A prodrug design for improved oral absorption and reduced hepatic interaction

Article abstract of DOI:10.1016/j.bmc.2017.08.027

A series of estradiol-17-β esters of N-(p-sulfomylbenzamide)-amino acids were prepared and evaluated for systemic and hepatic estrogenic activity after oral administration in ovariectomized rats. The alkyl substitution at nitrogen of amino acids such as proline or N-methyl-alanine produced compounds that exhibit potent oral activity. The proline analog (EC508) was further evaluated along with 17β-estradiol (E2) and ethinyl-estradiol (EE) and compared their effects on the uterus, angiotensin and HDL-cholesterol after oral administration to ovariectomized female rats. Orally administered EC508 produced systemic estrogenic activity 10 times greater than EE and a 100 times higher activity than E2 with no influence on levels of angiotensin and HDL-cholesterol, whereas EE and E2 reduced the HDL-cholesterol and increased the angiotensine plasma levels. EC508 might offer significant advantages in indications like fertility control and HRT based on its high oral bioavailability and lack of hepatic estrogenicity.

Full text of DOI:10.1016/j.bmc.2017.08.027

Accepted Manuscript
A prodrug design for improved oral absorption and reduced hepatic interaction
Gulzar Ahmed, Walter Elger, Frederick Meece, Hareesh B. Nair, Birgitt
Schneider, Ralf Wyrwa, Klaus Nickisch
PII:
S0968-0896(17)30977-X
http://dx.doi.org/10.1016/j.bmc.2017.08.027
BMC 13930
DOI:
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
10 May 2017
9 August 2017
15 August 2017
Please cite this article as: Ahmed, G., Elger, W., Meece, F., Nair, H.B., Schneider, B., Wyrwa, R., Nickisch, K., A
prodrug design for improved oral absorption and reduced hepatic interaction, Bioorganic & Medicinal Chemistry
(2017), doi: http://dx.doi.org/10.1016/j.bmc.2017.08.027
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A prodrug design for improved oral
absorption and reduced hepatic interaction
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Gulzar Ahmed, Walter Elger, Frederick Meece, Hareesh B Nair, Birgitt Schneider, Ralf Wyrwa,
Klaus Nickisch
SO2NH2
O
R3
N
Prodrug
Drug
O
OH
CAII binding
group
R1R2O
Active compound
Active compound
Amino acid
linker
Ester
hydrolysis

Bioorganic & Medicinal Chemistry
journal homepage: www.els evier.com
A prodrug design for improved oral absorption and reduced hepatic interaction
Gulzar Ahmed*, Walter Elger, Frederick Meece, Hareesh B. Nair, Birgitt Schneider, Ralf Wyrwa, Klaus
Nickisch*
Evestra, Inc., 6410 Tri County Parkway, San Antonio, Texas 78154, USA
ARTICLE INFO
ABSTRACT
Article history:
Received
A series of estradiol-17-β esters of N-(p-sulfomylbenzamide)-amino acids were prepared and
evaluated for systemic and hepatic estrogenic activity after oral administration in ovariectomized
rats. The alkyl substitution at nitrogen of amino acids such as proline or N-methyl-alanine
produced compounds that exhibit potent oral activity. The proline analog (EC508) was further
evaluated along with 17β-estradiol (E2) and ethinyl-estradiol (EE) and compared their effects on
the uterus, angiotensin and HDL-cholesterol after oral administration to ovariectomized female
rats. Orally administered EC508 produced systemic estrogenic activity 10 times greater than EE
and a 100 times higher activity than E2 with no influence on levels of angiotensin and HDL-
cholesterol, whereas EE and E2 reduced the HDL-cholesterol and increased the angiotensine
plasma levels. EC 508 might offer significant advantages in indications like fertility control and
HRT based on its high oral bioavailability and lack of hepatic estrogenicity.
Received in revised form
Accepted
Available online
Keywords:
Estradiol
Testosterone
Prodrug
Hepatic estrogenicity
Estradiol sulfonamide ester
2009 Elsevier Ltd. All rights reserved.
1. Introduction
O
O
OH
O
O
O
N
There are drugs on the market with sub-optimal
bioavailability or adverse effect, and there are many more
compounds in clinical development that fail due to hepatic
toxicity or low bioavailability1a. The pharmaceutical industry
generally focuses on improving the characteristic of these
molecules through chemical manipulation to convert them into
compounds with desirable pharmaceutical properties. Some of
these drugs or clinical compounds can be rescued by converting
them into covalent derivatives (prodrugs) with little or no
biological activity resulting in improved physicochemical
properties. Upon administration, these prodrug compounds can
undergo in vivo biochemical transformation to release the active
drug before reaching their therapeutic target site1. All the small
molecule drugs approved during 2000 to 2008 consisted of
approximately 20% of prodrugs while prodrugs accounted for
33% of all drugs approved in 2010 alone2b.
O
N
N
H
N
N
N
COOH
O
O
HN
N
Enalapril
Olmesartan medoxomil
O
O
O
O
O
O
H
O
H2N
N
N
O
O
N
H
Oseltamivir
S
O
NH2
Pivampicillin
Figure 1. Examples of prodrug esters, and arrows mark the
side where the ester function is hydrolyzed.
Acid linker
OH
Carbonic anhydrase
binding group
Hydrolysis by
esterases
SO2NH2
Prodrug concepts are utilized more and more in drug
development in order to attain desired pharmaceutical outcomes
such as improved permeability, absorption and distribution.
