MAKALAH KAPITA SELEKTA ORGANIK

SINTESIS PYRROLOPYRAZOL, EVALUASI DAN SKRINING

FARMAKOLOGIS SEBAGAI AGEN ANTIDEPRESAN

Samar Said Fatahala, Shahira Nofal, Eman Mahmoud dan Rania Helmy Abd El-hameed

OLEH:

Dita Putri Amalia 1803124172

Irda Putri Wahyuni 1803111587
Irfan Ari Kurniawan 1803112123
Vivi Meina 1803112177

DOSEN PENGAMPU :
Prof. Dr. Jasril, M.Si

PROGRAM STUDI S1 KIMIA

FAKULTAS MATEMATIKA DAN ILMU
PENGETAHUAN ALAM
UNIVERSITAS RIAU
PEKANBARU
2021
 KATA PENGANTAR

Puji syukur diucapkan kehadirat Allah SWT atas segala rahmatNya

sehingga makalah dengan judul “Sintesis Pyrrolopyrazol, Evaluasi dan
Skrining Farmakologis sebagai Agen Antidepresan” ini dapat selesai dengan
baik. Tidak lupa kami ucapkan terimaksih kepada pihak yang telah ikut
berkontribusi dalam pembuatan makalah ini.
Penulis sangat berharap semoga makalah ini dapat menambah
pengetahuan dan pengalaman bagi pembaca. Penulis merasa masih banyak
kekurangan dalam penyusunan makalah ini karena keterbatasan pengetahuan dan
pengalaman, untuk itu kami sangat mengharapkan kritik dan saran yang
membangun dari pembaca.

Pekanbaru, 21 September 2021

Penulis

i
 DAFTAR ISI

KATA PENGANTAR .................................................................................................................................... i

DAFTAR ISI.................................................................................................................................................. ii
DAFTAR GAMBAR ................................................................................................................................... iii
DAFTAR TABEL ........................................................................................................................................ iii
I. PENDAHULUAN ........................................................................................................................... 1
1.1 Latar Belakang ..................................................................................................................... 1
1.2 Tujuan Penulisan................................................................................................................ 2
1.3 Tinjauan Pustaka................................................................................................................ 2
1.3.1. Depresi .............................................................................................................................. 2
1.3.2. Antidepresi ...................................................................................................................... 2
1.3.3. Inhibitor monoamine oksidase (MAOIs) ............................................................... 3
1.3.4. Selective serotonin-reuptake inhibitor (SSRI) ..................................................... 3
1.3.5. Pyrrole dan Pyrrolopyrimidine.................................................................................. 3
II. TATA KERJA ...................................................................................................................................... 5
2.1 Alat ............................................................................................................................................. 5
2.2 Bahan ........................................................................................................................................ 5
2.3 Metodologi ............................................................................................................................. 5
2.3.1 Sintesis senyawa 2a-i ................................................................................................... 5
2.3.2 Prosedur Umum untuk Sintesis Senyawa 4a-i ..................................................... 5
2.3.3 Prosedur Umum untuk Sintesis Senyawa 5a-i ..................................................... 5
2.3.4 Uji Aktivitas Antidepresan ......................................................................................... 6
2.3.5 Uji Suspensi Ekor (Tail Suspension Test) ............................................................. 6
2.3.6 Analisis Serotonin pada Otak Tikus Menggunakan HPLC.............................. 6
2.3.7 Analisis Statistik ............................................................................................................ 7
III. HASIL DAN PEMBAHASAN ............................................................................................. 8
3.1 Hasil Kimia.............................................................................................................................. 8
a. Persiapan Pyrrolopyrazole .................................................................................................... 8
b. Mekanisme ................................................................................................................................... 9
3.2 Hasil Biologis .......................................................................................................................... 9
IV. KESIMPULAN......................................................................................................................... 14
DAFTAR PUSTAKA ..................................................................................................................15

ii
 DAFTAR GAMBAR
Gambar 1. Pengaruh Fluoxetine dan kemungkinan obat antidepresan pada
mobilitas tikus dalam uji suspensi ekor…………………...………10
Gambar 2. Pengaruh Fluoxetine dan kemungkinan obat antidepresan pada
tingkat 5HT di homogenat otak tikus………..……………,...……11
Gambar 3. Model TST ....................................................................................... 12

DAFTAR TABEL

Tabel 1. Uji coba aktivitas antidepresan senyawa dalam suspensi

ekor………………………………….……………………………………………………………10

iii
I. PENDAHULUAN
1.1 Latar Belakang
Depresi adalah penyebab utama kecacatan di seluruh dunia. Jumlah orang
yang mengalami depresi meningkat sekitar 18% antara tahun 2005 dan 2015, dan
depresi mempengaruhi 322 juta orang, atau sekitar 4% dari populasi dunia.
Depresi merupakan gangguan mood yang pada akhirnya dapat menyebabkan
defisiensi parah di berbagai area penting dari fungsi normal. Ini juga telah
dikaitkan dengan peningkatan angka kematian. Penyebab penyakit ini belum
sepenuhnya dapat dijelaskan sampai saat ini. Namun demikian, peristiwa
patofisiologis yang menyertai depresi dipelajari secara ekstensif. Belakangan ini
ditunjukkan bahwa meskipun 40 tahun penelitian ekstensif, hipotesis defisiensi
monoamina masih dapat menjelaskan beberapa aspek gejala depresi.
Neurotransmiter monoamina, norepinefrin (NE) dan serotonin (SER, 5-HT)
diidentifikasi sebagai target obat penting dari penyakit kejiwaan. Serotonin (SER,
5-HT) adalah salah satu monoamina yang terlibat dalam patofisiologi depresi.
Beberapa penelitian telah mengungkapkan bahwa penurunan tingkat otak 5-HT
terhubung dengan gejala perilaku negatif depresi. Oleh karena itu, agen
antidepresan yang berperan dalam meningkatkan kadar 5-HT menjadi perhatian
dalam pengobatan depresi. Antidepresan trisiklik (TCA) dan inhibitor monoamine
oksidase (MAOIs) juga dikembangkan sebagai agen antidepresan aktif meskipun
memiliki berbagai efek samping, mulut kering, penglihatan kabur, retensi urin,
hipotensi, dan insomnia. Baru-baru ini, Selective Serotonin Reuptake Inhibitors
(SSRIs) direkomendasikan sebagai antidepresan.
Selain itu, Antidepresan heterosiklik juga dianggap sebagai pengobatan
antidepresan utama. Senyawa heterosiklik telah dipelajari secara ekstensif baik
secara alami maupun sintetis selama beberapa tahun terakhir. Betacarbolines,
alkaloid alami di tanaman yang berbeda, ditemukan menjadi molekul yang
bekerja pada situs benzodiazepine dari reseptor kompleks. Di antara mereka,
alkaloid harmala, yang ditemukan di lianaBanisteriopsiscaapi, harmalineharmine
dan trahydroharmine, leburan pyrrole (indole) alkaloid, menunjukkan
benzodiazepine seperti efek agonis sistematis. Harmine dan harmaline, keduanya
menunjukkan MAOI selektif dan reversibel, sedangkan tetrahydroharmine

1
dianggap sebagai SSRI yang lemah.

1.2 Tujuan Penulisan

Penulisan makalah ini bertujuan untuk mempelajari dan memahami
penelitian yang dilakukan oleh Samar Said Fatahala, Shahira Nofal, Eman
Mahmoud dan Rania Helmy Abd El-hameed mengenai Sintesis Pyrrolopyrazol,
Evaluasi dan Skrining Farmakologis sebagai Agen Antidepresan.

1.3 Tinjauan Pustaka

1.3.1. Depresi
Depresi adalah terganggunya fungsi normal manusia yang berhubungan
dengan alam perasaan yang sedih dan gejala penyertanya, termasuk perubahan
pola tidur dan nafsu makan, psikomotor, konsentrasi, anhedonia, kelelahan, rasa
putus asa dan tidak berdaya serta bunuh diri (Sadock & Sadock, 2010). Menurut
WHO pada tahun 2017, gejala depresi dapat tandai dengan kesedihan, kehilangan
minat atau kesenangan, perasaan bersalah atau rendah diri, tidur terganggu atau
nafsu makan terganggu, perasaan lelah, dan menurunnya konsentrasi. Menurut
WHO, Depresi termasuk kontributor terbesar penyebab ketidakmampuan dan
penyebab utama bunuh diri hampir 800.000 per tahun. Jumlah orang yang hidup
dengan depresi di dunia sekitar 322 juta. Depresi lebih sering terjadi 2 kali lipat
pada wanita dibandingkan pria. Penyebab terjadinya perbedaan ini masih tidak
diketahui.

1.3.2. Antidepresi
Antidepresi adalah produk yang menguntungkan untuk industri farmasi
yang oleh karena itu mencurahkan banyak penelitian untuk pengembangan agen
baru. Obat-obatan baru ini seringkali mahal dan mewakili potensi yang signifikan
dan terus meningkat pada sumber daya layanan kesehatan kecuali jika dievaluasi
dengan benar. Karena obat baru telah dievaluasi dengan perbandingan dengan
standar TCA, hasil tinjauan ini juga memiliki implikasi untuk evaluasi peran obat
baru seperti SSRI (Donoghue dkk, 1996).

2
1.3.3. Inhibitor monoamine oksidase (MAOIs)
Inhibitor monoamine oksidase (MAOIs) adalah agen psikofarmakologi
modern pertama yang terbukti berkhasiat dalam memperbaiki gejala depresi
klinis, dan penemuan ini menyebabkan perubahan luar biasa dalam pengobatan
gangguan mood. Mirip dengan perawatan psikofarmakologi lainnya, penemuan
MAOI memiliki asal-usul yang kebetulan. Efek positif iproniazid pada suasana
hati pertama kali diamati pada pasien tuberkulosis dan sekitar waktu yang sama,
yang lain menemukan bahwa iproniazid menghambat MAO Selanjutnya, pasien
dengan depresi klinis melaporkan perbaikan suasana hati dengan iproniazid, yang
disebut 'energizer psikis' dan disetujui sebagai antidepresan. Penemuan klinis ini
mengarah pada pengembangan beberapa MAOI lain dan penelitian tentang "teori
depresi monoamina." Terlepas dari kemanjuran MAOI dalam mengobati depresi,
mereka tidak banyak digunakan karena perkembangan selanjutnya dari
pengobatan antidepresan lain yang relatif lebih baik ditoleransi dan kurang
dibatasi oleh interaksi potensial dengan diet dan obat lain (Suchting dkk, 2001).

1.3.4. Selective serotonin-reuptake inhibitor (SSRI)

Antidepresan inhibitor reuptake serotonin selektif (SSRI) telah
mendominasi pasar antidepresan sejak awal 1990-an. Pada awalnya, ada beberapa
ketidakpastian tentang kemanjuran relatif mereka dibandingkan dengan agen
standar yang lebih tua, terutama antidepresan trisiklik (TCA). Selective serotonin-
reuptake inhibitor (SSRI) merupakan jenis kelompok obat antidepresan yang
utamanya digunakan untuk mengatasi gangguan depresi dan kondisi psikologis.

