This document summarizes the synthesis and evaluation of novel pH-sensitive phosphoramidate-based linkers and inhibitors for controlled drug release and treatment of prostate cancer. A library of tunable phosphoramidate linkers was synthesized and their drug release rates were monitored between pH 3.0-7.4. Additionally, a series of phosphoramidate inhibitors for prostate-specific membrane antigen (PSMA) were prepared using constrained 4-trans-hydroxyproline to enhance stability while maintaining potency. These inhibitors exhibited improved stability under harsh radiolabeling conditions without loss of inhibition potency, with some having IC50 values in the low nanomolar range. Preliminary small animal imaging studies showed tumor targeting

1. Ley 1
Second-generation of tunable pH-sensitive phosphoramidate-based
linkers for controlled release (co-first author, manuscript submitted)
A library of phosphoramidate-based cleavable linkers was synthesized via
late-stage diversification for efficiency and using cost-effective amines as
representatives of cytotoxic drugs or self-immolative spacers. Drug
release of each linker was monitored by 31
P NMR at pH 3.0 – 7.4 at 37 o
C.
These stability studies prove that our hydrophilic cleavable linker is
tunable by adjusting the distance between the neighboring carboxylic acid
and phosphorus core. This pH-sensitive scaffold can release amine-
containing drugs or self-immolative spacers at varying rates for controlled
release applications, such as antibody-drug conjugates (ADCs) and small-
molecule drug conjugates (SMDCs).
Figure 2. (A) Raw
stacked NMR data for
2a (1.11 ppm) at pH
5.5. Peak at 1.74 ppm
is hydrolytic product.
(B) Compiled and
fitted data for area of
2a (blue) normalized
to standard and its
hydrolytic product
(red).
Table 1. Half-life in hours (% RSD) at 37 o
C
pH 3.0 4.5 5.5 6.5 7.4
1a 0.20 (9.0) 2.59 (1.5) 17.6 (3.2) stablea
stablea
2a 0b
0.66 (7.0) 2.13 (1.1) 7.78 (1.6) 29.6 (5.2)
3a 17.7 (3.9) stablea
stablea
stablea
stablea
5a 36.8 (4.5) stablea
stablea
stablea
stablea
6a stablea
stablea
stablea
stablea
stablea
1b 0b
0b
0.23 (3.2) 0.73 (5.5%) 6.02 (1.4)
2b 0b
0b
0b
0.08 (9.7) 0.84 (6.6)
3b 0.23 (3.0) 2.24 (1.4) 17.1 (1.4) stablea
stablea
5b 0.51 (3.7) 9.24 (2.1) 27.0 (4.8) stablea
stablea
6b 0.34 (3.2) 5.25 (1.2) 25.1 (5.6) stablea
stablea
1c 0b
0.08 (5.8) 0.79 (2.4) 2.16 (1.6) 13.1 (2.9)
2c 0b
0b
0.08 (17.7) 0.31 (9.9) 2.33 (1.2)
3c 0.96 (2.7) 7.60 (1.8) 29.8 (6.9) stablea
stablea
4c 1.98 (0.9) 17.0 (1.7) 29.8 (6.6) stablea
stablea
5c 2.71 (0.8) 22.1 (2.8) stablea
stablea
stablea
6c 2.27 (1.1) 18.4 (3.9) stablea
stablea
stablea
1d 0b
0b
0.07 (16.5) 0.19 (6.4) 1.57 (2.5)
3d 0.13 (3.9) 0.98 (1.6) 6.64 (1.5) 18.6 (4.3) stablea
4d 0.24 (3.8) 2.09 (1.2) 10.3 (2.0) 30.4 (6.0) stablea
5d 0.31 (2.3) 2.73 (0.8) 16.6 (2.4) stablea
stablea
6d 0.34 (4.7) 2.80 (8.7) 12.6 (3.9) stablea
stablea
a
No detectable decomposition over 8 h; b
Complete decomposition in << 5 min
Scheme 1. (i) (PhO)2P(O)H, py, -5 o
C to rt; (ii) BnOH; (iii) BrCCl3 or CCl4, CH2Cl2, -15 o
C; (iv) Amine.HCl,
Et3N, cat. 4-DMAP for 1c-d and 2a-c; (v) H2, 10% Pd/C, KHCO3 (aq), 1,4-dioxanes
O
N
H
R2
HO
R3
n
O
N
H
R2
O
R3
n
P
H
OBn
O
N
P
O
N
H
OBn
O
R3
R2 OR1
nm
i, ii iii, iv v
1a-d
2a-c
3a-d
4c-d
5a-d
6a-d
N
P
O
N
H
OH
O
R3
R2 OR1
nm
1a-d: m=0 or 2; n=1; R1=H or Me; R2=CO2H; R3=H
2a-c: m=0 or 2; n=1; R1=H or Me; R2=CO2H; R3=Me
3a-d: m=0 or 2; n=2; R1=H or Me; R2=CO2H; R3=H
4c-d: m=2; n=3; R1=H or Me; R2=CO2H; R3=H
5a-d: m=0 or 2; n=1; R1=H or Me; R2=R3=H
6a-d: m=0 or 2; n=2; R1=H or Me; R2=R3=H
Figure 1. Series of tunable pH-sensitive
phosphoramidate-based linkers
0.0
0.2
0.4
0.6
0.8
1.0
0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0
Norm. Int. Area
Time (h)
Linker 2a
Hydrolytic Product
H
N
P
O
N
H
OH
O
HO2C O
7a
HO
P
O
N
H
OH
O
HO2C O
hydrolytic
product
A B
2a

2. Ley 2
Synthesis and evaluation of
constrained phosphoramidate
inhibitors of prostate-specific
membrane antigen
A series of phosphoramidate-based
inhibitors for prostate cancer were
prepared based on 4-trans-
hydroxyproline to minimize interaction
between the α-carboxylate of the P1
residue and the phosphorus center.