Prodrugs can also alter solubility, metabolism, toxicity or
elimination. Prodrugs can be produced by modifying diverse
functional groups such as carboxylic acids, amines, sulfides and
alcohols. The esters are among the common functional groups
utilized in prodrug design (figure 1). These ester prodrugs usually
pass through the gastrointestinal (GI) track unaffected. Once
absorbed into the blood, the drugs can be released by the action
of esterases2.
O
R3
N
O
R1 R2
H2NO2
S
O
O
E2MATE
Carbonic anhydrase
binding group
Hydrolysis by
steroid sulfatase
HO
Figure 2. A sulfamate prodrug, E2MATE and a new
sulfonamide-ester prodrug.
Regardless of many efforts, it can be stated that a general
applicable prodrug concept which could be applied broadly in
different therapeutic classes is still missing. In our search for a
broadly applicable concept, we analyzed earlier work by Elger et.
———
* Corresponding authors. e-mail: gahmed@evestra.com; e-mail: knicknisch@evestra.com

al. utilizing the sulfamate moiety3. The sulfamate prodrug of
estradiol (E2MATE) exhibits high oral exposures which can be
rationalized by their reversible binding to carbonic anhydrases
(CA) located in high concentrations inside erythrocytes. The
reversible binding to CA is advantageous because once the
prodrug absorbs through the intestine and enters the portal vein,
these molecules bind specifically to CAII inside the erythrocytes
before entering the liver. This provides a shielding effect for
these prodrug molecules to avoid the interaction with the hepatic
enzymes or first pass effect4. A prodrug, E2MATE (also known
as J995 or ZK190628) was developed utilizing this concept and
was successful in preclinical studies (figure 2). However, it failed
in the human trials due to an adverse side effect resulting from
strong inhibition of human sulfatase5, which was also required
for the hydrolysis of the prodrug. It was therefore hypothesized
that separating the CAII binding function from the parent
molecule (modified as an ester) that could be cleaved in vivo to
release the parent molecule might be a useful approach (figure 2
& 3)6.
Plasma
I
n
t
e
s
t
i
n
e
Portal vein
Erythrocyte
L
i
v
e
r
Erythrocyte
Esterases
PD
PD
D
PD
PD
CA II
CA II
Hydrolysis by
esterases
OH
SO2NH2
O
R3
N
O
R1 R2
O
HO
Estradiol (Drug)
Prodrug
HO
Figure 3: Schematic for sulfonamide prodrug bypassing hepatic
interaction inside erythrocyte (PD= prodrug, D= drug).
2. Chemistry
Synthesis of our target molecules were carried out as detailed
in Scheme 1. The appropriate N-protected amino acids 3a-p were
esterified via dicyclopropylcarbodiimide (DIC) or N-(3-
Herein, we are reporting a prodrug concept by linking an
active drug estradiol (E2)7,8 with sulfonyl-amino acids to improve
bioavailability and bypass liver metabolism (figure 2).
dimethylaminopropyl)-N’-ethylcarbodiimide
hydrochloride
(EDCI) coupling with 3-tert-butyldimethylsilyl-estradiol 2 to
afford esters 4a-p. Subsequent deprotection of amines under
O
Z
OH
O
N
O
Z
R2
a
b
R1
N
HO
R2
R1
4
3
2
TBSO
TBSO
SO2NH2
O
O
O
CO2H
O
O
NH
R2
N
R2
c
R1
+
R1
SO2NH2
5
6
TBSO
TBSO
O
SO2NH2
O
O
N
d
R2
R1
7
HO
O
O
O
COCl
N
SO2NH2
1) DIEA, THF
2) NH4OH
R2
R1
5 +
SO2NH2
6
TBSO
Scheme 1. Method A (Z=Cbz) reagents and conditions: (a) DIC, DMAP, DCM, rt; (b) 10% Pd/C, 30 psi H2, ethyl acetate;
(c) DIC, DMAP, DCM, rt; (d) p-TsOH, DCM, acetone, MeOH, H2O, rt or TBAF, THF, rt; or Method B (Z=BOC): (a) EDCI,
DMAP, DCM, rt; (b) TFA, DCM.

usual palladium catalyzed hydrogenation for benzyloxycarbonyl
or triflouroacetic acid for tert-butoxycarbonyl groups rendered
amino-acid esters 5a-p. 4-Sulfamoylbenzoic acid was first treated
with DIC in DCM, followed by the amines 5 to afford the amides
6. The poor solubility of the compounds containing sulfamoyl
substituent in most organic solvents causes the coupling reaction
to be sluggish. The removal of the silyl group 6 under standard
TBAF/THF conditions worked, but we have observed cleaner
reaction profiles when the deprotection was carried out with p-
toluenesulfonic acid to afford phenols 7a-g (Table 1).
saponification is advantageous in order to avoid any unforeseen
clearance pathway of the prodrug.
Table 2: In vitro data for prodrug molecules
Compound
hCAII
inhibition
(IC50 nM)*
309
hPlasma/Esterase
(% saponified, 10
min)
-
Acetazol-
amide
7a
65
87
70
97
89
75
84
83
-
7b
7c
250
232
202
100
325
110
300
Target molecule with m-sulfamoyl group was synthesized by
treating amine 5 in THF with m-chlorosulphonylbenzoylchloride
followed by ammonium hydroxide, and subsequent silyl group
removal to afford the phenol 7h.
7d
7e
7f
7g
Table 1: Compound described in Scheme 1.