1.3.5. Pyrrole dan Pyrrolopyrimidine

Pirol dan pirol [2,3-D] pirimidin sangat menarik dalam penemuan obat.
Pirol adalah salah satu heterosiklik sederhana yang paling penting, yang
ditemukan dalam berbagai produk alami. Ini adalah fragmen struktural kunci dari
heme dan klorofil (dua pigmen penting untuk kehidupan), klorin, bakterioklorin,
corrins (vitamin B12) dan beberapa pigmen empedu (biliverdin dan bilirubin).
Pirol [2,3-D]pirimidin sebagai 7-deazapurin menunjukkan aktivitas biologis yang
luar biasa karena kemiripannya dengan purin seluler. sintesis pirol dan

3
pirolopirimidin memiliki beberapa aktivitas biologis seperti antimikroba,
analgesik, anti-inflamasi, anti-kanker, anti-virus, anti-konvulsan, anti-
hiperlipidemia, anti-depresan, antidiabetik, aktivitas anti-alergi (Mohamed dkk,
2017).

4
II. TATA KERJA
2.1 Alat
Peralatan yang digunakan dalam penelitian ini adalah refluks, spektrum IR
spektrofotometer Perkin-Elmer 1650 (AS), Spektrum H NMR [JOEL NMR FXQ-
300-500 MHz], Spektrum massa [70 EV EI Ms-QP 1000 EX, Shimadzu, Jepang].
Vario, peralatan Elmentar, analisis High Performance Liquid Chromatography
(HPLC), dan analisis statistic dengan Graphpad prisma 5 Software.

2.2 Bahan
Bahan yang digunakan dalam penelitian ini adalah tikus albino sebanyak
200 buah, hidroksilamin hidroklorida, asam asetat glasial, natrium astat anhidrat,
fenil isotiosianat, piridin, asam asetat, asetil aseton, etil sianoasetat, etanol,
fluoxetin dan DMSO (Dimetil Sulfoksida).

2.3 Metodologi
2.3.1 Sintesis senyawa 2a-i
Senyawa 1a-i disintesis dengan mencampurkan α-amino keton dengan
malononitrile, setelah itu campuran direfluks dalam keadaan medium basa. Hasil
turunan pirol 1a-i ditambahkan hidroksilamin hidroklorida dalam asam asetat
glasial yang mengandung natrium asetat anhidrat, setelah itu direfluks selama 10
jam. Campuran kemudian dibiarkan semalaman pada suhu kamar. Selanjutnya
campuran dituangkan ke dalam air. Padatan yang terbentuk disaring, dicuci
dengan air dan dikristalkan dari dioksan untuk menghasilkan senyawa 2a-i.

2.3.2 Prosedur Umum untuk Sintesis Senyawa 4a-i

Senyawa 2a-i ditambahkan dengan etanol, asam asetat dan asetilaseton.
Campuran reaksi direfluks selama 5 jam, kemudian dipekatkan dan dibiarkan
dingin. Endapan yang terbentuk dikumpulkan dan direkristalisasi dari etanol
untuk menghasilkan senyawa 4a-i.

2.3.3 Prosedur Umum untuk Sintesis Senyawa 5a-i

Campuran dari 2a-i ditambahkan dengan etil sianoasetat dan asam asetat glasial
direfluks selama 5 jam. Padatan yang terbentuk setelah pendinginan dikumpulkan dan

5
direkristalisasi dari etanol untuk menghasilkan senyawa 5a-i.

2.3.4 Uji Aktivitas Antidepresan

Hewan: Dua ratus dua puluh tikus albino, dengan berat 18-22g, terlibat dalam
penelitian ini. Hewan dipelihara dalam kondisi laboratorium normal dengan suhu
terkontrol (20 ± 2 °C) dan kelembaban (60%) dengan siklus cahaya teratur. Hewan-
hewan diadaptasi satu minggu sebelum penelitian dan memiliki pendekatan bebas untuk
makanan dan air laboratorium standar. Semua prosedur eksperimental dipandu sesuai
dengan etika untuk perawatan dan penggunaan hewan laboratorium dalam penelitian dan
diizinkan.
a. Obat dan Perawatan
Setiap kelompok terdiri dari sepuluh tikus: Kelompok control (Ctrl) menerima
DMSO sebagai pelarut, kelompok Fluoxetine (30 mg/kg) (Flx) digunakan sebagai kontrol
positif. Kelompok yang tersisa menerima senyawa yang berbeda. Senyawa yang berbeda
dan fluoxetine (30 mg/kg, IP, kontrol positif) dilarutkan dalam DMSO dan diberikan
melalui rute injeksi intraperitoneal (IP) 30 menit sebelum pengujian perilaku.
2.3.5 Uji Suspensi Ekor (Tail Suspension Test)
Tikus yang diisolasi secara akustik dan visual digantung lima puluh cm di atas
lantai dengan pita perekat yang ditempatkan kira-kira satu cm dari ujung ekor. Waktu
imobilitas diverifikasi selama periode enam menit. Tikus dianggap tidak bergerak hanya
jika mereka tergantung secara pasif atau tidak bergerak sama sekali. Antidepresan
konvensional menurunkan waktu imobilitas dalam tes ini.
2.3.6 Analisis Serotonin pada Otak Tikus Menggunakan HPLC
Otak tikus yang menunjukkan aktivitas antidepresan positif dikeluarkan dengan
cepat dan dibekukan pada suhu -80 C. Hari berikutnya setelah otak dibekukan, daerah
terpilih dibedah di bawah kontrol visual sebagai berikut: potongan pertama dibuat pada
tingkat kiasma optikum dan yang kedua setinggi badan mammillary. Kemudian daerah
otak yang dibedah ditempatkan ke dalam tabung Eppendorf dan ditimbang. Setelah itu,
setiap jaringan otak dihomogenisasi dengan pengganggu sel ultrasonik (Vibracell 72434,
Bioblock, Illkirch-Cedex) dalam 150μl 0,1 M asam perklorat yang mengandung 0,4 mM
natrium meta bisulfit. Homogenat kemudian disentrifugasi pada 10.000 xg selama 25
menit pada 4 C sampai analisis. Filtrat disuntikkan ke dalam sistem HPLC. Sistem HPLC
terdiri dari pompa pengiriman gradien kuaterner Model HP 1050 (Hewlett-Packard),
sampel injector Model 7125 (Rheodyne, Berkeley), dan kolom analitis ODS 2c 18, 4,6 x
250 mm, ukuran partikel 5 m (Hewlett-Packard). Model detektor elektrokimia HP 1049

6
A(Hewlett-Packard) dengan elektroda kerja karbon kaca digunakan pada pengaturan
tegangan positif. Konsentrasi monoamina dalam setiap sampel dihitung dari area puncak
kromatografi dan dinyatakan sebagai ng/g jaringan basah.
2.3.7 Analisis Statistik
Perangkat lunak GraphPad prisma 5 digunakan untuk analisis statistik hasil, yang
disajikan sebagai rata-rata ± kesalahan standar. Uji signifikansi antara kelompok yang
diuji dilewatkan menggunakan analisis varians satu arah (ANOVA) diikuti dengan uji
perbandingan ganda Tukey-Kramer. AP nilai <0,05 dianggap signifikan.

7
III. HASIL DAN PEMBAHASAN
3.1 Hasil Kimia
Tujuan dari penelitian ini adalah untuk mengembangkan pyrrolopyrazole
dan turunan pyrroloprazolopyrimidine. turunan pirol 1a-i dibuat dengan sintesis
aminocyanopyrroles melalui reaksi keton-amino dan malononitril dalam medium
dasar.

a. Persiapan Pyrrolopyrazole

8
 b. Mekanisme

Turunan pirol 1a-i diubah menjadi 3-amino-pirola [2,3-c] pirazol 2a-i

melalui kondensasi dengan hidroksilamin hidroklorida equimolar dalam asam
asetat glasial dengan natrium asetat anhidrat. Reaksi 2a-i dengan fenil
isothiocyanate menghasilkan turunan tiourea 3a-i. Pada perlakuan 2a-i dengan
asetil aseton, etil sianoasetat atau etil asetoasetat terjadi penutupan cincin dan
pyrrolo [2,3-c]pirazolo[1,5-a] pirimidin 4,5 dan 6 yang sesuai seperti mekanisme
yang terjadi.

3.2 Hasil Biologis

Nilai mobilitas untuk senyawa yang diperiksa, menunjukkan bahwa
menunjukkan aktivitas antidepresan positif seperti yang terungkap pada Tabel 1.

9
 Tabel 1. Uji coba aktivitas antidepresan senyawa dalam suspensi ekor
Senyawa Aktivitas antidepresan
Fluoxetine Positif
2d Positif
2h Positif
5h Positif
2i Positif
4h Positif

Nilai mobilitas dalam TST untuk fluoxetine, yang digunakan sebagai obat
antidepresan standar, dan senyawa 2d, 2h, 2i, 4h, dan 5h secara signifikan lebih
tinggi dari nilai kontrol, seperti yang diungkapkan pada Gambar.

Gambar 1. Pengaruh Fluoxetine dan kemungkinan obat antidepresan pada

mobilitas tikus dalam uji suspensi ekor

Nilai mobilitas ditunjukkan sebagai rata-rata dari total waktu mobilitas.

Fluoxetine menunjukkan 95% peningkatan mobilitas dibandingkan dengan hewan
kontrol sementara senyawa yang diteliti 2d, 2h, 2i, 4h, dan 5h menunjukkan hanya
sekitar 30% peningkatan mobilitas. Namun, waktu secara signifikan lebih rendah
dibandingkan dengan hewan yang diberikan Fluoxetine, seperti yang ditunjukkan
pada gambar.

10
Gambar 2. Pengaruh Fluoxetine dan kemungkinan obat antidepresan pada tingkat
5HT di homogenat otak tikus

Gambar diatas menggambarkan konsentrasi 5-HT di seluruh otak

homogenat. Konsentrasi serotonin secara signifikan lebih tinggi pada otak tikus
yang disuntik dengan Fluoxetine dan senyawa 2d, 2h, 2i, 4h, dan 5h
dibandingkan dengan hewan Kontrol. Peningkatan kadar 5-HT pada hewan yang
diberi fluoxetine adalah 66% dan peningkatan senyawa yang diteliti adalah 60%,
83%, 127%, 72%, dan 155%. Selain itu, konsentrasi 5-HT dalam senyawa 2i dan
5h secara signifikan lebih tinggi dari tingkat 5-HT pada hewan yang diberikan
Fluoxetine.

