mg, 0.15 mmol)，Et N (98
3
mg, 0.97 mmol) and compound 18 (50 mg, 0.10 mmol) was stirred in DMF (10 mL) for 12 h at
room temperature. After the solvent was evaporated under vacuum, CF COOH (6 mL) was
3
added and stirred at room temperature for 5 h. The solvent was removed under vacuum. 26 mg of
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L13 was obtained as a white solid after preparative HPLC purification. The yield is 43%. The
1
analysis HPLC retention time t = 11.2 min. H NMR (400 MHz, DMSO) δ 8.67 (d, J = 5.2 Hz,
R
1H), 8.32 (t, J = 5.6 Hz, 1H), 7.79 (d, J = 8.8 Hz, 2H), 7.41 - 7.30 (m, 5H), 6.98 (d, J = 8.8 Hz,
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
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5
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5
5
5
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8
9
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8
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H), 6.53 (d, J = 8.0 Hz, 1H), 6.39 (d, J = 7.9 Hz, 1H), 5.20 (s, 2H), 4.93 (s, 2H), 4.25 (q, J = 6.5
Hz, 1H), 4.06 (q, J = 7.8 Hz, 1H), 3.42 - 3.39 (m, 2H), 3.25 - 3.18 (m, 2H), 1.72 - 1.62 (m,
1
3
1
1
1
H), 1.59 - 1.45 (m, 3H), 1.36 - 1.31 (m, 2H). C NMR (100 MHz, DMSO) δ 174.48, 172.70,
69.97, 168.50, 165.49, 161.91, 159.68, 157.26, 135.65, 128.92, 128.48, 128.23, 128.08, 127.68,
14.06, 66.12, 64.66, 52.38, 52.29, 40.87, 31.86, 28.94, 22.73. HRMS calcd. for
+
C H N O [M+H] 617.2089, found 617.2074.
28 32
4
12
Synthesis of (S)-6-amino-2-(3-((S)-1-carboxy-2-(carboxyformamido)ethyl)ureido)hexanoic
acid. A mixture of compound 18 (80 mg, 0.16 mmol) was stirred in CF COOH (6 mL) at room
3
temperature for 5 h, the solvent was evaporated in vacuum to get crude product 43 mg as a colorless
thick oil, and used for next step without purification. The yield is 80%.
Synthesis of (4S,8S)-14-((3',6'-dihydroxy-3-oxo-3H-spiro[isob-enzofuran-1,9'-xanthen]-5-
yl)amino)-1,6-dioxo-14-thioxo-2,5,7,13-tetraazatetradecane-1,4,8-tricarboxylic acid (L14). A
mixture of compound fluorescein isothiocyanate isomer 1 (22 mg, 0.06 mmol)，Et N (29 mg,
3
0
.29 mmol) and compound 20 (20 mg, 0.06 mmol) was stirred in DMSO (1.5 mL) for 12 h at room
temperature in dark. Then water (1.5 mL) was added and the mixture was purified with preparative
HPLC to obtain L14 (23 mg, yield 54%) as a solid. The analysis HPLC retention time t = 10.8
R
1
min. H NMR (400 MHz, DMSO) δ 10.14 (s, 2H), 9.91 (s, 1H), 8.75 (s, 1H), 8.24 (s, 1H), 8.12 (s,
1
H), 7.74 (d, J = 8.6 Hz, 1H), 7.16 (d, J = 8.3 Hz, 1H), 6.67 (d, J = 2.1 Hz, 2H), 6.62 - 6.56 (m,
6H), 6.35 (d, J = 8.1 Hz, 1H), 4.31 - 4.25 (m, 1H), 4.12 - 4.03 (m, 1H), 3.25 - 3.21 (m, 4H), 1.73 -
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