R3
SO2NH2
7h
O
* Standard deviation (SD) ranges 5 to 11
N
O
O
Ovariectomized female rats were treated orally with the test
compounds and their uterine weight increase was reported as
compared to 100% for vehicle control (Table 3). The increase in
the uterine weight compared to vehicle indicates the efficacy of
the test compound. The compound 7a (glycine as amino acid
linker) caused minimal increase in uterine weight while
compound 7b with alanine linker had a positive effect on the
treated animals. The estrogenic effect noticeably dropped to
baseline when gem-dimethyl-glycine was employed as linker
compound 7c. We had further explored the size of alpha
substitution on glycine compound 7d and 7e. It is evident that
isopropyl group compound 7d is well tolerated but the benzyl 7e
has negative influence. The increase of systemic oral estrogenic
activities were highest when N-substituted amino acids were
employed. Compounds 7f (N-methyl-alanine) and 7g (proline)
were the most active prodrug molecules in this series. Compound
7h with 3-sulfamate-benzamide showed no improvement in
estrogenic activity. The effect of the enantiomer of amino acids
was also explored. The S-proline derived compound 7g exhibited
excellent estrogenic activity, while the R-proline derived
diastereomer of the compound 7g showed no improvement over
the vehicle control (compound not shown). In our opinion, the
differences in the estrogenic activities of these compounds
depend on oral bio-availability, otherwise there is not much
difference in their hydrolysis activity by esterases or carbonic
anhydrase binding affinity.
R2
R1
HO
Compound
R1
R2
H
R3
H
H
H
H
H
Me
-
H
7a
7b
7c
Me
Me
H
Me
H
Isopropyl
Benzyl
Me
7d
7e
H
7f
H
R1-R3 = propyl
R1-R3 = propyl
H
7g
7h*
H
-
* 3-sulfamoylbenzamide
3. Results
Our hypothesis was that an ideal estrogenic prodrug molecule
must bind to carbonic anhydrase (CA)9 inside erythrocytes and
consequently avoid exposure to metabolic effect of liver. Human
carbonic anhydrase II (hCAII) binding affinity was evaluated for
compounds 7a-t. It was encouraging to observe hCAII inhibition
in moderate ranges (IC50 > 50 nM to < 500 nM) as shown in
Table 3: In vivo ovariectomized rat’s uterine weight (po)
Compound
% uterine wt % uterine wt
1.0 µg dose
Table 2 & 4. It was assumed that strong binding affinity (IC50
<
10 µg dose
50 nM) towards CAII of these compounds might cause poor
efficacy because of slow release of the prodrug into plasma and a
weak binding compound (IC50 > 500 nM) might not protect these
prodrugs properly inside the erythrocytes. All the tested
compounds showed hCAII inhibition in the moderate range as
desired with the exception of compounds 7o, 7p and 7s (these
compounds possessed no affinity towards CAII).
Vehicle
7a
100
-
100
152
7b
163*
107
105
95
271’’
97
7c
In order to generate the active drug from the prodrug once it is
released into the plasma, compounds 7a-g were subjected to in
vitro human plasma incubation assay (ELISA) to monitor the
release of drug estradiol by hydrolysis of prodrug ester molecules
in the presence of plasma esterases. It is evident from the data in
Table 2 that our synthesized compounds readily hydrolyze once
exposed to esterases and over 80% of the parent compounds on
average converted to estradiol in about 10 minutes which is
primarily responsible for the efficacy. The high rate of
7d
267
7e
117
7f
145
244
-
289
7g
340
7h
108
* 3 µg dose; ‘’ 30 µg dose.

The activation of estrogen receptor by an agonist is
responsible for uterine weight increases6. Whether the prodrug or
estrogen (hydrolyzed parent drug) was causing this efficacy,
estrogen receptor (ER) agonist affinity was determined for
prodrug 7g and estradiol (E2). The compound 7g showed EC50
value equal to 432 nM for ER while estradiol had a value of 2.3
nM (determined by Life Technologies' SelectScreen® Profiling
Service). It is evident that the prodrug has poor affinity for ER,
so the in vivo activity is due to the hydrolyzed parent drug (E2).
Table 4: In vivo ovariectomized rat’s uterine weight (po) increase
of proline-linked prodrugs
R4
O
N
O
HO
Compound R4
% uterine
wt 10 µg
dose
hCAII
inhibition
(IC50 nM)
198
The effects of sulfonamide bearing moieties have been further
explored with fixing the linker as proline and the results are
summarized in table 4. The N-arylsulfonamide 7i resulted in no
positive effect on uterine growth when administered at 10 mg
dose, while N-benzylsulfonamide 7j exhibited an increase of
272% uterine growth. 4-Sulfomoylphenyl-acetamide 7k, the furyl
7l and 7m, the pyridyl 7n and 7o resulted in good to excellent
uterine weight increases. The sulfonamide 7p showed good
estrogenic effect without having any hCAII binding affinity,
which might not reduce hepatic toxicity. The methyl-7q (7g-
methyl) retained all the activity compared with 7g. The biaryl
analogs 7s, 7r and 7t resulted in a wide range of activity with the
para-para 7r as most active. This exercise shows, a wide variety
of sulfonamide bearing moieties can be employed without losing
efficacy which can be beneficial when working with other drug
targets.