11
 Gambar 3. Model TST

Beberapa model hewan percobaan untuk depresi telah disiapkan untuk

mempelajari depresi dan efek antidepresan dari obat-obatan baru. Salah satu
model yang sudah ada adalah tail suspension test (TST). Tes ini melibatkan
hewan pengerat ke rangsangan permusuhan yaitu suspensi oleh ekor. Prosedur ini
menghasilkan defisit jangka panjang dalam motivasi dan kemampuan untuk
melepaskan diri dari rangsangan negatif bila memungkinkan.. Hal ini juga
menyebabkan perubahan perilaku yang berkaitan dengan yang diamati pada
depresi. Keuntungan utama dari TST adalah dapat menentukan efek antidepresan
dari spektrum antidepresan yang luas terlepas dari mekanisme yang
mendasarinya. Didapat bahwa pengurangan periode imobilitas atau peningkatan
periode mobilitas di TST menunjukkan aktivitas antidepresan pada hewan
pengerat. Di sini, menunjukkan bahwa lima senyawa yang disintesis memiliki
pengaruh positif yang jelas pada hewan yang dikenai TST yang sebanding dengan
Fluoxetine standar.
Fluoxetine adalah antidepresan dari kelas selective serotonin reuptake
inhibitor (SSRI). Mekanisme kerjanya tergantung pada peningkatan kadar 5-HT di
otak. Mengikuti peningkatan level 5-HT, perilaku positif efeknya ditunjukkan
pada subjek depresi. Level 5-HT diukur pada hewan yang disuntik dengan
senyawa yang disintesis tidak mencapai tingkat yang sama yang dicapai oleh
mereka yang disuntik dengan Fluoksetin. Namun demikian, tingkat 5-HT pada
hewan yang menerima senyawa yang disinyesis secara signifikan lebih tinggi
dibandingkan dengan hewan kontrol. Oleh karena itu, hasil perilaku yang

12
ditunjukkan kita dapat menyimpulkan dengan aman bahwa senyawa yang
disintesis memiliki efek antidepresan yang menjanjikan

13
IV. KESIMPULAN
Penelitian melaporkan bahwa beberapa senyawa pirol kemungkinan
memiliki aktivitas antidepresan. Hasil biologis juga menunjukkan bahwa senyawa
fluoxetine, 2d, 2h, 2i, 4h, dan 5h menunjukkan aktivitas antidepresan. Konsentrasi
serotonin lebih tinggi pada otak tikus yang disuntik dengan Fluoxetine dan
senyawa 2d, 2h, 2i, 4h, dan 5h dibandingkan dengan hewan Kontrol. Oleh karena
itu, hasil perilaku yang ditunjukkan dari penelitian dapat disimpulkan bahwa
senyawa yang disintesis memiliki efek antidepresan yang menjanjikan.

14
 DAFTAR PUSTAKA

Donoghue, J, Tylee, A., dan Wildgust, H. 1996. Analisis database cross sectional
dari resep antidepresan dalam praktik umum di Inggris, 1993-5. BMJ.
313(7061):861–2
Mohamed, M, S., Rania, H, A, E, H., dan Amira I, S. 2017. Strategi Sintesis dan
Nilai Biologis Pyrrole dan Pyrrolopyrimidine. Journal of Advanced
Pharmacy Research. 1(1): 1-24
Sadock, B. J. & Sadock, V. A., 2010. Buku Ajar Psikiatri Klinis Edisi 2. Penerbit
Buku Kedokteran EGC, Jakarta.
Suchting, R., dkk. 2011. Revisiting monoamine oxidase inhibitors for the
treatment of depressive disorders: A systematic review and network
meta-analysis. Journal of Affective Disorders. 282:1153–1160

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See discussions, stats, and author profiles for this publication at: https://www.researchgate.net/publication/328811270

Pyrrolopyrazoles; Synthesis, Evaluation and Pharmacological Screening as

Antidepressant Agents

Article  in  Medicinal Chemistry · November 2018

DOI: 10.2174/1573406414666181108090321

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Medicinal Chemistry, 2018, 14, 1-12 1

RESEARCH ARTICLE

Pyrrolopyrazoles: Synthesis, Evaluation and Pharmacological Screening as

Antidepressant Agents

Samar Said Fatahala1*, Shahira Nofal2, Eman Mahmoud2 and Rania Helmy Abd El-hameed1

1
Pharmaceutical Organic Chemistry Department; 2Pharmacology and Toxicology Department, Faculty of Pharmacy,
Helwan University, Ain-Helwan, Postal code: 11795, Helwan, Cairo, Egypt

ARTICLE HISTORY
Received: June 04, 2018
Revised: September 08, 2018
Accepted: October 30, 2018
DOI:
10.2174/1573406414666181108090321
Abstract: Background: Pyrroles and fused pyrroles are of great interest as biologically active
compounds, among these activities; antidepressant activity is of special concern.
Objective: Synthesis of a series of pyrrolopyrazoles and their pyrimidine derivatives and their
characterization using spectral data to be monitored for antidepressant activity using behavioral
techniques.
Method: A control group was administered the vehicle i.p., positive control group received
fluoxetine as standard and all other groups were administered the tested compounds. The groups
were subjected to tail suspension test (TST) to determine the antidepressant activity compared with
fluoxetine as a standard drug. The compounds exhibiting antidepressant activity were then used to
analyze changes in serotonin (5HT) level in the brain of albino mice.
Results: TST results showed that both pyrazoles and pyrazolopyrimidines derivatives exhibit
promising anti-depressant activity.
Conclusion: Compounds [pyrazoles & pyrazlopyrimidines] showed promising antidepressant ac-
tivity possibly mediated by the increased levels of 5HT.

Keywords: Pyrrole, Pyrrolopyrazole, Pyrazolopyrimidine, Synthesis, SAR, Anti-depressant assay.

1. INTRODUCTION of interest in the treatment of depression. Tricyclic antide-

Depression is a mood disorder that may eventually lead
to a severe deficiency in various important areas of normal
functioning. It has also been associated with a rise in the
mortality rate [1]. The causes of this disease are not fully
elucidated until now [2]. Nevertheless, the pathophysiologi-
cal events accompanying depression are extensively studied.
It is latterly shown that despite 40 years of extensive re-
search, the monoamine deficiency hypotheses can still ex-
plain some aspects of depressive symptoms [3].
Monoamine neurotransmitters; norepinephrine (NE) and
serotonin (SER, 5-HT) were identified as important drug
targets of psychiatric diseases [4]. Serotonin (SER, 5-HT) is
one of the monoamines involved in the pathophysiology of
depression [5]. Several studies have revealed that the de-
crease in 5-HT brain level is connected to the negative
behavioural symptoms of depression [6, 7]. Hence, antide-
pressant agents that play a role in increasing 5-HT levels are
*Address correspondence to this author at the Pharmaceutical organic
chemistry department, Faculty of Pharmacy, Helwan University, Ain-
Helwan, Helwan, Cairo. Postal code: 1179, Egypt; Tel/Fax: 00202-2554-
1601; E-mail; samarradwan1@yahoo.com
pressants (TCAs) and monoamine oxidase inhibitors
(MAOIs) were also developed as active antidepressant
agents despite a wide range of side effects, dry mouth,
blurred vision, urinary retention, hypotension, and insomnia
[8-10]. Recently, Selective Serotonin Reuptake Inhibitors
(SSRIs) have been recommended as antidepressants [11, 12],
as revealed in Fig. (1a).
Heterocyclic antidepressants have been considered as the
principal line antidepressant treatment. Heterocyclic com-
pounds have been studied extensively both naturally and
synthetically during the past few years [12–15]. Beta-
carbolines, naturally occurring alkaloids in different plants,
were found to be molecules that act on the benzodiazepine
site of the GABA-A complex receptors [16, 17]. Among
them, harmala alkaloids, which were found in the liana Ban-
isteriopsiscaapi, harmalineharmine and trahydroharmine,
fused pyrrole (indole) alkaloid, show a benzodiazepine like
systematic agonist effect [16, 18]. Harmine and harmaline,
both showed selective and reversible MAOI, while tetrahy-
droharmine was considered as a weak SSRI, as revealed in
Fig. (1b).

1573-4064/18 $58.00+.00 © 2018 Bentham Science Publishers

2 Medicinal Chemistry, 2018, Vol. 14, No. 00 Fatahala et al.

Selective Serotonin Reuptake Inhibitors (SSRIs)

O
MeHNH2C

CF3 MeHN Cl
(Me)2NH2CH2C
Fluoxetine Cl
F Sertraline
NC O

(R,S)- Citalopram

Fig. (1a). First generation SSRI, Fluoxetine, Sertaline and citalopram have achieved great success in treating depression, they also have some
troublesome effects including sedation, anxiety, headache, tremor, and sexual dysfunction [11, 12].

Naturally occuring Pyrroles derivatives as antidepressant

MAOI SSRI

MeO NH
MeO N MeO N
N
N N H
H H
Harmine Harmaline Tetrahydroharmine

Fig. (1b).
-Carboline alkaloids, known as harmala alkaloids, acting as antidepressant agent in traditional medicine to improve mood and
other related effects [16-18].

Pyrroles containg compounds Pyrimidine containg compounds

F O N

N (CH2)2 O
N F
HN N
O2S N
N Risoperidone
LY367265
N

Pyrrole-3-carboxamides

CONH(CH2)2 N N Ar

Ph Me Ar= 2,3dichlorophenyl
N R= H, Me
R

Fig. (1c). Pyrroles, Indole and Pyrimidine compounds showing activity as antidepressant.

Novel pyrrole bearing antidepressant agents were synthe-
sized with multiple activities [4, 9, 19], as a way to
overcome numerous side effects associated with non-
selective binding at post-synaptic 5-HT receptors.
LY367265, indole derivative, has been proposed and
developed as potential antidepressants, with dual activity at
SERT while binding antagonistically to the 5-HT2A receptor
[20, 21]. Pyrimidine derivatives possessing antidepressant
derivatives possessing antidepressant and analgesic activities
have been itemised in literature [14, 22, 23]. Risperidone,
reported as an antipsychotic drug and also known as a struc-
tural hybrid of butyrophenone, is used as anxiolytic, anti-
depressant and antiparkinsonian [22], as revealed in Fig.
(1c).
Pyrroles as Anti-depressant Agents Medicinal Chemistry, 2018, Vol. 14, No. 00 3

2. EXPERIMENTAL 2.1.6. 3-Amino-6-(1,5-dimethyl-3-oxo-2-phenyl-2,3-

dihydro-1H-pyrazol-4-yl)-4,5-diphenyl-1H-pyrrolo[2,3-
2.1. Chemical Experimental
c]pyrazole (2d)
2.1.1. Experimental
Yield: 73%; m.p.: 189-191 °C; IR (KBr)  (cm-1): 3367,
2.1.2. Synthesis of Lead Compounds 3339 (NH2), 3212 (NH), 1732 (C=O), 1604 (C=N); MS (EI)
m/z: 460 (M+, 31.66 %); 1H NMR (DMSO-d6, 300 MHz)