These scaffolds were designed to
withstand harsher conditions required
for installing 68
Ga and 177
Lu in
radiotheranostic applications and proved
to exhibit enhanced stability without loss
of potency.
Table 3. Inhibition potency of
phosphoramidate inhibitorsa
Entry IC50 (nM)
3 27 (3)
4 19 (1)
5 1.3 (0.08)
6 0.4 (0.05)
7a 60 (11)
7b 357 (29)
8a 79 (6)
8b 112 (8)
9a 2.6 (0.2)
9b 3.0 (0.3)
10a 1.3 (0.2)
10b 11 (0.8)
a
Standard deviation in parentheses
Table 2. Comparative stability of
representative phosphoramidates at pH 4.5
Entry Temp (o
C) t1/2 (min)
6 50 75
6 70 12
10a 50 105
10a 70 32
Figure 4. PET/CT image (20 min static scan)
of male nude mouse bearing a CWR22Rv1
tumor xenograft at 2 h post-injection of
[18
F]10a. Arrow indicates tumor placement.
CO2H
N
O
∗∗
P
O
OH
H
N CO2H
CO2H
H
N
∗∗
O
CO2H
n
O
O
N
H
F
9a: *2S, *4R, n=0
9b: *2R, *4S, n=0
10a: *2S, *4R, n=1
10b: *2R, *4S, n=1
CO2H
N
O
∗∗
P
O
OH
H
N CO2H
CO2H
H
N
∗∗
O
CO2H
O
N
H
7a: *2S, *4R, n=0
7b: *2R, *4S, n=0
8a: *2S, *4R, n=1
8b: *2R, *4S, n=1
P
O
OH
H
N CO2H
CO2H
O
CO2H
N
H
O
H
N
CO2H
N
H
O
H
n
3: n=0
4: n=1
P
O
OH
H
N CO2H
CO2H
O
CO2H
N
H
O
H
N
CO2H
N
H
O
n
5: n=0
6: n=1
O
F
n
H
P
O
OH
H
N CO2H
CO2H
O
1
CO2H
N
H
O
H2N
CO2H
P
O
OH
H
N CO2H
CO2H
O
2
H
N CO2H
O
H2N
CO2H
A
B
Figure 3. (A) Current phosphoramidate inhibitors of PSMA 1-6. (B)
Phosphoramidate inhibitors of PSMA with enhanced stability 7a-10b.
HN
∗∗
∗∗
CO2H
OH
HN
∗∗
∗∗
CO2Bn
OH
CO2Bn
N
O
∗∗H
N
∗∗
OH
CO2Bn
R
CO2Bn
N
O
∗∗
P
O
BnO
H
N
∗∗
O
CO2Bn
R
H
CO2Bn
N
O
∗∗
P
O
BnO
H
N
∗∗
O
CO2Bn
R
H
N CO2Bn
CO2Bn
a b c,d e f, R = Boc
g, R = Cbz
7a: *2S, *4R
7b: *2R, *4S
CO2Bn
N
O
∗∗
P
O
BnO
H2N
∗∗
O
CO2Bn
H
N CO2Bn
CO2Bn
h
n
O
OH
O
N
H
F
O
OH
N
H
Cbz
h
CO2Bn
N
O
∗∗
P
O
BnO
H
N CO2Bn
CO2Bn
H
N
∗∗
O
CO2Bn
O
N
H
Cbz
CO2Bn
N
O
∗∗
P
O
BnO
H
N CO2Bn
CO2Bn
H
N
∗∗
O
CO2Bn
n
O
O
N
H
F
i
i
8a: *2S, *4R
8b: *2R, *4S
9a: *2S, *4R, n=0
9b: *2R, *4S, n=0
10a: *2S, *4R, n=1
10b: *2R, *4S, n=1
21a: *2S, *4R, n=0
21b: *2R, *4S, n=0
22a: *2S, *4R, n=1
22b: *2R, *4S, n=1
11a: *2S, *4R
11b: *2R, *4S
12a: *2S, *4R
12b: *2R, *4S
13a: *2S, *4R, R=Boc
13b: *2R, *4S, R=Boc
14a: *2S, *4R, R=Cbz
14b: *2R, *4S, R=Cbz
15a: *2S, *4R, R=Boc
15b: *2R, *4S, R=Boc
16a: *2S, *4R, R=Cbz
16b: *2R, *4S, R=Cbz
17a: *2S, *4R, R=Boc
17b: *2R, *4S, R=Boc
18a: *2S, *4R, R=Cbz
18b: *2R, *4S, R=Cbz
19a: *2S, *4R
19b: *2R, *4S
20a: *2S, *4R
20b: *2R, *4S
Scheme 2. (a) BnOH, p-toluene-SO3H, Benzene, 125 o
C, 20 h reflux; (b) R-Glu-OBzl (R=Cbz or Boc), HBTU, Et3N,
DMF; (c) (PhO)2P(O)H, pyridine, -5 o
C to rt, 2 h; (d) BnOH, rt, 3 h; (e) H-Glu(OBzl)-OBzl HCl, CCl4, Et3N, CH3CN; (f)
30% TFA/CH2Cl2, rt, 1.5 h; (g) H2, 10% Pd/C, K2CO3, ddH2O, 1,4-dioxanes; (h) HBTU, Et3N, DMF; (i) H2, 10% Pd/C,
K2CO3, ddH2O, 1,4-dioxanes