92
7i
*
SO2NH2
SO2NH2
*
272
267
129
500
7j
O
7k
*
SO2NH2
O
318
313
250
7
7l
O
SO2NH2
*
O
N
7m
O
SO2NH2
SO2NH2
SO2NH2
*
O
259
268
250
7n
*
In order to show the general scope of this strategy, the
compound 7g also known as EC508 was chosen for further
evaluation. A side by side study of 7g with estradiol (E2) and
ethinyl-estradiol (EE) (Figure 4) in ovariectomized rats has
demonstrated that at 10 µg doses 7g caused the uterine weight to
more than double, whereas under E2 treatment no effect was seen
at all and only a small weight increase could be observed at this
dose level for EE. These data suggest that EC 508 might be
around 100 times more potent than E2 and around 10 times more
potent than EE after oral treatment. The plasma of these rats was
also analyzed for HDL-cholesterol levels (Figure 5) and
angiotensin concentration (Figure 6) at the end of the studies.
Compound 7g did not impact in anyway at all doses on the level
of HDL studied while EE and E2 clearly reduced the level of
HDL indicating hepatic interaction with EE and E2. Hepatic
functions were also monitored by analyzing the angiotensin
concentration. Again compound 7g exerted no influence on
angiotensin production while EE and E2 showed elevated
angiotensin.
O
N
5000
7o
*
268
258
10000
210
7p
7q
* SO2NH2
O
SO2NH2
*
*
216
10000
7s
O
SO2NH2
*
309
153
290
120
7r
7t
SO2NH2
O
*
O
SO2NH2
900
800
700
600
500
400
300
200
100
0
0
0.1
1
10
100
1000
µg/animal/d
EE
vehicle
E2
EC508
Figure 5: Effects on HDL-cholesterol levels in plasma of the
studied rats.
Figure 4: Comparison of estrogenic action of estradiol (E2),
ethinyl estradiol (EE) and EC508.

4. Conclusion
A series of amino acid-linked benzenesulfonamide estradiol
compounds was prepared. These prodrugs possess moderate
affinity for carbonic anhydrase II (hCAII) which allows them to
hide inside the erythrocytes and avoid interaction with the liver
enzymes. Once released from erythrocytes into the plasma, they
are readily hydrolyzed by esterases to produce estradiol.
Optimization based on the amino acid substitutions led to EC508,
an orally efficacious compound capable of producing excellent
estrogenic activity, while producing no effects on plasma HDL-
cholesterol and angiotensin levels. Side by side comparison
studies of estradiol, ethynyl-estradiol and EC508 have shown that
EC508 offered superior estrogen dependent uterine weight
growth and no impact on the liver function as measured by the
HDL- cholesterine and angiotensine levels.
The presented findings might have two distinct application. A
new estrogenic principle that combines high oral bioavailability
with reduced hepatic estrogenicity could set new safety standards
in hormonal contraception and hormone replacement therapy.
Figure 6: Angiotensin concentration in the treated rats.
Pharmacokinetic (PK) properties of compound 7g was
evaluated in rat. We were please to find that this compound
displayed excellent oral bioavailability with prolonged half-life
and low in vivo clearance in blood (Table 5). We analyzed the
plasma as well as blood and found 20 times more compound 7g
concentration in blood compared to plasma as was found for
J99510. It is consistent with the argument that this compound bind
to carbonic anhydrase inside the erythrocytes and avoid the
metabolic action of hepatocytes. It is noteworthy to mention that
the clearance of compound 7g is high in plasma due to the fact of
its saponification by the plasma esterases.
In addition the concept of combining an ester linker with an
aromatic sulfonamide moiety should be applicable to other drug
classes that have problems with low or varying oral
bioavailability and high hepatic toxicity or clearance. The
testosterone prodrugs (e.g. EC586) study will be published in
detail soon. The finding supports that the concept might be
broadly applicable, but for each class of compounds a structural
optimization has to be performed. The work is ongoing to apply
this concept to other steroidal and non-steroidal drugs, and other
drugs incorporating this prodrug concept will be disclosed in due
time.
Table 5: Pharmacokinetics of EC508 dosed at 1.0 mg/kg iv and
5.0 mg/gk po Male SD rat in comparison to E2
5. Experimental
5.1 Chemistry
Entry
Plasma
62.76
5.68
Blood
Cla (mL/min/kg)
Vdb (L/kg)
T1/2 (h)c
3.86
Nuclear magnetic resonance spectra were recorded on a
Bruker ARX (300 MHz) spectrometer as deuterochloroform
(CDCl3) solutions using tetramethylsilane (TMS) as an internal
standard (δ = 0) unless noted otherwise. ‘Flash column’
chromatography was performed on 32-64 µM silica gel obtained
from EM Science, Gibbstown, New Jersey. Thin-layer
chromatography (TLC) analyses were carried out on silica gel GF
(Analtech) glass plates (2.5 cm x 10 cm with 250 µM layer and
pre-scored). Most chemicals and solvents were analytical grade
and used without further purification. Commercial reagents were
purchased from Aldrich Chemical Company (Milwaukee, WI).
0.24
4.58
4.90
F (%)d
102.08
122.08
a In vivo clearance after iv dosing.
b Volume of distribution at steady state after iv dosing.
c Half-life after oral dosing.
d Bioavailability after oral dosing.
In order to test the applicability of the concept to other drug
classes, a small pilot study was performed producing some
prodrugs of testosterone following the same concept. One
compound, EC586 (figure 7) showed a significantly enhanced
serum exposure of testosterone after oral administration in the
male rats compared to testosterone-propionate (TP, a marketed
prodrug).