Melting points were uncorrected and defined using E- (ppm): 2.4 (s, 3H, CH3), 3.4 (s, 3H, N-CH3), 5.38 (s, 2H,
lectro-thermal IA 9100 apparatus (Shimadzu, Japan). NH2, D2O exchangeable), 7.0-8.2 (m, 15H, Ar-H), 8.6 (s,
Spectral analysis was performed in Organic Microanalysis 1H, NH); Anal. Calcd for C28H24N6O (460.53): C, 73.04; H,
Unit, Faculty of Science, Cairo University, Cairo, Egypt. 5.22, N, 18.26, O, 3.48 %. Found: C, 72.96; H, 5.48; N,
First, IR spectra were predicted as potassium bromide pellets 18.54, O, 3.40%.
on a Perkin-Elmer 1650 spectrophotometer (USA), 1H NMR
spectra [JOEL NMR FXQ-300 MHz and JOEL NMR FXQ- 2.1.7. 3-Amino-6-benzyl-4,5-diphenyl-1H-pyrrolo[2,3-
500 MHz spand], chemical shifts were stated as ppm against c]pyrazole (2e)
TMS as internal reference. Mass spectra [70 EV EI Ms-QP
Yield: 56%; m.p.: 193-195 °C; IR (KBr)  (cm-1): 3414,
1000 EX, Shimadzu, Japan]. Finally, Microanalyses were
3399 (NH2), 3220 (NH), 1587 (C=N); MS (EI) m/z: 364
performed using Vario, Elmentar apparatus, Shimadzu, Ja-
(M+, 33.5 %); 1H NMR (DMSO-d6, 300 MHz)
(ppm):
pan. The literature reported the synthesis of compounds 1a-i
4.45 (s, 2H, NH2, D2O exchangeable), 5.15 (s, 2H, CH2),6.7-
[24–28]. All novel compounds obtained spectral data reliable 7.9 (m, 15H, Ar-H), 9.04 (s, 1H, NH); Anal. Calcd for
with the suggested structure. Microanalyses were found
C24H20N4 (364.44): C, 79.12; H, 5.49, N, 15.38%. Found: C,
within ±0.4% of the theoretical values.
79.05; H, 5.66; N, 15.14%.
2.1.3. General Procedure for the Synthesis of Compounds
2.1.8. 3-Amino-6-(4-methylphenyl)-4-phenyl-1H-
2a-i
pyrrolo[2,3-c]pyrazole (2f)
A mixture of 1a-i (10 mmol) and hydroxylamine hydro-
Yield: 64%; m.p.: 182-184 °C; IR (KBr)  (cm-1): 3529,
chloride (0.60 g, 10 mmol) in glacial acetic acid (30 mL) 3418 (NH2), 3236 (NH), 1621 (C=N); MS (EI) m/z: 288
containing anhydrous sodium acetate (1g) was boiled under
(M+, 68.2%); 1H NMR (DMSO-d6, 300 MHz)
(ppm): 2.3
reflux for 10 hrs. The reaction mixture was left overnight at
(s, 3H, CH3), 4.6 (s, 2H, NH2, D2O exchangeable), 6.7-7.5
room temperature and then poured onto water. The solid
(m, 10H, Ar-H), 8.05 (s, 1H, NH); ,Anal. Calcd for C18H16N4
precipitate was filtered off, washed with water, and crystalli-
(288.36): C, 74.98; H, 5.59; N, 19.43%. Found: C, 75.16; H,
zed from dioxane to yield 2a-i.
5.81; N, 19.60%.
2.1.4. 3-Amino-6-(4-methylphenyl)-4,5-diphenyl-1H-
2.1.9. 3-Amino-6-(4-methoxyphenyl)-4-phenyl-1H-
pyrrolo[2,3-c]pyrazole (2a)
pyrrolo[2,3-c]pyrazole (2g)
Yield: 80%; m.p.: 209-211 °C; IR (KBr)  (cm-1): 3567,
Yield: 73%; m.p.: 186-188 °C; IR (KBr)  (cm-1): 3456,
3526 (NH2), 3166 (NH), 1576 (C=N); MS (EI) m/z: 364
3449 (NH2), 3212 (NH), 1510 (C=N), 1322 (C-O); MS (EI)
(M+, 75%); 1H NMR (DMSO-d6, 300 MHz)
(ppm): 2.24
m/z: 304 (M+, 42%); 1H NMR (DMSO-d6, 300 MHz)

(s, 3H, CH3), 4.99 (s, 2H, NH2, D2O exchangeable), 6.8-7.9
(ppm): 3.69 (s, 3H, OCH3), 5.35 (s, 2H, NH2, D2O ex-
(m, 14H, Ar-H), 8.5 (s, 1H, NH); ,Anal. Calcd for C24H20N4 changeable), 6.8-7.9 (m, 10H, Ar-H), 8.5 (s, 1H, NH); ,Anal.
(364.45): C, 79.10; H, 5.53; N, 15.37%. Found: C, 78.86; H,
Calcd for C18H16N4O (304.35): C, 71.04; H, 5.30; N, 18.41;
5.84; N, 15.68%.
O, 5.26%. Found: C, 71.09; H, 5.12; N, 18.78; O, 5.59%.
2.1.5. 3-Amino-6-(4-methoxyphenyl)-4,5-diphenyl-1H-
2.1.10. 3-Amino-6-(3,4-dichlorophenyl)-4-phenyl-1H-
pyrrolo[2,3-c]pyrazole (2b)
pyrrolo[2,3-c]pyrazole (2h)
Yield: 87%; m.p.: 226-228 °C; IR (KBr)  (cm-1): 3417,
Yield: 72%; m.p.: 217-219 °C; IR (KBr)  (cm-1): 3405,
3402 (NH2), 3194 (NH), 1536 (C=N), 1315 (C-O); MS (EI) 3396 (NH2), 3217 (NH), 1568 (C=N); MS (EI) m/z: 342
m/z: 380 (M+, 58.4%); 1H NMR (DMSO-d6, 300 MHz)

(M+, 50%); 1H NMR (DMSO-d6, 300 MHz)
(ppm): 5.32
(ppm): 3.49 (s, 3H, OCH3), 5.17 (s, 2H, NH2, D2O ex-
(s, 2H, NH2, D2O exchangeable), 6.87-8.0 (m, 9H, Ar-H),
changeable), 6.9-8.2 (m, 14H, Ar-H), 8.8 (s, 1H, NH); ,Anal.
8.4 (s, 1H, NH); Anal. Calcd for C17H12Cl2N4 (343.21): C,
Calcd for C24H20N4O (380.45): C, 75.77; H, 5.30; N, 14.73;
59.65; H, 3.51; Cl, 20.47, N, 16.37%. Found: C, 59.43; H,
O, 4.21%. Found: C, 76.06; H, 5.14; N, 14.38; O, 4.55%.
3.68; Cl, 20.81, N, 16.51%.
2.1.5. 3-Amino-6-(3,4-dichlorophenyl)-4,5-diphenyl-1H-
2.1.11. 3-Amino-6-(1,5-dimethyl-3-oxo-2-phenyl-2,3-
pyrrolo[2,3-c]pyrazole (2c)
dihydro-1H-pyrazol-4-yl)-4-phenyl-1H-pyrrolo[2,3-
Yield: 68%; m.p.: 156-158 °C; IR (KBr)  (cm-1): 3391, c]pyrazole (2i)
3366 (NH2), 3195 (NH), 1593 (C=N); MS (EI) m/z: 418
Yield: 86%; m.p.: 178-180 °C; IR (KBr)  (cm-1): 3388,
(M+, 33.6%); 1H NMR (DMSO-d6, 300 MHz)
(ppm): 5.4
3345 (NH2), 3203 (NH), 1704 (C=O), 1576 (C=N); MS (EI)
(s, 2H, NH2, D2O exchangeable), 6.7-8.1 (m, 13H, Ar-H),
m/z: 384 (M+, 62 %); 1H NMR (DMSO-d6, 300 MHz)

8.68 (s, 1H, NH); Anal. Calcd for C23H16Cl2N4 (419.30): C, (ppm): 2.31 (s, 3H, CH3), 3.38 (s, 3H, N-CH3), 5.27 (s, 2H,
66.03; H, 3.83; Cl, 16.75, N, 13.39%. Found: C, 66.16; H,
NH2, D2O exchangeable), 7.1-8.2 (m, 11H, Ar-H), 8.4 (s,
3.64; Cl, 16.38, N, 13.58%.
1H, NH); Anal. Calcd for C22H20N6O (384.43): C, 68.75; H,
4 Medicinal Chemistry, 2018, Vol. 14, No. 00
5.21, N, 21.88, O, 4.17 %. Found: C, 68.90; H, 5.03; N,
22.14, O, 3.89%.
2.2. General Procedure for the Synthesis of Compounds
3a-i
A mixture of 2a-i (5 mmol), phenyl isothiocyanate (0.66
mL, 5 mmol) and pyridine (30 mL) was refluxed for 3 hrs,
then concentrated and finally was allowed to cool. The for-
med precipitate was collected and recrystallized from dioxa-
ne to yield compounds 3a-i.
2.2.1. 1[6-(4-Methylphenyl)-4,5-diphenyl-1H-pyrrolo[2,3-
c]pyrazol-3-yl]-3-phenyl thiourea (3a)
Yield: 29%; m.p.: 280-282 °C; IR (KBr)  (cm-1):
3421,3361 (NH), 1566 (C=N), 1240 (C=S); MS (EI) m/z:
499 (M+, 47.3%); 1H NMR (DMSO-d6, 300 MHz)
(ppm):
2.31 (s, 3H, CH3), 4.99, 5.3 (s, 2H, 2NH-thiourea, D2O ex-
changeable), 6.8-7.8 (m, 19H, Ar-H), 8.4 (s, 1H, NH); Anal.
Calcd for C31H25N5S (499.64): C, 74.52; H, 5.04; N, 14.02;
S, 6.42%. Found: C, 74.89; H, 5.38; N, 13.86; S, 6.31%.
2.2.2. 1[6-(4-Methoxyphenyl)-4,5-diphenyl-1H-pyrrolo[2,3-
c]pyrazol-3-yl]-3-phenyl thiourea (3b)
Yield: 60%; m.p.: 266-268 °C; IR (KBr)  (cm-1): 3422,
3340 (NH), 1613 (C=N), 1322 (C-O), 1236 (C=S); MS (EI)
m/z: 515 (M+, 25.8%); 1H NMR (DMSO-d6, 300 MHz)

(ppm): 3.56 (s, 3H, OCH3), 5.13, 5.4 (s, 2H, 2NH-thiourea,
D2O exchangeable), 7.0-8.2 (m, 19H, Ar-H), 8.4 (s, 1H,
NH); Anal. Calcd for C31H25N5OS (515.64): C, 72.21; H,
4.89; N, 13.58; O, 3.10; S, 6.22%. Found: C, 72.46; H, 5.04;
N, 13.87; O, 3.19; S, 6.38%.
2.2.3. 1[6-(3,4-Dichlorophenyl)-4,5-diphenyl-1H-
pyrrolo[2,3-c]pyrazol-3-yl]-3-phenyl thiourea (3c)
Yield: 44%; m.p.225-227: °C; IR (KBr)  (cm-1):
3415,3395 (NH), 1578 (C=N), 1266 (C=S); MS (EI) m/z:
553 (M+, 69%); 1H NMR (DMSO-d6, 300 MHz)
(ppm):
4.6, 5.1 (s, 2H, 2NH-thiourea, D2O exchangeable), 6.8-7.9
(m, 18H, Ar-H), 8.2 (s, 1H, NH); Anal. Calcd for
C30H21Cl2N5S (554.49): C, 65.09; H, 3.80; Cl, 12.66, N,
12.66; S, 5.78%. Found: C, 65.23; H, 3.88; Cl, 12.82, N,
12.46; S, 5.91%.
2.2.4. 1[6-(1,5-dimethyl-3-oxo-2-phenyl-2,3-dihydro-1H-
pyrazol-4-yl)-4,5-diphenyl-1H-pyrrolo[2,3-c]pyrazol-3-yl]-
3-phenyl thiourea (3d)
Yield: 60%; m.p.: 215-217 °C; IR (KBr)  (cm-1): 3392,
3364 (NH), 1722 (C=O), 1601 (C=N), 1251 (C=S); MS (EI)
m/z: 595 (M+, 18%); 1H NMR (DMSO-d6, 300 MHz)