General Method B: Boc-glycine-OH 3a (2.0 g, 2 eq.) was
treated with 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide
(EDCI, 2.2 g, 2 eq.) in DCM (30 mL) for 1 hour at rt. Estradiol 2
(2.1 g, 1 eq.) and 4-dimethylaminopyridine (DMAP, 0.7 g, 1 eq)
were then added and the resulting mixture was stirred at rt for 20
h. The reaction was concentrated and the residue was purified by
silica gel chromatography using 60 – 100 % DCM in hexanes as
eluent to afford the ester product as white solid 4a (2.1 g, 68%
yield). The ester 4a (2.0 g) was treated with triflouroacetic acid
(TFA, 6 mL) in DCM (30 mL) at rt for 24 h. After the
completion of reaction, the reaction was diluted with toluene (30
mL) and triflouroacetic acid was removed under anhydrous
conditions to yield the amine compound 5a (2.1 g as TFA salt).
The compound 5a (1.0 g, 1 eq.) was then treated with p-
aminosulfamoyl-benzoic acid (0.54 g, 1.5 eq.) in the presence of
DIC (0.42 mL, 1.5 eq.), HOBt (0.41 g, 1.5 eq.), and Hunig’s base
(DIEA, 1.3 mL, 4 eq.) in DCM (20 mL) at rt for 72 h. The
reaction was concentrated and the residue was purified by silica
gel chromatography using 15% acetone in DCM as eluent to
O
O
O
N
O
O
N
SO2NH2
O
O
EC586
AUC0-3h = 330 ng/mL
dose = 3.0 mg/rat, n = 5 rats
Testosterone-propionate (TP)
AUC0-3h = 2.5 ng/mL
dose = 3.0 mg/rat, n = 5 rats
Figure 7: Examples of testosterone prodrugs with PK data.

afford the desired product 6a as white solid (0.4 g, 36 % yield).
The compound 6a (0.4 g, eq.) was reacted with
(s, 1H), 4.95 (s, 2H, NH2), 4.80 (m, 1H), 3.00 (s, 1H, CH3,
rotamer), 2.90 (s, 2H, CH3, rotamer), 0.85 (s, 2H, CH3, rotamer),
0.83 (s, 1H, CH3, rotamer). IR (cm-1): 3353, 3257, 2924, 2862,
1731, 1617. [α]D24 = -10o (c=0.5, 1,4-dioxane).
1
tetrabutylammonium fluoride trihydrate (TBAF, 0.2 g, 1 eq.) in
THF (20 mL) at rt for 60 min. The reaction was quenched with
aq. ammonium chloride, extracted with ethyl acetate (3 x 25 mL),
the combined organic was dried under sodium sulfate, filtered
and concentrated. This residue was purified by silica gel
chromatography using 5 % acetone in 1:1 hexanes:ethyl acetate
to afford (13S,17S)-3-hydroxy-13-methyl-7,8,9,11,12,13,14,15-
[(13S,17S)-3-hydroxy-13-methyl-6,7,8,9,11,12,14,15,16,17-
decahydrocyclopenta[a]phenanthren-17-yl]
sulfamoylbenzoyl)amino]propanoate was synthesized according
2-methyl-2-[(4-
1
to method A as white solid 7c (0.61 g): H NMR (δ, DMSO-d6
300 MHz): 8.98 (s, 1H), 8.78 (s, 1H), 7.97 (d, 2H, ArH, J= 8.4
Hz), 7.89 (d, 2H, ArH, J= 8.1 Hz), 7.48 (s, 2H, NH2), 7.02 (d,
1H, ArH, J= 8.7 Hz), 6.48 (dd, 1H, ArH, J= 2.4, 8.1 Hz), 6.41 (d,
1H, ArH, J= 2.1 Hz), 4.55 (t, 1H, J= 8.4 Hz), 1.47 (s, 6H, di-
CH3), 0.66 (s, 3H, CH3). IR (cm-1): 3504, 3355, 3168, 3067,
2922, 2867, 1727, 1648. [α]D24 = +48o (c=0.5, 1,4-dioxane).
,16,17-decahydro-6H-cyclopenta[a]phenan-thren-17-yl
2-(4-
sulfamoylbenzamido)acetate (0.15 g, 46% yield) as white solid
7a: 1H NMR (δ, DMSO-d6 300 MHz): 9.15 (t, 1H, -NH, J= 5.82
Hz), 8.01 (d, 2H, ArH, J= 8.56 Hz), 7.91 (d, 2H, ArH, J= 8.54
Hz), 7.50 (bs, 1H, ArOH), 7.02 (d, 1H, ArH, J= 8.46 Hz), 6.49
(dd, 1H, ArH, J= 2.46, 8.37 Hz), 6.42 (d, 1H, ArH, J= 2.42 Hz),
4.67 (t, 1H, 17-CH, J=7.08 Hz), 4.02 (d, 2H, J= 5.75 Hz), 0.73 (s,
[(13S,17S)-3-hydroxy-13-methyl-6,7,8,9,11,12,14,15,16,17-
decahydrocyclopenta[a]phenanthren-17-yl] (2S)-3-methyl-2-[(4-
sulfamoylbenzoyl)amino]butanoate was synthesized according to
method A as white solid 7d (1.90 g): 1H NMR (δ, 5 CDCl3 300
MHz): 7.97 (d, 2H, ArH, J= 8.7 Hz), 7.91 (d, 2H, ArH, J= 8.4
Hz), 7.13 (d, 1H, ArH, J= 8.1 Hz), 6.81 (d, 1H, J= 8.4 Hz), 6.63
(dd, 1H, ArH, J= 2.7, 8.4 Hz), 6.56 (d, 1H, ArH, J= 2.1 Hz), 5.01
(s, 2H, NH2), 4.79 (m, 3H), 1.04 (dd, 6H, J= 2.7, 6.9 Hz), 0.87 (s,
3H, CH3). IR (cm-1): 3338, 3252, 2967, 2924, 1722, 1710, 1657.