(ppm): 2.3 (s, 3H, CH3), 3.3 (s, 3H, N-CH3), 4.87, 5.3 (s, 2H,
2NH-thiourea, D2O exchangeable), 6.9-8.1 (m, 20H, Ar-H),
8.5 (s, 1H, NH); ,Anal. Calcd for C35H29N7OS (595.72): C,
70.59; H, 4.87; N, 16.47; O, 2.69; S, 5.38%. Found: C,
70.41; H, 5.07; N, 16.71; O, 2.88; S, 5.73%.
2.2.5. 1[6-benzyl-4,5-diphenyl-1H-pyrrolo[2,3-c]pyrazol-3-
yl]-3-phenyl thiourea (3e)
Yield: 54%; m.p.: 226-228 °C; IR (KBr)  (cm-1): 3412,
3388 (NH), 1621 (C=N), 1267 (C=S); MS (EI) m/z: 499
(M+, 38.7%); 1H NMR (DMSO-d6, 300 MHz)
(ppm): 5.1
(s, 2H, CH2), 5.8 (s, 2H, 2NH-thiourea, D2O exchangeable),
Fatahala et al.
6.9-7.6 (m, 20H, Ar-H), 8.5 (s, 1H, NH); ,Anal. Calcd for
C31H25N5S (499.63): C, 74.55; H, 5.01; N, 14.03; S, 6.41%.
Found: C, 74.49; H, 5.33; N, 13.78; S, 6.71%.
2.2.6. 1[6-(4-Methylphenyl)-4-phenyl-1H-pyrrolo[2,3-
c]pyrazol-3-yl]-3-phenyl thiourea (3f)
Yield: 48%; m.p.: 228-230 °C; IR (KBr)  (cm-1): 3429,
3238 (NH), 1636 (C=N), 1256 (C=S); MS (EI) m/z: 423
(M+, 42%); 1H NMR (DMSO-d6, 300 MHz)
(ppm): 2.43
(s, 3H, CH3), 4.8, 5.0 (s, 2H, 2NH-thiourea, D2O ex-
changeable), 6.9-7.8 (m, 15H, Ar-H), 8.3 (s, 1H, NH); ,Anal.
Calcd for C25H21N5S (423.54): C, 70.90; H, 5.00; N, 16.54;
S, 7.57%. Found: C, 70.86; H, 5.35; N, 16.76; S, 7.32%.
2.2.7. 1[6-(4-Methoxyphenyl)-4-phenyl-1H-pyrrolo[2,3-
c]pyrazol-3-yl]-3-phenyl thiourea (3g)
Yield: 54%; m.p.: 199-201 °C; IR (KBr)  (cm-1): 3451,
3342 (NH), 1625 (C=N), 1326 (C-O), 1243 (C=S); MS (EI)
m/z: 439 (M+, 33.5%); 1H NMR (DMSO-d6, 300 MHz)

(ppm): 3.5 (s, 3H, OCH3), 4.5, 4.9 (s, 2H, 2NH-thiourea,
D2O exchangeable), 7.0-8.1 (m, 15H, Ar-H), 8.72 (s, 1H,
NH); ,Anal. Calcd for C25H21N5OS (439.54): C, 68.32; H,
4.82; N, 15.93; O, 3.64; S, 7.29%. Found: C, 68.56; H, 5.09;
N, 15.87; O, 3.39; S, 7.58%.
2.2.8. 1[6-(3,4-Dichlorophenyl)-4-phenyl-1H-pyrrolo[2,3-
c]pyrazol-3-yl]-3-phenyl thiourea (3h)
Yield: 51%; m.p.:266-268 °C; IR (KBr)  (cm-1):
3419,3399 (NH), 1577 (C=N), 1260 (C=S); MS (EI) m/z:
477 (M+, 64.3%); 1H NMR (DMSO-d6, 300 MHz)
(ppm):
4.9, 5.23 (s, 2H, 2NH-thiourea, D2O exchangeable), 6.88-7.9
(m, 14H, Ar-H), 8.12 (s, 1H, NH); Anal. Calcd for
C24H17Cl2N5S (478.40): C, 60.38; H, 3.56; Cl, 14.68, N,
14.68; S, 6.71%. Found: C, 60.23; H, 3.81; Cl, 14.82, N,
14.99; S, 6.98%.
2.2.9. 1[6-(1,5-dimethyl-3-oxo-2-phenyl-2,3-dihydro-1H-
pyrazol-4-yl)-4-phenyl-1H-pyrrolo[2,3-c]pyrazol-3-yl]-3-
phenyl thiourea (3i)
Yield: 67%; m.p.: 283-285 °C; IR (KBr)  (cm-1): 3390,
3344 (NH), 1725 (C=O), 1619 (C=N), 1246 (C=S); MS (EI)
m/z: 519 (M+, 25%); 1H NMR (DMSO-d6, 300 MHz)

(ppm): 2.23 (s, 3H, CH3), 3.3 (s, 3H, N-CH3), 4.7, 5.23 (s,
2H, 2NH-thiourea, D2O exchangeable), 6.9-8.0 (m, 16H, Ar-
H), 8.45 (s, 1H, NH); Anal. Calcd for C29H25N7OS (519.62):
C, 67.05; H, 4.82; N, 18.88; O, 3.08; S, 6.17%. Found: C,
67.31; H, 5.02; N, 18.79; O, 3.27; S, 6.30%.
2.3. General Procedure for the Synthesis of Compounds
4a-i
Ethanol (25 mL) and few drops of acetic acid were added
to a mixture of 2a-i (5 mmol), acetylacetone (0.5 mL, 5
mmol) . The reaction mixture was refluxed for 5 hrs, then
concentrated and allowed to cool. The formed precipitate
was collected and recrystallized from ethanol to yield com-
pounds 4a-i.
2.3.1. 2,4-Dimethyl-7-(4-methylphenyl)-8,9-diphenyl-1H-
pyrrolo[2,3-c]pyrazolo[1,5-a] pyrimidine (4a)
Yield: 56%; m.p.: 251-253 °C; IR (KBr)  (cm-1): 1597
(C=N); MS (EI) m/z: 428 (M+, 31.7%); 1H NMR (DMSO-
Pyrroles as Anti-depressant Agents
d6, 300 MHz)
(ppm): 2.37 (s, 3H, CH3), 2.46 (s, 3H, CH3),
2.6 (s, 3H, CH3), 6.8-7.6 (m, 15H, Ar-H); Anal. Calcd for
C29H24N4 (428.54): C, 81.28; H, 5.65; N, 13.07%. Found: C,
80.96; H, 5.82; N, 13.34%.
2.3.2. 2,4-Dimethyl-7-(4-methoxyphenyl)-8,9-diphenyl-1H-
pyrrolo[2,3-c]pyrazolo[1,5-a]pyrimidine (4b)
Yield: 68%; m.p.: 237-239 °C; IR (KBr)  (cm-1): 1616
(C=N), 1333 (C-O); MS (EI) m/z: 444 (M+, 81%); 1H NMR
(DMSO-d6, 300 MHz)
(ppm): 2.3 (s, 3H, CH3), 2.6 (s, 3H,
CH3), 3.52 (s, 3H, OCH3), 6.9-8.0 (m, 15H, Ar-H); ,Anal.
Calcd for C29H20N4O (444.54): C, 78.36; H, 5.44; N, 12.60;
O, 3.60%. Found: C, 78.16; H, 5.67; N, 12.85; O, 3.51%.
2.3.3. 2,4-Dimethyl-7-(3,4-dichlorophenyl)-8,9-diphenyl-
1H-pyrrolo[2,3-c]pyrazolo[1,5-a]pyrimidine (4c)
Yield: 48%; m.p.: 174-176 °C; IR (KBr)  (cm-1): 1583
(C=N); MS (EI) m/z: 482 (M+, 29%); 1H NMR (DMSO-d6,
300 MHz)
(ppm): 2.21 (s, 3H, CH3), 2.4 (s, 3H, CH3), 6.8-
8.1 (m, 14H, Ar-H); Anal. Calcd for C28H20Cl2N4 (483.39):
C, 69.71; H, 4.15; Cl, 14.52, N, 11.62%. Found: C, 69.46; H,
3.99; Cl, 14.67, N, 11.81%.
2.3.4. 2,4-Dimethyl-7-(1,5-dimethyl-3-oxo-2-phenyl-2,3-
dihydro-1H-pyrazol-4-yl)-8,9-diphenyl-1H-pyrrolo[2,3-
c]pyrazolo[1,5-a]pyrimidine (4d)
Yield: 60%; m.p.: 204-206 °C; IR (KBr)  (cm-1): 1742
(C=O), 1556 (C=N); MS (EI) m/z: 524 (M+, 60 %); 1H
NMR (DMSO-d6, 300 MHz)
(ppm): 2.2 (s, 3H, CH3), 2.29
(s, 3H, CH3), 2.34 (s, 3H, CH3), 3.2 (s, 3H, N-CH3), 7.1-8.2
(m, 16H, Ar-H); Anal. Calcd for C33H28N6O (524.62): C,
75.57; H, 5.34, N, 16.03, O, 3.05 %. Found: C, 75.76; H,
5.13; N, 15.84, O, 3.35%.
2.3.5. 2,4-Dimethyl-7-benzyl-8,9-diphenyl-1H-pyrrolo[2,3-
c]pyrazolo[1,5-a]pyrimidine (4e)
Yield: 51%; m.p.: 208-210 °C; IR (KBr)  (cm-1): 1596
(C=N); MS (EI) m/z: 428 (M+, 82 %); 1H NMR (DMSO-d6,
300 MHz)
(ppm): 2.25 (s, 3H, CH3), 2.39 (s, 3H, CH3), 5.2
(s, 2H, CH2), 7.1-8.0 (m, 16H, Ar-H); Anal. Calcd for
C29H24N4 (428.53): C, 81.31; H, 5.61, N, 13.08,%. Found: C,
81.66; H, 5.02; N, 13.22%.
2.3.6. 2,4-Dimethyl-7-(4-methylphenyl)-9-phenyl-1H-
pyrrolo[2,3-c]pyrazolo[1,5-a] pyrimidine (4f)
Yield: 42%; m.p.: 197-199 °C; IR (KBr)  (cm-1): 1562
(C=N); MS (EI) m/z: 352 (M+, 23%); 1H NMR (DMSO-d6,
300 MHz)
(ppm): 2.3 (s, 3H, CH3), 2.43 (s, 3H, CH3), 2.7
(s, 3H, CH3), 6.9-7.7 (m, 11H, Ar-H); ,Anal. Calcd for
C23H20N4 (352.44): C, 78.38; H, 5.72; N, 15.90%. Found: C,
78.19; H, 5.90; N, 16.06%.
2.3.7. 2,4-Dimethyl-7-(4-methoxyphenyl)-9-phenyl-1H-
pyrrolo[2,3-c]pyrazolo[1,5-a] pyrimidine (4g)
-1
Yield: 73%; m.p.: 203-205 °C; IR (KBr)  (cm ): 1612
(C=N), 1334 (C-O); MS (EI) m/z: 368 (M+, 26.1%); 1H
NMR (DMSO-d6, 300 MHz)
(ppm): 2.23 (s, 3H, CH3), 2.4
(s, 3H, CH3), 3.8 (s, 3H, OCH3), 7.0-8.2 (m, 11H, Ar-H);
,Anal. Calcd for C23H20N4O (368.44): C, 74.98; H, 5.47; N,
15.21; O, 4.34%. Found: C, 72.17; H, 5.16; N, 15.07; O,
4.57%.
Medicinal Chemistry, 2018, Vol. 14, No. 00 5
2.3.8. 2,4-Dimethyl-7-(3,4-dichlorophenyl)-9-phenyl-1H-
pyrrolo[2,3-c]pyrazolo[1,5-a] pyrimidine (4h)
Yield: 71%; m.p.: 232-234 °C; IR (KBr)  (cm-1): 1616
(C=N); MS (EI) m/z: 406 (M+, 48%); 1H NMR (DMSO-d6,
300 MHz)
(ppm): 2.72 (s, 3H, CH3), 2.88 (s, 3H, CH3),
7.1-8.1 (m, 10H, Ar-H); Anal. Calcd for C22H16Cl2N4
(407.3): C, 65.02; H, 3.94; Cl, 17.24, N, 13.79%. Found: C,
65.26; H, 3.81; Cl, 17.07, N, 13.86%.
2.3.9. 2,4-Dimethyl-7-(1,5-dimethyl-3-oxo-2-phenyl-2,3-
dihydro-1H-pyrazol-4-yl)-9-phenyl-1H-pyrrolo[2,3-
c]pyrazolo[1,5-a]pyrimidine (4i)
Yield: 85%; m.p.: 209-211 °C; IR (KBr)  (cm-1): 1733
(C=O), 1593 (C=N); MS (EI) m/z: 448 (M+, 52.5 %); 1H
NMR (DMSO-d6, 300 MHz)
(ppm): 2.17 (s, 3H, CH3),
2.26 (s, 3H, CH3), 2.3 (s, 3H, CH3), 3.38 (s, 3H, N-CH3),
6.9-7.9 (m, 12H, Ar-H); Anal. Calcd for C27H24N6O
(448.52): C, 72.32; H, 5.36, N, 18.75, O, 3.57 %. Found: C,
72.56; H, 5.13; N, 18.94, O, 3.77%.
2.4. General Procedure for the Synthesis of Compounds
5a-i
A mixture of 2a-i (5 mmol), ethyl cyanoacetate (0.65
mL, 5 mmol) and glacial acetic acid (20 mL) was refluxed
for 5 hrs. The formed solid product after cooling was
collected and recrystallized from ethanol to yield compounds
5a-i.
2.4.1. 2-Amino-7-(4-methylphenyl)-8,9-diphenyl-1H-
pyrrolo[2,3-c]pyrazolo[1,5-a] pyrimidin-4-one (5a)
Yield: 33%; m.p.: 287-289 °C; IR (KBr)  (cm-1): 3437,
3352 (NH2), 3226 (NH), 1728 (C=O), 1588 (C=N); MS (EI)
m/z: 431 (M+, 61%); 1H NMR (DMSO-d6, 300 MHz)

(ppm): 2.51 (s, 3H, CH3), 4.59 (s, 2H, NH2, D2O ex-
changeable), 6.8-7.6 (m, 15H, Ar-H), 8.22 (s, 1H, NH); A-
nal. Calcd for C27H21N5O (431.52): C, 75.18; H, 4.87; N,
16.24; O, 3.71%. Found: C, 74.98; H, 5.08; N, 16.36; O,
3.52%.
2.4.2. 2-Amino-7-(4-methoxyphenyl)-8,9-diphenyl-1H-
pyrrolo[2,3-c]pyrazolo[1,5-a] pyrimidin-4-one (5b)
Yield: 49%; m.p.: 294-296 °C; IR (KBr)  (cm-1): 3482,
3418 (NH2), 3281 (NH), 1703 (C=O), 1666 (C=N), 1307 (C-
O); MS (EI) m/z: 447 (M+, 38%); 1H NMR (DMSO-d6, 300
MHz)
(ppm): 3.65 (s, 3H, OCH3), 4.22 (s, 2H, NH2, D2 O
exchangeable), 6.8 -7.9 (m, 15H, Ar-H), 8.5 (s, 1H, NH);
Anal. Calcd for C27H21N5O2 (447.52): C, 72.48; H, 4.70; N,
15.66; O, 7.16%. Found: C, 72.41; H, 4.56; N, 15.89; O,
7.34%.
2.4.3. 2-Amino-7-(3,4-dichlorophenyl)-8,9-diphenyl-1H-
pyrrolo[2,3-c]pyrazolo[1,5-a] pyrimidin-4-one (5c)
Yield: 38%; m.p.: 171-173 °C; IR (KBr)  (cm-1): 3493,
3405 (NH2), 3271 (NH), 1727 (C=O), 1634 (C=N); MS (EI)
m/z: 485 (M+, 35%); 1H NMR (DMSO-d6, 300 MHz)

(ppm): 4.9 (s, 2H, NH2, D2O exchangeable), 6.7 -7.8 (m,
14H, Ar-H), 8.2 (s, 1H, NH); Anal. Calcd for C26H17Cl2N5O
(486.35): C, 64.33; H, 3.51; Cl, 14.43; N, 14.43; O, 3.29%.
Found: C, 63.98; H, 3.59; Cl, 14.67; N, 14.39; O, 3.44%.
6 Medicinal Chemistry, 2018, Vol. 14, No. 00
2.4.4. 2-Amino-7-(1,5-dimethyl-3-oxo-2-phenyl-2,3-
dihydro-1H-pyrazol-4-yl)-8,9-diphenyl-1H-pyrrolo[2,3-
c]pyrazolo[1,5-a] pyrimidin-4-one (5d)
Yield: 85%; m.p.: 213-215 °C; IR (KBr)  (cm-1): 3394,
3355 (NH2), 3176 (NH), 1730, 1712 (C=O), 1587 (C=N);
MS (EI) m/z: 527 (M+, 73.8%); 1H NMR (DMSO-d6, 300
MHz)
(ppm): 2.27 (s, 3H, CH3), 3.5 (s, 3H, N-CH3), 4.96
(s, 2H, NH2, D2O exchangeable), 7.1 -8.2 (m, 16H, Ar-H),
8.6 (s, 1H, NH); Anal. Calcd for C31H25N7O2 (527.58): C,
70.59; H, 4.74; N, 18.59; O, 6.07%. Found: C, 70.91; H,
4.36; N, 18.28; O, 5.77%.
2.4.5. 2-Amino-7-benzyl-8,9-diphenyl-1H-pyrrolo[2,3-
c]pyrazolo[1,5-a] pyrimidin-4-one (5e)
-1
Yield: 61%; m.p.: 233-235 °C; IR (KBr)  (cm ): 3422,
3385 (NH2), 3310 (NH), 1706 (C=O), 1594 (C=N); MS (EI)
m/z: 431 (M+, 51.6%); 1H NMR (DMSO-d6, 300 MHz)

(ppm): 4.82 (s, 2H, NH2, D2O exchangeable), 5.42 (s, 2H,
CH2), 6.8 -7.9 (m, 16H, Ar-H), 8.42 (s, 1H, NH); Anal.
Calcd for C27H21N5O (431.49): C, 75.17; H, 4.87; N, 16.24;
O, 3.71%. Found: C, 75.43; H, 4.52; N, 16.61; O, 3.54%.
2.4.6. 2-Amino-7-(4-methylphenyl)-9-phenyl-1H-
pyrrolo[2,3-c]pyrazolo[1,5-a] pyrimidin-4-one (5f)
-1
Yield: 67%; m.p.: 237-239 °C; IR (KBr)  (cm ): 3572,
3433 (NH2), 3169 (NH), 1693 (C=O), 1612 (C=N); MS (EI)
m/z: 355 (M+, 52.2%); 1H NMR (DMSO-d6, 300 MHz)

(ppm): 2.43 (s, 3H, CH3), 4.77 (s, 2H, NH2, D2O ex-
changeable), 7.0-8.1 (m, 11H, Ar-H), 8.5 (s, 1H, NH); Anal.
Calcd for C21H17N5O (355.62): C, 70.99; H, 4.79; N, 19.72;
O, 4.51%. Found: C, 70.81; H, 5.06; N, 19.67; O, 4.67%.
2.4.7. 2-Amino-7-(4-methoxyphenyl)-9-phenyl-1H-
pyrrolo[2,3-c]pyrazolo[1,5-a]pyrimidin-4-one (5g)
-1
Yield: 56%; m.p.: 212-214 °C; IR (KBr)  (cm ): 3489,
3403 (NH2), 3183 (NH), 1715 (C=O), 1637 (C=N), 1321 (C-
O); MS (EI) m/z: 371 (M+, 19.5%); 1H NMR (DMSO-d6,
300 MHz)
(ppm): 3.48 (s, 3H, OCH3), 5.09 (s, 2H, NH2,
D2O exchangeable), 6.7 -7.8 (m, 11H, Ar-H), 8.32 (s, 1H,
NH); Anal. Calcd for C21H17N5O2 (371.42): C, 67.92; H,
4.58; N, 18.87; O, 8.63%. Found: C, 68.26; H, 4.69; N,
18.97; O, 8.32%.
2.4.8. 2-Amino-7-(3,4-dichlorophenyl)-9-phenyl-1H-
pyrrolo[2,3-c]pyrazolo[1,5-a]pyrimidin-4-one (5h)
-1
Yield: 50%; m.p.: 282-284 °C; IR (KBr)  (cm ): 3509,
3462 (NH2), 3287 (NH), 1727 (C=O), 1617 (C=N); MS (EI)
m/z: 409 (M+, 27.4%); 1H NMR (DMSO-d6, 300 MHz)