[α]D24 = +40o (c=0.6, 1,4-dioxane).
3H, CH3). IR (cm-1): 3369, 3278, 2933, 1736, 1656, 1529. [α]D
23
= +18o (c=0.5, 1,4-dioxane).
(2S)-((13S,17S)-3-hydroxy-13-methyl-7,8,9,11,12,13,14,15,-
16,17-decahydro-6H-cyclopenta[a]phenan-thren-17-yl)
2-(4-
sulfamoylbenzamido)propanoate (7b) was synthesized according
to method B starting with Boc-alanine 3b, as white solid (75
mg): 1H NMR (δ, 5:1 CDCl3:DMSO_d6 300 MHz): 8.54 (s, 1H,
ArOH), 8.18 (d, 1H, NH, J= 7.03 Hz), 7.99 (dd, 4H, ArH, J=
2.4, 8.88 Hz), 7.07 (d, 1H, ArH, J= 8.35 Hz), 6.97 (s, 2H, NH2),
6.62 (dd, 1H, ArH, J= 2.60, 8.39 Hz), 6.54 (d, 1H, ArH, J= 2.46
Hz), 4.77 (t, 1H, 17-CH, J=7.99 Hz), 4.71 (t, 1H, 17-CH, J=7.30
Hz), 1.54 (d, 3H, CH3, J= 7.29 Hz), 0.75 (s, 3H, CH3). IR (cm-1):
3496, 3381, 2930, 22911, 1743, 1706, 1650. [α]D23 = +34o (c=0.5,
1,4-dioxane).
[(13S,17S)-3-hydroxy-13-methyl-6,7,8,9,11,12,14,15,16,17-
decahydrocyclopenta[a]phenanthren-17-yl] (2S)-3-phenyl-2-[(4-
sulfamoylbenzoyl)amino]propanoate was synthesized according
1
to method A as white solid 7e (0.44 g): H NMR (δ, DMSO-d6
300 MHz): 9.04 (d, 1H, NH, J= 7.5 Hz), 9.00 (s, 1H, OH), 7.94
(d, 2H, ArH, J= 8.4 Hz), 7.89 (d, 2H, ArH, J= 8.1 Hz), 7.49 (s,
2H, NH2), 7.30 (m, 5H), 7.02 (d, 1H, ArH, J= 8.4 Hz), 6.49 (dd,
1H, ArH, J= 1.8, 8.1 Hz), 6.42 (d, 1H, ArH, J= 1.8 Hz), 4.67 (m,
2H), 0.68 (s, 3H, CH3). IR (cm-1): 3351, 2924, 2868, 1722, 1650.
[α]D24 = +42o (c=0.5, 1,4-dioxane).
General Method A: N-Cbz-methylalanine 3f (2.45 g, 2 eq.)
was treated with DIC (1.6 mL, 2 eq.) in DCM (33 mL) for 30
min under nitrogen at rt. TBS-Estradiol 2 (2.0 g, 1 eq.) and
DMAP (0.063 g, 0.1 eq.) were then added and the resulting white
mixture was stirred for 16 h rt. After filtration, the filtrate was
concentrated and the residue was purified by silica gel
chromatography using 5 – 40 % ethyl acetate in hexanes as eluent
to afford the white solid product 4f (2.85 g, 91% yield). The Cbz
group of ester 4f (2.85 g) was then removed using Pd/C (10% Pd,
0.51 g) and hydrogen (30 psi) on a Parr shaker in ethyl acetate
(35 mL) as solvent for 16 h. After filtration through celite and
concentration of the solvent, the white solid product 5f (2.2 g,
quantitative) was obtained. The amide formation of the amine 5
was carried out using p-sulfamoylbenzoic acid (2.22 g, 2.4 eq.)
and DIC (1.7 mL, 2.4 eq.) in DCM (75 mL) followed by the
addition of the amine 5f (2.2 g, 1 eq.) and DMAP (56 mg, 0.1
eq.) . The reaction was not soluble initially so ethyl acetate (35
mL) was added and the reaction mixture was stirred at rt for 3
days. The reaction was filtered and the filtrate was concentrated.
The residue was purified by silica gel chromatography using 10
% ethyl acetate in hexanes as eluent to afford the white solid
product 6f (1.90 g, 63% yield). The silyl ether 6f (1.90 g) was
then removed using p-toluenesulphonic acid (1.16 g, 2 eq.) in
DCM (14 mL), acetone (14 mL), methanol (0.4 mL) and water
(0.3 mL) at rt for 16 h. The reaction was quenched with aq.
sodium bicarbonate and extracted with ethyl acetate (2 x 60 mL).