(ppm): 5.2 (s, 2H, NH2, D2O exchangeable), 6.9 -8.1 (m,
10H, Ar-H), 8.4 (s, 1H, NH); Anal. Calcd for C20H13Cl2N5O
(410.26): C, 58.68; H, 3.18; Cl, 17.11; N, 17.11; O, 3.91%.
Found: C, 58.57; H, 3.49; Cl, 16.88; N, 16.98; O, 3.72%.
2.4.9. 2-Amino-7-(1,5-dimethyl-3-oxo-2-phenyl-2,3-
dihydro-1H-pyrazol-4-yl)-9-phenyl-1H-pyrrolo[2,3-
c]pyrazolo[1,5-a]pyrimidin-4-one (5i)
Yield: 81%; m.p.: 272-274 °C; IR (KBr)  (cm-1): 3487,
3422 (NH2), 3190 (NH), 1718,1709 (C=O), 1604 (C=N);
MS (EI) m/z: 451 (M+, 43%); 1H NMR (DMSO-d6, 300
MHz)
(ppm): 2.23 (s, 3H, CH3), 3.4 (s, 3H, N-CH3), 5.0 (s,
2H, NH2, D2O exchangeable), 6.8 -8.0 (m, 12H, Ar-H), 8.3
Fatahala et al.
(s, 1H, NH); Anal. Calcd for C25H21N7O2 (451.48): C, 66.52;
H, 4.66; N, 21.73; O, 7.09%. Found: C, 66.23; H, 4.65; N,
21.84; O, 6.82%.
2.5. General Procedure for the Synthesis of Compounds
6a-i
A mixture of 2a-i (5 mmol), ethyl acetoacetate (0.65 mL,
5 mmol) and glacial acetic acid (20 mL) was refluxed for 5
hrs. The solid product that formed after cooling was collec-
ted and recrystallized from ethanol to yield compounds 6a-i.
2.5.1. 2-Methyl-7-(4-methylphenyl)-8,9-diphenyl-1H-
pyrrolo[2,3-c]pyrazolo[1,5-a]pyrimidin-4-one (6a)
Yield: 68%; m.p.: 263-265 °C; IR (KBr)  (cm-1): 3114
(NH), 1706 (C=O), 1592 (C=N); MS (EI) m/z: 430 (M+,
47.3%); 1H NMR (DMSO-d6, 300 MHz)
(ppm): 2.26 (s,
3H, CH3), 2.4 (s, 3H, CH3), 7.05-7.9 (m, 15H, Ar-H), 8.25
(s, 1H, NH); Anal. Calcd for C28H22N4O (430.53): C, 78.14;
H, 5.12; N, 16.47; O, 4.71%. Found: C, 78.48; H, 5.37; N,
16.72; O, 4.78%.
2.5.2. 2-Methyl-7-(4-methoxyphenyl)-8,9-diphenyl-1H-
pyrrolo[2,3-c]pyrazolo[1,5-a]pyrimidin-4-one (6b)
Yield: 76%; m.p.: 283-285 °C; IR (KBr)  (cm-1): 3219
(NH), 1688 (C=O), 1582 (C=N), 1317 (C-O); MS (EI) m/z:
446 (M+, 26%); 1H NMR (DMSO-d6, 300 MHz)
(ppm):
2.35 (s, 3H, CH3), 3.54 (s, 3H, OCH3), 7.0 -8.0 (m, 15H, Ar-
H), 8.3 (s, 1H, NH); Anal. Calcd for C28H22N4O2 (446.53):
C, 75.34; H, 4.93; N, 12.56; O, 7.17%. Found: C, 75.11; H,
4.86; N, 12.84; O, 7.37%.
2.5.3. 2-Methyl-7-(3,4-dichlorophenyl)-8,9-diphenyl-1H-
pyrrolo[2,3-c]pyrazolo[1,5-a]pyrimidin-4-one (6c)
Yield: 36%; m.p.: 199-201 °C; IR (KBr)  (cm-1): 3196
(NH), 1718 (C=O), 1613 (C=N); MS (EI) m/z: 484 (M+,
62.8%); 1H NMR (DMSO-d6, 300 MHz)
(ppm): 2.28 (s,
3H, CH3), 6.7 -8.0 (m, 14H, Ar-H), 8.4 (s, 1H, NH); Anal.
Calcd for C27H18Cl2N4O (485.36): C, 66.94; H, 3.72; Cl,
14.46; N, 11.57; O, 3.31%. Found: C, 66.65; H, 3.81; Cl,
14.71, N, 11.49; O, 3.69%.
2.5.4. 2-Methyl-7-(1,5-dimethyl-3-oxo-2-phenyl-2,3-
dihydro-1H-pyrazol-4-yl)-8,9-diphenyl-1H-pyrrolo[2,3-
c]pyrazolo[1,5-a]pyrimidin-4-one (6d)
Yield: 77%; m.p.: 215-217 °C; IR (KBr)  (cm-1): 3216
(NH), 1691, 1707 (C=O), 1621 (C=N); MS (EI) m/z: 526
(M+, 51%); 1H NMR (DMSO-d6, 300 MHz)
(ppm): 2.21
(s, 3H, CH3), 2.27 (s, 3H, CH3), 3.4 (s, 3H, N-CH3), 6.8 -7.9
(m, 16H, Ar-H), 8.34 (s, 1H, NH); Anal. Calcd for
C32H26N6O2 (526.59): C, 73.00; H, 4.94; N, 15.97; O, 6.08%.
Found: C, 72.75; H, 5.23; N, 15.82; O, 6.35%.
2.5.5. 2-Methyl-7-benzyl-8,9-diphenyl-1H-pyrrolo[2,3-
c]pyrazolo[1,5-a]pyrimidin-4-one (6e)
Yield: 30%; m.p.: 239-241 °C; IR (KBr)  (cm-1): 3177
(NH), 1712 (C=O), 1609 (C=N); MS (EI) m/z: 430 (M+,
38.4%); 1H NMR (DMSO-d6, 300 MHz)
(ppm): 2.28 (s,
3H, CH3), 5.4 (s, 2H, CH2), 6.9 -7.8 (m, 16H, Ar-H), 8.28 (s,
1H, NH); Anal. Calcd for C28H22N4O (430.50): C, 78.14; H,
5.12; N, 13.02; O, 3.72%. Found: C, 78.02; H, 5.34; N,
13.37; O, 3.41%.
Pyrroles as Anti-depressant Agents
2.5.6. 2-Methyl-7-(4-methylphenyl)-9-phenyl-1H-
pyrrolo[2,3-c]pyrazolo[1,5-a]pyrimidin-4-one (6f)
Yield: 81%; m.p.: 219-221 °C; IR (KBr)  (cm-1): 3184
(NH), 1736 (C=O), 1627 (C=N); MS (EI) m/z: 354 (M+,
39.8%); 1H NMR (DMSO-d6, 300 MHz)  (ppm): 2.31 (s,
3H, CH3), 2.43 (s, 3H, CH3), 6.9-7.9 (m, 11H, Ar-H), 8.2 (s,
1H, NH); Anal. Calcd for C22H18N4O (354.43): C, 74.58; H,
5.08; N, 15.82; O, 4.52%. Found: C, 74.71; H, 5.27; N,
15.61; O, 4.77%.
2.5.7. 2-Methyl-7-(4-methoxyphenyl)-9-phenyl-1H-
pyrrolo[2,3-c]pyrazolo[1,5-a]pyrimidin-4-one (6g)
-1
Yield: 56%; m.p.: 243-245 °C; IR (KBr)  (cm ): 3263
(NH), 1752 (C=O), 1593 (C=N), 1314 (C-O); MS (EI) m/z:
370 (M+, 71%); 1H NMR (DMSO-d6, 300 MHz)  (ppm):
2.3 (s, 3H, CH3), 3.59 (s, 3H, OCH3), 6.8 -8.0 (m, 11H, Ar-
H), 8.34 (s, 1H, NH); Anal. Calcd for C22H18N4O2 (370.43):
C, 71.35; H, 4.86; N, 15.14; O, 8.65%. Found: C, 71.21; H,
4.63; N, 15.37; O, 8.92%.
2.5.8. 2-Methyl-7-(3,4-dichlorophenyl)-9-phenyl-1H-
pyrrolo[2,3-c]pyrazolo[1,5-a]pyrimidin-4-one (6h)
Yield: 46%; m.p.: 271-273 °C; IR (KBr)  (cm-1): 3199
(NH), 1735 (C=O), 1612 (C=N); MS (EI) m/z: 408 (M+,
29%); 1H NMR (DMSO-d6, 300 MHz)  (ppm): 2.31 (s, 3H,
CH3), 6.8 -8.1 (m, 10H, Ar-H), 8.4 (s, 1H, NH); Anal. Calcd
for C21H14Cl2N4O (409.27): C, 61.76; H, 3.43; Cl, 17.16, N,
13.73; O, 3.92%. Found: C, 61.41; H, 3.66; Cl, 17.39, N,
13.62; O, 4.17%.
2.5.9. 2-Methyl-7-(1,5-dimethyl-3-oxo-2-phenyl-2,3-
dihydro-1H-pyrazol-4-yl)-9-phenyl-1H-pyrrolo[2,3-
c]pyrazolo[1,5-a]pyrimidin-4-one (6i)
Yield: 90%; m.p.: 233-235 °C; IR (KBr)  (cm-1): 3244
(NH), 1723, 1741 (C=O), 1590 (C=N); MS (EI) m/z: 450
(M+, 66.4%); 1H NMR (DMSO-d6, 300 MHz)  (ppm): 2.3
(s, 3H, CH3), 2.44 (s, 3H, CH3), 3.5 (s, 3H, N-CH3), 6.9 -
8.0 (m, 12H, Ar-H), 8.4 (s, 1H, NH); Anal. Calcd for
C26H22N6O2 (450.49): C, 69.33; H, 4.89; N, 18.67; O, 7.11%.
Found: C, 68.98 H, 4.67; N, 18.93; O, 7.47%.
2.6. Antidepressant Activity
Animals:Two hundred and twenty albino mice, weighing
18–22g, were involved in this study. The animals were kept
under normal laboratory conditions with controlled tempera-
ture (20 ± 2 °C) and humidity (60%) with regular light cycle
(12 light/12 dark). The animals were adapted one week befo-
re the study and had a free approach to standard laboratory
food and water ad libitum. All experimental procedures were
guided in accord with the ethics for the care and use of labo-
ratory animals in the study and permitted by the local ethics
committee in the Faculty of Pharmacy- Helwan University.
2.6.1. Drugs and Treatment
Each group consisted of ten mice: Control group (Ctrl)
received DMSO as a vehicle, Fluoxetine group (30 mg/kg)
(Flx) was used as positive control [29]. The remaining
groups received the different compounds. The different
compounds and fluoxetine (30 mg/kg, I.P., positive control)
were dissolved in DMSO and administered by intraperito-
neal injection route (I.P.) 30 min before behavioral testing.
Medicinal Chemistry, 2018, Vol. 14, No. 00 7
The groups receiving the synthesized compounds were ad-
ministered an equivalent weight of the compound to the
standard dissolved in DMSO.
2.6.2. Materials
The different compounds were synthesized by organic
chemistry department faculty of Pharmacy Helwan Universi-
ty. Fluoxetine and DMSO were obtained from Sigma-
Aldrich St. Louis, USA.
2.6.3. Methods
2.6.3.1.Tail Suspension Test (TST)
The total duration of immobility prompted by tail sus-
pension was determined according to the method previously
described [30]. Mice both acoustically and visually isolated
were suspended fifty cm above the floor by adhesive tape
placed approximately one cm from the tip of the tail. Immo-
bility time was verified during a six-min period. Mice were
considered immobile only when they hung passively or stay-
ed completely motionless. Conventional antidepressants dec-
reased the immobility time in this test [30-32].
2.6.4. Analysis of Serotonin in Rats Brains Using HPLC
Brains of mice showing positive antidepressant activity
were rapidly removed and kept frozen at -80C. Next day
after the brains were frozen, selected regions were dissected
under visual control as follows: the first cut was made at the
level of the optic chiasm and the second one at the level of
the mammillary body. Then the dissected brain regions were
placed into the Eppendorf tubes and weighed. Afterward,
each brain tissue was homogenized with an ultrasonic cell
disrupter (Vibracell 72434, Bioblock, Illkirch-Cedex) in
150μl 0.1 M perchloric acid containing 0.4 mM sodium meta
bisulphite. The homogenates were then centrifuged at 10.000
x g for 25 min at 4C until analysis. Filtrates were injected
into HPLC system. The HPLC system consisted of a quater-
nary gradient delivery pump Model HP 1050 (Hewlett-
Packard), a sample injector Model 7125 (Rheodyne, Ber-
keley), and analytical column ODS 2c 18, 4.6 x 250 mm,
particle size 5 μm (Hewlett-Packard). The electrochemical
detector model HP 1049 A(Hewlett-Packard) with glassy
carbon working electrode was used at a positive voltage
setting. The concentration of monoamines in each sample
was calculated from the chromatographic peak area and ex-
pressed as ng/g wet tissue [33].
2.6.5. Statistical Analysis
GraphPad prism 5 software was used for statistical analy-
sis of the results, which were presented as means ± standard
errors. Tests of significance between the tested groups were
passed out using one-way analysis of variance (ANOVA)
followed by Tukey-Kramer multiple comparison test. A p
value of <0.05 was considered to be significant.
3. RESULTS AND DISCUSSION
3.1. Chemistry
The aim of this work was to develop new pyrrolo-
pyrazole and pyrroloprazolopyrimidine derivatives; the pyr-
roles derivatives 1a-i were prepared by the same methods as
8 Medicinal Chemistry, 2018, Vol. 14, No. 00 Fatahala et al.