The combined organic layer was dried with sodium sulfate,
filtered and concentrated. The residue was purified by silica gel
chromatography using 10 - 30 % acetone in DCM as eluent to
afford [(13S,17S)-3-hydroxy-13-methyl-6,7,8,9,11,12,14,15,16,-
17-decahydrocyclopenta[a]phenanthren-17-yl](2S)-2-[methyl-(4-
sulfamoylbenzoyl)amino]propanoate as white solid 7f (1.47 g,
94% yield): 1H NMR (δ, CDCl3 300 MHz): 7.96 (d, 2H, J= 8.1
Hz), 7.99 (d, 2H, J= 8.1 Hz), 7.13 (d, 1H, ArH, J= 8.1 Hz), 6.62
(dd, 1H, ArH, J= 2.7, 8.4 Hz), 6.54 (d, 1H, ArH, J= 2.7 Hz), 5.30
(2S)-((13S,17S)-3-hydroxy-13-methyl-7,8,9,11,12,13,14,15,-
16,17-decahydro-6H-cyclopenta[a]-phenanthren-17-yl) 1-(4-
sulfamoylbenzoyl)pyrrolidine-2-carboxylate was synthesized
1
according to method A as white solid 7g (0.51 g): H NMR (δ,
CDCl3 300 MHz): 7.96 (d, 2H, ArH, J= 7.25 Hz), 7.66 (d, 1.6H
rotamer, ArH, J= 7.28 Hz), 7.47 (d, 0.4H rotamer, ArH, J= 7.28
Hz), 7.40 (s, 1H, ArOH), 7.10 (d, 1H, ArH, J= 8.43 Hz), 6.64 (d,
1H, ArH, J= 8.31 Hz), 6.58 (s, 1H, ArH), 6.04 (bs, 2H, NH2),
4.81 (t, 1H, 17-CH), 4.67 (m, 1H, CH), 0.84 (s, 3H, CH3). IR
(cm-1): 3378, 3218, 2925, 1738, 1615, 1498. [α]D23 = -12o (c=0.5,
1,4-dioxane).
Method C: The steroid 5g (0.7 g) and diisopropylethylamine
o
(DIEA, 1 mL) in THF (20 mL) were chilled to -50 C, followed
by the addition of m-chlorosulfonyl-benzoyl chloride (0.9 mL).
o
The resulting yellow mixture was slowly warm to 0 C over 40
min., and 28% ammonium hydroxide (7 mL) was then added.
The resulting mixture was then warmed to rt over 30 min., the
reaction was diluted with aq. ammonium chloride, extracted with
ethyl acetate (3 x 25 mL), the combined organic was dried under
sodium sulfate, filtered and concentrated. This residue was
purified by silica gel chromatography using 15 % acetone in
dichloromethane to afford the white solid 6h (0.53 g, 67% yield).
The compound 6h (0.52 g,
1 eq.) was treated with
tetrabutylammonium fluoride trihydrate (TBAF, 0.32 g, 1.2 eq.)
in THF (30 mL) at rt for 60 min. The reaction was quenched with
aq. ammonium chloride, extracted with ethyl acetate (3 x 25 mL),
the combined organic was dried under sodium sulfate, filtered
and concentrated. This residue was purified by silica gel
chromatography using 5 % acetone in 1:1 hexanes:ethyl acetate

to (2S)-((13S,17S)-3-hydroxy-13-methyl-7,8,9,11,12,13,14,15,-
16,17-decahydro-6H-cyclopenta[a]-phenanthren-17-yl) 1-(3-
sulfamoyl-benzoyl)pyrrolidine-2-carboxylate (0.31 g, 73
female Wistar rats, 2 weeks after ovariectomy were used in the
experiments. The systemic estrogenicity was evaluated. Animal
were treated with the test substance from day 1-3, sacrificed on
day 4 of the study. The number of rats per group was 5. The
weight of the uterus was measured and reported as a percentage
of weight increase compared to control. Ethinyl estradiol and
estradiol were used as positive controls.
%
yield) as white solid 7h: ): 1H NMR (δ, CDCl3 300 MHz): 8.12
(s, 1H, ArH), 7.99 (d, 1H, ArH, J= 7.71 Hz), 7.76 (d, 1H, ArH,
J= 7.62 Hz), 7.57 (t, 1H, ArH, J= 7.7 Hz), 7.37 (s, 1H, ArOH),
7.11 (d, 1H, ArH, J= 8.64 Hz), 6.65 (d, 1H, ArH, J= 5.76 Hz),
6.58 (s, 1H, ArH), 5.85 (bs, 2H, NH2), 4.80 (t, 1H, 17-CH,
J=8.29 Hz), 4.66 (m, 1H), 0.84 (s, 3H, CH3). IR (cm-1): 3341,
3249, 2924, 2866, 1727, 1612, 1499. [α]D23 = -16o (c=0.5, 1,4-
dioxane).
Acknowledgments
Dr. Sabine Bischoff from the Institute of Animal Science and
Animal Welfare, Friedrich- Schiller-University Jena, were
helpful supporting the animal studies in the ethical committee
and providing the facilities for their performance, which is
gratefully acknowledged.
5.2. Biological Evaluation
In Vitro Studies
5.2.1 Carbonic anhydrase II (CAII) inhibition assay
References and notes
The catalytic activity of human CA II was monitored by the
hydrolysis of the non-physiological but commercially available
ester, 4-NPA (Sigma-Aldrich, St. Louis, MO).
1. (a) Waterbeemd, H.; Smith, D.A.; Beaumont, K.; Walker, D.K. J.
Med. Chem. 2001, 44, 1313-1333; (b) Stella, V.J.; Charman,
W.N.; Naringrekar, V.H. Drugs 1985, 29, 455-473; (c) Lin, C.;
Sunkara, G.; Cannon, J.B.; Ranade, V. Am. J. Ther. 2012, 19, 33-
43; (d) Huttunen, K.M.; Rautio, J. Curr. Top Med. Chem. 2011,
11, 2265-2287.
The assay solution (100 uL) containing 1 uM purified CA II in
50 mM, pH 7.5 MOPS, 33 mM Na2SO4, 1.0 mM EDTA buffer
was dispensed into flat-bottom 96-well plate. Each compound
plate was tested in triplicate. Compounds were allowed to
equilibrate with the enzyme for 15 min at room temperature. On
each plate, the CA II inhibitor and acetazolamide, was added in
duplicate (10 uM final concentration) as a positive control (100%
inhibition), and 100% DMSO was included as a negative control
(0% inhibition). The reaction was initiated by addition of 10 uL
of 5 mM 4-NPA solution in 10% DMSO, 90% assay buffer. The
absorbance of each well was measured at 348 nm every 12 to 15
2. (a) Rautio, J.; Kumpulainen, H.; Heimbach, R.; Oliyai, R.; Oh, D.;
Jarvinen, T.; Savolainen, J. Nat. Rev. Drug Disc. 2008, 7, 255-
270; (b) Huttunen, K.M.; Raunio, H.; Rautio, J. Pharmacol. Rev.
2011, 63, 750-771.
3. Elger, W.; Schwarz, S.; Hedden, A.; Reddersen, G.; Schneider, B.
J. Steroid Biochem. Molec. Biol. 1995, 55, 395-403.
4. (a)Elger, W.; Palme, H.J.; Schwarz, S. Exp. Opin. Invest. Drugs
1998, 7, 575-589; (b) Von Schoultz B.; Carlstroem K.; Eriksson
A.; Hendriksson P.; Pousette A.; Stege R. The Prostate 1989, 14,
389-395.
s
for
5
min with SpectraMax Plus 384 microplate
5. Colette, S.; Defrere, S.; Lousse, C.; Van Langendonckt, A.;
Gottland, J.P.; Loumaye, E.; Donnez, J. Hum. Reprod. 2011, 26,
1362-1370.
6. Elger, W.; Wyrwa, R.; Ahmed, G,; Meece, F.; Nair, H.B.;
Santhamma, B.; Kileen, Z.; Schneider, B.; Meister, R.; Schubert,
H.; Nickisch, K. J. Steroid Biochem. Mol. Biol. 2017, 165, 305-
311.
spectrophotometer, running SoftMax Pro software. Initial
substrate concentration was 450 uM 4-NPA and final volume of
111 uL per well and 18 uM final compound concentration11. Each
data points obtained is an average of triplicate per compound
tested.
5.2.2. Esterase activity assay
7. (a) Van Hylckama Vlieg A.; Middeldorp S. Journal of Thrombosis
and Hemostasis 2011, 9, 257-266; (b) Elger, W.; Palme, H.J.;
Schwarz, S. Exp. Opin. Invest. Drugs 1998, 7, 575-589; (c) Jouin,
P.; Busquet, J.; Magali, A.; Ghanem, P.; Pierre, A. Fran. Pat.
1999, 2774989; (d) Zhenzhong, Z.; Xiufang, S.; Chongwei, Z.;
Hongling, Z,; Yan, M.A. Can. Pat. 102079771 A 20110601; (e)
Goldzieher, J.W. Am. J. Gynec. 1990, 163, 318-322.
8. Stanczyk F.Z.; Archer D.F.; Bhavnani B.R. Contraception 2013,
87, 706-727.
The amount of estradiol (E2) released was measured by
ELISA kit (Calbiotech, Spring Valley, CA; cat#ES180S) based
on the principle of competitive binding between E2 in the test
specimen and E2-enzyme conjugate for a constant amount of
anti-estradiol conjugated polyclonal antibody. Anti-E2 coated
wells were incubated with E2 standards, controls, samples and
E2 enzyme conjugates. A fixed amount of HRP- labelled E2
competes with the endogenous E2 in the standard or samples. E2
peroxidase conjugate immunologically bound to the well
progressively decreases as the concentration of E2 in the
specimen increases and that will be measured spectrophoto-
metrically at 450nm. The mean absorbance value for each
specimen to determine the corresponding concentration of E2 in
pg/mL against the standard curve prepared from known estradiol
standard solutions provided12. Each compound was run in
duplicate and reported as a percent average.
9. Iyer, , R.; Barrese, A.A. 3rd; Parakh, S.; Parker, C.N.; Tripp, B.C. J
Biomol Screen 2006, 11, 782-91.
10. (a) Elger W.; Schwarz S.; Hedden A.; Reddersen G.; Schneider B.
Steroid Biochem. Molec. Biol. 1995, 55, No.3/4, pp. 395-403; (b)
Maschak, C.A.; Lobo, R.A.; Dozono-Takano, R..; Eggena, P.;
Nakamura, R.M.; Brenner, P.F.; Mishell, D.R. Am.J.Obstt.Gynec.
1982, 144, 511-518; (c)
11. Judd, H.L. Am. Obstet. Gynecol. 1987, 157, 1326-1331.
12. Elkik, F.; Gompel, A.; Mercier Bodard, C.; Kuttenn, F.; Guyenne,
P.N.; Carvol, P.; Mauvis-Jarvis, P. Am. J. Obstet. Gynecol. 1982,
143, 888-892.
5.2.3. In vivo uterine weight increase studies
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The rat investigations were done with some modifications of
the procedure described elsewhere10. Ovariectomized adult