Ph CN
Ph O
CH2(CN)2
B1
X NHAr Base /Reflux X NH2
N


amino ketones Ar
1a-i

Cpd X Ar e Ph Benzyl
a Ph pTolyl f H p-Tolyl
b Ph 4-OCH3-C6H4 g H p-Tolyl
c Ph 3,4-Cl2-C6H3 h H 3,4-Cl2-C6H3
d Ph antipyrinyl i H antipyrinyl

Fig. (2a). Synthesis of aminocyanopyrroles; via the reaction of
-amino ketones and, malononitriles in basic medium.

NH2 NH2
Ph CN Ph
NH2OH.HCl Ph AcOH. AcONa
B2 N
NOH
X N NH2 NH2 NH
X X N
N
Ar
Ar Ar
1a-i
2a-i

Fig. (2b). Predicated mechanism for preparation of pyrrolopyrazole 2a-i.

Ph CN

X NH2
N NHCSNHPh
Ar Ph
N
1a-i Cpd X Ar
NH
X N a Ph 4-CH3-C6H4
AcOH. AcONa
NH2OH.HCl

Ar b Ph 4-OCH3-C6H4
c Ph 3,4-Cl2-C6H3
S CH3
NC 3a-i d Ph antipyrinyl
PH
e Ph CH2-C6H5
N
f H 4-CH3-C6H4
Ph
g H 4-OCH3-C6H4
NH2 CH2(COCH3)2 N CH3 h H 3,4-Cl2-C6H3
Ph N i H antipyrinyl
X N
N
Ar O
NH
X NH2
N 4a-i
N Ph
Ar HN
NCCH2CO2Et N
Ph H3C
2a-i
N CH3
O
N antipyrinyl
X N
H Ar
3 CC
OC
H
2 CO 5a-i CH3
2 Et

HN
Ph
N O
N
X N
Ar

6a-i

Scheme 1.

previously mentioned in our work [34–37], and revealed in Pyrrole derivatives 1a-i were converted into the cor-
Fig. (2a). responding 3-amino-pyrrole[2,3-c]pyrazoles 2a-i via con-
Pyrroles as Anti-depressant Agents
densation with an equimolar hydroxylamine hydrochloride in
glacial acetic acid in the presence of anhydrous sodium ace-
tate [38]. The predicted mechanism for this conversion was
discussed in a study [39], and also revealed in Fig. (2b).
The reaction of 2a-i with phenyl isothiocyanate afforded
thiourea derivatives 3a-i. On treatment of 2a-i with acetyl
acetone, ethyl cyanoacetate and/or ethyl acetoacetate ring
closure occurred and the corresponding pyrrolo[2,3-
c]pyrazolo[1,5-a]pyrimidines 4,5 and 6 were afforded
respectively, as revealed in Scheme 1.
3.2. Biological Results
Mobility values; for twenty examined compounds,
showed that only five compounds; (namely; 2d, 2h, 2i, 4h,
and 5h) showed positive antidepressant activity as revealed
in Table 1.
Table 1. Antidepressant activity of compounds in tail suspen-
sion test.
Compound Antidepressant activity
Fluoxetine positive
2d positive
2h positive
5h positive
2i positive
4h positive
1b,c,d,e,g; 2e,g; 3a,d,e; 4g;5b,e & 6a,e showed no activ-
ity and considered as in active compounds.
Mobility values in the TST for fluoxetine, which was
used as a standard antidepressant drug, and compounds 2d,
2h, 2i, 4h, and 5h were significantly higher than control val-
ues, as revealed in Fig. (3a). Mobility values were shown as
Fig. (3a). Effect of Fluoxetine and of probable antidepressant drugs on the mobility of mice in the tail suspension test. Results were ex-
pressed as mean ± standard error of time in seconds spent in the mobile phase. ANOVA was performed followed by Tukey-Kramer post-hoc
(n=10). (*) significantly different from the control group (Ctrl). (#) significantly different from Fluoxetine administered group (Flx). p<0.05.
Medicinal Chemistry, 2018, Vol. 14, No. 00 9
the average of the total time of mobility. Fluoxetine showed
95% increase in mobility as compared to control animals
while the compounds under investigation 2d, 2h, 2i, 4h, and
5h showed around only 30% increase in mobility. However,
the time was significantly lower matched to Fluoxetine ad-
ministered animals, as revealed in Fig. (3a).
Fig. (3b) illustrates 5-HT concentration in whole brain
homogenate. Serotonin concentrations were significantly
higher in the brains of mice injected with Fluoxetine and
compounds 2d, 2h, 2i, 4h, and 5h compared to the Control
animals. The increase of 5-HT level in fluoxetine adminis-
tered animals was of 66% and the increase in the compounds
under investigation was of 60%, 83%, 127%, 72%, and
155%, respectively compared to control animals. Addition-
ally, 5-HT concentrations in compounds 2i and 5h were sig-
nificantly higher than 5-HT level in the Fluoxetine adminis-
tered animals, as revealed in Fig. (3b).
To discuss the above-mentioned data, several experimen-
tal animal models for depression have been settled to study
depression and the antidepressant effect of novel drugs. One
of these established models is the tail suspension test (TST).
This test involved exposing rodents to aversive stimuli i.e.
suspension by the tail [40]. This procedure results in long-
lasting deficits in the motivation and the capability to escape
the negative stimulus whenever possible. It also led to
behavioural changes that are related to those observed in
depression [41]. The main advantage of TST was that, it
could determine the antidepressant effect of a broad spec-
trum of antidepressants irrespective of their underlying
mechanisms [42].
It was established that reduced periods of immobility or
increased periods of mobility in TST indicate an antidepres-
sant activity in rodents [30,42]. Here, we show that our five
newly synthesized compounds had a clear positive influence
on animals subjected to TST that was comparable to that of
the standard Fluoxetine.
Fluoxetine is an antidepressant from the class of selective
serotonin reuptake inhibitors (SSRIs). Its mechanism of ac-
tion depends on the increase of 5-HT levels in the brain. Fol-
10 Medicinal Chemistry, 2018, Vol. 14, No. 00 Fatahala et al.

Fig. (3b). Effect of Fluoxetine and of probable antidepressant drugs on 5HT levels in brain homogenates of mice. Results were expressed as
mean ± standard error of ng/g wet brain tissue. ANOVA was performed followed by Tukey-Kramer post-hoc (n=10). (*) significantly differ-
ent from the control group (Ctrl). (#) significantly different from Fluoxetine administered group (Flx). p<0.05.
CH3

N Cyclization of 4-Me pyrimidine

also destroy activity for all
Decrease polarity of (2) NH2 by NHCSNHPh Ph
derivatives
adding CSPh qp shifted cpd to be Ph N O
in active NH
N X N
NH
X N Ar
Ar 6
3
NH2
Ph ** Effect ofN-aryl group occured in cpds 2d, 2h, 2i
Ph CN
N Ar= antipyrinyl (2d , 2i)
Ar= dichlorophenyl (2h)
NH increase activity either where X=H or Ph
X N
X NH2 Cyclization shift activity ***In case of ONLY aliphatic ring (Ar= Benzyl ) effect of X show no increase in
N
towards pyrazoles Ar activity
Ar derivatives 2 X=Ph & Ar= Benzyl (2e)
1
CH3
All aminocyano pyrroles
show no activity NH2
N
Ph N
N CH3 Ph
NH N O
H N
NH
4h H N
Ar
5h
Ar= Dichlorophenyl Ar

Pyrimidine ring restore activity ONLY in case of Dichlorophenyl (X= H)

indicated that activity related to haloaryl ring

Fig. (4). Structure-activity relationship for all tested compounds.

lowing this increase in 5-HT levels, positive behavioural CONCLUSION

effects are demonstrated in depressive subjects. 5-HT levels
As stated in our research, we prepared some pyrroles1
measured in animals injected with our novel compounds did
and fused Pyrroles and examined their possible antidepres-
not reach the same level reached by those injected with
sant activity. The biological result revealed that both fused
Fluoxetine. Nevertheless, 5-HT levels in animals who re-
ceived the new compounds were significantly higher than pyrrolo[2,3-c]pyrazoles and pyrrolo[2,3-c]pyrazolo[1,5-a]
pyrimidines showed promising activity, and may be consid-
those in control animals. Therefore, and in addition to the
ered as antidepressant agents, as revealed in Fig. (4). Fur-
behavioural results shown above, we can safely conclude
thermore, this activity may be modulated by a serotonergic
that our compounds have a promising antidepressant effect
pathway, but further investigation is required to characterize
and that this effect may be due to a serotonergic pathway but
the molecular mechanism involved. These outcomes afford
maybe not a serotonin reuptake inhibitory pathway such as
that of the standard Fluoxetine. guidance for the design and structural alterations of these
Pyrroles as Anti-depressant Agents
derivatives for better antidepressant activity, which was im-
portant for the improvement of a novel class of entry inhibi-
tors.
ETHICS APPROVAL AND CONSENT TO PARTICI-
PATE
Not applicable.
HUMAN AND ANIMAL RIGHTS
No Animals/Humans were used for studies that are the
basis of this research.
CONSENT FOR PUBLICATION
Not applicable.
CONFLICT OF INTEREST
We wish to confirm that there are no known conflicts of
interest associated with this publication and there has been
no significant financial support for this work that could have
influenced its outcome. We confirm that we have given due
consideration to the protection of intellectual property asso-
ciated with this work and that there are no impediments to
publication, including the timing of publication, with respect
to intellectual property. In doing so, we confirm that we have
followed the regulations of our institutions concerning intel-
lectual property.
ACKNOWLEDGEMENTS
We like to convey our grateful thanks to prof. Dr. Mos-
sad Said Mohamed (professor of Pharmaceutical Organic
Chemistry) and the staff members of Pharm. Org. Chem.
Dep. (Faculty of Pharmacy, Helwan University) for their
assistance.
SUPPLEMENTARY MATERIAL
Supplementary material is available on the publisher’s
website along with the published article.
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